Roxana N. Beladi1,3,†, Kyle S. Varkoly1,3,†, Lauren Schutz1,†, Liqiang Zhang1, Jordan R. Yaron1,
Qiuyun Guo1,4, Michelle Burgin1, Ian Hogue2, Wesley Tierney2, Wojciech Dobrowski5 and
Alexandra R. Lucas1,2,*
1Center for Personalized Diagnostics and 2Center for Immunotherapy, Vaccines and Virotherapy Boiodesign Institute,
Arizona State University, Tempe, AZ, USA; 3Kansas City University, College of Medicine, Kansas City, Missouri, USA;
4Tongji Medical College, Huazhong University, Huazhong, China; 5Lublin Medical University, Lublin, Poland
Abstract: Progressive neurological damage after brain or spinal cord trauma causes loss of motor
function and treatment is very limited. Clotting and hemorrhage occur early after spinal cord (SCI)
and traumatic brain injury (TBI), inducing aggressive immune cell activation and progressive neu-
ronal damage. Thrombotic and thrombolytic proteases have direct effects on neurons and glia, both
healing and also damaging bidirectional immune cell interactions. Serine proteases in the thrombo-
lytic cascade, tissue- and urokinase-type plasminogen activators (tPA and uPA), as well as the clot-
ting factor thrombin, have varied effects, increasing neuron and glial cell growth and migration
(tPA), or conversely causing apoptosis (thrombin) and activating inflammatory cell responses. tPA
and uPA activate plasmin and matrix metalloproteinases (MMPs) that break down connective tissue
allowing immune cell invasion, promoting neurite outgrowth. Serine proteases also activate chemo-
kines. Chemokines are small proteins that direct immune cell invasion but also mediate neuron and
glial cell communication. We are investigating a new class of therapeutics, virus-derived immune
modulators; One that targets coagulation pathway serine proteases and a second that inhibits chem-
okines. We have demonstrated that local infusion of these biologics after SCI reduces inflammation
providing early improved motor function. Serp-1 is a Myxomavirus-derived serine protease inhibi-
tor, a serpin, that inhibits both thrombotic and thrombolytic proteases. M-T7 is a virus-derived
Here we review the roles of thrombotic and thrombolytic serine proteases and chemoattractant pro-
teins, chemokines, as potential therapeutic targets for SCI. We discuss virus-derived immune modu-
lators as treatments to reduce progressive inflammation and ongoing nerve damage after SCI.
Keywords: Neurotrauma, spinal cord injury, immune, inflammation, serine protease, serpin, thrombosis, thrombolysis, chemo-
To touch to feel, to move, Pain, pleasure known, Moving, then still, All lost, To hope,
But then to save, To grow and grow again, Anew, Alexandra Lucas, MD, 2020.
1.1. Trauma in the Central Nervous System; Spinal Cord
Injury (SCI) and Traumatic Brain Injury (TBI)
Damage after traumatic brain injury (TBI) or spinal cord
injury (SCI) can be extraordinarily severe, causing prolonged
morbidity and mortality and often leaving victims with sus-
tained neurological deficits [1-6]. It is estimated that approx-
imately 288,000 people in the United States are currently
living with severe penetrating and / or compressive
*Address correspondence to this author at the Center for Personalized
Diagnostics and Center for Immunotherapy, Vaccines and Virotherapy
Biodesign Institute, Arizona State University, Tempe, AZ, USA;
Tel: (352)672-2301; E-mail firstname.lastname@example.org
†To be considered co-first authors.
neuronal damage after SCI . The World Health Organiza-
tion (WHO) estimates that approximately 250,000-500,000
people worldwide suffer a spinal cord injury each year .
Injury to the central nervous system is associated with initial
bleeding and / or clotting, followed by an aggressive in-
flammatory response and a final organization of the damaged
area into cystic spaces and scarring, which can be both pro-
tective and healing or cause further damage [8-14]. Neuronal
damage is extensive and progressive with SCI and TBI, due
to ongoing hemorrhage as well as micro-thrombotic changes
in small vessels with secondary persistent immune damage
limiting recovery. High-dose methylprednisolone (steroid) is
given in some centers for SCI; however, a significant amount
of published data has indicated that the risk of adverse ef-
fects and morbidity consistently outweighs benefit. Further,
many clinical trials have indicated that steroids for SCI lead
A R T I C L E H I S T O R Y
Received: October 25, 2020
Revised: December 28, 2020
Accepted: February 24, 2021
1875-6190/21 $65.00+.00 © 2021 Bentham Science Publishers
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Current Neuropharmacology, 2021, 19, 1835-1854
Serine Proteases and Chemokines in Neurotrauma: New Targets for
Immune Modulating Therapeutics in Spinal Cord Injury
1836 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
to variable results that are dependent upon individual pa-
tients [15-25]. Thus, current cervical SCI guidelines strongly
advise against the use of steroids and many experts advocate
against their use [26-28]. The American Association of
Neurosurgeons issued a Level 1 recommendation against
the use of methylprednisolone to treat acute SCI, cautioning
about the use of high-dose steroids due to their negative side
effect profile . The use of steroids is thus now at the dis-
cretion of treating physicians, remaining a topic for debate
[15-30]. There is a clear and current need for effective treat-
ment of SCI and for a standard of medical care for these pa-
tients. Many new treatments are under active investigation
and some hold great promise, but none have proven clinical
benefit at this time. Patients with SCI are often left with
permanent disabilities, such as paralysis, painful radiculopa-
thy, and paresthesia. In some cases, patients have ongoing
progressive damage requiring mechanical or motorized sup-
port for mobility. Newer experimental approaches to treat-
ment include stem cells, either implanted alone or implanted
in hydrogels. These hydrogels are designed to provide a sur-
face for neuronal outgrowth across cystic spaces [31-57].
Other treatments have been developed that range from modi-
fication of local electrolyte composition [54-56] to altering
protease activation. Some approaches specifically target ser-
ine proteases in the thrombotic and thrombolytic cascades, in
addition to modulation of proteases that induce inflammatory
damage or cause fibrous scar formation and further oxidative
damage. Conversely, other potentially beneficial treatments
include the release of growth factors that encourage neuronal
growth and extension [58-61]. These newer treatments re-
An improved understanding of the basic cellular and mo-
lecular mechanisms that induce progressive neurological
damage after SCI is central to developing effective treat-
ments that will improve short- and long-term outcomes after
CNS injury. In this review, we begin with an overview of
two pathways that have been implicated in early injury after
spinal cord trauma and that are also implicated in the ongo-
ing damage leading to worsening function and pain. We will
discuss the roles of activation and suppression of the clot-
dissolving (thrombolytic, also termed fibrinolytic) and also
the clot-forming (thrombotic) serine proteases after SCI [62,
63], as well as the roles of the small proteins that act to direct
immune cell migration and invasion to sites of trauma, the
chemokines, in SCI [62-68]. We will discuss studies relevant
to these specific pathways in SCI and TBI as well as relevant
findings after ischemic damage in strokes (cerebrovascular
accidents, CVA). We will then review the potential for treat-
ing early SCI with drugs that target serine protease and
chemokine pathways, as well as a new class of virus-derived
immune-modulating proteins that selectively target coagula-
tion (thrombosis and thrombolysis) and also chemokine
pathways [62-67]. Two such virus-derived immune modula-
tors have been tested for efficacy in rat spinal cord compres-
sion models for SCI and have demonstrated some early ben-
efit. Other mammalian proteins that target these same path-
ways are also under investigation and have shown promise in
preclinical studies. These findings underscore the central
roles that these molecular pathways have in SCI and the po-
tential to design new approaches to the treatment of SCI that
modulate these pathways and improve long term outcomes.
