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Fibrinolytic Serine Proteases, Therapeutic Serpins and Inflammation: Fire Dancers and Firestorms

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The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, “off-the-shelf” system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are the ser ine p rotease in hibitors, termed serpins . Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a “respiratory” coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.
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REVIEW
published: 25 March 2021
doi: 10.3389/fcvm.2021.648947
Frontiers in Cardiovascular Medicine | www.frontiersin.org 1March 2021 | Volume 8 | Article 648947
Edited by:
Christian Schulz,
Ludwig Maximilian University of
Munich, Germany
Reviewed by:
Toni M. Antalis,
University of Maryland, United States
Robert Lindsay Medcalf,
Monash University, Australia
*Correspondence:
Alexandra R. Lucas
arlucas5@asu.edu
Specialty section:
This article was submitted to
Atherosclerosis and Vascular
Medicine,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 02 January 2021
Accepted: 17 February 2021
Published: 25 March 2021
Citation:
Yaron JR, Zhang L, Guo Q, Haydel SE
and Lucas AR (2021) Fibrinolytic
Serine Proteases, Therapeutic Serpins
and Inflammation: Fire Dancers and
Firestorms.
Front. Cardiovasc. Med. 8:648947.
doi: 10.3389/fcvm.2021.648947
Fibrinolytic Serine Proteases,
Therapeutic Serpins and
Inflammation: Fire Dancers and
Firestorms
Jordan R. Yaron1, 2, Liqiang Zhang 1, Qiuyun Guo 1, Shelley E. Haydel 3, 4 and
Alexandra R. Lucas 1
*
1Center for Personalized Diagnostics and Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute,
Arizona State University, Tempe, AZ, United States, 2School for Engineering of Matter, Transport and Energy, Ira A. Fulton
Schools of Engineering, Arizona State University, Tempe, AZ, United States, 3Center for Bioelectronics and Biosensors, The
Biodesign Institute, Arizona State University, Tempe, AZ, United States, 4School of Life Sciences, Arizona State University,
Tempe, AZ, United States
The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine
protease cascades are critical determinants of morbidity and mortality during infection
and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving
(thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex
relationship of sequentially activated serine proteases and their regulatory inhibitors in
the circulating blood. The proteases and inhibitors interact continuously throughout all
branches of the cardiovascular system in the human body, representing one of the
most abundant groups of proteins in the blood. There is an intricate interaction of the
coagulation cascades with endothelial cell surface receptors lining the vascular tree,
circulating immune cells, platelets and connective tissue encasing the arterial layers.
Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic
cascades initiate immune cell responses, representing a front line, “off-the-shelf” system
for inducing inflammatory responses. These hemostatic pathways are one of the first
response systems after injury with the fibrinolytic cascade being one of the earliest to
evolve in primordial immune responses. An equally important contributor and parallel
ancient component of these thrombotic and thrombolytic serine protease cascades are
the serine protease inhibitors, termed serpins. Serpins are metastable suicide inhibitors
with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory
pathways throughout the body. Serpins are now known to also modulate the immune
response, either via control of thrombotic and thrombolytic cascades or via direct effects
on cellular phenotypes, among many other functions. Here we review the co-evolution
of the thrombolytic cascade and the immune response in disease and in treatment.
We will focus on the relevance of these recent advances in the context of the ongoing
COVID-19 pandemic. SARS-CoV-2 is a “respiratory” coronavirus that causes extensive
cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting
in severe and potentially fatal coagulopathies.
Keywords: serpin, thrombolysis, fibrinolysis, coagulation, inflammation, serine protease, infection, virus
Yaron et al. Fibrinolysis, Inflammation and Serpins
INTRODUCTION
Hemostatic control of bleeding by clot formation (thrombosis)
and the subsequent dissolution through fibrinolysis (also termed
thrombolysis) are essential components in the front line response
to trauma (1). In the past decade, intensive research has
revealed that thrombosis and fibrinolysis are extensively involved
in immune pathologies not directly linked to clotting or
hemorrhage, including disorders related to sterile inflammatory
diseases and microbial and viral infections (2). Interactions
between coagulation pathways and the inflammatory immune
response are now known to be essential to maintaining health
and limiting disease.
The interaction between coagulation and inflammation is
bidirectional, a “two-way street,” and one begets the other
(3). Coagulation is used here to refer to thrombosis (clot
formation) and thrombolysis (clot breakdown). It is well-
understood that unregulated clotting or bleeding can have
severe adverse consequences. Too much clotting causes occlusion
of circulating blood (e.g., blocking the circulation), while
too much fibrinolysis leads to hemorrhage and blood loss,
and an excess of either can be fatal. To further complicate
this interaction, in some cases severe vascular damage or
infection causes excess thrombosis, consumption of clotting
factors and eventual deficit in the homeostatic balance and
excess fibrinolysis. This consumptive coagulopathy (CC) or
disseminated intravascular coagulation (DIC) markedly increases
mortality in viral and bacterial sepsis. Unregulated inflammation
also causes severe tissue disruption, endothelial damage,
microthrombotic occlusions, vascular leakage, hemorrhage, and
shock with death. Elucidation of the cross-talk of these
pathways (termed “thromboinflammation”) makes eminently
clear that inflammation can cause clotting, and clotting can cause
inflammation, thus the regulation of one pathway affects the
other (4).
Serine proteases are highly active and one of the most
prevalent protease classes, driving the thrombotic and
thrombolytic cascades. Dysregulation of these coagulation
pathway proteases leads to onset and/or exacerbation of
numerous diseases, including rare bleeding disorders, chronic
lung disease, septic shock (whether viral, bacterial, or fungal in
origin), DIC (or CC), and neurodegeneration. The thrombotic
and thrombolytic cascades are intrinsically regulated by the
serine protease inhibitor (serpin) superfamily (5). Recent
investigations have identified numerous immune modulating
functions for serpins, clearly demonstrating that these complex
inhibitors directly interact with and influence immune cell
responses and regulate inflammation beyond direct effects on
the thrombotic and thrombolytic pathways (6). Thus, a complete
understanding of the role of the thrombotic and thrombolytic
proteases, and the serpins that regulate their function in the
circulating blood, may lead to novel therapeutic avenues for
treating a diverse array of immunopathologies.
The role of the thrombotic pathway in inflammation has
been extensively highlighted in numerous reviews (4,710). In
this review, we will discuss the interaction of the fibrinolytic
pathway with inflammatory responses and the bidirectional
regulation of these responses, both fibrinolytic and inflammatory,
by serpins. We begin with an exploration of the evolutionary
roots of the coagulation-associated pathways, both thrombotic
and thrombolytic, and the serpins that regulate these pathways,
as evidence for their origins in primordial immune responses. We
then focus specifically on the fibrinolytic/thrombolytic pathway,
the interaction of serine protease activity in fibrinolysis and
inflammation, and their contributing roles in disease. Next, we
discuss the evidence for utilizing serpins as therapeutics designed
to modulate the fibrinolytic response in disease. We will conclude
with a brief discussion of the fibrinolytic pathway and serpins
in the pathogenesis (and potentially treatment) of the ongoing
COVID-19 pandemic caused by SARS-CoV-2.
IMMUNE ORIGINS AT THE ANCIENT
ROOTS OF COAGULATION PROTEASES
AND SERPINS
Thrombosis and
Thrombolysis–Evolutionary Roots
Serine proteases involved in coagulation are functionally
conserved across the Kingdom Animalia and represent
an ancient class of proteins. Emerging evidence suggests
that independent evolution has occurred for at least
two separate functions for these pathways: (i) control of
thrombotic/thrombolytic responses and (ii) regulation of
the immune response. While some pro-thrombotic (clotting)
enzymes appear to have emerged as early as 700 million years ago,
the genes and proteins required for the conversion of fibrinogen
to fibrin did not appear until 500–600 million years ago (11).
This timeline appears to have coincided with the emergence of
fibrinolysis 570 million years ago (in the Precambrian period)
(12). While much has been discovered in the developmental
biology-based study of clotting across Animalia, the clotting
“toolkit” has been found to differ greatly amongst animals.
Exploration of the ancient roots of clotting now reveals that the
coagulation pathways may originally have had central roles in
innate immune responses, or inflammation (Figure 1).
Mammals (Class Mammalia) harbor the most complex
coagulation system, with the classically defined contact activation
or “intrinsic” pathway and the tissue factor/factor VII system
or the “extrinsic” pathway (13,14). The pathways converge,
resulting in a complex downstream cascade of protease activation
events leading to the activation of factor X and thrombin,
conversion of fibrinogen to fibrin, and ultimate generation of
a stable fibrin clot (Figure 1). In the vascular system, thrombi
form on the surface of activated platelets, damaged endothelium
in the lining of the arterial wall, and activated macrophage
cells adherent to damaged endothelium. This entire interactive
complex both activates and is activated by the kallikrein-
kinin system. While apparently important to the activation of
thrombosis, deficiency of this pathway affects thrombosis and
modifies immune responses.
A careful examination of other animals reveals a distinct
role for coagulation in the immune response. For example,
despite the presence of components of the kallikrein-kinin system
Frontiers in Cardiovascular Medicine | www.frontiersin.org 2March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
FIGURE 1 | The thrombotic and thrombolytic cascades and primordial immune response. The thrombotic pathways (intrinsic and extrinsic) and the thrombolytic
(fibrinolysis) pathway involve a complex cascade of protease activation. Solid arrows indicate the conversion to an active protease, while dotted line arrows indicate
the activity of the activating upstream protease. A variety of inhibitors are shown, with serpin inhibitors denoted by a serpin protein structural image. Examples of early
primordial immune response origins are noted in context of the pathways. MYA, million years ago.
in birds (Class Aves), amphibians (Class Amphibia) and fish
(Class Actinopterygii), evidence suggests these components do
not drive clot formation, but rather, regulate angiogenesis and
the immune response system (Figure 1). The process of clotting
in these Classes is regulated by the extrinsic tissue factor-
directed pathway (15). Looking further, lower-level animals
(e.g., invertebrates) also contain mechanisms for regulating
clotting. Due to their open circulatory system and propensity
for massive loss of hemolymph (the equivalent of combined
blood and lymphatic fluid), clotting evolved very efficiently in
insects (Class Insecta) and is central to innate immunity in
Drosophila (16). Class Ascidiacea, which include sac-like marine
invertebrate filter feeders, is among the most ancient coagulation
systems investigated. While their plasma contains some blood
clotting components (such as von Willebrand factor, vWF),
blood in Ascidacea animals does not clot, and these components
are predominantly used to regulate innate immune responses
(Figure 1) (17,18).
A similar primordial role in innate immunity can be found
for fibrinolysis, a cascade that balances and is complementary
to the coagulation cascade which is responsible for dissolution
of a fibrin clot. In Mammalia, fibrinolysis is initiated by
the conversion of inactive, circulating plasminogen into active
plasmin by serine proteases referred to as the plasminogen
activators, tissue- and urokinase-type plasminogen activators
(tPA and uPA, respectively) (Figure 1). Activated plasmin then
breaks down cross-linked fibrin, resulting in dissolution of the
thrombus. Investigations into lower-level organisms distinctly
reveal the immune response origins of the thrombolysis pathway.
For example, Caenorhabditis elegans (Class Chromadorea),
despite its lack of vasculature, blood, or hemolymph, expresses
a functional plasminogen-like protease required for organ
development and innate immunity (19,20). Thus, fibrinolysis
represents another component of the primordial innate immune
response that has been preserved for millions of years.
Serpins–Evolutionary Roots
As counterparts to the proteases that mediate fibrinolysis, serpins
are the equally-ancient, intrinsic protease inhibitors that arose
in Animalia some 650–700 million years ago. Serpins are
highly metastable proteins characterized by two key structural
components: a reactive center loop (RCL) and a 4-stranded, core
beta-sheet (termed the “A” beta-sheet). The reactive center loop
contains a protease recognition sequence which acts as a bait for
activated serine proteases (Figure 2). Cleavage of the protease
recognition sequence by the appropriate protease creates a
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Yaron et al. Fibrinolysis, Inflammation and Serpins
FIGURE 2 | Serpin-dependent mechanism of protease inhibition. (A) Natively
folded serpins present a reactive center loop (RCL) which acts as a bait for
interaction with active serine proteases. Upon association, initiation of
proteolytic activity by the protease triggers the formation of a Michaelis
complex and covalent bonding of the serpin with the protease. (B) The primary
outcome of Michaelis complex formation is reconfiguration of the serpin, with
the RCL inserting as the third of five strands in the core beta sheet and
permanent denaturation of the target serine protease. (C) The secondary and
less frequent outcome is completion of proteolytic activity and dissociation of
the active serine protease, while the RCL continues to insert as the third of five
strands in the serpin core beta sheet.
transient Michaelis complex, as occurs in any generalized
enzyme-substrate interaction, where the protease and the serpin
are covalently bonded at the active site of the protease (Figure 2).
Upon cleavage of the recognition site, the metastability inherent
in the serpin is released and the serpin-protease pair initiates
a dramatic (on a molecular scale) conformational change
(Figure 2). The reactive center loop “swings” 70 angstroms
across the protein and inserts itself as the third strand in a
now five-stranded beta sheet. The conformational change of the
serpin induces a deformation of the active site in the protease,
disallows completion of the protease-substrate interaction, and
permanently denatures both the serpin and protease in what is
referred to as a “suicide complex” (Figure 2). Because serpins are
tuned to their inhibitor by changes in the protease recognition
sequence in their RCL, multiple gene duplications have resulted
in the ability for serpins to become tailored to a wide variety
of proteases. Interestingly, in some cases individual serpins have
lost their canonical protease inhibitory function, as in the case of
Maspin, which does not form suicide complexes upon protease
digestion with any tested protease and which is associated with
tumor suppression by a mechanism that is still poorly defined
(21). Accordingly, the genomes of most members of Animalia
contain a multitude of serpins with human and mouse genomes
encoding 37 and 60 serpins, respectively (22,23). At the other
end of the evolutionary spectrum, the genome of Caenorhabditis
elegans encodes nine serpins (24).
The evolutionary origins of immune regulation by serpins
are exemplified by Drosophila. Persephone is a circulating
serine protease in Drosophila upstream of the fly Toll pattern
recognition receptor pathway and activates the protease Spätzle
(25). Deficiency in the Spn43Ac serpin, which regulates
Persephone, leads to constitutive activation of Spätzle
in the Drosophila innate immune response and results in
developmental lethality (the nec phenotype) (25). Thus, both
serine proteases and their serpins have evolutionary roots in
regulating inflammation and the innate immune response.
OVERVIEW OF THE FIBRINOLYTIC
MACHINERY
Serine Proteases of the Fibrinolysis System
Fibrinolysis is ultimately mediated by the activity of the serine
protease plasmin (Figure 3). Initiation of fibrinolysis requires
the conversion of the inactive zymogen plasminogen to active
plasmin, which then subsequently breaks down cross-linked clots
(Figure 3). Plasminogen is predominantly synthesized in the liver
and secreted at a plasma concentration of 150 µg/mL (26).
Plasminogen is converted by plasmin primarily by the activity
of two plasminogen activators, tPA and uPA, as discussed in
the next paragraph. Forward feedback of plasmin activity on its
own activators results in increased processing of plasminogen
to accelerate the generation of additional active plasmin (27).
Plasmin activation occurs in situ when plasminogen co-localizes
with its activators in a “ternary complex” at C-terminal lysine
residue binding sites on fibrin (28). Active plasmin then directly
cleaves the cross-linked fibrin to dissociate the clot. Plasminogen
coordination at the cell membrane, and therefore localized
generation of plasmin at cell surfaces, is mediated by a number of
receptors, with the predominant plasminogen receptor being Plg-
RKT (29,30). Plg-RKT is expressed by a wide variety of cell types
in all tissues, including migrating immune cells, and co-localizes
with the receptor for uPA (discussed below) (29). The expression
of Plg-RKT enhanced plasminogen activation by more than 12-
fold, in part by coordinating its localization with uPA and tPA at
the cell surface (29).
The serine proteases tissue- and urokinase-type plasminogen
activators, tPA and uPA, respectively, are the key components
of the plasminogen activation system. While tPA and uPA share
only about 40% amino acid similarity, their basic structure is
highly similar (31). The basal circulating levels of tPA and uPA
are low compared to other circulating proteins, with tPA reported
in the range of 1–10 ng/mL and uPA at 2–10x lower levels
(0.1–0.3 ng/mL) (3236). Synthesis of tPA occurs in abundance
in both the vasculature and the central nervous system. In the
nervous system, tPA is synthesized and released by neurons and
glial cells and is constitutively active in a number of regions
of the brain where its activity has been associated with neural
plasticity (37). tPA has been identified in secretary vesicles after
membrane depolarization and is rapidly localized to neuronal
synapses (38). Further studies have identified that the activity
of tPA is essential for the late phase of long-term potentiation
and is a driver of synaptic growth (39). In the vasculature,
synthesis of tPA occurs predominantly in endothelial cells
and is stored in granules called regulated secretory organelles
(RSOs). In vitro and in vivo experiments indicate that RSOs are
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Yaron et al. Fibrinolysis, Inflammation and Serpins
FIGURE 3 | Canonical signaling of the fibrinolysis pathway. Fibrinolysis is characterized by the degradation of a fibrin clot into degradation products by plasmin.
