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

Abstract

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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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 (4,7–10). 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
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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) (32–36). 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 (68–70). 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 (84–86). PAI-1 is an efficient inhibitor of both
uPA and tPA (2 ×107M−1s−1each) while PAI-2 effectively
inhibits uPA (2 ×106M−1s−1) and is a poor inhibitor of tPA
(2 ×105M−1s−1) (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 (113–115). 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 (119–121). 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 (138–140). 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
(185–188). 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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Yaron et al. Fibrinolysis, Inflammation and Serpins
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 (204–207).
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
(212–214).
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
(242–244). 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
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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.
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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