1.2. Coagulation Pathway Activation; Thrombosis and
Thrombolysis after Neurotrauma
When there is trauma in the mammalian body, the initial
response is hemorrhage due to vessel damage and leak.
There is also a risk for vascular thrombotic occlusion in larg-
er or smaller vessels (macro- and micro-thrombotic occlu-
sion) due to compression and / or stasis of blood flow. Each
of these initial clot-forming breakdown responses is de-
signed to prevent excess bleeding by activating clot-forming
proteases, and to prevent excess clotting by activating
thrombolysis, representing a natural balance of thrombosis
and thrombolysis throughout the body and in the CNS. The
activation of these clot forming and clot-dissolving cascades,
in turn, can induce innate (inflammatory) and (antibody me-
diated) immune cell responses. Thus, initial trauma is fol-
lowed by repair responses through activation of both the
coagulation pathways and through innate and adaptive im-
mune pathways, the restorative inflammatory and immune
responses. In contrast, the persistence of inappropriate or
excess thrombosis, thrombolysis or immune responses can
also lead to damage. Persistent hemorrhage or clotting can
cause further tissue destruction and activation of immune
cells that invade nervous system tissues, causing escalating
damage to the CNS. Thrombosis, hemorrhage and excess
immune cell activation can initiate further progressive dam-
age after brain or spinal cord trauma.
Sequential coagulation pathway and immune responses
to injury are seen throughout the mammalian body and cause
ongoing severe damage in clotting or inflammatory disor-
ders, as is seen in inflammatory atherosclerosis and vascu-
litis, or infectious disorders, such as acute respiratory distress
syndromes in SARS-CoV-2 infections or bacterial sepsis
with disseminated intravascular coagulation. This response
to injury has been described for hundreds of years by two
well-established medical axioms; 1) the inflammatory re-
sponse to trauma referred to as ‘tumor, rubor, calor and dol-
or’ in Latin, representing swelling, erythema or redness,
warmth, and pain respectively and 2) Virchow’s triad, spe-
cifically venous stasis, activation of coagulation, and venous
damage. The inflammatory response after injury was first
described by Galen and recorded by Celsus the Roman
scholar in the first century AD and more recently ‘functio
laesa’, or loss of function, has been added as the fifth sign of
inflammation and seems very appropriate when discussing
damage to the CNS . Virchow’s triad was described by
Rudolf Virchow in the 1800’s in Berlin, Germany. This triad
is described in greater depth as transient hypercoagulability
with platelet activation, venous stasis from paralyzed venous
muscle pumps, and vascular endothelial damage from ac-
companying injury, venous dilation, and pressure on the
veins, which all contribute to venous thromboembolism
(VTE). Patients who suffer from acute cervical SCI have a
very high risk of systemic venous thromboembolism, and
spinal trauma guidelines recommend both mechanical and
chemical blood clot prophylaxis [70, 71], with the potential to
increase bleeding at the site of SCI. In SCI, these elements of
hemorrhage and thrombosis, and the subsequent aggressive
Serine Proteases and Chemokines in Neurotrauma Current Neuropharmacology, 2021, Vol. 19, No. 11 1837
inflammation are exaggerated in the restricting space of the
thecal sac in primary injury for SCI, or in the skull for TBI.
It is becoming increasingly evident that the coagulation
pathways, both thrombotic and thrombolytic, can alter neu-
ronal damage and neuronal regrowth after trauma. These
interactions are extraordinarily complex, with multiple layers
of direct interactions on neurons and glial cells as well as
secondary activation of immune responses, interactions that
can promote repair and regrowth or conversely lead to fur-
ther damage . An added layer of complexity ensues
when one considers the initial response to any trauma will
involve the innate (inflammatory) immune response that acts
to break down damaged tissue and provide an arena for re-
growth of healthy tissues, an essential natural repair process
[72, 73]. The blood brain barrier (BBB) also modifies these
repair and damage reactions. The BBB provides a second
unique area of protection in the CNS, preventing excess tox-
ins and cytokine-mediated immune cell activation and inva-
sion into neural tissue in the spine or brain, and preventing
damaging immune cell activation. The integrity of the BBB
can also be lost after severe neurotrauma, decreasing protec-
tive functions when broken down by physical trauma, prote-
ase, hypoxia induced by a prothrombotic factors, and plate-
let-activating factor causing further injury to the spinal cord
or brain . Tissue and urokinase-type plasminogen activa-
tors (tPA and uPA, respectively) are acute phase reactants
[74, 75] that are released by both injured neurons and glial
cells in the CNS as well as from endothelium in blood and
vessels supplying the CNS [72-75]. The plasminogen activa-
tors (PAs) are frequently deemed only primary fibrinolytic
factors that break down fibrin in clots, eg. clot-dissolving
reagents. However, both PAs have a wide range of functions
that include direct effects on neural plasticity, neurite out-
growth, glial cell activity, the endothelium in the vascula-
ture, the BBB and both circulating and resident immune cells
in the CNS. Similarly, thrombin is a central mediator of fibrin
deposition and clot formation and is often considered solely
as a thrombotic agent released with endothelial damage and
platelet activation. However, as for tPA and uPA, thrombin
also has a wide array of functions outside of clot formation,
including activation of immune cell responses and direct
effects on neurons. Thus, both thrombolytic and thrombotic
serine proteases have direct effects on neuron growth, exten-
sion, and neuroplasticity after CNS damage [71-75] as well
as indirect effects on immune cell responses to injury.
Current understanding of the roles of serine proteases and
serine protease inhibitors, termed serpins, in neurotrauma
after SCI and, more generally in TBI, is reviewed in the fol-
1.3. General Overview of Immune Cell Responses to CNS
The first stage in the immune response to spinal cord
injury is infiltrating neutrophils and monocytes that enter the
spine from circulating blood and are recruited to sites of SCI
through glial chemokine and cytokine release. Subsequent
upregulation of chemotactic adhesion molecules such as
ICAMs and VCAMs which are Ig-superfamily cell adhesion
molecules as well as upregulation of selectins such as P-
selectins and L-selectins on endothelial cells attracts cells in
the circulating blood (the arterial lumen) to migrate into sites
of neurological damage. This is a complex response in that
both peripheral immune cells from the circulating blood,
along with endogenous microglia, drive the inflammatory
response to SCI . If the immune or inflammatory re-
sponse is prolonged or excessive, this can cause progressive
damage. Damage is produced by cellular invasion, break-
down of cells, connective tissue and vascular barriers and
also activation of coagulation pathway responses (Figs. 1-3).
However, the innate immune responses are also the first-line
response to injury and healing and can enable cellular and
neurological tissue recovery, leading to nervous system tis-
sue repair through this mixed reparative and pathological
responses . The immune response related to circulating
blood mononuclear cells invading at sites of trauma may
provide an early response that is lost once the area of damage
is sealed off, leading to a reestablished predominant role of
the intrinsic glial cell responses.