Plasmin is generated from plasminogen by uPA and tPA. Several serpins and other inhibitors provide a tight regulation of this cascade. Substantial promiscuity exists
across multiple elements of the pathway, providing redundant controls against inappropriate activation. AIIt and uPAR are shown as representative canonical
fibrinolytic receptors for brevity. AIIt, Annexin II tetramer; PAI-1,2,3, Plasminogen Activator Inhibitor-1, 2, 3; PN-1, Protease Nexin-1; TAFI, Thrombin activatable
fibrinolysis inhibitor; tPA, Tissue-type plasminogen activator; uPA, Urokinase-type plasminogen activator; uPAR, Urokinase-type plasminogen activator receptor.
trafficked and secreted rapidly in response to diverse physiologic
stimuli, such as histamine (40), activated thrombin (41) and
bradykinin, a metabolic product of the kinin-kallikrein system
(42). While secreted tPA is active in the open circulation, its
plasmin generation activity is enhanced by interaction with its
receptor, the annexin A2 heterotetramer (AIIt), composed of two
units of annexin A2 bound by a dimer of S100A10 (43). Binding
of tPA to AIIt enhanced plasmin generation by 77-fold and
mice deficient in S100A10, and therefore deficient in functional
AIIt receptors, have 40% reductions in plasmin generation
and reduced clearance of batroxobin-induced vascular
thrombi (43,44).
In contrast to the activity of tPA, uPA functions
predominantly in innate, inflammatory immune responses
and the tissue responses to injury, rather than the coagulation
response (45). uPA is synthesized in a wide variety of tissues and
cell types including vascular endothelial cells (46), hepatocytes
(47), keratinocytes (48), renal tubular epithelial cells (49),
neurons (50), and immune cells of both monocytic (51) and
lymphocytic (52) lineages. Basal release of uPA is in the form of
a single-chain zymogen with little or no proteolytic activity. uPA
is further processed to a double-chain active enzyme that has a
several hundred-fold higher activity by plasmin (53), kallikrein
(54), activated factor XII (54), or trypsin-like proteases, for
example, as released from tumors (55). While uPA can act in the
open circulation, its enzymatic efficiency substantially increases
by interaction with the urokinase-type plasminogen activator
receptor (uPAR, also called CD87) (56). Interestingly, the activity
of uPA itself is partially lowered by interaction with uPAR,
but complex local coordination of uPA and its substrates (e.g.,
plasminogen) results in a net total increase in processivity by
means of concentration and spacial protein-protein coordination
effects (57). Interestingly, uPAR interacts promiscuously with
components of the kallikrein pathway, FXII and AIIt, suggesting
a broader, yet unexplored, role in regulating fibrinolytic and
immune responses (43,58,59).
In addition to their function in fibrinolysis, plasmin, tPA,
uPA and their receptors—Plg-Rs, the LDL receptor-related
protein-1 (LRP1), and uPA receptor (uPAR), respectively,—have
important functions in tissue remodeling and cell invasion.
These thrombolytic proteases alter the extracellular matrix
and modify cellular phenotype conversion via induction of
intracellular signaling cascades. Thus far there have been 12 Plg-
Rs identified, some of which are expressed on the cell surface
and others are localized intracellularly (60). The dominant Plg-
R expressed by macrophages is the plasminogen receptor Plg-
RKT, which is a surface expressed receptor where plasminogen
binds and is activated (61). The activation of plasminogen
when bound to the macrophage-expressed receptor is required
for efficient invasion and clearance of dead cells (62,63).
uPAR is moderately expressed in most tissues in a healthy
organism, including the lungs, kidneys, spleen, blood vessels,
uterus, bladder, thymus, heart, liver, and testes (64). However,
uPAR expression is strongest in tissue actively undergoing
extensive remodeling (65). For example, keratinocytes at the
migrating edge of cutaneous wounds exhibit potent upregulation
of uPAR and wounds heal poorly in uPAR-deficient mice (66,67).
Upon immunological activation, neutrophils, monocytes and T
cells markedly upregulate uPAR expression (6870). Exposure
of uPAR to uPA enhances the differentiation of monocytes
to macrophages (71). Expression of uPAR also dictates the
interaction of macrophages and neutrophils in efferocytosis (the
clearance of apoptotic cells), as well as phagocytosis of viable cells
by macrophages. Deficiency of uPAR in either macrophages or
viable neutrophils enhances phagocytosis, but deficiency of uPAR
in both cell types blocks phagocytosis (72). Independent of its
fibrinolytic activity, recombinant uPA elicits an anti-apoptotic
response in cultured endothelial cells by specific induction of the
X-linked inhibitor of apoptosis (XIAP) protein (73). Similarly,
uPA attenuates macrophage apoptosis induced by Ox-LDL and
ER stress by activation of ERK1/2 and downregulation of
Bim (71). Likewise, recombinant tPA dose-dependently rescued
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Yaron et al. Fibrinolysis, Inflammation and Serpins
cultured neurons from serum deprivation-induced apoptosis via
a mechanism involving the PI-3 kinase pathway (74). Thus, serine
proteases of the fibrinolytic cascade have essential fibrinolysis-
independent functions in tissues and the immune system.
Regulators of Fibrinolysis in Mammals
Serpins have evolved into a large class of regulatory proteins
with extensive functions throughout circulating hematological
and immune pathways in a wide range of organ systems from
the cardiovascular tree to endocrine and neurological organs
(75). As our knowledge of serpin sequences and structures
have progressed, it is evident that widespread exchanges and
combinations enabled serpins to target more than one pathway.
As serpin functionality has evolved, some serpins retained
immune regulating functions while concurrently expanding to
target the thrombolytic cascades. In this section, we will discuss
the known regulators of the thrombolytic/fibrinolytic cascades
and in subsequent sections discuss their additional capacity
to cross interaction and regulate immune and inflammatory
responses with potential for providing new therapeutic reagents.
Serpin-dependent regulation of the fibrinolytic serine
proteases in mammals is mediated by the plasminogen activator
inhibitor (PAI)-1 (SERPINE1), PAI-2 (SERPINB2), and PAI-3
(SERPINA5; also called Protein C inhibitor, PCI) and protease
nexin-1 (PN-1 or SERPINE2) against uPA and tPA, and by
alpha-2-antiplasmin (SERPINF2) against activated plasmin. tPA
is also regulated by a central nervous system-specific serpin,
neuroserpin (SERPINI1). Two other serpins, alpha-1-antitrypsin
(AAT or A1AT, SERPINA1) and Complement C1 inhibitor
(C1INH, SERPING1), target fibrinolytic proteases but are
better known for inhibition of other protease and inflammatory
systems outside of the coagulation pathways will be briefly
discussed.
Classical serpin-mediated inhibition of fibrinolytic serine
proteases leads to permanent inactivation of both the serpin and
protease via formation of a classic suicide complex (described
above). Thus, tight control of fibrinolysis requires that circulating
serpins are present in molar excess, or pre-synthesized and
rapidly released from stores, without the need for transcription
and translation. Accordingly, serpins account for up to 10% of
circulating proteins in the circulation (76).
PAI-1, the principal member of the PAI protein family,
is primarily produced by hepatocytes and secreted into the
circulation by the liver. To a lesser extent, PAI-1 is synthesized
and secreted by the kidney, spleen, heart, lung and adipose
tissues (77). Additionally, circulating platelets continuously
synthesize PAI-1, which is actively and rapidly released upon
platelet activation and contributes to the stability of clots by
limiting fibrinolysis (78). Circulating PAI-1 is usually present
in a concentration range of 20–30 ng/mL, which is in three-
fold excess of basal circulating tPA and up to 300-fold excess
of basal circulating uPA (79). PAI-2 expression is restricted to
keratinocytes, macrophages, activated monocytes, the placenta,
and adipocytes (80). Circulating PAI-2 in healthy individuals is
essentially undetectable, but drastically increases in pregnancy
to over 250 ng/mL and rapidly declines postpartum (81). The
skin is a major site for PAI-2 expression, where PAI-2 cross-
links to the cornified cell membrane via transglutaminase during
the terminal differentiation of keratinocytes to inhibit over-
proliferation (82,83). PAI-2 has been called the “undecided
serpin” because its specific endogenous biological role has
remained elusive, despite associations with regulating fibrinolysis
and inflammation (8486). PAI-1 is an efficient inhibitor of both
uPA and tPA (2 ×107M1s1each) while PAI-2 effectively
inhibits uPA (2 ×106M1s1) and is a poor inhibitor of tPA
(2 ×105M1s1) (87,88). PCI is predominantly an inhibitor
of proteases in the thrombotic pathway, but detection of kidney-
derived PCI complexed with uPA in the urine resulted in its
identification as the third member of the PAI family as PAI-
3 (89). Further justification for designating PCI as PAI-3 is
the ability to inhibit plasma kallikrein and activated Factor XI
(90), which are alternative activators of plasminogen (91,92).
Subsequent discussion of the roles of PAI serpins in disease will
focus on PAI-1, as it is the principal serpin inhibitor of the
fibrinolytic cascade.
Protease nexin-1 (PN-1 or SERPINE2) is expressed in diverse
tissues during development, including cartilage, lung, skin, the
urogenital tract and the nervous system, where it was originally
identified as Glia-derived nexin (93). PN-1 is nearly undetectable
in circulating plasma, amounting to 1 ng/mL or 20 picomolar
amounts (94). In contrast to the low levels in circulation,
PN-1 is endogenously synthesized and stored in abundance
in the alpha-secretory granules of platelets, from which it is
rapidly released upon platelet activation (95). As the second-
order rate constant of PN-1-tPA interaction is three orders
of magnitude lower than PAI-1-tPA, it was expected that PN-
1 is not the primary inhibitor of fibrinolysis (96). However,
systematic in vivo studies by Boulaftali et al. demonstrated
that PN-1 inhibits both fibrin-bound tPA and auto-activation
of plasminogen by fibrin-bound plasmin and is an important
regulator of fibrinolysis (97).
Alpha-2-antiplasmin (SERPINF2) is the major serpin
inhibitor of activated plasmin. Alpha-2-antiplasmin
is synthesized in the liver and kidney at nearly
equivalent levels (98). Alpha-2-antiplasmin is present
at significantly higher concentrations than the PAI
serpins, with circulating levels at 70 µg/mL (99,100).
Thus, alpha-2-antiplasmin, in coordination with
the PAI family and PN-1, mediate the multi-stage,
tightly controlled serpin-dependent regulation of the
fibrinolytic proteases.
Neuroserpin (SERPINI1) is expressed predominantly in
the central nervous system and was originally isolated from
chicken ventral spinal cord neurons (101). Neuroserpin, a
highly specific inhibitor of tPA, is expressed from the growth
cone of neurons and is a poor inhibitor of uPA and plasmin
(102). Neuroserpin is found at a concentration of 7.4 µg/L
in the cerebrospinal fluid (CSF) and is significantly elevated
in the CSF of patients with Alzheimer’s Disease (103). Indeed,
numerous neuropathologies are associated with dysregulation
of neuroserpin. For example, familial encephalopathies with
neuroserpin inclusion bodies are associated with mutations such
as S49P-Syracuse (late onset encephalopathy) and S52R-Portland
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Yaron et al. Fibrinolysis, Inflammation and Serpins
(early onset encephalopathy) (104). Polymers of misfolded
neuroserpin stimulate inflammation via NF-kappaB and
oxidative stress signaling in an unfolded protein response-
independent manner and may contribute to neurodegeneration
(105,106). The spatiotemporal patterning of neuroserpin
suggests a role in neuronal development and synaptogenesis via
homeostatic maintenance of tissue by limiting excess tPA activity
which can lead to cerebral ischemia and epilepsy (104). The
critical and sensitive role of neuroserpin in regulating tPA activity
outside of the circulating blood highlights the importance of
effective control of these serine proteases in diverse tissues.
C1INH and A1AT act primarily as regulators of complement
and neutrophil elastase, and mutations in both of these serpins
cause severe genetic disorders (107,108). C1INH deficiency
causes angiogenic edema which can be life threatening and
A1AT deficiency resulting from aggregating mutations which
deplete circulating levels of A1AT causes severe lung damage
and emphysema. Both serpins are believed to primarily regulate
serine proteases outside of the coagulation cascades and have
established inhibitory functions for uPA and plasmin (109,110).
In addition to serpin-dependent regulation of fibrinolysis,
there are other non-serpin regulators/inhibitors of fibrinolysis.
Thrombin-activatable fibrinolysis inhibitor (TAFI; also called
carboxypeptidase B2, CPB2) is a non-serpin negative inhibitor
of plasmin activity and acts as the terminal enzyme of the
thrombotic cascade. TAFI is synthesized by the liver and
megakaryocytes as an inactive zymogen (111,112) and is
processed to the functional form by thrombin, the thrombin-
thrombomodulin complex, or plasmin (113115). TAFI is
secreted at circulating concentrations of 4–15 µg/mL (116).
Processed TAFI exerts its anti-fibrinolytic effect by cleaving the
C-terminal lysine residues which act as the plasminogen-binding
site mediating plasminogen-to-plasmin conversion (117). Thus,
active TAFI reduces fibrinolysis by suppressing the in situ
activation of plasmin.
Alpha-2-macroglobulin (A2M) is a large (720 kDa) broad-
spectrum inhibitor of an expansive array of proteases across
all catalytic classes, including trypsin, chymotrypsin, thrombin,
plasmin, kallikrein, uPA, cathepsins, papain, and matrix
metalloproteinases among others (118). Accordingly, A2M can
act as an inhibitor of both thrombosis and fibrinolysis. A2M
is primarily synthesized in the liver, but in vitro experiments
indicate that cultured cells from the lung as well as macrophages
and microglia can also synthesize and secrete A2M (119121). In
the fibrinolytic cascade, A2M inhibits the activity of plasmin and
its upstream activator uPA (122).
FIBRINOLYSIS PATHWAY-ASSOCIATED
SERPINS AS THERAPEUTICS IN
INFLAMMATORY DISEASE
The dysregulation of fibrinolytic signaling is now identified
as an important component of numerous pathologies. Genetic
deficiencies in fibrinolytic regulation lead to bleeding disorders,
organ dysfunction, and damage, and acquired disorders cause
tissue fibrosis, dysregulated bleeding, cirrhosis, amyloidosis, and
certain cancers (123). Several recent reviews discuss the role of
the fibrinolytic system in inflammation and the immune response
(124,125). Similarly, small molecules inhibitors of fibrinolytic
serine proteases such as tranexamic acid are under investigation
for therapeutic modulation of the fibrinolytic processes that are
associated with effects on the immune response (126). However,
the highly evolved and potent inhibitory mechanisms of serpins
have led to a growing interest in using serpins themselves as
therapeutics. In the following section, we will discuss examples
of the therapeutic use of fibrinolysis pathway-associated serpins,
limiting our overview to examples which have been tested in
preclinical models.
PAI-1: Therapeutic Applications
Considering the delicate balance of the canonical targets of
PAI-1—tPA and uPA—and the detrimental consequences of
their dysregulation after injury, PAI-1 is a natural choice
for therapeutic modulation of fibrinolysis dysregulation and
has been demonstrated as such in numerous studies. PAI-
1 is frequently seen as a mediator of injury and has been
experimentally targeted to limit disease, especially cancer (127).
The use of PAI-1 as a therapeutic at first may be unexpected.
A large portion of studies investigating the delivery of PAI-1
for therapeutic purposes center on the cardiovascular system
where thrombotic occlusion of infarction is treated with tPA, a
target for PAI-1. However, PAI-1 has been extensively studied
in numerous preclinical non-thrombotic animal disease models
with demonstrated benefit. Unexpectedly, Carmeliet et al.
demonstrated that adenovirus-mediated gene transfer of human
PAI-1 in mice prior to induction of electric or mechanical
vascular injury of the femoral or carotid arteries, respectively,
reduced arterial neointima formation, a precursor to occlusive
arterial plaque (128). This finding was supported by work from
Schäfer et al. showing that bone marrow-derived PAI-1 reduces
neointimal formation and luminal stenosis in bone marrow
transplantation after carotid injury with ferric chloride (129).
In subsequent work, Wu et al. demonstrated that recombinant
PAI-1 prevented intimal hyperplasia in a model of carotid artery
injury in rats, thus further supporting a potential therapeutic
role for PAI-1 to prevent vascular restenosis (130). Interestingly,
their systematic investigation involving constitutively active,
inhibition-defective, and vitronectin binding-deficient forms of
PAI-1 demonstrated that the ability to therapeutically limit
intimal hyperplasia was mediated by either the ability for the
serpin to inhibit proteases, or to bind to vitronectin, but did
not require both. Zhong et al. similarly demonstrated that
recombinant PAI-1, differentially through binding to vitronectin
or protease inhibitory activity, mediates a therapeutic reduction
of cardiac fibrosis in a model of cardiac fibrosis in uni-
nephrectomized mice fed a high salt diet and angiotensin II
(131). Using an adenovirus-5 (Ad5) with a CMV promoter, Qian
et al. found that overexpression of human PAI-1 protected ApoE-
deficient mice from abdominal aortic aneurysm induced by
angiotensin II when delivered directly into the perivascular tissue
of the aorta, but not when delivered systemically by tail-vein
injection (132). Ad5-mediated gene transfer of human PAI-1 was
also shown by Heymans et al. to preserve pump function in mice
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Yaron et al. Fibrinolysis, Inflammation and Serpins
after acute pressure overload by attenuation of left ventricular
remodeling (129). In a highly rational translation of its natural
function, Jankun et al. reported that a modified version of PAI-
1 with a half-life increased from 2 h to more than 700 h (very
long half-life, termed VLHL PAI-1) was therapeutically effective
at promoting hemostasis in reducing total blood loss induced by
tail clipping when given by systemic or topical administration in
mice (133).
PAI-1 has also demonstrated therapeutic efficacy in models
outside of the conventional cardiovascular system. Yang et al.
identified a role for tPA in the breakdown of the blood-
brain barrier during neonatal cerebral hypoxia-ischemia in rats
(134). Administration of recombinant PAI-1 dose-dependently
preserved brain tissue and reduced edema, axonal degeneration
and cortical cell death. The same group further adapted the
treatment to an intranasal delivery format, which reached the
cerebral cortex and reduced 75% of brain atrophy in hypoxic-
ischemic brain injury of newborns and in lipopolysaccharide-
sensitized hypoxic-ischemic brain injury (135). Swiercz et al.
demonstrated that exogenous delivery of the 14-1b active mutant
of PAI-1 with an extended half-life limited angiogenesis and
LNCaP prostate cancer tumor growth in SCID mouse xenografts
when delivered by continuous infusion with subcutaneously
implanted osmotic pumps (136). Praus et al. reported that
liver adenoviral delivery of PAI-2, but not PAI-1, reduced the
incidence of lung and brain liver tumor metastases but did not
increase mouse survival (137).