The general role of glial cells, which includes multiple
cell subsets in the CNS, is to support signaling and neuron
activity, as well as acting as a resident immune response
network within the CNS (Fig. 1). Microglia are CNS im-
mune cells that police the CNS environment, phagocytosing
pathogens and secreting cytokines and growth factors [76-
80]. Oligodendrocytes produce myelin sheaths that insulate
axons, creating quick and efficient action potential conduc-
tion. Satellite oligodendrocytes are situated in the grey mat-
ter of the CNS and do not myelinate axons; however, during
demyelinating injuries, satellite oligodendrocytes are recruit-
ed to aid in remyelinating damaged axons. This oligodendro-
cyte recruitment is advantageous in times of injury because
oligodendrocytes support the metabolic needs of neurons and
protect against neuronal apoptosis . Astrocytes act as
links between the vasculature and neurons, providing inter-
faces between the vasculature and neurons in the brain and
spinal column. Astrocytes provide neurotransmitters, ions,
and nutrients for neuronal signaling. Pericytes sheath endo-
thelial cells in capillaries in the vascular networks and modu-
late capillary diameter and alter vascular coupling and func-
2. THROMBOLYTIC SERINE PROTEASES
2.1. Tissue-type Plasminogen Activator, tPA
Tissue-type plasminogen activator, tPA, has displayed
both beneficial and also harming actions in the CNS after
neurotrauma. tPA is a highly effective thrombolytic, well-
known for clinical applications as a highly effective treat-
ment, improving lives and cardiac function as well as recov-
ery after strokes by dissolving clots and opening arterial oc-
clusions after acute thrombotic occlusion. tPA, as well as
urokinase and streptokinase, act rapidly to dissolve sudden
coronary occlusions in ST elevation myocardial infarctions
(STEMI), disrupting occlusive coronary arterial clot and
more recently tPA has been used for cerebrovascular throm-
botic occlusions, in strokes, termed cerebrovascular acci-
dents (CVA or stroke) . The fibrinolytic (thrombolytic)
plasminogen activator, termed fibrokinase, was first reported
by Astrup and Permin in 1947 . Later Pennica, et al.
1838 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
cloned and expressed tPA in Escherichia coli in 1983, lead-
ing to clinical applications for treatment in ST Elevation
Myocardial Infarction (STEMI heart attack) . Thrombo-
lysis for heart attacks is now widely displaced by direct me-
chanical revascularization, termed primary percutaneous
coronary intervention (PCI) with stent implants for heart
attacks. However, tPA is used and is highly effective, when
PCI is not accessible. tPA is also used in acute thrombotic
CVAs, if detected early within a narrow therapeutic window.
In treatment for early-detection of CVA, tPA is highly bene-
ficial, but does have the attendant risk of bleeding. Not only
can tPA treatment lead to reperfusion of an ischemic cerebral
vascular occlusion, but tPA also has a risk of secondary
hemorrhage, due in part to increased collateral supply to the
ischemic area or disruption of the BBB at sites of infarction
with hemorrhagic transformation. Thus, the use of tPA is
highly beneficial in reducing ischemic thrombotic infarction
in the brain (CVA) and restoring blood flow to the brain, but
tPA can also cause harm due to excess bleeding produced by
an imbalance in the thrombotic and thrombolytic pathways.
Thus, the use of tPA for CVA has remained a subject of de-
bate, and this risk of hemorrhage after tPA treatment for
CVA further illustrates the risks of excess damage when
there is ongoing bleeding in the CNS.
tPA is a 69kDa protease that interacts with low-density
lipoprotein receptor-related protein (LRPR) and NMDA (N-
Methyl-D-Aspartate) receptors that modify cell activation
. tPA not only functions to break down blood clots [82-
85], increasing oxygen and nutrient supply for neuronal
health, but tPA is also associated with directly aiding in neu-
ronal growth (Fig. 2) and has shown early benefit in neuro-
trauma with initial effects of improved motor function in
induced pluripotent stem cell treatments in rats  tPA has
pleiotropic, wide-ranging effects in the CNS including bene-
Fig. (1). Neuronal and non-neuronal glial cells depicting their infrastructure and modulation of the blood-brain barrier. (A higher resolution /
colour version of this figure is available in the electronic copy of the article).
Serine Proteases and Chemokines in Neurotrauma Current Neuropharmacology, 2021, Vol. 19, No. 11 1839
ficial effects on neuronal development with enhanced neuro-
plasticity, axonal regeneration, neuronal excitation, neuron to
vascular connections, excitotoxicity, axon regeneration, neu-
roprotection, microglial activation and also enhanced in-
flammation and altering BBB integrity [86-93]. How tPA
mediates these diverse activities is dependent upon the pres-
ence of targeted cell receptors, growth factors, matrix metal-
loproteinases (MMPs), as well as apparent dependence of
tPA function on the presentation of tPA as either a monomer
or dimer . The mechanisms by which tPA exerts its ac-
tions in the CNS and the role of the very divergent beneficial
and / or damaging molecular actions remain topics of debate.
With neurotrauma and specifically SCI, tPA is reported
to produce early beneficial effects after SCI with improved
motor function (Fig. 2). tPA is also reported to lead to a loss
of integrity of the BBB, the cerebrovascular barrier that pro-
vides functional protection against excess toxicity, systemic
[94-98] immune responses, as well as local aggressive im-
mune damage and toxicity . It has been demonstrated
that tPA deficient (tPA-/-) mice have smaller stroke volumes
suggesting that tPA also has damaging functions after CVA.
Supportive studies have demonstrated that tPA treatment
leads to adverse effects in tPA-/- mice by increasing stroke
volumes after middle cerebral artery occlusion (Fig. 3) [96-
100]. There are reports that tPA breaks down endothelial
cell-to-cell connections, the basis for the protective BBB.
This is supported by studies demonstrating that tPA-/- mice
have higher preserved integrity of the BBB [82, 83]. tPA
regulation of the BBB is controlled through activation of
Fig. (2). Postulated protective effects of serine proteases after SCI. Thrombin leads to decreased bleeding through thrombus formation. tPA
and uPA breakdown excessive clotting. tPA enhances neurite outgrowth. Serp-1 balances excess thrombotic or thrombolytic serine protease
activity by targeting activated proteases in both pathways and can reduce inflammatory cell activation and invasion. (A higher resolution /
colour version of this figure is available in the electronic copy of the article).
1840 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
platelet-derived growth factor receptor alpha (PDGFRα) on
perivascular astrocytes mediating increased vascular perme-
ability . The endothelial cell layer with smooth muscle
cells and perivascular astrocytes maintains the integrity of
the BBB, maintaining tight control over concentrations of
molecules in the CNS.