PAI-1 therapy may also have a role in treating non-sterile
conditions, as the pro-inflammatory functions of PAI-1 are
found to be critical in numerous animal models of difficult-
to-treat infections (138140). Renckens et al. identified an
essential role for PAI-1 in the host response to the respiratory
pathogen Klebsiella pneumoniae, a Gram-negative bacteria,
demonstrating an enhanced immune response, reduced lethality
and prevention of sepsis and distal organ injury in mice
transgenically overexpressing human PAI-1 delivered by Ad5
vector (141). Interestingly, the authors found that intranasal Ad5
delivery of PAI-1, but not Ad5 delivery alone in healthy mice also
induced pulmonary inflammation and suggested that increased
PAI-1 levels in the lungs may prime a protective inflammatory
response in the context of infection.
PN-1: Therapeutic Applications
PN-1 is expressed by many tissues in the body and thus
exhibits broad potential regulatory functions outside of a role
in modulating fibrinolysis in the circulation. Several features
of PN-1 function have been translated to a potential for
therapeutic development. Activation of fibrinolytic machinery,
such as uPA and tPA, in tissues activates downstream matrix
metalloproteinases (MMPs), leading to degradation of collagen
and elastin in the extracellular matrix architecture (142).
Stevens et al. demonstrated that intraarticular administration of
recombinant PN-1 prevents articular cartilage degradation in
rabbits subjected to interleukin-1 beta/basic fibroblast growth
factor insult (143). Curiously, McKee et al. discovered that the
PN-1 can engage the canonical serpin-enzyme complex receptor,
lipoprotein receptor-related protein-1 (LRP-1), in the form of
a PN-1:uPA complex to downregulate the activity of the sonic
hedgehog (SHH) pathway which is involved in the malignant
transformation of numerous tissues (144). Treatment of PC3
prostate cancer cells with recombinant PN-1 or combinatorial
treatment with SHH pathway inhibitors significantly reduced
xenograft tumor growth in SCID mice and was associated
with alterations in angiogenesis (144). In a subsequent study,
the same group identified that PN-1 may act via inhibition
of X-chromsome-linked inhibitor of apoptosis (XIAP) and
reported that therapeutic treatment of xenograft tumor growth
in SCID mice was also synergistic with XIAP inhibitors (145).
A potential limitation of both of these studies, however, is the
use of a pre-treatment regimen for investigation. It will be
valuable to know whether PN-1 has anti-tumor activity after
tumor engraftment and growth. The utility of PN-1-mediated
targeting of the SHH pathway was recently demonstrated by
Li and Wang et al. using a model of Alzheimer’s disease
in APP/PS1 transgenic mice (146). Hippocampal delivery of
lentivirus particles to overexpress PN-1 resulted in improved
cognitive function, reduced amyloid deposition and preserved
neuronal cell viability.
Alpha-2-Antiplasmin (A2AP): Therapeutic
Applications
Owing to the fact that alpha-2-antiplasmin (A2AP) is the primary
inhibitor of plasmin, the majority of research on A2AP in the
therapeutic context has focused on diminishing the inhibitor
activity of A2AP, thereby enhancing fibrinolysis (147). In
contrast, less focus has been spent on investigating A2AP to limit
bleeding. In early work, Weitz et al. found that supplementation
with A2AP inhibited tPA-induced fibrinogenolysis and bleeding,
but did not affect thrombolysis in a model of jugular vein
thrombosis in rabbits (148). Nieuwenhuizen et al. investigated
the therapeutic administration of A2AP in a model of joint
bleeding-induced arthropathy, which can persist even after the
administration of clotting factor (149). Using a model of needle-
induced arthropathy in Factor VIII-deficient mice, the authors
found a reduction in both synovitis and cartilage damage
over a period of 5 weeks when antiplasmin was given by
direct intraarticular administration, whereas the uPA inhibitor
amiloride was ineffective (149,150). A2AP has also been
found to have efficacy in limiting cancer burden. Hayashido
et al. reported a drastic reduction of SCCKN squamous
cell carcinoma tumor growth in SCID mice when the cells
overexpressed A2AP vs. a mock-expressing control. A2AP-
mediated reductions in tumor growth occurred by limiting E-
cadherin processing by the fibrinolysis pathway (151). Similarly,
Paquet-Fifield et al. reported restricted lymphatic remodeling
and reduced metastases in SCID mice harboring 293-EBNA
cells overexpressing A2AP and VEGF-D vs. LacZ (152). A
limitation in these studies investigating the role of A2AP on
tumorigenesis is again the prior delivery of A2AP to cells before
implantation. Investigations determining if post-implantation
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Yaron et al. Fibrinolysis, Inflammation and Serpins
delivery of the A2AP gene sequence or recombinant protein will
have a similar effects could advance A2AP as a therapeutic.
Neuroserpin: Therapeutic Applications
The therapeutic use of neuroserpin has primarily been
investigated in neuropathologies. In early studies Yepes
et al. found that intracerebral administration of neuroserpin
was protective after in a model of middle cerebral artery
occlusion (MCAO) stroke and prevented basement membrane
proteolysis and cellular apoptosis in the ischemic penumbra
of rats by more than 50% (153). In subsequent work, Zhang
et al. used neuroserpin as an adjuvant treatment and found that
intracisternally-injected neuroserpin increased the therapeutic
window for therapeutic tPA administration after MCAO in
rats by as much as 4 h with reduced brain edema and ischemic
lesion volume (154). This work was recently confirmed in
a similar study by Cai et al. (155). Interestingly, Wu et al.
showed that the neuroprotective effect of neuroserpin in
experimental MCAO was independent of its ability to inhibit
tPA because protection was observed even in tPA-deficient
mice, suggesting broader protective mechanisms in cerebral
ischemia potentially involving less efficiently-inhibited serine
proteases such as plasmin (156). Yepes et al. further reported that
administration of neuroserpin into the ipsilateral hippocampus
enhances neuronal survival and delays the progression of
seizure activity in rats and mice subjected to kainic acid-induced
seizures (157). Labeurrier et al. also showed neuroprotection
against NMDA-induced excitotoxicity when neuroserpin was
co-injected with NMDA into the left striatum or left cortex of
mice (158).
Leveraging the neuroserpin therapeutic benefits on
brain-associated pathologies, several studies have expanded
investigations into the broader therapeutic effects of neuroserpin.
In a rat model of spinal cord injury induced by clip compression,
neuroserpin immediately injected intrathecally increased
numbers of anterior horn motor neurons associated with
restoration of autophagy and improved functional recovery
as determined by the Basso Beattie Bresnahan scoring system
(159). Upon intravitreal administration, neuroserpin protected
against retinal ischemia-reperfusion injury induced by elevated
intraocular pressure associated with attenuation of apoptosis
(160). In other studies examining neuroserpin as an immune
modulating therapeutic, Munuswamy-Ramanujam et al.
reported that intravenous administration of recombinant
neuroserpin prevented vasculopathy in a mouse aortic allograft
transplant model. In this model, neuroserpin reduced plaque
growth and T-cell invasion and T helper cell responses (161).
In contrast, neuroserpin was ineffective when evaluated as a
treatment for severe gammaherpesviral (MHV68) infection and
associated vasculitis in interferon gamma receptor-deficient
(IFNγR/) mice (162). The highly specific endogenous
sequestration of neuroserpin to the central nervous system may
provide certain advantages to its therapeutic administration as it
may not be subjected to the same degree of negative regulation
in extra-neural tissues. While currently under-explored, studies
investigating neuroserpin efficacy in other serpin-sensitive
therapeutic scenarios are warranted.
ALPHA-1-ANTITRYPSIN: A
PROMISCUOUS SERPIN WITH POTENT
THERAPEUTIC PROPERTIES
Alpha-1-antitrypsin (A1AT or AAT, SERPINA1) is the
prototypical and best studied member of the serpin superfamily
(163). While neutrophil elastase is the prominent and most
characterized target of A1AT, leading to a reduction in
neutrophilic inflammation (164), early investigations of
A1AT activity identified a broad serine protease reactivity
with cathepsins, caspases, metalloproteases, and coagulation
cascade-associated serine proteases thrombin and plasmin
(165). Interestingly, Talens et al. reported that A1AT is the
most abundant non-covalently bound protein in fibrin clots
and remains functionally active as a serpin in situ (166). The
exclusion of A1AT from classical descriptions of fibrinolytic
regulation may thus be an oversight, due to a focus on current
understanding of therapeutic benefit in lung disease and
underrepresenting the local control of plasmin activity by A1AT
directly in the fibrin clot.
A1AT deficiency is a potentially severe, chronic condition
characterized by unregulated inflammation primarily in the
lungs, leading to COPD and emphysema, and in the liver
leading to cirrhosis. Given A1AT potency as an inhibitor of
serine proteinases, A1AT deficiencies may also be associated
with other under recognized complications, such as during
post-surgical healing (167,168). A1AT recombinant protein
therapy (augmentation therapy) is clinically eficacious, thereby
prompting A1AT gene therapy, systemically administered via
viral vectors, to advance into clinical trials and yield promising
results (169).
Beyond treatment of serpin genetic deficiencies, A1AT is a
broad and potent immune modulator. In early work, Libert
et al. demonstrated a protective role for A1AT, which they first
identified as an acute phase reactant, in lethal TNF insult in mice
by a mechanism dependent on reducing platelet-activating factor
and associated with reversals of body temperature drop, liver
injury and increased clotting time when given recombinantly by
intraperitoneal or intravenous administration (170). Later, the
therapeutic effect of A1AT was demonstrated by Churg et al.
in a model of cigarette smoke-induced emphysema in mice,
which the authors suggested may be related to inhibition of
both matrix metalloproteinases as well as TNF signaling (171).
The protective effect of A1AT in the lungs may underscore
the distinct physiological role observed with genetic deficiency.
Similarly, Wang et al. found that A1AT treatment limited
pulmonary apoptosis and necrosis in a rat model of ventilator-
associated acute respiratory distress syndrome (ARDS) (172).
Akbar et al. found that gene therapy with A1AT delivered
by adeno-associated virus-8 (rAAV8) ameliorated bone loss in
an ovariectomy-induced osteoporosis mouse model of post-
menopause osteoporosis which was associated with inhibition of
IL-6 and RANK levels (173).
A1AT has demonstrated repeated therapeutic efficacy in
various models of cellular transplantation. Lewis et al. reported
pancreatic islet transplantation survival was extended by
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Yaron et al. Fibrinolysis, Inflammation and Serpins
treatment with recombinant clinical-grade human A1AT,
associated with a reduction of inflammatory cell infiltration and
abrogation of TNF signaling (174). This effect was extended to
preservation of islet cell viability in streptozotocin-treated mice.
In a related study, Zhang et al. described that A1AT-dependent
protection of islet viability after cytokine- and streptozotocin-
induced diabetes in mice was in part due to dramatic reduction
of beta cell apoptosis (175). Recently, the protective effect of
A1AT in islet cell transplantation was demonstrated by Gou
et al. in an intrahepatic transplant model in NOD-SCID mice
by suppressing macrophage activation with reduced TNF, iNOS,
IL-6, and CD11c signals (176). A1AT has been effective in
preventing graft rejection in other cellular transplant models, as
well. Marcondes et al. showed that A1AT prevented graft-vs.-host
disease (GVHD) in an allogeneic murine transplantation model
in both a preconditioning and post-conditioning treatment
regimen (177). In a similar follow-up study, Tawara et al.
confirmed the protective effect of A1AT in bone marrow
transplant GVHD and described an associated reduction in TNF,
IL1b, IL-6, and NF-kappaB signaling (178). Lee et al. showed
that intravenous administration of A1AT reduced short-term
engraftment of hepatocytes in rats which remained significant at
48 h (179). While significance was lost at longer timepoints, there
was evidence of viable engrafted hepatocytes up to 1 month after
transplant, which was not apparent in control mice. Recent work
by Emtiazjoo et al. showed a remarkable reduction of acute lung
allograft injury in an orthotopic single left lung transplantation
model from Lewis to Sprague-Dawley rats at 8 days post-
transplantation (180). Of crucial importance, protection was
achieved in the absence of any systemic immunosuppression.
Addressing another complication of diabetes, Ortiz et al.
reported that A1AT treatment alleviated the progression of
diabetic retinopathy in mice by suppressing TNF signaling in
both the serum and retina and promoting an M2-polarized
macrophage population, which ultimately delayed ganglion cell
loss and retinal thinning (181). In another study examining A1AT
efficacy for ophthalmological disorders, Yang et al. demonstrated
protection of iPSC grafts after subretinal transplantation into
the eye of mice with preexisting ocular hypertension by
inhibition of microglial activation (182). Interestingly, Zhou
et al. reported that suppression of microglial inflammation and
neurodegeneration in the eye was achieved by intraperitoneal
injection of A1AT in a Rd1(FVB/N) mouse model of retinal
degeneration (183).
Ischemia-reperfusion injury is characterized by a transient
loss of blood and oxygen to a tissue, followed by a period of
reoxygenation which paradoxically accelerates damage caused
during the hypoxic period (184). Ischemia-reperfusion injury
can occur in any tissue, whether by pathogenic etiology
or by complications of surgical procedure, and there is an
unmet need for novel therapeutics to address the condition
(185188). Moldthan et al. first demonstrated the therapeutic
efficacy of A1AT therapy in a rat model of ischemic stroke
which resulted in a drastic reduction of infarct volume and
preservation of sensory motor system function (189). Toldo
et al. generated a recombinant A1AT-Fc fusion protein and
demonstrated efficacy in reducing inflammation following
myocardial ischemia-reperfusion in mice which they found
was independent of the capacity to inhibit elastase (190).
In translation of this work, the VCU-a1RT clinical trial
(NCT01936896) was undertaken to investigate the potential
protective effect of A1AT therapy (Prolastin R
) in patients
with ST-segment elevation myocardial infarction (STEMI) (191).
Abbate et al. reported that the VCU-a1RT trial found no in-
hospital adverse effects of A1AT therapy and that a blunted
initial inflammatory response resulted in significantly reduced
CRP levels 14 days after admission. In further analysis of the
trial, Abouzaki et al. also described a shorter time-to-peak in CK-
MB levels indicating an inhibition of the onset of inflammatory
injury (192). In investigations of other tissues, Maicas et al. found
a limited therapeutic efficacy for clinical grade human A1AT
(Prolastin R
) in a mouse model of renal ischemia-reperfusion
injury where they reported a significant decrease in kidney injury
molecule-1 levels in urine but no effect on renal fibrosis (193).
This finding contrasts later work by Jeong et al. who reported a
significant protection against renal ischemia-reperfusion injury
upon treatment with A1AT, including attenuated tubular injury
and fibrosis (194). The design of these two contrasting studies
are similar in the use of FDA-approved clinical grade A1AT at a
dose of 80 mg/kg/day, however Jeong et al. administrated A1AT
for 3 days prior to surgery and only followed up at 24 h post-
procedure, whereas Maicas et al. first administrated A1AT at 24 h
pre-procedure and followed up at 8 and 15 days post-procedure.
Thus, the fibrotic phenotype likely developed over a longer time
course than observed by Jeong et al.
Lupus is an autoimmune condition characterized by
dysregulated adaptive and innate immune responses in tissues
and the vasculature which can have damaging and potentially
lethal effects on end organs such as the kidneys and lungs
(195). Elshikha et al. investigated the protective effects of A1AT
therapy in a series of preclinical studies. In the first of the
series, they showed that A1AT inhibits plasmacytoid dendritic
cell activation and protects against nephritis in the MRL/lpr
spontaneous lupus model (196). They went on to describe that
gene therapy with A1AT delivered by rAAV8 prolongs lifespan
in NZM2410 mice which develop spontaneous lupus with early-
onset glomerulonephritis and that the protection was associated
with reduced autoantibody levels (197). Recently, Elshikha et al.
described that treatment with recombinant A1AT limited disease
progression and suppressed TNF signaling in a pristane-induced
model of acute lupus diffuse alveolar lung hemorrhage (DAH)
(198). Thus, the broad immune modulating effects of A1AT
highlight the significant therapeutic potential of this serpin with
broad activity against a wide range of serine proteases in diverse
diseases driven by dysregulated inflammatory processes.
SERP-1: A VIRUS-DERIVED
COAGULATION REGULATOR AND
THERAPEUTIC IMMUNE MODULATOR
Viruses, especially DNA viruses with large genomes such as
poxviruses and herpesviruses, have expertly evolved highly
effective and potent immune modulating protein machinery that
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evade host immune defenses (199). These proteins have become
the focus of a growing field of research around the development
of virus-derived therapeutics (200), some of which have led
to increased interest in mammalian serpin therapeutics. The
most thoroughly investigated virus-derived therapeutic protein is
Serp-1, a serpin from Myxoma virus which targets the fibrinolytic
serine proteases uPA, tPA, and plasmin as well as the thrombotic
proteases FXa and thrombin (in the presence of heparin) (201,
202). The first demonstration of Serp-1 therapeutic efficacy
was in a rabbit model of aortic balloon angioplasty injury,
where protection was characterized by significantly reduced
inflammation and plaque growth (203). The doses used in
that original study were single intravenous injections in the
picogram range given immediately after angioplasty injury.
This therapeutic effect against inflammation and vasculopathy
with plaque growth was further demonstrated in models of
aortic, renal and heterotopic heart transplants in mice and
rats and in a mouse carotid compression model (204207).