Thus, tPA is highly multifunctional, with multiple and
extensive activities separated from plasmin activation and
clot breakdown and separate from the dissolution of fibrin
degradation in clots. There is a high expression of tPA in
vascular endothelium, as well as throughout the brain in the
microglia, astrocytes, oligodendrocytes and neurons [84, 87].
tPA is considered an acute phase reactant, upregulated with
increased secretion in the endothelium after CNS injury, but
also upregulated in neurons, microglia, astrocytes and oli-
godendrocytes after CNS injury . In neurons, tPA is ex-
pressed by excitatory neurons as well as perivascular inter-
neurons . tPA is reported to activate growth factors such
as nerve growth factors (pro-NGF and pro-BDNF), activated
protein C, platelet-derived growth factor (PDGF) and matrix
metalloproteinases (MMPs) (Fig. 2) . tPA binds and/or
activates multiple receptors such as low-density lipoprotein,
receptor-related protein (LRP), NMDA-receptor, annexin-II,
and epidermal growth factor receptors (EGFRs) [82, 89-1].
tPA is cleared from the blood by the low-density lipoprotein
receptor-related protein (LRP1), with this interaction activat-
ing microglial cells in the ischemic brain . tPA also acti-
vates matrix metalloproteinases (MMPs) that break down
Fig. (3). Postulated potential adverse effects produced by serine proteases after SCI. Thrombin can increase neuronal apoptosis, cell death,
and increase local inflammation. tPA and uPA can induce excess bleeding and breakdown of the BBB as well as enhancing immune cell
activation and invasion. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
Serine Proteases and Chemokines in Neurotrauma Current Neuropharmacology, 2021, Vol. 19, No. 11 1841
local connective tissue, allowing both innate inflammatory
cell and neuron migration or invasion .
tPA is reported to release connective tissue stores of
growth factors leading to enhanced, directed neuronal migra-
tion, growth, and rejuvenation. It is hypothesized that tPA
leads to neuronal cell growth and migration through the
breakdown of connective tissue (CT) . tPA is detected as
a protein released in the neuron growth cone, altering neurite
outgrowth and remodeling (Fig. 2) . tPA treatment and
tPA deficient mouse models have thus demonstrated a role
for tPA in responses to neuron excitation by altering cere-
brovascular integrity and neurovascular blood flow, altering
BBB permeability and providing energy supplies necessary
for active neurons. tPA is thus believed to play a role in
maintaining neuronal activity, potentially through control of
the BBB and local energy supply to neurons, termed neu-
rometabolic coupling [95-97]. Plasminogen activators and
plasmin are closely linked to progressive hemorrhagic con-
version (PHC) after TBI or SCI, again illustrating the dual
roles of tPA (and their receptors) in outcomes after CNS injury
. tPA and uPA also activate plasmin which in turn breaks
down fibrin clots. tPA and uPA also act individually via
plasmin to increase matrix metalloproteinase (MMPs) activa-
tion. MMPs break down the connective tissue surrounding
endothelial cells and in surrounding neurons to allow for
immune cell invasion. As noted, tPA also separates endothelial
cell connections leading to increased breakdown of the BBB
and vascular leak . These effects of tPA on connective
tissue breakdown and immune cell invasion are also reported
with the urokinase-type plasminogen activator, uPA.
2.2. Urokinase-type Plasminogen Activator, uPA
Similar to tPA, uPA also has many diverse functions,
acting as a thrombolytic to break down clots, but with a re-
ported greater role in cell activation and enabling immune
cell migration. uPA is now considered to act predominantly
in cellular activation and migration, as opposed to acting as a
primary thrombolytic, particularly in tumor cells . uPA
interacts with a differing set of cell surface receptors. uPA
binds to the uPA receptor (uPAR), a non-transmembrane,
glycosylphosphatidylinositol (GPI) linked protein receptor
that sits in a large lipid raft of proteins on the surface of in-
flammatory cells. uPAR interacts with circulating uPA and
tissue vitronectin, but also has cis interactions with proteins
in the uPAR lipid raft, specifically integrins, lipoprotein
LDL-receptor- related protein (LRP), and chemokine recep-
tors . uPAR alters cell activity via interactions with
other members of the lipid raft as well as via GPI linking.
uPAR interacts with the actin motility machinery in cells and
alters gene expression, signaling and cell activation via JAK-
STAT and other intracellular pathways . uPAR increas-
es chemotaxis and beta2-integrin-dependent adhesion. Mon-
ocyte recruitment and neutrophil migration are significantly
impaired in uPAR-deficient (uPAR-/-) mice . uPAR
alters cell migration and adhesion via interactions with the
extracellular matrix, specifically collagens I, III and IV, fi-
bronectin, fibrin and vitronectin. uPA and uPAR act in con-
cert to alter uPA, PAI-1(Plasminogen Activator Inhibitor-1)
and uPAR expression [104, 105] uPA also has the capacity
to activate pro-neurotrophic factors, including the hepatocyte
growth factor (HGF), a motor neuron survival factor [106,
107], or to activate pro-BDNF (brain-derived neurotrophic
factor) and pro-NGF (nerve growth factor) via plasmin
. Furthermore, both uPA and tPA have been implicated
in synaptic remodeling associated with cerebellar motor
learning, visual cortex ocular dominance columns, and both
hippocampal and corticostriatal long-term potentiation (LTP)
uPA, when bound to the uPAR on the cell surface, acti-
vates plasmin, similar to tPA activation of plasmin. uPA,
together with plasmin, activates MMPs as reported for tPA
leading to the breakdown of collagen and elastin in the con-
nective tissue (CT) and the endothelial glycocalyx, allowing
cell invasion [112, 113] from the vasculature. With connec-
tive tissue breakdown, immune response cells can more
readily invade connective tissue surrounding cells leading to
either cell and tissue repair, disruption of the BBB, or further
immune-mediated damage. This CT breakdown is also posit-
ed to enhance neuron outgrowth after damage to the spine
. CT breakdown also releases stores of growth factors
such as PDGF and TGF, further potentiating neurite out-
growth . Of interest, proteases activate chemokines and
release them from cell surfaces initiating neuronal migration
as further discussed in the section on chemokines.
3. THROMBOTIC SERINE PROTEASES, THROMBIN
Thrombin, factor two in the coagulation cascade, is similarly
acutely activated with endothelial damage. Clots form on
damaged endothelium in the inner arterial layer as well as on
the surface of activated platelets and adherent macrophages.
α-Thrombin is a 36 kDA serine protease composed of two
chains linked by a disulfide bond [115, 116]. Thrombin is a
central mediator of intrinsic and extrinsic clotting cascades,
initiating fibrinogen conversion to fibrin with deposition at
sites of endothelial damage and platelet activation and adhe-
sion along damaged endovascular and platelet cell surfaces
. Thrombin acts through four protease-activated recep-
tors (PARs 1,2,3, and 4), with prothrombin and PAR1 being
upregulated at sites of SCI and TBI .
In addition to clot formation detected after spinal cord
injury (SCI) and traumatic brain injury (TBI), thrombin is
also increased in Experimental Autoimmune Encephalomye-
litis (EAE) models in mice models, another inflammatory
disease of the CNS. Thrombin was dramatically elevated
during the peak of EAE, before the impaired motor function
is detected . Thrombin has reported effects on neurons
after neurotrauma, post-stroke ischemia, and degenerative
nerve disease. Thrombin acts on the endothelial cells of the
BBB, astrocytes, and microglia as a pro-inflammatory medi-
ator, promoting vascular dysfunction and neurodegeneration
. Thrombin is associated with membrane lipid peroxi-
dation (MLP), reactive oxygen species (ROS), and platelet
Thrombin has direct adverse effects on neurons. At select
concentrations, thrombin induces apoptosis in neurons via
PAR-1 and MLP via caspase 3 (Figs. 2 and 3). When bound
to PAR-1, thrombin induces apoptosis in motor neurons and
can induce neurite retraction as well as astrogliosis .
PAR-1 also induces the expression of adhesion molecules
1842 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
such as P-selectin, leading to immune/inflammatory cell
recruitment and secondary vascular tissue damage .
Thrombin-induced secondary injury is mediated through
its receptor, protease-activated receptor-1 (PAR-1), by "bi-
ased agonism" . Activated protein C (APC) acts
through the same PAR-1 receptor but functions as an antico-
agulant and anti-inflammatory protein, which counteracts
many of the effects of thrombin and is neuroprotective .