Serp-1 was also demonstrated to be protective in a collagen-
induced arthritis model in rats, with generalized immune
modulation outside of transplant and pro-atherogenic disease
states (208). In more recent work, Serp-1 was found to be an
effective therapeutic against severe vasculitis in both human
temporal artery biopsy transplants from patients suspected to
have Giant cell arteritis into SCID mice and in the lethal
MHV68 gammaherpesvirus-induced vasculitis in interferon
gamma receptor-deficient mice (162,209). Interestingly, peptides
derived from Serp-1 are also therapeutically effective in the
gammaherpesvirus-induced vasculitis model and protection
imparted by both the full protein and the peptide derivatives
are dependent on composition of the gut microbiome (202,
210,211). In vitro studies demonstrated that, these reactive
center loop (RCL) peptides bound and inhibited mammalian
serpins (202). While these studies all used an intraperitoneal
or intravenous delivery of naked recombinant protein, Serp-
1 is also amenable to drug delivery vehicles and is currently
the only serpin demonstrated for delivery by such approaches.
Serp-1 is capable of sustained delivery in a chitosan-collagen
biocompatible hydrogel and has demonstrated therapeutic
efficacy in models of full-thickness cutaneous wound healing in
mice and in spinal cord injury in rats, with efficacy dependent
on engagement of uPAR in the fibrinolytic signaling pathway
(212214).
Serp-1 is a First-in-Class therapeutic and the first virus-
derived protein given to humans in an FDA-overseen Phase
IIa clinical trial (NCT00243308) for patients with unstable
coronary syndromes, unstable angina and small heart attacks
(215). Serp-1 was safe and well-tolerated with a major adverse
cardiac event score (MACE) of zero and with a dose-dependent
reduction in heart damage markers Troponin and CK-MB.
Importantly, there were no detectable neutralizing antibodies
against Serp-1. Thus, Serp-1 represents a cross-pathway serpin
targeting thrombolytic and thrombotic cascades which regulates
inflammatory responses in part by engaging signaling in the
fibrinolytic pathway with potent therapeutic efficacy in a wide
variety of disease states.
FIBRINOLYSIS AND SERPINS IN
SARS-COV-2 INFECTION AND THE
COVID-19 PANDEMIC
Understanding the bidirectional activation of the coagulation
proteases and activation of immune responses indicates a
clear target for serpin therapeutics in severe infections where
both coagulopathy as well as excessive and damaging immune
response cause increased damage and mortality. Serpins have
been examined in preclinical models of severe viral infections
with coagulopathy, one notable example being the use of Serp-
1 treatment in the severe vasculitis/lung hemorrhage model in
IFNγR/mouse models. In these models, as noted, Serp-1
improves survival and reduces both lung consolidation as well
as vascular inflammation. Similarly, PAI-1 has proven beneficial
in models of severe Klebsiella pneumoniae. Antithrombin III
(ATIII), a serpin inhibiting the clotting pathway and activated by
heparin infusions, also has had variable benefit in clinical trials of
bacterial sepsis in man.
In December 2019, a pneumonia of unknown origin was
identified in Wuhan, the capital of Hubei province in China
and identified as a Severe acute respiratory distress syndrome
(SARS) coronavirus denoted as SARS-CoV-2 (216). The disease
caused by SARS-CoV-2, is referred to as coronavirus disease 2019
(COVID-19) and is now a worldwide pandemic as declared by
the World Health Organization (217). As of March 10th, 2021,
>118 million cases of COVID-19 has been reported globally with
a death toll >2.6 million worldwide (Worldmeters.info).
Symptomatically, COVID-19 commonly causes severe
coughing and hemoptysis, shortness of breath and hypoxemia
accompanied by widespread lung infiltrates, consolidation and
in some cases hemorrhage, with fever, weakness and confusion.
SARS-CoV-2 is therefore identified, along with SARS-CoV-1,
as a severe acute respiratory disease (218). However, despite
the commonality of ARDS in COVID-19 patients, mounting
evidence suggests that infection with SARS-CoV-2 induces a
hypercoagulable state (219). Spiezia et al. reported that severe
hypercoagulability, but not a clearly defined consumptive
coagulopathy (or disseminated intravascular coagulopathy,
DIC), is present in COVID-19 patients with acute respiratory
failure (220). Endothelial injury and dysfunctional coagulation,
with associated resistance against fibrinolysis, may thus reclassify
COVID-19 as a vascular disorder complicated by widespread
microthrombotic occlusions rather than a respiratory disease
(221). We direct readers to a thorough description of this
hypothesis in a recent review by Siddiqi, Libby and Ridker (222).
The apparent dysfunction of fibrinolysis during COVID-
19 infection would suggest that therapeutic administration of
serpins of the fibrinolytic pathway against SARS-CoV-2 may not
be beneficial. However, many factors are worth consideration in
the use of serpins in severe viral septic states with imbalance
in coagulation as well as excess aggressive immune responses
in accompanying cytokine storm. First, on a molecular level,
SARS-CoV-2, like other coronaviruses, requires proteolytic
processing of its Spike (S) protein in order to appropriately
dock with and enter host cells (223,224). It is now known
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Yaron et al. Fibrinolysis, Inflammation and Serpins
that the SARS-CoV-2 S protein is processed sequentially by the
subtilisin-like peptidase furin and the transmembrane serine
protease TMPRSS2 (225). Indeed, mechanistic studies have
shown that a clinically approved TMPRSS2 inhibitor, camostat
mesylate, inhibited SARS-CoV-2 S-driven infection in vitro (226).
A recent retrospective observational case series on a small
cohort of ICU patients in Germany found reduced severity in
patients treated with camostat mesylate vs. those who received
hydroxychloroquine (227).
Several fibrinolysis pathway serpins may interfere with these
mechanisms (Figure 4). A preprint by Azouz et al., first deposited
in May 2020, demonstrated that A1AT inhibits TMPRSS2 in an
HEK-293T overexpression system (228). In an important follow-
up study deposited in July 2020, where Wettstein et al. produced
SARS-CoV-2 S-protein pseudoparticles and performed in vitro
infection of Caco2 cells in the presence of chromatographically
fractioned bronchoalveolar lavage samples they found that the
highest inhibition of infection occurred in the fraction containing
A1AT (229). These findings agree with prior reports of the
ability for A1AT to inhibit infectivity of TMPRSS2-dependent
viruses. Beard et al. reported that A1AT inhibits in vitro and
in vivo mouse infection by H1N1 Influenza, which requires
hemagglutinin processing via TMPRSS2 (230). Similarly, Esumi
et al. reported that hepatitis C virus infection proceeds by the
activity of TMPRSS2, which was dose-dependently inhibited in
vitro by A1AT (231).
Second, on a coagulation systemic level, serpins target areas
with activated serine proteases. Thus serpins are predicted
to target areas with active thrombosis and or thrombolysis.
Dysregulated thrombosis and thrombolysis are now clearly an
important component of COVID-19 disease (232). What is not
clear is how or why these pathways become dysregulated, and
some have proposed that increased amounts of active PAI-1
may induce a feed-forward loop of inflammatory events (233).
This idea has led to the recent initiation of a clinical trial to
test the PAI-1 inhibitor, TM5614, for treating high-risk patients
hospitalized with severe COVID-19 (NCT04634799). Similarly,
tranexamic acid, a uPA inhibitor, is under investigation to
combat COVID-19 (NCT04338074, NCT04338126). In further
support of this, several groups have suggested modulation of the
fibrinolytic pathway by targeting plasmin/plasminogen based on
imbalances in protease levels in COVID-19 patients (234,235).
However, sensitivity in the pathway imbalance urges caution
in administration, and the dynamics of the disease may dictate
appropriate timing for intervention (236,237).
While it is firmly established that coagulopathy occurs in
COVID-19 patients, there is a possibility that PAI-1 may in fact
be a protective host factor against SARS-CoV-2. Dittmann et al.
reported that PAI-1 can dose-dependently inhibit Influenza A
infection by preventing hemagglutinin processing by TMPRSS2
(238). Thus, the ability for PAI-1 to inhibit TMPRSS2 suggests
that the problem of dysregulation of fibrinolysis may be more
complex than focusing on PAI-1 may solve. Colling and Kanthi
propose that the ratios of active PAI-1 and tPA may be more
indicative of the pathway activity in COVID-19 patients due to
ongoing consumption and microvascular thromboses (239). In
support of this hypothesis, a recent preprint by Zuo et al. on 118
hospitalized COVID-19 patients and 30 healthy controls found
that patients who died had significantly higher levels of both PAI-
1 and tPA (240). Importantly, the authors found that a higher
ratio of tPA vs. PAI-1 was indicative of potential mortality, and
was driven by an increase in tPA, not PAI-1.
Furin is a second protease involved in S-protein priming
during SARS-CoV-2 infection, and also has a role in intracellular
processing (241). Furin is present in both membrane-bound
and secreted, soluble states with the latter usually associated
with a variety of pathologies, such as diabetes or infection
(242244). Cheng et al. recently reported that small molecule
inhibitors of furin prevent SARS-CoV-2 infection as well as
intracellular processing in vitro (245). However, the significant
role of furin in normal tissue development and homeostasis
make it a difficult target for therapeutic modulation and there
are no FDA-approved furin inhibitors for clinical use. On the
other hand, there is precedent for experimentally targeting
furin with serpins to limit viral infection. For example, Shapiro
et al. reported A1AT-mediated inhibition of HIV infection in
vitro, which is dependent on furin-mediated processing of the
membrane protein gp160 (246,247). Numerous groups have
engineered A1AT to fine-tune its properties, such as the A1AT
Portland variant with increased specific and activity against furin
described by Jean et al. to have anti-pathogenic properties (248).
Similarly, Anderson et al. reported another A1AT variant (α1-
PDX) with 3,000-fold higher anti-furin activity which potently
inhibited HIV gp160-dependent infection in vitro (249). Furin
is also inhibited by PAI-1 (intracellular furin) and endothelial
PN-1 (extracellular furin), but their ability to limit infection via
furin inhibition-dependent mechanisms remains to be explored
(250,251).
Third, the now understood evolution of serine proteases
and serpins as regulators of both thrombosis and thrombolysis,
and also of inflammation, would suggest a potential for the
use of serpins that target both coagulation as well as immune
responses in severe viral infections. The immune system response
to SARS-CoV-2 reveals a different perspective for COVID-
19 and fibrinolysis. Numerous inflammatory pathologies are
associated with increased circulating levels of soluble uPAR
(suPAR), produced by the cleavage of the C-terminal glycosyl-
phosphatidylinositol linker by phospholipases (252). Growing
evidence has established suPAR as a useful diagnostic and
prognostic indicator of severe, acute pathologies including sepsis
(253). Based on similarities of COVID-19 complications with
diseases associated with or exacerbated by elevated suPAR,
D’Alonzo et al. proposed suPAR as a therapeutic target
for treating SARS-CoV-2 infection (254). In support of this
proposition, early in the COVID-19 pandemic Rovina et al.
identified elevated suPAR in 57 Greek patients and 15 American
patients as a highly significant early prognostic indicator of
severe outcomes in SARS-CoV-2 infection (255). More recently,
Azam et al. investigated the association of acute kidney injury
(AKI) in COVID-19 patients with suPAR (256). AKI occurs
in up to 50% of severe COVID-19 patients and significantly
increases morbidity and mortality. Azam et al. found that the
highest tertile of suPAR levels was associated with a more
than nine-fold increase in AKI in COVID-19 patients and
Frontiers in Cardiovascular Medicine | www.frontiersin.org 12 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
FIGURE 4 | Inhibition of SARS-CoV-2 infection and processing by fibrinolysis pathway-associated serpins. SARS-CoV-2 entry is dependent on proteolytic processing
of the spike protein in order to engage the ACE2 receptor and internalize. Processing of the spike protein is performed primarily by TMPRSS2 and Furin. PAI-1 and
A1AT are known inhibitors of TMPRSS2 and experimental evidence demonstrates that A1AT can inhibit SARS-CoV-2 infection in vitro. Based on similarities in
inhibition properties, it may be predicted that Serp-1 will also inhibit TMPRSS2 with similar results. Furin has both an extracellular and intracellular role in the
SARS-CoV-2 life cycle and there is evidence for extracellular furin inhibition by A1AT and A1AT variants (Portland and α-PDX) as well as endothelial PN-1, and for
intracellular furin inhibition by PAI-1.
was independent of inflammatory markers or demographic
subgroups. The uPAR system has other roles in SARS-CoV-2
infection in addition to suPAR elevation. Ly6E is a member of
the uPAR family of proteins and a lymphocyte marker associated
with immunological regulation and also recently associated with
host responses to viral infection (257). Zhao et al. demonstrated
that Ly6E restricts the entry of human coronaviruses in an ectopic
expression model using both the common HCoV-O43 as well
as SARS-CoV-2 (258). Importantly, Pfaender et al. performed
crucial in vivo experiments in wildtype and Ly6E-deficient mice
that revealed a critical Ly6E-dependent host defense against
coronaviruses, including MERS-CoV, SARS-CoV, and SARS-
CoV-2 (259). Mechanistic studies performed by the authors
demonstrate that Ly6E prevents coronavirus entry into host cells
by preventing S-protein-mediated membrane fusion. Of interest,
the virus derived serpin that we have studied extensively, Serp-1,
and also PAI-1 bind and block the uPA/uPAR complex (212,213).
With Serp-1 this leads to marked anti-inflammatory function.
Thus, the uPA/uPAR/suPAR system represents an attractive
therapeutic target for modulation to treat or limit the severity
of immune disorders as well as potentially specific treatment for
COVID-19 disease progression.
The role of serpins and serine proteases of the fibrinolytic
system in COVID-19 is complex and investigations on the
potential therapeutic modulation of these processes with natural,
virus-derived or engineered serpins, expanding the consideration
of these proteins beyond only regulation of the fibrinolytic
system, may be a valuable pursuit as many of these modulators
are already found to be safe and effective, and in some cases FDA-
approved.
DISCUSSION
The ancient roots of clot formation and clot dissolution speaks
to the necessary role these pathways play in protection of the
host from excess thrombosis and thrombolysis to immune-based
disorders. In simplest terms, protection against loss of blood
(or hemolymph in the case of lower organisms) after traumatic
injury is an essential component of survival. However, the greater
understanding of the role of these pathways in host responses to
infection point to a more complex role for the coagulation and
fibrinolysis cascades.
Focusing on the fibrinolytic cascade, we have reviewed the
diversity of serine protease and serpin control of this essential
pathway. The complexity of the pathway also underscores how
critical this pathway is to maintain a normal homeostatic balance:
dysregulation may easily lead to disease and therefore redundant
control has evolved to maintain homeostasis and preserve host
viability. A striking consequence of the evolution of serpin
regulators of fibrinolysis is their potency and general safety. As
we have discussed, these factors have led to the investigation of
fibrinolysis-associated serpins as therapeutics. However, despite
the dense and growing body of work justifying the application of
serpins as therapeutics there are few, if any, indications outside
of augmentation therapy for A1AT deficiency and related lung
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Yaron et al. Fibrinolysis, Inflammation and Serpins
and liver diseases which have received FDA approval. There is
a growing need for novel therapeutics for inflammation-related
disorders and naturally-evolved or improved, engineered serpins
may provide higher potency and lower off-target selectivity
than small molecule inhibitors. We would suggest that more
attention should be given to the investigation of serpins as
new potential therapeutics. This is particularly urgent in the
context of severe cases of ARDS and microthrombotic vascular
complications in the COVID-19 Pandemic (and potentially
other viral pandemics), where serpins may provide a uniquely
multifunctional role in modulating the host immune response as
well as the virus life cycle toward improved outcomes.
AUTHOR CONTRIBUTIONS
JRY wrote the first draft of the manuscript and
produced all figures. LZ, QG, SEH, and ARL revised
the manuscript. All authors approved the final version of
the manuscript.
FUNDING
This work was supported by AHA and NIH grants as well as
ASU/Biodesign startup funding to ARL.
ACKNOWLEDGMENTS
We thank Dr. Grant McFadden for helpful conversations
and are grateful for the critical work of the Serpin Biology
research community that has been essential to delineate the
structure, function, and applied science of the serpin superfamily.
Figures were created with BioRender.com and exported under a
paid subscription.
REFERENCES
1. Moore HB, Moore EE. Temporal changes in fibrinolysis following injury.
Semin Thromb Hemost. (2020) 46:189–98. doi: 10.1055/s-0039-1701016
2. Weidmann H, Heikaus L, Long AT, Naudin C, Schlüter H, Renné T. The
plasma contact system, a protease cascade at the nexus of inflammation,
coagulation and immunity. Biochim Biophys Acta Mol Cell Res. (2017)
1864:2118–27. doi: 10.1016/j.bbamcr.2017.07.009
3. Levi M, Van Der Poll T. Two-way interactions between inflammation
and coagulation. Trends Cardiovasc Med. (2005) 15:254–9.
doi: 10.1016/j.tcm.2005.07.004
4. Jackson SP, Darbousset R, Schoenwaelder SM. Thromboinflammation:
challenges of therapeutically targeting coagulation and other host defense
mechanisms. Blood. (2019) 133:906–18. doi: 10.1182/blood-2018-11-882993
5. Mkaouar H, Akermi N, Kriaa A, Abraham AL, Jablaoui A, Soussou S, et al.
Serine protease inhibitors and human wellbeing interplay: new insights for
old friends. PeerJ. (2019) 2019:1–21. doi: 10.7717/peerj.7224
6. Wojta J. Macrophages and Thrombin—Another Link between
Inflammation and Coagulation. Thromb Haemost. (2020) 120:537–537.
doi: 10.1055/s-0040-1708551
7. Foley JH, Conway EM. Cross talk pathways between
coagulation and inflammation. Circ Res. (2016) 118:1392–408.
doi: 10.1161/CIRCRESAHA.116.306853
8. Maas C, Renne T. Coagulation factor XII in thrombosis and inflammation.
Blood. (2018) 131:1903–9. doi: 10.1182/blood-2017-04-569111
9. Nurden AT. Platelets, inflammation and repair. Front Biosci. (2018) 23:726–
51. doi: 10.2741/4613
10. Dzik S. Complement and coagulation: cross talk through time. Transfus Med
Rev. (2019) 33:199–206. doi: 10.1016/j.tmrv.2019.08.004
11. Anitua E, Nurden P, Nurden AT, Padilla S. More than 500 million years of
evolution in a fibrin-based therapeutic scaffold. Regen Med. (2020) 15:1493–
8. doi: 10.2217/rme-2020-0049
12. Chana-Muñoz A, Jendroszek A, Sønnichsen M, Wang T, Ploug M, Jensen JK,
et al. Origin and diversification of the plasminogen activation system among
chordates. BMC Evol Biol. (2019) 19:1–17. doi: 10.1186/s12862-019-1353-z
13. Schmaier AH. The contact activation and kallikrein/kinin systems:
pathophysiologic and physiologic activities. J Thromb Haemost. (2016)
14:28–39. doi: 10.1111/jth.13194
14. Visser M, Heitmeier S, Ten Cate H, Spronk HMH. Role of factor xia
and plasma kallikrein in arterial and venous thrombosis. Thromb Haemost.
(2020) 120:883–993. doi: 10.1055/s-0040-1710013
15. Pavlopoulou A, Pampalakis G, Michalopoulos I, Sotiropoulou G.
Evolutionary history of tissue kallikreins. PLoS ONE. (2010) 5:e13781.
doi: 10.1371/journal.pone.0013781
16. Govind S. Innate immunity in Drosophila: pathogens and pathways. Insect
Sci. (2008) 15:29–43. doi: 10.1111/j.1744-7917.2008.00185.x
17. Wright RK, Cooper EL. Inflammatory reactions of the protochordata. Integr
Comp Biol. (1983) 23:205–11. doi: 10.1093/icb/23.1.205
18. Davidson B, Swalla BJ. A molecular analysis of ascidian metamorphosis
reveals activation of an innate immune response. Development.
(2002) 129:4739–51.
19. Styer KL, Hopkins GW, Bartra SS, Plano GV, Frothingham R, Aballay
A. Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent
process that involves novel virulence factors. EMBO Rep. (2005) 6:992–7.
doi: 10.1038/sj.embor.7400516
20. Hisamoto N, Li C, Yoshida M, Matsumoto K. The C.elegans
HGF/Plasminogen-like protein SVH-1 has protease-dependent
and -independent functions. Cell Rep. (2014) 9:1628–34.
doi: 10.1016/j.celrep.2014.10.056
21. Bailey CM, Khalkhali-Ellis Z, Seftor EA, Hendrix MJC. Biological functions
of maspin. J Cell Physiol. (2006) 209:617–24. doi: 10.1002/jcp.20782
22. Heit C, Jackson BC, McAndrews M, Wright MW, Thompson DC, Silverman
GA, et al. Update of the human and mouse SERPIN gene superfamily. Hum
Genomics. (2013) 7:1–14. doi: 10.1186/1479-7364-7-22
23. Sanrattana W, Maas C, de Maat S. SERPINs-From trap to treatment. Front
Med. (2019) 6:1–8. doi: 10.3389/fmed.2019.00025
24. Luke CJ, Pak SC, Askew DJ, Askew YS, Smith JE, Silverman GA.
Selective conservation of the RSL-encoding, proteinase inhibitory-type,
clade L serpins in Caenorhabditis species. Front Biosci. (2006) 11:581–94.
doi: 10.2741/1820
25. Levashina EA, Langley E, Green C, Gubb D, Ashburner M,
Hoffmann JA, et al. Constitutive activation of toll-mediated antifungal
defense in serpin-deficient Drosophila. Science. (1999) 285:1917–9.
doi: 10.1126/science.285.5435.1917
26. Cederholm-Williams SA. Concentration of plasminogen and antiplasmin
in plasma and serum. J Clin Pathol. (1981) 34:979–81. doi: 10.1136/jcp.34.
9.979
27. Cesarman-Maus G, Hajjar KA. Molecular mechanisms of fibrinolysis.
Br J Haematol. (2005) 129:307–21. doi: 10.1111/j.1365-2141.2005.0
5444.x
28. Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and
two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem.
(1982) 257:2920–5. doi: 10.1016/S0021-9258(19)81052-9
29. Andronicos NM, Chen EI, Baik N, Bai H, Parmer CM, Kiosses
WB, et al. Proteomics-based discovery of a novel, structurally unique,
and developmentally regulated plasminogen receptor, Plg-RKT, a major
regulator of cell surface plasminogen activation. Blood. (2010) 115:1319–30.
doi: 10.1182/blood-2008-11-188938
Frontiers in Cardiovascular Medicine | www.frontiersin.org 14 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
30. Miles LA, Vago JP, Sousa LP, Parmer RJ. Functions of the plasminogen
receptor Plg-R KT. J Thromb Haemost. (2020) 18:2468–81.
doi: 10.1111/jth.15014
31. Friezner Degen SJ, Rajput B, Reich E. The human tissue
plasminogen activator gene. J Biol Chem. (1986) 261:6972–85.
doi: 10.1016/S0021-9258(19)62711-0
32. Margaglione M, Di Minno G, Grandone E, Vecchione G, Celentano E,
Cappucci G, et al. Abnormally high circulation levels of tissue plasminogen
activator and plasminogen activator inhibitor-1 in patients with a history
of ischemic stroke. Arterioscler Thromb Vasc Biol. (1994) 14:1741–5.
doi: 10.1161/01.ATV.14.11.1741
33. Geier B, Großefeld M, Barbera L, Mumme A. Pharmacokinetics of tissue
plasminogen activator in an isolated extracorporeal circuit. J Vasc Surg.
(2001) 33:165–9. doi: 10.1067/mva.2001.109765
34. Segarra A, Chacón P, Martinez-Eyarre C, Argelaguer X, Vila J, Ruiz P,
et al. Circulating levels of plasminogen activator inhibitor type-1 tissue
plasminogen activator, and thrombomodulin in hemodialysis patients:
biochemical correlations and role as independent predictors of coronary
artery stenosis. J Am Soc Nephrol. (2001) 12:1255–63.
35. Shariat SF, Roehrborn CG, McConnell JD, Park S, Alam N, Wheeler
TM, et al. Association of the circulating levels of the urokinase system
of plasminogen activation with the presence of prostate cancer and
invasion, progression, and metastasis. J Clin Oncol. (2007) 25:349–55.
doi: 10.1200/JCO.2006.05.6853
36. Schmedes CM, Grover SP, Hisada YM, Goeijenbier M, Hultdin J, Nilsson S,
et al. Increased circulating extracellular vesicle tissue factor activity during
orthohantavirus infection is associated with intravascular coagulation. J
Infect Dis. (2019) 222:1392–9. doi: 10.1093/infdis/jiz597
37. Melchor JP, Strickland S. Tissue plasminogen activator in central nervous
system physiology and pathology. Thromb Haemost. (2005) 93:655–60.
doi: 10.1160/TH04-12-0838
38. Gualandris A, Jones TE, Strickland S, Tsirka SE. Membrane depolarization
induces calcium-dependent secretion of tissue plasminogen activator. J
Neurosci. (1996) 16:2220–5. doi: 10.1523/JNEUROSCI.16-07-02220.1996
39. Baranes D, Lederfein D, Huang VY, Chen M, Bailey CH, Kandel ER. Tissue
plasminogen activator contributes to the late phase of LTP and to synaptic
growth in the hippocampal mossy fiber pathway. Neuron. (1998) 21:813–25.
doi: 10.1016/S0896-6273(00)80597-8
40. Knipe L, Meli A, Hewlett L, Bierings R, Dempster J, Skehel P, et al. A revised
model for the secretion of tPA and cytokines from cultured endothelial cells.
Blood. (2010) 116:2183–91. doi: 10.1182/blood-2010-03-276170
41. Emeis JJ, Van Den Eijnden-Schrauwen Y, Van Den Hoogen CM, De
Priester W, Westmuckett A, Lupu F. An endothelial storage granule
for tissue-type plasminogen activator. J Cell Biol. (1997) 139:245–56.
doi: 10.1083/jcb.139.1.245
42. Delacretaz E, Ganz LI, Soejima K, Friedman PL, Walsh EP, Triedman
JK, et al. Bradykinin stimulates the release of tissue plasminogen
activator in human coronary circulation: effects of angiotensin-
converting enzyme inhibitors. J Am Coll Cardiol. (2001) 37:1565–70.
doi: 10.1016/S0735-1097(01)01202-5
43. Madureira PA, Surette AP, Phipps KD, Taboski MAS, Miller VA, Waisman
DM. The role of the annexin A2 heterotetramer in vascular fibrinolysis.
Blood. (2011) 118:4789–97. doi: 10.1182/blood-2011-06-334672
44. Surette AP, Madureira PA, Phipps KD, Miller VA, Svenningsson P, Waisman
DM. Regulation of fibrinolysis by S100A10 in vivo.Blood. (2011) 118:3172–
81. doi: 10.1182/blood-2011-05-353482
45. Rosso M, Del Margheri F, Serrati S, Chilla A, Laurenzana A, Fibbi G.
The urokinase receptor system, a key regulator at the intersection between
inflammation, immunity, and coagulation. Curr Pharm Des. (2011) 17:1924–
43. doi: 10.2174/138161211796718189
46. Takahashi K, Uwabe Y, Sawasaki Y, Kiguchi T, Nakamura H, Kashiwabara K,
et al. Increased secretion of urokinase-type plasminogen activator by human
lung microvascular endothelial cells. Am J Physiol Lung Cell Mol Physiol.
(1998) 275:47–54. doi: 10.1152/ajplung.1998.275.1.L47
47. Shanmukhappa K, Sabla GE, Degen JL, Bezerra JA. Urokinase-type
plasminogen activator supports liver repair independent of its cellular
receptor. BMC Gastroenterol. (2006) 6:1–9. doi: 10.1186/1471-230X.-6-40
48. Ghosh S, Brown R, Jones JCR, Ellerbroek SM, Stack MS. Urinary-type
plasminogen activator (uPA) expression and uPA receptor localization are
regulated by α3β1 integrin in oral keratinocytes. J Biol Chem. (2000)
275:23869–76. doi: 10.1074/jbc.M000935200
49. Roelofs JJTH, Rowshani AT, Van Den Berg JG, Claessen N, Aten J, Ten
Berge IJM, et al. Expression of urokinase plasminogen activator and its
receptor during acute renal allograft rejection. Kidney Int. (2003) 64:1845–53.
doi: 10.1046/j.1523-1755.2003.00261.x
50. Diaz A, Merino P, Manrique LG, Ospina JP, Cheng L, Wu F, et al. A
cross talk between neuronal urokinase-type plasminogen activator (uPA)
and astrocytic uPA receptor (uPAR) promotes astrocytic activation and
synaptic recovery in the ischemic brain. J Neurosci. (2017) 37:10310–22.
doi: 10.1523/JNEUROSCI.1630-17.2017
51. Gyetko MR, Wilkinson CC, Sitrin RG. Monocyte urokinase expression:
modulation by interleukins. J Leukoc Biol. (1993) 53:598–601.
doi: 10.1002/jlb.53.5.598
52. Gundersen D, Trân-Thang C, Sordat B, Mourali F, Rüegg C. Plasmin-
induced proteolysis of tenascin-C: modulation by T lymphocyte-derived
urokinase-type plasminogen activator and effect on T lymphocyte adhesion,
activation, and cell clustering. J Immunol. (1997) 158:1051–60.
53. Nielsen LS, Hansen JG, Skriver L, Wilson EL, Kaltoft K, Zeuthen J, et al.
Purification of zymogen to plasminogen activator from human glioblastoma
cells by affinity chromatography with monoclonal antibody. Biochemistry.
(1982) 21:6410–5. doi: 10.1021/bi00268a014
54. Ichinose A, Fujikawa K, Suyama T. The activation of pro-urokinase by
plasma kallikrein and its inactivation by thrombin. J Biol Chem. (1986)
261:3486–9. doi: 10.1016/S0021-9258(17)35674-0
55. Koivunen E, Huhtala ML, Stenman UH. Human ovarian tumor-associated
trypsin. Its purification and characterization from mucinous cyst fluid
and identification as an activator of pro-urokinase. J Biol Chem. (1989)
264:14095–9. doi: 10.1016/S0021-9258(18)71648-7
56. Behrendt N, Stephens RW. The urokinase receptor. Fibrinolysis Proteolysis.
(1998) 12:191–204. doi: 10.1016/S0268-9499(98)80013-1
57. Higazi AAR, Cohen RL, Henkin J, Kniss D, Schwartz BS, Cines
DB. Enhancement of the enzymatic activity of single-chain urokinase
plasminogen activator by soluble urokinase receptor. J Biol Chem. (1995)
270:17375–80. doi: 10.1074/jbc.270.29.17375
58. Colman R. Regulation of angiogenesis by the kallikrein-kinin system. Curr
Pharm Des. (2006) 12:2599–607. doi: 10.2174/138161206777698710
59. Stavrou EX, Fang C, Bane KL, Long AT, Naudin C, Kucukal E, et al. Factor
XII and uPAR upregulate neutrophil functions to influence wound healing. J
Clin Invest. (2018) 128:944–59. doi: 10.1172/JCI92880
60. Plow EF, Doeuvre L, Das R. So many plasminogen receptors: why? J Biomed
Biotechnol. (2012) 2012:1–7. doi: 10.1155/2012/141806
61. Miles LA, Lighvani S, Baik N, Andronicos NM, Chen EI, Parmer CM, et al.
The plasminogen receptor, Plg-RKT, and macrophage function. J Biomed
Biotechnol. (2012). doi: 10.1155/2012/250464
62. Miles LA, Baik N, Lighvani S, Khaldoyanidi S, Varki NM, Bai H, et al.
Deficiency of plasminogen receptor, Plg-RKT, causes defects in plasminogen
binding and inflammatory macrophage recruitment in vivo.J Thromb
Haemost. (2017) 15:155–62. doi: 10.1111/jth.13532
63. Vago JP, Sugimoto MA, Lima KM, Negreiros-Lima GL, Baik N, Teixeira
MM, et al. Plasminogen and the plasminogen receptor, PLG-RKT, regulate
macrophage phenotypic, and functional changes. Front Immunol. (2019)
10:1–16. doi: 10.3389/fimmu.2019.01458
64. Blasi F, Sidenius N. The urokinase receptor: focused cell surface
proteolysis, cell adhesion and signaling. FEBS Lett. (2010) 584:1923–30.
doi: 10.1016/j.febslet.2009.12.039
65. Solberg H, Ploug M, Høyer-Hansen G, Nielsen BS, Lund LR. The murine
receptor for urokinase-type plasminogen activator is primarily expressed
in tissues actively undergoing remodeling. J Histochem Cytochem. (2001)
49:237–46. doi: 10.1177/002215540104900211
66. Rømer J, Lund LR, Eriksen J, Pyke C, Kristensen P, Dano K. The receptor
for urokinase-type plasminogen activator is expressed by keratinocytes
at the leading edge during re-epithelialization of mouse skin wounds.
J Invest Dermatol. (1994) 102:519–22. doi: 10.1111/1523-1747.ep1237
3187
Frontiers in Cardiovascular Medicine | www.frontiersin.org 15 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
67. d’Alessio S, Gerasi L, Blasi F. uPAR-deficient mouse keratinocytes fail to
produce EGFR-dependent laminin-5, affecting migration in vivo and in vitro.
J Cell Sci. (2008) 121:3922–32. doi: 10.1242/jcs.037549
68. Semnani R, Mizukami IF, Watt K, Todd RF, Liu DY. cDNA for Mo3, a
monocyte activation antigen, encodes the human receptor for urokinase
plasminogen activator. J Immunol. (1992) 148:3636–42.
69. Nykjaer A, Møller, B., Todd, R. F., Christensen, T., Andreasen, P. A.,
Gliemann, J., et al. (1994). Urokinase receptor. An activation antigen in
human T lymphocytes. J. Immunol. 152, 505–16.
70. Plesner T, Ploug M, Ellis V, Rønne E, Høyer-Hansen G, Wittrup M, et al.
The receptor for urokinase-type plasminogen activator and urokinase is
translocated from two distinct intracellular compartments to the plasma
membrane on stimulation of human neutrophils. Blood. (1994) 83:808–15.
doi: 10.1182/blood.V83.3.808.808
71. Paland N, Aharoni S, Fuhrman B. Urokinase-type plasminogen activator
(uPA) modulates monocyte-to-macrophage differentiation and prevents
Ox-LDL-induced macrophage apoptosis. Atherosclerosis. (2013) 231:29–38.
doi: 10.1016/j.atherosclerosis.2013.08.016
72. Park YJ, Liu G, Tsuruta Y, Lorne E, Abraham E. Participation of the
urokinase receptor in neutrophil efferocytosis. Blood. (2009) 114:860–70.
doi: 10.1182/blood-2008-12-193524
73. Prager GW, Mihaly J, Brunner PM, Koshelnick Y, Hoyer-Hansen G,
Binder BR. Urokinase mediates endothelial cell survival via induction of
the X-linked inhibitor of apoptosis protein. Blood. (2009) 113:1383–90.
doi: 10.1182/blood-2008-06-164210
74. Liot G, Roussel BD, Lebeurrier N, Benchenane K, López-Atalaya JP,
Vivien D, et al. Tissue-type plasminogen activator rescues neurones
from serum deprivation-induced apoptosis through a mechanism
independent of its proteolytic activity. J Neurochem. (2006) 98:1458–64.
doi: 10.1111/j.1471-4159.2006.03982.x
75. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PGW,
et al. The serpins are an expanding superfamily of structurally similar but
functionally diverse proteins. Evolution, mechanism of inhibition, novel
functions, and a revised nomenclature. J Biol Chem. (2001) 276:33293–6.
doi: 10.1074/jbc.R100016200
76. Lucas A, Yaron JR, Zhang L, Ambadapadi S. Overview of serpins and their
roles in biological systems. In: Methods in Molecular Biology. New York, NY:
Humana Press (2018) doi: 10.1007/978-1-4939-8645-3_1
77. Sawdey MS, Loskutoff DJ. Regulation of murine type 1 plasminogen
activator inhibitor gene expression in vivo. Tissue specificity and induction
by lipopolysaccharide, tumor necrosis factor-α, and transforming growth
factor-β. J Clin Invest. (1991) 88:1346–53. doi: 10.1172/JCI115440
78. Brogren H, Karlsson L, Andersson M, Wang L, Erlinge D, Jern S. Platelets
synthesize large amounts of active plasminogen activator inhibitor 1. Blood.