Effects of thrombin on recovery after neurotrauma have been
linked to interaction with PAR-1 receptors, and detrimental
functions of thrombin/PAR-1 binding are reported to be
opposed by APC, also working via the PAR-1 receptor.
Thrombin concentrations above 100U/mL have been report-
ed to directly induce neuronal apoptosis [122, 123].
4. SERINE PROTEASE INHIBITORS, SERPINS THAT
REGULATE THROMBOLYTIC AND THROMBOTIC
Serine proteases in the thrombotic and thrombolytic cas-
cades, in addition to balancing clot formation and dissolu-
tion, are, regulated by serine proteinase inhibitors, termed
serpins. [63, 124-129], as reviewed in the following section.
These regulators of the thrombolytic and thrombotic serine
proteases in the coagulation pathways have also reported
differing effects on recovery after neurotrauma as for their
targeted serine proteases. Serpins are suicide inhibitors that
bind to activated serine proteases, forming inactivated sui-
cide complexes where the activity of both the protease and
the serpin are lost. The serine protease binds and cleaves the
P1-P1’sequnce in the reactive center loop (RCL) of the ser-
pin and forming a covalent bond between the cleaved RCL
and the protease. The protease is then dragged to the oppo-
site pole of the serpin molecule, and the cleaved RCL be-
comes part pf the A beta sheet in the serpin. These serpins
regulate clotting throughout the circulating blood and repre-
sent up to 2-10% of the proteins in the circulating blood.
Additionally, some genetic disorders are caused by serpin
deficiencies; for example 1) alpha-1-antitrypsin (α1AT,
SERPINA1) deficiency, which causes severe pulmonary
disease, chronic hepatitis, cirrhosis, and hepatocellular carci-
noma, 2) C1 Esterase Inhibitor (C1INH, SERPING1) defi-
ciency which causes complement pathway activation and
excess immune cell activation, and 3) antithrombin (AT,
SERPINC1) deficiency which leads to excess thrombosis.
Serpins are necessary for control of these pathways and ser-
pin replacement therapy has been developed for some of
these serpin deficiency syndromes [124-130]. Serpins repre-
sent vital controls for the regulation of normal human home-
ostasis and function.
Not only can serpins be used for replacement therapy, but
they have also shown additional potential as therapeutics,
αlAT has been assessed for the treatment of sepsis caused by
Pseudomonas aeruginosa sepsis. C1INH has been developed
for inflammation-related complications as a replacement for
complement-activated inflammatory state and associated
metabolic acidosis. C1INH has been reported to decrease
tumor necrosis factor α and to attenuate renal, intestinal, and
lung injury in a dose-dependent manner. Pretreatment of
Wistar rats with human plasma-derived C1INH exhibited
protective effects in ischemia/ reperfusion injury of lower
extremities and associated lung damage, significantly reduc-
ing edema in re-perfused muscle and lungs, improved mus-
cle viability, and decreased plasma levels of pro-
4.1. Plasminogen Activator Inhibitor Type I (PAI-1),
PAI-2, and PEDF
Plasminogen activator inhibitor-1 (PAI-1 or SERPIN E1)
is only weakly expressed in a healthy brain, whether from
man or mouse. However, PAI-1 expression is markedly in-
creased after TBI or SCI or other pathologic neurologic con-
ditions, such as cerebral ischemia [124, 125]. PAI-1, as well
as PAI-2 and neuroserpin (NSP), regulate tPA and uPA ac-
tivity in the CNS, and PAI-1 and NSP are now considered
important in the regulation of tPA in the CNS [83, 126-132].
As noted above, tPA-/- mice have smaller stroke volumes.
However, as a principle regulator with wide-ranging plei-
otropic effects, tPA in the CNS has a neuroprotective func-
tion, promoting axonal generation in mice. tPA/PAI-1 com-
plexes increase BBB permeability after TBI in mouse mod-
els . Of interest, when the serpin PAI-1 binds to uPA /
uPAR complexes on the cell surface, detachment of cell sur-
face integrins from extracellular matrix (ECM) ligands oc-
curs, leading to internalization in an LRP1-uPA / uPAR-
dependent manner. This alters intracellular signaling such as
the Jak/STAT pathway, with subsequent changes in cell ac-
tivation, motility and response and ultimately culminating in
either enhanced or suppressed cell migration. When PAI-I
binds in a non-uPA/uPAR-dependent manner to LRP1, this
also triggers Jak/STAT cellular signaling events that culmi-
nate in enhanced cell migration [124, 125]. This increased
cell migration is hypothesized to increase BBB permeability
following SCI and TBI. Treatment with glucagon and a PAI-1
peptide is also reported to improve outcomes after SCI
through inhibition of ERK and JNK/MAPK intracellular
pathways [123, 131]. Another serpin, Pigment Epithelial
Derived Factor (PEDF or SERPIN F1) has angiogenic, anti-
apoptotic, and neurotrophic functions, with many effects
seen in the retina. PEDF also has been reported to alter PAI-1
activity and to be regulated by plasminogen .
4.2. Neuroserpin (Nsp)
Another mammalian serpin, neuroserpin (SERPIN II),
also binds to and inhibits tPA and uPA and is considered one
of the main regulators for thrombolytic proteases [59-61].
Neuroserpin (Nsp) has been assessed in models of vascular
inflammation after aortic allograft transplant in mice and
reduced inflammation, altering Th2 to Th1 ratios, in these
vascular models, nsp has known effects after injury to the
CNS with reported protective functions . Mice with
neuroserpin deficiency have larger stroke volumes after cer-
ebrovascular occlusion, eg. strokes. In serpinopathies caused
by genetic anomalies in the NSP RCL sequence, clumps of
serpins become adherent to one another in inactive deposits,
termed inclusion bodies, believed to be caused by the inser-
tion of the RCL of one serpin into an adjacent A beta-sheet
on adjacent serpin molecules [126-129]. NSP deficiency due
to aggregation and loss of function leads to epilepsy and neu-
rodegenerative disorders. Studies have also detected im-
proved outcomes with neuroserpin after neurotrauma [59-
Serine Proteases and Chemokines in Neurotrauma Current Neuropharmacology, 2021, Vol. 19, No. 11 1843
61]. NSP is reported to reduce autophagy and decrease neu-
ron apoptosis in rat models of SCI.
4.3. Antithrombin III (AT-III)
Anti-thrombin III (termed AT or AT-III, SERPIN C1) is
a serpin that has also been assessed in SCI models. When
treating patients with arterial or venous thrombosis, atrial
fibrillation (risk of transient ischemic strokes), or implant
(placement) of percutaneous coronary stent implants, heparin
is routinely administered as an anticoagulant. Heparin is de-
rived from tissue extracts of heparan sulfate, which is a mix-
ture of differing length glycosaminoglycans or polysaccha-
rides termed heparin, extracted form mammalian tissues.
Heparan sulfate is the predominant endothelial glycocalyx in
arterial wall connective tissue. Heparin, as a therapeutic,
functions by increasing the activity of the mammalian serpin,
AT. AT binds and inhibits factor IIa, IXa, XIa, XIIa, and Xa,
in particular factors II and X, in the clotting cascade .
Three studies have reported improved outcomes in rodent
models treated with AT-III after SCI [134-137]. AT-III may
improve functional outcome after compression injury to the
SC due to decreased tumor necrosis factor-alpha (TNF-α)
and increased prostaglandin-I 2 (PGI2) expression and re-
lease in the vascular endothelium, with reduced inflammato-
ry neutrophil responses. AT’s anti-inflammatory effects are
not detectable after treatment with Indomethacin, a non-
steroidal anti-inflammatory drug (NSAID) that blocks pros-
taglandin (PG) activity [136, 137]. AT has also been found
to reduce ischemia reperfusion injury (IRI) in models of is-
chemic neuronal damage. In this IRI model, AT reduced the
numbers of microthrombi and increased the numbers of mo-
tor neurons in animals subjected to SCI .
5. CHEMOKINES IN CNS INJURY
Chemokines are small 8-12 kDa proteins classified into 4
chemokine classes: C, CC, CXC, and CX3C based upon the
sequence of amino acids interposed between the first two CC
amino acids in the sequence. CXC chemokines are often
linked to neutrophil and macrophage activation and migra-
tion, while CC chemokines are linked to monocyte and lym-
phocyte activation/ migration [138-140]. Chemokines form
chemoattractant gradients by binding to tissue glycosamino-
glycans (GAGs) and lining up in the tissue to attract leuko-
cytes. Leukocytes bind to chemokines via cellular chemokine
receptors, 7 transmembrane G protein-coupled chemokine
receptors  that bind to a differing area on the chemo-
kine. These are the so-termed “chemokine gradients” that
direct innate and acquired immune cells into areas of tissue
damage or infection . It is important to note that the
interaction of chemokines with both GAGs and also with
leukocyte receptors is not a selective, one-to-one interaction.
There is extensive promiscuity among chemokines with
chemokine cross-reactivity with multiple GPCR’s, chemokine
receptors, as well as individual receptors. The combined in-
teraction of chemokines with GAGs and with receptors may
provide a more specific interaction, but this remains to be
determined. These chemokine GAG receptor interactions
attract leukocytes to areas of tissue damage, and in some
cases, chemokine also activates cells outside of their binding
It is also now known that chemokines are involved in the
CNS response to injury. Chronic neurodegeneration has been
shown to prime astrocytes to release large amounts of chem-
okines when acutely stimulated with cytokines . Chem-
okines have been identified as markers for neurodegenerative
changes in the CNS [142-144]. Altered chemokine expression
is also reported in other neurological disorders, including
stroke, schizophrenia, and depression, in addition to neuro-
degenerative disorders [145-150]. Chemokine signaling is
now reported to play a key role in astrocyte, glial cell and
neuronal responses to injury and well as functioning as neu-
rotransmitters [145-148]. Of great interest, serine proteases
can release cell surface chemokines and activate chemokines
in the nervous system linking chemokine activity back to the
thrombolytic serine proteases. Chemokines may also direct
neuronal extension and growth following spinal cord injury
[147-148], and thus may have some beneficial functions sim-
ilar to what is seen with the serine protease tPA. In SCI, the
CC chemokine subtype CCL2 has been associated with neu-
roprotective mechanisms of mesenchymal stem cell implants
after SCI. CCL2 provides a protective function by driving
macrophage recruitment to sites of neurotrauma and increas-
ing conversion of macrophage to an M2 anti-inflammatory,
neuroprotective phenotype. In a mouse model, human CCL2
prevented motor neuron degeneration in vitro and delivered
in mice after SCI is reported to improve motor performance
[147, 148]. In contrast, others have reported that blockade of
the chemokine MCP-1 reduced inflammation and neuronal
damage after SCI in a rat model. siRNA blockade of MCP-1
expression in this SCI model reduced neuronal apoptosis
. CCL20 is also linked to aggressive neuroinflammation
after SCI. Neutralizing antibodies reduced edema and in-
flammation and improved motor function after compressive
injury in a rat model .
Thus chemokine activation is a double-edged sword,
chemokines as noted above, are closely associated with im-
mune cell activation as well as inflammatory cell invasion
after SCI, which can lead to damage with excessive inflam-
mation (Fig. 3) while other studies have demonstrated pro-
tective functions [134-152]. It should also be noted that the
innate and acquired immune responses are a protective
mechanism designed to protect and heal after severe trauma
or infections. The innate immune, the inflammatory response
is activated early and leads to the initial healing or tissue
repair responses. Excess immune cell activation can be dam-
aging, but the mammalian body relies on these early innate
immune responses to allow for the rebuilding of damaged
6. CONNECTIVE TISSUE (CT)
The brain is a soft tissue, lacking much of the usual con-
nective tissue and collagen scaffolding seen in other struc-
tures. The one obvious exception being the external layers
surrounding the brain, such as the meninges or the vascular
structures associated with arteries, veins and the blood brain
barrier. Thus the brain soft tissue lacks typical collagen scaf-
folding but does have glycosaminoglycan and proteoglycan
in the supportive tissues. Collagen IV is seen in soft nervous
system scarring. Mechanical stresses are the subject of ongo-
ing investigations [153-157]. All of these changes occurring
1844 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
in the brain however, may differ from the connective tissue
changes seen in damaged or inflamed arteries where a more
typical connective tissue containing collagen I, II, IV and VI
around the damaged artery will differ from that seen in the
brain and can alter immune cell responses and invasion after
injury. Altering the endothelial layer and damage or leaking
in the BBB also alters both connective tissue composition
and immune responses at sites of trauma.
Fibrous or glial scars build-up at sites of SCI, walling off
the area of damage and ongoing inflammation and lead to a
central cystic space in the spine. This scar and the central
cystic space is a soft tissue and is reported to prevent neuron
growth across cystic areas of damage and necrosis due to a
lack of support for axon extension. However, walling off this
space together with the gradual removal of the encased fluid
in the damaged area can also prevent extension of damage
beyond the original injury site protecting adjacent areas of
the spine not initially damaged by the original trauma.
Glial scars have long been thought to form physical and
chemical barriers to nerve growth. These physical barriers
encompass deposits of the extracellular matrix that interfere
with axonal growth [150, 151], barriers that include chon-
droitin sulfate proteoglycans and other inhibitory matrix
molecules that bind to receptors and can inhibit axonal
growth [146-155]. Fibronectin together with chondroitin
sulfate (CS GAG) inhibits axonal outgrowth in vitro. CS
proteoglycan-rich regions also block axonal regeneration in
vivo [151-157]. Furthermore, intrathecal injection of Chon-
droitinase ABC, an enzyme that degrades CS GAG at sites
of nervous system injury, upregulates a regeneration-
associated protein in injured neurons and promotes regenera-
tion of both ascending sensory projections and descending
In contrast, transgenic manipulation of SOX9 and N-
acetylgalactosaminyl transferase are reported to reduce
GAGs after SCI, neuroprotection and axon regeneration.
Laminin supports neuron outgrowth both in vitro and sup-
ports outgrowth in human embryonic stem cells [156-159].
Chemokines are guided along glycosaminoglycans (GAGs)
to form gradients that direct leukocyte trafficking into sites
of damage. As noted above, these chemokine and immune
cell responses can provide both protective actions to enhance
nerve growth, but also can lead to progressive nerve damage
in the case of excess immune cell activation and aggression.
As with thrombotic and thrombolytic serine proteases and
chemokines, the connective tissues at sites of spinal cord
injury can have both beneficial reparative functions as well
as functions that limit regrowth and healing after SCI.