(2004) 104:3943–8. doi: 10.1182/blood-2004-04-1439
79. Simpson AJ, Booth NA, Moore NR, Bennett B. Distribution of plasminogen
activator inhibitor (PAI-1) in tissues. J Clin Pathol. (1991) 44:139–43.
doi: 10.1136/jcp.44.2.139
80. Medcalf RL. Plasminogen Activator Inhibitor Type 2: Still an EnigmaticSer pin
But a Model for Gene Regulation. 1st ed. San Diego, CA: Elsevier Inc. (2011).
81. Kruithof E, Baker M, Bunn C. Biological and clinical aspects of
plasminogen activator inhibitor type 2. Blood. (1995) 86:4007–24.
doi: 10.1182/blood.V86.11.4007.bloodjournal86114007
82. Hibino T, Matsuda Y, Takahashi T, Goetinck PF. Suppression of keratinocyte
proliferation by plasminogen activator inhibitor-2. J Invest Dermatol. (1999)
112:85–90. doi: 10.1046/j.1523-1747.1999.00466.x
83. Oji V, Oji ME, Adamini N, Walker T, Aufenvenne K, Raghunath M,
et al. Plasminogen activator inhibitor-2 is expressed in different types of
congenital ichthyosis: in vivo evidence for its cross-linking into the cornified
cell envelope by transglutaminase-1. Br J Dermatol. (2006) 154:860–7.
doi: 10.1111/j.1365-2133.2005.07109.x
84. Medcalf RL, Stasinopoulos SJ. The undecided serpin: the ins and outs
of plasminogen activator inhibitor type 2. FEBS J. (2005) 272:4858–67.
doi: 10.1111/j.1742-4658.2005.04879.x
85. Schroder WA, Major L, Suhrbier A. The role of serpinB2 in immunity. Crit
Rev Immunol. (2011) 31:15–30. doi: 10.1615/CritRevImmunol.v31.i1.20
86. Schroder WA, Hirata TD, Le TT, Gardner J, Boyle GM, Ellis J,
et al. SerpinB2 inhibits migration and promotes a resolution phase
signature in large peritoneal macrophages. Sci Rep. (2019) 9:1–15.
doi: 10.1038/s41598-019-48741-w
87. Thorsen S, Philips M, Selmer J, Lecander I, Åstedt B. Kinetics of inhibition
of tissue-type and urokinase-type plasminogen activator by plasminogen-
activator inhibitor type 1 and type 2. Eur J Biochem. (1988) 175:33–9.
doi: 10.1111/j.1432-1033.1988.tb14162.x
88. Andreasen PA, Georg B, Lund LR, Riccio A, Stacey SN. Plasminogen
activator inhibitors: hormonally regulated serpins. Mol Cell Endocrinol.
(1990) 68:1–19. doi: 10.1016/0303-7207(90)90164-4
89. Stief TW, Radtke KP, Heimburger N. Inhibition of urokinase by
protein C-inhibitor (PCI): evidence for identity of PCI and plasminogen
activator inhibitor 3. Biol Chem Hoppe Seyler. (1987) 368:1427–34.
doi: 10.1515/bchm3.1987.368.2.1427
90. Meijers JCM, Meijers J Kanters DHA, Meijers J Kanters Vlooswijk RA, Van
Erp HE, Hessing M, Bouma BN. Inactivation of human plasma kallikrein
and factor XIa by protein c inhibitor. Biochemistry. (1988) 27:4231–7.
doi: 10.1021/bi00412a005
91. Colman RW. Activation of plasminogen by human plasma
kallikrein. Biochem Biophys Res Commun. (1969) 35:273–9.
doi: 10.1016/0006-291X(69)90278-2
92. Miles LA, Greengard JS, Griffin JH. A comparision of the abilities of plasma
kallikrein, β-factor XIIa, factor XIa and urokinase to activate plasminogen.
Thromb Res. (1983) 29:407–17. doi: 10.1016/0049-3848(83)90244-X
93. Mansuy IM, Van Der Putten H, Schmid P, Meins M, Botteri FM,
Monard D. Variable and multiple expression of Profease Nexin-1 during
mouse organogenesis and nervous system development. Development.
(1993) 119:1119–34.
94. Baker JB, Gronke RS. Protease nexins and cellular regulation. Semin Thromb
Hemost. (1986) 12:216–20. doi: 10.1055/s-2007-1003554
95. Boulaftali Y, Adam F, Venisse L, Ollivier V, Richard B, Taieb S, et al.
Anticoagulant and antithrombotic properties of platelet protease nexin-1.
Blood. (2010) 115:97–106. doi: 10.1182/blood-2009-04-217240
96. Eaton DL, Scott RW, Baker JB. Purification of human fibroblast urokinase
proenzyme and analysis of its regulation by proteases and protease nexin. J
Biol Chem. (1984) 259:6241–7. doi: 10.1016/S0021-9258(20)82132-2
97. Boulaftali Y, Ho-Tin-Noe B, Pena A, Loyau S, Venisse L, François
D, et al. Platelet protease nexin-1, a serpin that strongly influences
fibrinolysis and thrombolysis. Circulation. (2011) 123:1326–34.
doi: 10.1161/CIRCULATIONAHA.110.000885
98. Menoud PA, Sappino N, Boudal-Khoshbeen M, Vassalli JD, Sappino AP. The
kidney is a major site of α2-antiplasmin production. J Clin Invest. (1996)
97:2478–84. doi: 10.1172/JCI118694
99. Bjorn W, Torbjorn Nilsson BC. Studies on a form of alpha2-antiplasmin in
plasma which does not interact with the lysine binding sites in plasminogen.
Thromb Res. (1982) 28:193–9. doi: 10.1016/0049-3848(82)90261-4
100. Abdul S, Leebeek FWG, Rijken DC, De Willige SU. Natural heterogeneity
of α2-antiplasmin: functional and clinical consequences. Blood. (2016)
127:538–45. doi: 10.1182/blood-2015-09-670117
101. Osterwalder T, Contartese J, Stoeckli ET, Kuhn TB, Sonderegger P.
Neuroserpin, an axonally secreted serine protease inhibitor. EMBO J. (1996)
15:2944–53. doi: 10.1002/j.1460-2075.1996.tb00657.x
102. Krueger SR, Ghisu GP, Cinelli P, Gschwend TP, Osterwalder T, Wolfer DP,
et al. Expression of neuroserpin, an inhibitor of tissue plasminogen activator,
in the developing and adult nervous system of the mouse. J Neurosci. (1997)
17:8984–96. doi: 10.1523/JNEUROSCI.17-23-08984.1997
103. Nielsen HM, Minthon L, Londos E, Blennow K, Miranda E,
Perez J, et al. Plasma and CSF serpins in Alzheimer disease
and dementia with Lewy bodies. Neurology. (2007) 69:1569–79.
doi: 10.1212/01.wnl.0000271077.82508.a0
104. Galliciotti G, Glatzel M, Kinter J, Kozlov SV, Cinelli P, Rülicke T,
et al. Accumulation of mutant neuroserpin precedes development of
clinical symptoms in familial encephalopathy with neuroserpin inclusion
bodies. Am J Pathol. (2007) 170:1305–13. doi: 10.2353/ajpath.2007.06
0910
105. Davies MJ, Miranda E, Roussel BD, Kaufman RJ, Marciniak SJ, Lomas
DA. Neuroserpin polymers activate NF-κB by a calcium signaling pathway
that is independent of the unfolded protein response. J Biol Chem. (2009)
284:18202–9. doi: 10.1074/jbc.M109.010744
Frontiers in Cardiovascular Medicine | www.frontiersin.org 16 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
106. Guadagno NA, Moriconi C, Licursi V, D’Acunto E, Nisi PS, Carucci
N, et al. Neuroserpin polymers cause oxidative stress in a neuronal
model of the dementia FENIB. Neurobiol Dis. (2017) 103:32–44.
doi: 10.1016/j.nbd.2017.03.010
107. Carugati A, Pappalardo E, Zingale LC, Cicardi M. C1-inhibitor
deficiency and angioedema. Mol Immunol. (2001) 38:161–73.
doi: 10.1016/S0161-5890(01)00040-2
108. Stoller JK, Aboussouan LS. A review of α1-antitrypsin deficiency. Am J
Respir Crit Care Med. (2012) 185:246–59. doi: 10.1164/rccm.201108-1428CI
109. Clemmensen I, Christensen F. Inhibition of urokinase by complex
formation with human α1-antitrypsin. BBA - Enzymol. (1976) 429:591–9.
doi: 10.1016/0005-2744(76)90307-7
110. Pannell R, Kung W, Gurewich V. C1-inhibitor prevents non-specific
plasminogen activation by a prourokinase mutant without impeding
fibrin-specific fibrinolysis. J Thromb Haemost. (2007) 5:1047–54.
doi: 10.1111/j.1538-7836.2007.02453.x
111. Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular
cloning, and partial characterization of a novel carboxypeptidase
B from human plasma. J Biol Chem. (1991) 266:21833–8.
doi: 10.1016/S0021-9258(18)54713-X
112. Mosnier LO, Buijtenhuijs P, Marx PF, Meijers JCM, Bouma BN.
Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in
human platelets. Blood. (2003) 101:4844–6. doi: 10.1182/blood-2002-09-
2944
113. Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI,
a thrombin-activable fibrinolysis inhibitor. J Biol Chem. (1995) 270:14477–
84. doi: 10.1074/jbc.270.24.14477
114. Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase
B, couples the coagulation and fibrinolytic cascades through the
thrombin-thrombomodulin complex. J Biol Chem. (1996) 271:16603–8.
doi: 10.1074/jbc.271.28.16603
115. Marx PF, Dawson PE, Bouma BN, Meijers JCM. Plasmin-mediated activation
and inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry.
(2002) 41:6688–96. doi: 10.1021/bi015982e
116. Heit JA. Thrombophilia: Clinical and Laboratory Assessment and
Management. Third Edit. Philadelphia, PA: Elsevier Inc. (2013).
117. Foley JH, Cook PF, Nesheim ME. Kinetics of activated thrombin-activatable
fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine
residues of fibrin degradation products and removal of plasminogen-binding
sites. J Biol Chem. (2011) 286:19280–6. doi: 10.1074/jbc.M110.215061
118. Barrett AJ, Starkey PM. The interaction of α2 macroglobulin with
proteinases: characteristics and specificity of the reaction, and a hypothesis
concerning its molecular mechanism. Biochem J. (1973) 133:709–24.
doi: 10.1042/bj1330709
119. Dziegielewska KM, Saunders NR, Schejter EJ, Zakut H, Zevin-Sonkin
D, Zisling R, et al. Synthesis of plasma proteins in fetal, adult,
and neoplastic human brain tissue. Dev Biol. (1986) 115:93–104.
doi: 10.1016/0012-1606(86)90231-9
120. Bouma ME, Pessah M, Renaud G, Amit N, Catala D, Infante R. Synthesis and
secretion of lipoproteins by human hepatocytes in culture. Vitr Cell Dev Biol.
(1988) 24:85–90. doi: 10.1007/BF02623884
121. Lysiak JJ, Hussaini IM, Gonias SL. α2-macroglobulin
synthesis by the human monocytic cell line THP-1 is
differentiation state-dependent. J Cell Biochem. (1997) 67:492–7.
doi: 10.1002/(SICI)1097-4644(19971215)67:4<492::AID-JCB7>3.0.CO;2-N
122. Holmberg L, Lecander I, Astedt B. Binding of urokinase to plasma
proteinase inhibitors. Scand J Clin Lab Invest. (1980) 40:743–7.
doi: 10.3109/00365518009095590
123. Francis RB. Clinical disorders of fibrinolysis: a critical review. Blut. (1989)
59:1–14. doi: 10.1007/BF00320240
124. Draxler D, Sashindranath M, Medcalf R. Plasmin: a modulator
of immune function. Semin Thromb Hemost. (2016) 43:143–53.
doi: 10.1055/s-0036-1586227
125. Heissig B, Salama Y, Takahashi S, Osada T, Hattori K. The multifaceted
role of plasminogen in inflammation. Cell Signal. (2020) 75:109761.
doi: 10.1016/j.cellsig.2020.109761
126. Draxler DF, Yep K, Hanafi G, Winton A, Daglas M, Ho H,
et al. Tranexamic acid modulates the immune response and
reduces postsurgical infection rates. Blood Adv. (2019) 3:1598–609.
doi: 10.1182/bloodadvances.2019000092
127. Lin H, Xu L, Yu S, Hong W, Huang M, Xu P. Therapeutics
targeting the fibrinolytic system. Exp Mol Med. (2020) 52:367–79.
doi: 10.1038/s12276-020-0397-x
128. Carmeliet P, Moons L, Lijnen R, Janssens S, Lupu F, Collen D,
et al. Inhibitory role of plasminogen activator inhibitor-1 in arterial
wound healing and neointima formation. Circulation. (1997) 96:3180–91.
doi: 10.1161/01.CIR.96.9.3180
129. Schäfer K, Schroeter MR, Dellas C, Puls M, Nitsche M, Weiss
E, et al. Plasminogen activator inhibitor-1 from bone marrow-
derived cells suppresses neointimal formation after vascular
injury in mice. Arterioscler Thromb Vasc Biol. (2006) 26:1254–9.
doi: 10.1161/01.ATV.0000215982.14003.b7
130. Wu J, Peng L, McMahon GA, Lawrence DA, Fay WP. Recombinant
plasminogen activator inhibitor-1 inhibits intimal hyperplasia. Arterioscler
Thromb Vasc Biol. (2009) 29:1565–70. doi: 10.1161/ATVBAHA.109.189514
131. Zhong J, Yang HC, Kon V, Fogo AB, Lawrence DA, Ma J. Vitronectin-
binding PAI-1 protects against the development of cardiac fibrosis
through interaction with fibroblasts. Lab Investig. (2014) 94:633–44.
doi: 10.1038/labinvest.2014.51
132. Qian HS, Gu JM, Liu P, Kauser K, Halks-Miller M, Vergona R, et al.
Overexpression of PAI-1 prevents the development of abdominal aortic
aneurysm in mice. Gene Ther. (2008) 15:224–32. doi: 10.1038/sj.gt.3303069
133. Jankun J, Keck R, Selman SH, Skrzypczak-Jankun E. Systemic or topical
application of plasminogen activator inhibitor with extended half-life (VLHL
PAI-1) reduces bleeding time and total blood loss. Int J Mol Med. (2010)
26:501–4. doi: 10.3892/ijmm_00000491
134. Yang D, Nemkul N, Shereen A, Jone A, Scott Dunn R, Lawrence DA, et al.
Therapeutic administration of plasminogen activator inhibitor-1 prevents
hypoxic-ischemic brain injury in newborns. J Neurosci. (2009) 29:8669–74.
doi: 10.1523/JNEUROSCI.1117-09.2009
135. Yang D, Sun YY, Lin X, Baumann JM, Warnock M, Lawrence DA,
et al. Taming neonatal hypoxic-ischemic brain injury by intranasal
delivery of plasminogen activator inhibitor-1. Stroke. (2013) 44:2623–7.
doi: 10.1161/STROKEAHA.113.001233
136. Jankun J. Recombinant PAI-1 inhibits angiogenesis and reduces size of
LNCaP prostate cancer xenografts in SCID mice. Oncol Rep. (2001) 8:463–70.
doi: 10.3892/or.8.3.463
137. Praus M, Wauterickx K, Collen D, Gerard RD. Reduction of tumor cell
migration and metastasis by adenoviral gene transfer of plasminogen
activator inhibitors. Gene Ther. (1999) 6:227–36. doi: 10.1038/sj.gt.3300802
138. Kager LM, Wiersinga WJ, Roelofs JJTH, Meijers JCM, Levi M, Van’T Veer
C, et al. Plasminogen activator inhibitor type I contributes to protective
immunity during experimental Gram-negative sepsis (melioidosis). J
Thromb Haemost. (2011) 9:2020–8. doi: 10.1111/j.1538-7836.2011.04473.x
139. Lim JH, Woo CH, Li JD. Critical role of type 1 plasminogen activator
inhibitor (PAI-1) in early host defense against nontypeable Haemophilus
influenzae (NTHi) infection. Biochem Biophys Res Commun. (2011) 414:67–
72. doi: 10.1016/j.bbrc.2011.09.023
140. Harslund J, Frees D, Leifsson PS, Offenberg H, Rømer MU, Brünner N, et al.
The role of Serpine-1 and tissue inhibitor of metalloproteinase type-1 in early
host responses to Staphylococcus aureus intracutaneous infection of mice.