7. VIRUS-DERIVED IMMUNE-MODULATING
Viruses have studied mammalian immune response sys-
tems, developing highly effective immune-modulating pro-
teins through trial and error as they have evolved to highly
effective pathogens [63, 160-162]. These millions of years of
evolution have led to the development of potent immune
modulators that target central pathways. The viral immune
modulators regulate immune responses, allowing viruses to
invade and replicate by blocking or evading the host re-
sponses designed to inhibit the viral infection. Thus, evolu-
tion has allowed viruses to design reagents that very effec-
tively inhibit host immune responses often targeting key
steps in immune reactions and working ta picomolar or low-
er concentrations, allowing the virus to fight back against a
host immune response that attacks the virus. Viruses have
been used as therapeutics as vaccines and as agents for gene
expression. We have been developing these naturally
evolved viral immune-modulating proteins as a new class of
therapeutics. Two of the most effective pathways around
which virus-derived immune modulators have been devel-
oped are the serpins [160-174] and the chemokine modula-
Poxviruses are large DNA viruses and highly effective
pathogens. Myxoma virus is a poxvirus that selectively in-
fects and kills European rabbits but does not infect other
mammals [63, 161-181]. Several genes identified in Myx-
omavirus enhance virus pathogenicity and have proven high-
ly effective in targeting key immune response pathways. The
immune response pathways targeted by viruses are often
central regulatory pathways, allowing the virus to effectively
escape the host immune response [63,160-174].
Serp-1 is a secreted Myxomavirus serpin that inhibits
tPA, uPA, plasmin, as well as factor X and thrombin, prote-
ases in both thrombolytic and thrombotic cascades, respec-
tively. Poxviruses as well as herpesviruses, both large DNA
viruses, have also developed chemokine modulating proteins
(CMPs), immune modulators which inhibit chemokines.
We have examined both virus-derived serpins and chem-
okine modulators as new therapeutic approaches to improve
outcomes after SCI, testing these in rat models of SCI.
7.1. Virus-derived Serpins
Serpins expressed by poxviruses target thrombotic and
thrombolytic proteases as noted above, as well as apoptotic
pathways, often displaying marked efficacy and potency.
Myxoma virus encodes two such serpins, Serp-1 and Serp-2
that have been studied in animal models of inflammatory
disease [160-174]. Serp-1 is a 55 kDa glycosylated secreted
serpin that binds tPA, uPA, plasmin, factor X, and thrombin
inhibiting immune responses driven by both clots forming
and clot-dissolving cascades (Fig. 4). Serp-2 is a cross-class
serpin that blocks both serine and cysteine proteases, inhibit-
ing both apoptosis and the inflammasome through binding
granzyme B as well as caspases 1 and 8. Cowpox virus en-
codes a similar cross-class serpin known as Crm A that also
binds and inhibits caspases 1 and 8 and granzyme B. Over
millions of years of evolution, virus-derived serpins have
developed the advantage of targeting coagulation, inflam-
masome, and apoptotic pathways, blocking these host im-
mune responses essential to effective viral infections, allow-
ing the virus to survive and infect other cells (Fig. 4)
[63,163-174]. We have examined the efficacy of blocking
both the thrombotic and thrombolytic pathways as well as
associated aggressive inflammation in multiple animal mod-
els of disease [63, 163-174]. Serp-1 improved outcomes in
preclinical animal models of vascular disease, including ath-
erosclerosis, restenosis after angioplasty and stent implant,
allograft vasculopathy after organ transplant, and rare in-
Serine Proteases and Chemokines in Neurotrauma Current Neuropharmacology, 2021, Vol. 19, No. 11 1845
flammatory vasculitic syndromes (IVS) such as Takayasu’s
and Giant cell arteritis. Serp-2 also improved outcomes in
models of atherosclerosis in Apolipoprotein E (ApoE)
knockout mice with carotid cuff injury, as well as aortic al-
lograft transplants and liver ischemia models . In more
recent work, we have demonstrated accelerated wound heal-
ing when applying Serp-1 with saline through a collagen-
chitosan hydrogel .
Of interest, Serp-1 has also been assessed as a treatment
to reduce arterial inflammation in patients with acute unsta-
ble coronary syndromes with coronary stent implants. A ran-
domized, double-blinded Phase 2a clinical trial performed at
7 sites in Canada and the US demonstrated an early and sig-
nificant reduction in markers of myocardial damage. In addi-
tion, no significant adverse events were demonstrated with
zero Major Adverse Cardiovascular Effects (MACE) and no
detected neutralizing antibodies .
Both the preclinical and the Phase 1 safety trial with
Serp-1 in healthy volunteers also demonstrated remarkable
safety. The fact that these findings have been proven in a
wide range of animal vascular and inflammatory models as
well as up to phase 2 clinical trial ascertains that these virus-
derived immune modulators can be used safely as therapeu-
tics in human diseases .
Prolonged inflammation leads to continued damage after
SCI, providing an avenue for Serp-1 as a potential therapeu-
tic [173-175]. Serp-1 has been shown to be neuroprotective
with subdural infusion and when introduced after SCI in a
chitosan collagen hydrogel. Serp-1 treatment reduced tissue
damage with associated early benefit, improving neurologi-
cal function [173-175]. While Serp-1 has also been tested in
rat models of SCI, other virus-derived serpins such as Serp-2
or CrmA have yet to be examined in SCI models.
7.2. Virus-derived Chemokine Modulating Proteins
Myxomavirus has also developed two highly potent
chemokine modulating proteins, M-T1 and M-T7 [63, 176-
183]. The murine gamma Herpesvirus (MHV68) expresses a
chemokine modulating protein (CMP), M3, required to es-
tablish a normal latent viral load [176-183]. M-T1 binds at
the receptor binding domain of chemokines in the CC chem-
okine class. M-T7 is a 37kDa glycosylated protein that
shares sequence homology with the rabbit interferon gamma
(IFN-) receptor gamma (IFN-) and selectively binds rabbit
IFNJ. M-T7 inhibits chemokines in rats, mice, and humans
through binding and inhibition of the glycosaminogycan
(GAG) binding domain of C, CC, and CXC chemokines
[176-183]. M-T7 also inhibits rabbit IFN-, but this is spe-
cies-specific to rabbits and does not bind IFN- from other
species. M3 from MHV68 blocks both chemokine receptor
binding as well as chemokine GAG binding [184-187]. M-
T7 and M3, through blocking chemokine binding to GAGs
in the glycocalyx around endothelial cells in arteries, can
effectively inhibit immune cell activation.
M-T1, M-T7 and M3 reduce vascular inflammation in
simple rodent balloon injury models [63, 176-183]. M-T7
has further demonstrated improved outcomes in aortic and
renal transplants in mouse models and after stent implant in
rabbit models of restenosis. M-T7 has more recently been
demonstrated to accelerate wound healing and to reduce SCI
after balloon crush injury in a rat SCI model .
Fig. (4). Effects of virus-derived immune modulating proteins on cellular activation and invasion. A. HEC (Hoechst) stain of Myxomavirus
infected transformed Monkey kidney cells. Myxoma is a poxvirus that infects cells and the virus-infected cells express virus-derived im-
mune-modulating proteins that block host immune attacks against the infected cells. Two such Myxomavirus -derived proteins include the
serpin, Serp-1, and the chemokine modulating protein(CMP), M-T7. B. M-T7 blocks chemokine binding to tissue GAGs, thus decreasing
chemotaxis. C. Serp-1 binds and inhibits uPA/ uPAR complexes on mononuclear cells, blocking cell activation. (A higher resolution / colour
version of this figure is available in the electronic copy of the article).