Pathog Dis. (2013) 68:96–104. doi: 10.1111/2049-632X.12055
141. Renckens R, Roelofs JJTH, Bonta PI, Florquin S, De Vries CJM,
Levi M, et al. Plasminogen activator inhibitor type 1 is protective
during severe Gram-negative pneumonia. Blood. (2007) 109:1593–601.
doi: 10.1182/blood-2006-05-025197
142. Kim KS, Lee YA, Choi HM, Yoo MC, Yang HI. Implication of MMP-
9 and urokinase plasminogen activator (uPA) in the activation of pro-
matrix metalloproteinase (MMP)-13. Rheumatol Int. (2012) 32:3069–75.
doi: 10.1007/s00296-011-2095-4
143. Stevens P, Scott RW, Shatzen EM. Recombinant human protease nexin-1
prevents articular cartilage-degradation in the rabbit. Agents Actions Suppl.
(1993) 39:173–7. doi: 10.1007/978-3-0348-7442-7_20
144. McKee CM, Xu D, Cao Y, Kabraji S, Allen D, Kersemans V, et al. Protease
nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma. J Clin
Invest. (2012) 122:4025–36. doi: 10.1172/JCI59348
Frontiers in Cardiovascular Medicine | www.frontiersin.org 17 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
145. McKee CM, Ding Y, Zhou J, Li C, Huang L, Xin X, et al. Protease
nexin 1 induces apoptosis of prostate tumor cells through inhibition of
X-chromosome-linked inhibitor of apoptosis protein. Oncotarget. (2015)
6:3784–96. doi: 10.18632/oncotarget.2921
146. Li XL, Wang P, Xie Y. Protease nexin-1 protects against Alzheimer’s disease
by regulating the sonic hedgehog signaling pathway. Int J Neurosci. (2020)
2020:7454. doi: 10.1080/00207454.2020.1773821
147. Lee KN, Jackson KW, Christiansen VJ, Chung KH, McKee PA.
Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and
removal. Curr Med Chem Cardiovasc Hematol Agents. (2004) 2:303–10.
doi: 10.2174/1568016043356228
148. Weitz JI, Leslie B, Hirsh J, Klement P. α2-Antiplasmin supplementation
inhibits tissue plasminogen activator- induced fibrinogenolysis and bleeding
with little effect on thrombolysis. J Clin Invest. (1993) 91:1343–50.
doi: 10.1172/JCI116335
149. Nieuwenhuizen L, Roosendaal G, Mastbergen SC, Coeleveld K, Biesma DH,
Lafeber FPJG, et al. Antiplasmin, but not amiloride, prevents synovitis and
cartilage damage following hemarthrosis in hemophilic mice. J Thromb
Haemost. (2014) 12:237–45. doi: 10.1111/jth.12467
150. Vassalli JD, Belin D. Amiloride selectively inhibits the urokinase-
type plasminogen activator. FEBS Lett. (1987) 214:187–91.
doi: 10.1016/0014-5793(87)80039-X
151. Hayashido Y, Hamana T, Ishida Y, Shintani T, Koizumi KI, Okamoto T.
Induction of α2-antiplasmin inhibits E-cadherin processing mediated by the
plasminogen activator/plasmin system, leading to suppression of progression
of oral squamous cell carcinoma via upregulation of cell-cell adhesion. Oncol
Rep. (2007) 17:417–23. doi: 10.3892/or.17.2.417
152. Paquet-Fifield S, Roufail S, Zhang YF, Sofian T, Byrne DJ, Coughlin PB, et al.
The fibrinolysis inhibitor α2-antiplasmin restricts lymphatic remodelling
and metastasis in a mouse model of cancer. Growth Factors. (2017) 35:61–75.
doi: 10.1080/08977194.2017.1349765
153. Yepes M, Sandkvist M, Wong MKK, Coleman TA, Smith E, Cohan
SL, et al. Neuroserpin reduces cerebral infarct volume and protects
neurons from ischemia-induced apoptosis. Blood. (2000) 96:569–76.
doi: 10.1182/blood.V96.2.569
154. Zhang Z, Zhang L, Yepes M, Jiang Q, Li Q, Arniego P, et al. Adjuvant
treatment with neuroserpin increases the therapeutic window for tissue-
type plasminogen activator administration in a rat model of embolic stroke.
Circulation. (2002) 106:740–5. doi: 10.1161/01.CIR.0000023942.10849.41
155. Cai L, Zhou Y, Wang Z, Zhu Y. Neuroserpin extends the time window of tPA
thrombolysis in a rat model of middle cerebral artery occlusion. J Biochem
Mol Toxicol. (2020) 34:1–8. doi: 10.1002/jbt.22570
156. Wu J, Echeverry R, Guzman J, Yepes M. Neuroserpin protects neurons
from ischemia-induced plasmin-mediated cell death independently of tissue-
type plasminogen activator inhibition. Am J Pathol. (2010) 177:2576–84.
doi: 10.2353/ajpath.2010.100466
157. Yepes M, Sandkvist M, Coleman TA, Moore E, Wu JY, Mitola D, et al.
Regulation of seizure spreading by neuroserpin and tissue-type plasminogen
activator is plasminogen-independent. J Clin Invest. (2002) 109:1571–8.
doi: 10.1172/JCI0214308
158. Lebeurrier N, Liot G, Lopez-Atalaya JP, Orset C, Fernandez-Monreal
M, Sonderegger P, et al. The brain-specific tissue-type plasminogen
activator inhibitor, neuroserpin, protects neurons against excitotoxicity
both in vitro and in vivo.Mol Cell Neurosci. (2005) 30:552–8.
doi: 10.1016/j.mcn.2005.09.005
159. Li Z, Liu F, Zhang L, Cao Y, Shao Y, Wang X, et al. Neuroserpin restores
autophagy and promotes functional recovery after acute spinal cord injury
in rats. Mol Med Rep. (2018) 17:2957–63. doi: 10.3892/mmr.2017.8249
160. Gu RP, Fu LL, Jiang CH, Xu YF, Wang X, Yu J. Retina is protected
by neuroserpin from ischemic/reperfusion-induced injury independent
of tissue-type plasminogen activator. PLoS ONE. (2015) 10:e0130440.
doi: 10.1371/journal.pone.0130440
161. Munuswamy-Ramanujam G, Dai E, Liu L, Shnabel M, Sun YM, Bartee M,
et al. Neuroserpin, a thrombolytic serine protease inhibitor (serpin), blocks
transplant vasculopathy with associated modification of T-helper cell subsets.
Thromb Haemost. (2010) 103:545–55. doi: 10.1160/TH09-07-0441
162. Chen H, Zheng D, Abbott J, Liu L, Bartee MY, Long M, et al. Myxomavirus-
derived serpin prolongs survival and reduces inflammation and hemorrhage
in an unrelated lethal mouse viral infection. Antimicrob Agents Chemother.
(2013) 57:4114–27. doi: 10.1128/AAC.02594-12
163. Huntington JA. Serpin structure, function and dysfunction. J Thromb
Haemost. (2011) 9(Suppl 1):26–34. doi: 10.1111/j.1538-7836.2011.04360.x
164. Stockley RA. The multiple facets of alpha-1-antitrypsin. Ann Transl Med.
(2015) 3:130. doi: 10.3978/j.issn.2305-5839.2015.04.25
165. Janciauskiene S, Welte T. Well-known and less well-known functions of
Alpha-1 antitrypsin: its role in chronic obstructive pulmonary disease
and other disease developments. Ann Am Thorac Soc. (2016) 13:S280–8.
doi: 10.1513/AnnalsATS.201507-468KV
166. Talens S, Malfliet JJMC, van Hal PTW, Leebeek FWG, Rijken DC.
Identification and characterization of α1-antitrypsin in fibrin clots. J Thromb
Haemost. (2013) 11:1319–28. doi: 10.1111/jth.12288
167. Cathomas M, Schüller A, Candinas D, Inglin R. Severe postoperative
wound healing disturbance in a patient with alpha-1-antitrypsin deficiency:
the impact of augmentation therapy. Int Wound J. (2015) 12:601–4.
doi: 10.1111/iwj.12419
168. Torres-Durán M, Lopez-Campos JL, Barrecheguren M, Miravitlles M,
Martinez-Delgado B, Castillo S, et al. Alpha-1 antitrypsin deficiency:
outstanding questions and future directions. Orphanet J Rare Dis. (2018)
13:1–15. doi: 10.1186/s13023-018-0856-9
169. Lorincz R, Curiel DT. Advances in Alpha-1 Antitrypsin Gene Therapy. Am J
Respir Cell Mol Biol. (2020) 63:560–70. doi: 10.1165/rcmb.2020-0159PS
170. Libert C, Van Molle W, Brouckaert P, Fiers W. alpha1-Antitrypsin inhibits
the lethal response to TNF in mice. J Immunol. (1996) 157:5126–9.
171. Churg A, Wang RD, Xie C, Wright JL. A-1-antitrypsin ameliorates cigarette
smoke-induced emphysema in the mouse. Am J Respir Crit Care Med. (2003)
168:199–207. doi: 10.1164/rccm.200302-203OC
172. Wang X, Gong J, Zhu J, Jin Z, Gao W. Alpha 1-antitrypsin for treating
ventilator-associated lung injury in acute respiratory distress syndrome rats.
Exp Lung Res. (2019) 45:209–19. doi: 10.1080/01902148.2019.1642968
173. Akbar MA, Cao JJ, Lu Y, Nardo D, Chen M-J, Elshikha AS, et al.
Alpha-1 antitrypsin gene therapy ameliorates bone loss in ovariectomy-
induced osteoporosis mouse model. Hum Gene Ther. (2016) 27:679–86.
doi: 10.1089/hum.2016.029
174. Lewis EC, Shapiro L, Bowers OJ, Dinarello CA. A1-antitrypsin monotherapy
prolongs islet allograft survival in mice. Proc Natl Acad Sci USA. (2005)
102:12153–8. doi: 10.1073/pnas.0505579102
175. Zhang B, Lu Y, Campbell-Thompson M, Spencer T, Wasserfall C, Atkinson
M, et al. A1-antitrypsin protects B-cells from apoptosis. Diabetes. (2007)
56:1316–23. doi: 10.2337/db06-1273
176. Gou W, Wang J, Song L, Kim D, Cui W, Strange C, et al. Alpha-
1 antitrypsin suppresses macrophage activation and promotes islet graft
survival after intrahepatic islet transplantation. Am J Transplant. (2020)
1–12. doi: 10.1111/ajt.16342
177. Marcondes AM, Li X, Tabellini L, Bartenstein M, Kabacka J, Sale GE, et al.
Inhibition of IL-32 activation by α-1 antitrypsin suppresses alloreactivity and
increases survival in an allogeneic murine marrow transplantation model.
Blood. (2011) 118:5031–9. doi: 10.1182/blood-2011-07-365247
178. Tawara I, Sun Y, Lewis EC, Toubai T, Evers R, Nieves E, et al.
Alpha-1-antitrypsin monotherapy reduces graft-versus-host disease after
experimental allogeneic bone marrow transplantation. Proc Natl Acad Sci
USA. (2012) 109:564–9. doi: 10.1073/pnas.1117665109
179. Lee C, Dhawan A, Filippi C, Mitry R, Iansante V, Dacosta RF,
et al. Improving the efficacy of hepatocyte transplantation using alpha-
1 antitrypsin as an immune modulator. J Hepatol. (2018) 68:S130–S131.
doi: 10.1016/S0168-8278(18)30472-0
180. Emtiazjoo AM, Hu H, Lu L, Brantly ML. Alpha-1 antitrypsin attenuates acute
lung allograft injury in a rat lung transplant model. Transplant Direct. (2019)
5:1–6. doi: 10.1097/TXD.0000000000000898
181. Ortiz G, Lopez ES, Salica JP, Potilinski C, Fernández Acquier M,
Chuluyan E, et al. Alpha-1-antitrypsin ameliorates inflammation and
neurodegeneration in the diabetic mouse retina. Exp Eye Res. (2018) 174:29–
39. doi: 10.1016/j.exer.2018.05.013
182. Yang S, Xian B, Li K, Luo Z, Liu Y, Hu D, et al. Alpha 1-
antitrypsin inhibits microglia activation and facilitates the survival of iPSC
grafts in hypertension mouse model. Cell Immunol. (2018) 328:49–57.
doi: 10.1016/j.cellimm.2018.03.006
Frontiers in Cardiovascular Medicine | www.frontiersin.org 18 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
183. Zhou T, Huang Z, Zhu X, Sun X, Liu Y, Cheng B, et al. Alpha-
1 antitrypsin attenuates M1 microglia-mediated neuroinflammation
in retinal degeneration. Front Immunol. (2018) 9:1–13.
doi: 10.3389/fimmu.2018.01202
184. Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury: pathogenic
mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol. (2003)
18:891–902. doi: 10.1046/j.1440-1746.2003.03056.x
185. Wu MY, Yiang GT, Liao WT, Tsai APY, Cheng YL, Cheng PW, et al.
Current mechanistic concepts in ischemia and reperfusion injury. Cell
Physiol Biochem. (2018) 46:1650–67. doi: 10.1159/000489241
186. Yaron JR, Chen H, Ambadapadi S, Zhang L, Tafoya AM, Munk BH,
et al. Serp-2, a virus-derived apoptosis and inflammasome inhibitor,
attenuates liver ischemia-reperfusion injury in mice. J Inflamm. (2019) 16:12.
doi: 10.1186/s12950-019-0215-1
187. Yaron JR, Zhang L, Guo Q, Chen H, Lucas AR. A mouse model of
acute liver injury by warm, partial ischemia-reperfusion for testing the
efficacy of virus-derived therapeutics. In: Lucas AR, editors. Viruses as
Therapeutics. Methods in Molecular Biology, vol. 2225. Humana; New York,
NY. doi: 10.1007/978-1-0716-1012-1_16
188. Li J, Zhao J, Xu M, Li M, Wang B, Qu X, et al. Blocking GSDMD
processing in innate immune cells but not in hepatocytes protects
hepatic ischemia–reperfusion injury. Cell Death Dis. (2020) 11:244.
doi: 10.1038/s41419-020-2437-9
189. Moldthan HL, Hirko AC, Thinschmidt JS, Grant MB, Li Z, Peris
J, et al. Alpha 1-antitrypsin therapy mitigated ischemic stroke
damage in rats. J Stroke Cerebrovasc Dis. (2014) 23:e355–63.
doi: 10.1016/j.jstrokecerebrovasdis.2013.12.029
190. Toldo S, Mauro AG, Marchetti C, Rose SW, Mezzaroma E, Van Tassell
BW, et al. Recombinant human alpha-1 antitrypsin-fc fusion protein
reduces mouse myocardial inflammatory injury after ischemia-reperfusion
independent of elastase inhibition. J Cardiovasc Pharmacol. (2016) 68:27–32.
doi: 10.1097/FJC.0000000000000383
191. Abbate A, Van Tassell BW, Christopher S, Abouzaki NA, Sonnino C, Oddi C,
et al. Effects of Prolastin C (plasma-derived alpha-1 antitrypsin) on the acute
inflammatory response in patients with ST-segment elevation myocardial
infarction (from the VCU-alpha 1-RT pilot study). Am J Cardiol. (2015)
115:8–12. doi: 10.1016/j.amjcard.2014.09.043
192. Abouzaki NA, Christopher S, Trankle C, Van Tassell BW, Carbone S,
Mauro AG, et al. Inhibiting the inflammatory injury after myocardial
ischemia reperfusion with plasma-derived alpha-1 antitrypsin. J Cardiovasc
Pharmacol. (2018) 71:375–9. doi: 10.1097/FJC.0000000000000583
193. Maicas N, Van Der Vlag J, Bublitz J, Florquin S, Bebber MB, Dinarello
CA, et al. Human Alpha-1-Antitrypsin (hAAT) therapy reduces renal
dysfunction and acute tubular necrosis in a murine model of bilateral
kidney ischemia-reperfusion injury. PLoS ONE. (2017) 12:e0168981.
doi: 10.1371/journal.pone.0168981
194. Jeong KH, Lim JH, Lee KH, Kim MJ, Jung HY, Choi JY, et al. Protective effect
of alpha 1-antitrypsin on renal ischemia-reperfusion injury. Transplant Proc.
(2019) 51:2814–22. doi: 10.1016/j.transproceed.2019.04.084
195. Xu T, Zhang G, Lin H, Xie Y, Feng Y, Zhang X, et al. Clinical
characteristics and risk factors of diffuse alveolar hemorrhage in systemic
lupus erythematosus: a systematic review and meta-analysis based on
observational studies. Clin Rev Allergy Immunol. (2019) 59:295–303.
doi: 10.1007/s12016-019-08763-8
196. Elshikha AS, Lu Y, Chen MJ, Akbar M, Zeumer L, Ritter A, et al.
Alpha 1 antitrypsin inhibits dendritic cell activation and attenuates
nephritis in a mouse model of lupus. PLoS ONE. (2016) 11:e0156583.
doi: 10.1371/journal.pone.0156583
197. Elshikha AS, Yuan Y, Lu Y, Chen MJ, Abboud G, Akbar MA, et al. Alpha 1
antitrypsin gene therapy extends the lifespan of lupus-prone mice. Mol Ther
Methods Clin Dev. (2018) 11:131–42. doi: 10.1016/j.omtm.2018.10.007
198. Elshikha AS, Abboud G, van der Meijden-Erkelens L, Lu Y, Chen M-J, Yuan
Y, et al. Alpha-1-antitrypsin ameliorates pristane induced diffuse alveolar
hemorrhage in mice. J Clin Med. (2019) 8:1341. doi: 10.3390/jcm8091341
199. Finlay BB, McFadden G. Anti-immunology: evasion of the host immune
system by bacterial and viral pathogens. Cell. (2006) 124:767–82.
doi: 10.1016/j.cell.2006.01.034
200. Yaron JR, Zhang L, Guo Q, Burgin M, Schutz LN, Awo E, et al. Deriving
immune modulating drugs from viruses—a new class of biologics. J Clin
Med. (2020) 9:972. doi: 10.3390/jcm9040972
201. Nash P, Whitty A, Handwerker J, Macen J, McFadden G. Inhibitory specificity
of the anti-inflammatory myxoma virus serpin, SERP-1. J Biol Chem. (1998)
273:20982–91. doi: 10.1074/jbc.273.33.20982
202. Mahon BP, Ambadapadi S, Yaron JR, Lomelino CL, Pinard MA, Keinan S,
et al. Crystal structure of cleaved serp-1, a myxomavirus-derived immune
modulating serpin: structural design of serpin reactive center loop peptides
with improved therapeutic function. Biochemistry. (2018) 57:1096–107.
doi: 10.1021/acs.biochem.7b01171
203. Lucas A, Liu LY, Macen J, Nash P, Dai E, Stewart M, et al. Virus-
encoded serine proteinase inhibitor SERP-1 inhibits atherosclerotic plaque
development after balloon angioplasty. Circulation. (1996) 94:2890–900.
doi: 10.1161/01.CIR.94.11.2890
204. Bot I, Von der Thüsen JH, Donners MMPC, Lucas A, Fekkes ML, De Jager
SCA, et al. Serine protease inhibitor Serp-1 strongly impairs atherosclerotic
lesion formation and induces a stable plaque phenotype in ApoE-/- mice.