1846 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
7.3 Treatment with Virus-derived Immune Modulators
Both Serp-1 and M-T7 have been examined as potential
therapeutics in rat models of balloon crush spinal cord inju-
ry. Each protein was tested individually as a local subdural
infusion started at the time of SC crush injury . Serp-1
has been more extensively assessed in the rat SCI model.
When Serp-1 was administered as a short-term local infusion
at sites of distal T12 SCI crush injury, there was reduced
inflammation and improved motor function as assessed by a
modified motor scale devised by Kwiecien et al. [1745, 175].
Fig. (5). Upper panel - Illustration of potential therapeutic targets for Serp-1 and M-T7 in areas of neurotrauma with neuronal damage and
excess progressive immune-mediated damage. Bottom panel - Micrographs demonstrate reduced tissue damage and reduced inflammation
after SCI in the rat model of lower thoracic forceps crush injury; 7 days follow up after implant of Serp-1 in a chitosan-collagen hydrogel.
Implant was at the site of injury immediately after crush injury [174-176]. Left - Control CCH - chitosan collagen hydrogel illustrates large
area damage and inflammation. Right - CCH Serp-1 - hydrogel implant illustrates reduced area of damage and inflammation. 10X Magnifi-
cation. H&E Staining. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
Serine Proteases and Chemokines in Neurotrauma Current Neuropharmacology, 2021, Vol. 19, No. 11 1847
Longer-term infusions also displayed benefit, as did a chi-
tosan-collagen hydrogel implant after forceps crush injury
. However, systemic or intraperitoneal injections did
not improve outcomes nor reduce inflammation at the site of
balloon crush injury. More prolonged local SC infusions of
Serp-1 again displayed early benefit, however, this benefit
was lost after 14-21 days postinjury. Application of a hydro-
gel that provided local Serp-1 releases again demonstrated
improved early motor function after balloon crush SCI.
As noted, the administration of Serp-1 via a chitosan-
collagen hydrogel in rats demonstrated a significant reduc-
tion of the extent of spinal cord damage and reduced in-
flammation. Analysis by immunohistochemical staining for
Neurofilament Medium polypeptide (NF-M) at day 7 post-
injury indicated that high-dose Serp-1 (delivered through the
hydrogel) significantly reduced the area of neuronal loss in
comparison to the control (hydrogel alone). This treatment
also limited neuronal damage at day 28 post-injury when
compared to the hydrogel alone. These areas of NF-M stain-
ing displayed reduced apoptosis as indicated by fewer caspa-
se-3 positive cells for rats treated with Serp-1. This suggests
that the reduced area of injury may have been mediated
through suppression of apoptosis of neuronal cells. Addi-
tionally, immunohistochemical staining for CD3+ T-cells
indicated that treatment with Serp-1 also significantly re-
duced the number of immune cells in the area of injury at 28
days postinjury (Fig. 5) [173-175].
In our Serp-1 model, the effects on the extent of astro-
gliosis were determined by staining for glial fibrillary acidic
protein (GFAP). On day 7 post-injury, the number of GFAP-
positive cells proximal to the injury was significantly in-
creased for rats treated with Serp-1 via the chitosan-collagen
hydrogel. A histological scoring system for GFAP-positive
compartmentalization also demonstrated that the Serp-1 chi-
tosan-collagen hydrogel (CCH) stimulated earlier protection
of spinal cord lesions (Fig. 5). This indicates that treatment
with Serp-1 may promote earlier astrogliosis and compart-
mentalization of the injury.
Astrocytes are critical inflammatory regulators in the
CNS. Astrogliosis is the process by which trauma, ischemia,
infection, stroke, autoimmune, or neurodegenerative change
induce an increase in the number of astrocytes. Astrogliosis
has a dual role in spinal cord injury with both detrimental
effects as well as recent discovery of its protective effects in
limiting damage and extent of damage. It is important to note
that astrogliosis does not occur in isolation but is a coordi-
nated part to a multicellular reaction to CNS trauma involv-
ing other glial cells, neurons, and non-neuronal cells. More
recently, scientists recognize that the process of astrogliosis
is not a simple generic response, it is rather a fine-tuned
unique response that occurs on a spectrum from reversible
alterations in gene expression to pronounced cell prolifera-
tion with compact scar formation and permanent tissue rear-
rangement . Astrocyte scarring plays a critical role in
walling off inflammatory cells from the area of trauma or
autoimmune damage into nearby healthy tissue.
The mechanism involves a wide range of specific molec-
ular signaling mechanisms that regulate specific aspects of
astrogliosis. Thus, it is not a simple on-off reaction. Astro-
cytes not only attract inflammatory cells by opening the
blood-brain barrier through the release of chemokines but
also directly have strong immunosuppressive effects on in-
flammatory constituents. In fact, more recent studies are re-
vealing that CNS autoimmune diseases that target astrocytes
have a more severe course than autoimmune diseases that
attack other cell lines.
This improved function and healing with Serp-1 hydrogel
may also be the end result of inhibition of excess inflamma-
tion and tPA or uPA mediated immune cell responses and /
or inhibition of thrombin-mediated inflammation and apop-
tosis. Serp-1 is a serpin and thus acts at sites where serine
proteases are activated. With the combined activation of
thrombotic (thrombin) and thrombolytic (tPA and uPA)
Serp-1, my function to balance and or damp down excess
protease activation. Much further work will be required to
identify the specific pathways targeted by Serp-1 and M-T7
as well as the optimal timing for treatment in the benefits
detected with treatments for acute SCI and to determine
methods by which longer term improvement can be identi-
fied and developed.
One small study has also demonstrated the benefit with
M-T7 when infused locally at sites of balloon crush SCI in
rats. We are currently investigating the local effects of Serp-1
and M-T7 treatment on dorsal root ganglion neuronal out-
growth in vitro. Effects of Serp-1 or M-T7, when applied in
the presence and absence of the thrombolytic and thrombotic
proteases, will be assessed in vitro, allowing an analysis of
the potential molecular mechanisms of action of each im-
mune modulator when used after SCI.
What has become clear in reviewing these specific serine
proteases, serpin, and chemokine pathways in CNS injury is
that these pathways have proven to be highly active, with
significant effects on the responses in the CNS to injury and
healing. It is also evident that many of the agents functioning
in these protease and chemokine responses have both direct
effects on neurons and glial cells as well as indirect effects
on the systemic and locally mediated immune responses to
injury. Each of these pathways has been demonstrated to
have some benefits and also some adverse effects dependent
upon the specific neurological and immunological responses.
With two very powerful immune modulators derived from
viruses we have seen some early benefits with reduced dam-
age and size of cystic spaces as well as reduced inflamma-
tion and preserved neuronal tissue and function. However,
these effects are demonstrated as an early benefit, but not a
long term benefit, as has been seen with many treatment ap-
proaches. This suggests that these newer biologics targeting
serine protease and chemokine pathways may well provide
an early benefit that would allow more prolonged neuronal
regrowth when combined in the future with other approaches
such as stem cell implants and hydrogels to support axon
extension and growth. Treatments for SCI may thus require a
staged approach. A greater understanding of the molecular
responses and their specific effects on repair after neuro-
trauma is in great need, and focused work on these areas will
benefit future approaches to new treatments.
1848 Current Neuropharmacology, 2021, Vol. 19, No. 11 Beladi et al.
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
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