Circ Res. (2003) 93:464–71. doi: 10.1161/01.RES.0000090993.01633.D4
205. Bedard EL, Jiang J, Arp J, Qian H, Wang H, Guan H, et al. Prevention of
chronic renal allograft rejection by SERP-1 protein. Transplantation. (2006)
81:908–14. doi: 10.1097/01.tp.0000203141.02725.8a
206. Dai E, Viswanathan K, Sun YM, Li X, Liu LY, Togonu-Bickersteth B,
et al. Identification of myxomaviral serpin reactive site loop sequences
that regulate innate immune responses. J Biol Chem. (2006) 281:8041–50.
doi: 10.1074/jbc.M509454200
207. Jiang J, Arp J, Kubelik D, Zassoko R, Liu W, Wise Y, et al.
Induction of indefinite cardiac allograft survival correlates with
toll-like receptor 2 and 4 downregulation after serine protease
inhibitor-1 (Serp-1) treatment. Transplantation. (2007) 84:1158–67.
doi: 10.1097/01.tp.0000286099.50532.b0
208. Brahn E, Lee S, Lucas A, McFadden G, Macaulay C. Suppression of
collagen-induced arthritis with a serine proteinase inhibitor (serpin)
derived from myxoma virus. Clin Immunol. (2014) 153:254–63.
doi: 10.1016/j.clim.2014.05.003
209. Chen H, Zheng D, Ambadapadi S, Davids J, Ryden S, Samy H, et al.
Serpin treatment suppresses inflammatory vascular lesions in temporal
artery implants (TAI) from patients with giant cell arteritis. PLoS ONE.
(2015) 10:e0115482. doi: 10.1371/journal.pone.0115482
210. Ambadapadi S, Munuswamy-Ramanujam G, Zheng D, Sullivan C, Dai E,
Morshed S, et al. Reactive Center Loop (RCL) peptides derived from serpins
display independent coagulation and immune modulating activities. J Biol
Chem. (2016) 291:2874–87. doi: 10.1074/jbc.M115.704841
211. Yaron JR, Ambadapadi S, Zhang L, Chavan RN, Tibbetts SA, Keinan
S, et al. Immune protection is dependent on the gut microbiome in
a lethal mouse gammaherpesviral infection. Sci Rep. (2020) 10:2371.
doi: 10.1038/s41598-020-59269-9
212. Viswanathan K, Richardson J, Togonu-Bickersteth B, Dai E, Liu L, Vatsya
P, et al. Myxoma viral serpin, Serp-1, inhibits human monocyte adhesion
through regulation of actin-binding protein filamin B. J Leukoc Biol. (2009)
85:418–26. doi: 10.1189/jlb.0808506
213. Zhang L, Yaron JR, Tafoya AM, Wallace SE, Kilbourne J, Haydel S,
et al. A virus-derived immune modulating serpin accelerates wound
closure with improved collagen remodeling. J Clin Med. (2019) 8:1626.
doi: 10.3390/jcm8101626
214. Kwiecien JM, Zhang L, Yaron JR, Schutz LN, Kwiecien-Delaney CJ, Awo EA,
et al. Local serpin treatment via chitosan-collagen hydrogel after spinal cord
injury reduces tissue damage and improves neurologic function. J Clin Med.
(2020) 9:1221. doi: 10.3390/jcm9041221
215. Tardif JC, L’Allier PL, Grégoire J, Ibrahim R, McFadden G, Kostuk
W, et al. A randomized controlled, phase 2 trial of the viral serpin
Serp-1 in patients with acute coronary syndromes undergoing
percutaneous coronary intervention. Circ Cardiovasc Interv. (2010)
3:543–8. doi: 10.1161/CIRCINTERVENTIONS.110.953885
216. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus
from patients with pneumonia in China, 2019. N Engl J Med. (2020) 382:727–
33. doi: 10.1056/NEJMoa2001017
Frontiers in Cardiovascular Medicine | www.frontiersin.org 19 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
217. Cucinotta D, Vanelli M. WHO declares COVID-19 a pandemic. Acta Biomed.
(2020) 91:157–60. doi: 10.23750/abm.v91i1.9397
218. Gattinoni L, Chiumello D, Caironi P, Busana M, Romitti F, Brazzi
L, et al. COVID-19 pneumonia: different respiratory treatments
for different phenotypes? Intensive Care Med. (2020) 46:1099–102.
doi: 10.1007/s00134-020-06033-2
219. Giannis D, Ziogas IA, Gianni P. Coagulation disorders in coronavirus
infected patients: COVID-19, SARS-CoV-1, MERS-CoV and lessons from
the past. J Clin Virol. (2020) 127:104362. doi: 10.1016/j.jcv.2020.104362
220. Spiezia L, Boscolo A, Poletto F, Cerruti L, Tiberio I, Campello E,
et al. COVID-19-related severe hypercoagulability in patients admitted to
intensive care unit for acute respiratory failure. Thromb Haemost. (2020)
120:998–1000. doi: 10.1055/s-0040-1714350
221. Weiss E, Roux O, Moyer JD, Paugam-Burtz C, Boudaoud L, Ajzenberg
N, et al. Fibrinolysis resistance: a potential mechanism underlying
COVID-19 coagulopathy. Thromb Haemost. (2020) 120:1343–5.
doi: 10.1055/s-0040-1713637
222. Siddiqi HK, Libby P, Ridker PM. COVID-19 – a vascular disease. Trends
Cardiovasc Med. (2020) 31:1–5. doi: 10.1016/j.tcm.2020.10.005
223. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure,
function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell.
(2020) 181:281–92.e6. doi: 10.1016/j.cell.2020.02.058
224. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, et al.
Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
Science. (2020) 367:1260–63. doi: 10.1126/science.abb2507
225. Hoffmann M, Kleine-Weber H, Pöhlmann S. A multibasic cleavage site in the
spike protein of SARS-CoV-2 is essential for infection of human lung cells.
Mol Cell. (2020) 78:779–84.e5. doi: 10.1016/j.molcel.2020.04.022
226. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen
S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is
blocked by a clinically proven protease inhibitor. Cell. (2020) 181:271–80.e8.
doi: 10.1016/j.cell.2020.02.052
227. Hofmann-Winkler H, Moerer O, Alt-Epping S, Bräuer A, Büttner B,
Müller M, et al. Camostat mesylate may reduce severity of coronavirus
disease 2019 sepsis: a first observation. Crit Care Explor. (2020) 2:e0284.
doi: 10.1097/CCE.0000000000000284
228. Azouz NP, Klingler AM, Callahan V, Akhrymuk IV, Elez K, Raich L, et al.
Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-Priming Protease
TMPRSS2. bioRxiv. (2020) 2:1–16. doi: 10.1101/2020.05.04.077826
229. Wettstein L, Conzelmann C, Müller J, Weil T, Groß R, Hirschenberger M,
et al. Alpha-1 antitrypsin inhibits SARS-CoV-2 infection. bioRxiv. (2020).
doi: 10.1101/2020.07.02.183764
230. Beard KS, MaWhinney S, Zamora M, Oberley-Deegan RE, Crapo JD, Pott
GB, et al. ALPHA-1-Antitrypsin inhibits influenza in vitro, reduces influenza
disease in vivo, and genetic deficiency is a risk factor for human influenza
infection. Cytokine. (2009) 48:38. doi: 10.1016/j.cyto.2009.07.347
231. Esumi M, Ishibashi M, Yamaguchi H, Nakajima S, Tai Y, Kikuta
S, et al. Transmembrane serine protease TMPRSS2 activates hepatitis
C virus infection. Hepatology. (2015) 61:437–46. doi: 10.1002/hep.
27426
232. Seheult JN, Seshadri A, Neal MD. Fibrinolysis shutdown and
thrombosis in severe COVID-19. J Am Coll Surg. (2020) 231:203–4.
doi: 10.1016/j.jamcollsurg.2020.05.021
233. Matsuyama T, Kubli SP, Yoshinaga SK, Pfeffer K, Mak TW. An aberrant
STAT pathway is central to COVID-19. Cell Death Differ., (2020) 3209–25.
doi: 10.1038/s41418-020-00633-7
234. Ji H-L, Zhao R, Matalon S, Matthay MA. Elevated Plasmin(ogen) as a
Common Risk Factor for COVID-19 Susceptibility. Physiol Rev. (2020)
100:1065–75. doi: 10.1152/physrev.00013.2020
235. Thierry AR. Anti-protease treatments targeting plasmin(ogen) and
neutrophil elastase may be beneficial in fighting COVID-19. Physiol Rev.
(2020) 100:1597–8. doi: 10.1152/physrev.00019.2020
236. Medcalf RL, Keragala CB, Myles PS. Fibrinolysis and COVID-19: a
plasmin paradox. J Thromb Haemost. (2020) 18:2118–22. doi: 10.1111/jth.
14960
237. Ogawa H, Asakura H. Consideration of tranexamic acid
administration to COVID-19 patients. Physiol Rev. (2020) 100:1595–6.
doi: 10.1152/physrev.00023.2020
238. Dittmann M, Hoffmann H, Scull MA, Gilmore RH, Bell KL, Ciancanelli M,
et al. A serpin shapes the extracellular environment to prevent influenza a
virus maturation. Cell. (2015) 160:631–43. doi: 10.1016/j.cell.2015.01.040
239. Colling ME, Kanthi Y. COVID19-associated coagulopathy: an exploration
of mechanisms. VascMed. (2020 ) 25:471–8. doi: 10.1177/1358863X20932640
240. Zuo Y, Warnock M, Harbaugh A, Yalavarthi S, Gockman K, Zuo M,
et al. Plasma tissue plasminogen activator and plasminogen activator
inhibitor-1 in hospitalized COVID-19 patients. medRxiv. (2020).
doi: 10.1101/2020.08.29.20184358
241. Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG, Decroly E.
The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-
like cleavage site absent in CoV of the same clade. Antiviral Res. (2020)
176:104742. doi: 10.1016/j.antiviral.2020.104742
242. Fernandez C, Rysä J, Almgren P, Nilsson J, Engström G, Orho-
Melander M, et al. Plasma levels of the proprotein convertase furin and
incidence of diabetes and mortality. J Intern Med. (2018) 284:377–87.
doi: 10.1111/joim.12783
243. Braun E, Sauter D. Furin-mediated protein processing in infectious diseases
and cancer. Clin Transl Immunol. (2019) 8:1–19. doi: 10.1002/cti2.1073
244. Adu-Agyeiwaah Y, Grant MB, Obukhov AG. The potential role of
osteopontin and furin in worsening disease outcomes in COVID-19 patients
with pre-existing diabetes. Cells. (2020) 9. doi: 10.3390/cells9112528
245. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, et al.
Furin inhibitors block SARS-CoV-2 spike protein cleavage to suppress
virus production and cytopathic effects. Cell Rep. (2020) 33:108254.
doi: 10.1016/j.celrep.2020.108254
246. Hallenberger S, Bosch V, Angliker H, Shaw E, Klenk HD, Garten W.
Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein
gp160. Nature. (1992) 360:358–61. doi: 10.1038/360358a0
247. Shapiro L, Pott GB, Hralston A. Alpha-1-antitrypsin inhibits
human immunodeficiency virus type 1. FASEB J. (2001) 15:115–22.
doi: 10.1096/fj.00-0311com
248. Jean F, Stella K, Thomas L, Liu G, Xiang Y, Reason AJ, et al. α1-Antitrypsin
Portland, a bioengineered serpin highly selective for furin: application
as an antipathogenic agent. Proc Natl Acad Sci USA. (1998) 95:7293–8.
doi: 10.1073/pnas.95.13.7293
249. Anderson ED, Thomas L, Hayflick JS, Thomas G. Inhibition of HIV-1 gp160-
dependent membrane fusion by a furin-directed α1-antitrypsin variant. J Biol
Chem. (1993) 268:24887–91. doi: 10.1016/S0021-9258(19)74548-7
250. Bernot D, Stalin J, Stocker P, Bonardo B, Scroyen I, Alessi MC,
et al. Plasminogen activator inhibitor 1 is an intracellular inhibitor
of furin proprotein convertase. J Cell Sci. (2011) 124:1224–30.
doi: 10.1242/jcs.079889
251. Boulaftali Y, Francois D, Venisse L, Jandrot-Perrus M, Arocas V, Bouton
MC. Endothelial protease nexin-1 is a novel regulator of a disintegrin and
metalloproteinase 17 maturation and endothelial protein c receptor shedding
via furin inhibition. Arterioscler Thromb Vasc Biol. (2013) 33:1647–54.
doi: 10.1161/ATVBAHA.113.301494
252. Ploug M, Ronne E, Behrendt N, Jensen AL, Blasi F, Dano K. Cellular
receptor for urokinase plasminogen activator. Carboxyl-terminal processing
and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem.
(1991) 266:1926–33. doi: 10.1016/S0021-9258(18)52382-6
253. Donadello K, Scolletta S, Covajes C, Vincent JL. SuPAR as a prognostic
biomarker in sepsis. BMC Med. (2012) 10:1–9. doi: 10.1186/1741-7015-10-2
254. D’Alonzo D, De Fenza M, Pavone V. COVID-19 and
pneumonia: a role for the uPA/uPAR system. Drug Discov
Today. (2020) 25:1528–34. doi: 10.1016/j.drudis.2020.
06.013
255. Rovina N, Akinosoglou K, Eugen-Olsen J, Hayek S, Reiser J, Giamarellos-
Bourboulis EJ. Soluble urokinase plasminogen activator receptor (suPAR)
as an early predictor of severe respiratory failure in patients with
COVID-19 pneumonia. Crit Care. (2020) 24:4–6. doi: 10.1186/s13054-020-0
2897-4
256. Azam TU, Shadid HR, Blakely P, O’Hayer P, Berlin H, Pan M, et al. Soluble
urokinase receptor (SuPAR) in COVID-19-Related AKI. J Am Soc Nephrol.
(2020) 31:2725–35. doi: 10.1681/ASN.2020060829
257. Yu J, Liu SL. Emerging role of LY6E in virus-host interactions. Viruses. (2019)
11:1–11. doi: 10.3390/v11111020
Frontiers in Cardiovascular Medicine | www.frontiersin.org 20 March 2021 | Volume 8 | Article 648947
Yaron et al. Fibrinolysis, Inflammation and Serpins
258. Zhao X, Zheng S, Chen D, Zheng M, Li X, Li G, et al. LY6E restricts entry of
human coronaviruses, including currently pandemic SARS-CoV-2. J Virol.
(2020) 94:1–17. doi: 10.1128/JVI.00562-20
259. Pfaender S, Mar KB, Michailidis E, Kratzel A, Boys IN, V’kovski P, et al. LY6E
impairs coronavirus fusion and confers immune control of viral disease. Nat
Microbiol. (2020) 5:1330–9. doi: 10.1038/s41564-020-0769-y
Conflict of Interest: JRY, LZ, and ARL are inventors on several patents and patent
applications relating to the use of Myxoma virus Serp-1 as a therapeutic. ARL is
a co-founder of a small spin-out biotechnology company, Serpass Biologics Inc.
which is developing Serp-1 as a therapeutic.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2021 Yaron, Zhang, Guo, Haydel and Lucas. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
Frontiers in Cardiovascular Medicine | www.frontiersin.org 21 March 2021 | Volume 8 | Article 648947
... The role of serpins in the regulation of inflammation is well known because the most abundant serpin in human serum is alpha-1-antitrypsin, which is a major protective factor against the damaging effects of neutrophil elastase (Mangan et al., 2008;Yaron et al., 2021). Other human serpins, such as antichymotrypsin, also have an anti-inflammatory function. ...
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Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.
... A PEGylated version of the Myxomavirus derived SERPIN Serp-1 has also been developed demonstrating improved efficacy in a mouse model of diffuse alveolar hemorrhage (259). Serp-1 is a broad acting SERPIN with anti-inflammatory and antifibrinolytic activities (260). In addition, however, Serp-1 also functions as a heparin dependent inhibitor of thrombin (261,262). ...
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Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies.
... Interest in these hydrolytic enzymes is due to their well-characterized, widespread, and diverse roles, in a host of physiological and pathological processes. For example, it has long been established, that in addition to their fibrinolytic role in clot dissolution (Yaron et al., 2021), the trypsin-like serine proteinases urokinase (uPA), tissue-type plasminogen activator (tPA) and plasmin play critical roles in a number of processes including extracellular matrix remodelling (Lu et al., 2011), wound healing and carcinogenesis (Aimes et al., 2003;Nyberg et al., 2006;Pawar et al., 2019). ...