Thrombin: A Gas Pedal Driving Innate Immunity.

Hemostasis and immunity were long considered entirely separate entities. In this issue of Immunity, Burzynski et al. (2019) find that thrombin, the key enzyme within the coagulation cascade, activates IL-1α, a central pleiotropic pro-inflammatory cytokine, to promote wound healing and platelet production following ectoderm injury.

The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin.

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.

Biology of factor XI.

ABSTRACT: Unique among coagulation factors, the coagulation factor XI (FXI) arose through a duplication of the gene KLKB1, which encodes plasma prekallikrein. This evolutionary origin sets FXI apart structurally because it is a homodimer with 2 identical subunits composed of 4 apple and 1 catalytic domain. Each domain exhibits unique affinities for binding partners within the coagulation cascade, regulating the conversion of FXI to a serine protease as well as the selectivity of substrates cleaved by the active form of FXI. Beyond serving as the molecular nexus for the extrinsic and contact pathways to propagate thrombin generation by way of activating FIX, the function of FXI extends to contribute to barrier function, platelet activation, inflammation, and the immune response. Herein, we critically review the current understanding of the molecular biology of FXI, touching on some functional consequences at the cell, tissue, and organ level. We conclude each section by highlighting the DNA mutations within each domain that present as FXI deficiency. Together, a narrative review of the structure-function of the domains of FXI is imperative to understand the etiology of hemophilia C as well as to identify regions of FXI to safely inhibit the pathological function of activation or activity of FXI without compromising the physiologic role of FXI.

De novo development of small cyclic peptides that are orally bioavailable.

Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as with biological drugs, most cyclic peptides cannot be applied orally because they are rapidly digested and/or display low absorption in the gastrointestinal tract, hampering their development as therapeutics. In this study, we developed a combinatorial synthesis and screening approach based on sequential cyclization and one-pot peptide acylation and screening, with the possibility of simultaneously interrogating activity and permeability. In a proof of concept, we synthesized a library of 8,448 cyclic peptides and screened them against the disease target thrombin. Our workflow allowed multiple iterative cycles of library synthesis and yielded cyclic peptides with nanomolar affinities, high stabilities and an oral bioavailability (%F) as high as 18% in rats. This method for generating orally available peptides is general and provides a promising push toward unlocking the full potential of peptides as therapeutics.

Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

C1 inhibitor deficiency enhances contact pathway-mediated activation of coagulation and venous thrombosis.

C1 inhibitor (C1INH) is a multifunctional serine protease inhibitor that functions as a major negative regulator of several biological pathways, including the contact pathway of blood coagulation. In humans, congenital C1INH deficiency results in a rare episodic bradykinin-mediated swelling disorder called hereditary angioedema (HAE). Patients with C1INH deficiency-associated HAE (C1INH-HAE) have increased circulating markers of activation of coagulation. Furthermore, we recently reported that patients with C1INH-HAE had a moderate but significant increased risk of venous thromboembolism. To further investigate the impact of C1INH deficiency on activation of coagulation and thrombosis, we conducted studies using patient samples and mouse models. Plasmas from patients with C1INH-HAE had significantly increased contact pathway-mediated thrombin generation. C1INH-deficient mice, which have been used as a model of C1INH-HAE, had significantly increased baseline circulating levels of prothrombin fragment 1+2 and thrombin-antithrombin complexes. In addition, whole blood from C1INH-deficient mice supported significantly increased contact pathway-mediated thrombin generation. Importantly, C1INH-deficient mice exhibited significantly enhanced venous, but not arterial, thrombus formation. Furthermore, purified human C1INH normalized contact pathway-mediated thrombin generation and venous thrombosis in C1INH-deficient mice. These findings highlight a key role for endogenous C1INH as a negative regulator of contact pathway-mediated coagulation in humans and mice. Further, this work identifies endogenous C1INH as an important negative regulator of venous thrombus formation in mice, complementing the phenotype associated with C1INH-HAE.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin.

BACKGROUND: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. METHODS: We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. RESULTS: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a ß-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both ß-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by ß-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. CONCLUSIONS: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

Steric hindrance and structural flexibility shape the functional properties of a guanine-rich oligonucleotide.

Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.

Factor XI as a therapeutic target in neuroinflammatory disease.

PURPOSE OF REVIEW: This review summarizes the pathophysiology and potential therapeutic options for treatment of multiple sclerosis, a common neuronal demyelinating disorder affecting 2.2 million people worldwide. As an autoimmune disorder, multiple sclerosis is associated with neuroinflammation and increased permeability of the blood-brain barrier (BBB), although the cause linking multiple sclerosis with compromised barrier function remains ill-defined. It has been previously shown that coagulation factors, including thrombin and fibrin, exacerbate the inflammatory processes and permeability of the BBB. RECENT FINDINGS: Increased levels of the coagulation factor (F) XII have been found in patients presenting with relapsing-remitting multiple sclerosis, with a deleterious role for FXII being validated in murine model of multiple sclerosis, experimental autoimmune encephalitis (EAE). Recent work has uncovered a role for the major substrate activated by FXII and thrombin, FXI, in the disorder of EAE. The study found that pharmacological targeting of FXI decreased clinical symptoms, lymphocyte invasion, and white matter destruction in a multiple sclerosis model. SUMMARY: This review emphasizes the role of FXII and FXI in regulating barrier function and the immune response in neuroinflammation. These new findings broaden the potential for therapeutic utility of FXI inhibitors beyond thrombosis to include neuroinflammatory diseases associated with compromised BBB function, including multiple sclerosis.

A mechanical modeling framework to study endothelial permeability.

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Cytoprotective E-WE thrombin reduces disease severity in a murine model of relapsing-remitting multiple sclerosis.

The blood-brain barrier is composed of microvascular endothelial cells, immune cells, and astrocytes that work in concert with the coagulation cascade to control inflammation and immune cell infiltration into the central nervous system. Endothelial cell dysfunction leading to increased permeability and compromised barrier function are hallmarks of neuroinflammatory and autoimmune disorders, including multiple sclerosis (MS). Therapeutic strategies that improve or protect endothelial barrier function may be beneficial in the treatment or prevention of neuroinflammatory diseases. We therefore tested the hypothesis that biasing thrombin toward anticoagulant and cytoprotective activities would provide equivalent or even additive benefit compared with standard-of-care therapeutic strategies, including corticosteroids. In a mouse model of relapsing-remitting MS, treatment with the thrombin mutant, E-WE thrombin, an engineered thrombin mutant with cytoprotective activities that is biased toward anticoagulant and cytoprotective activity, reduced neuroinflammation and extracellular fibrin formation in SJL mice inoculated with proteolipid protein (PLP) peptide. When administered at the onset of detectable disease, E-WE thrombin significantly improved the disease severity of the initial attack as well as the relapse and delayed the onset of relapse to a similar extent as observed with methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment. These results provide rationale for considering engineered forms of thrombin biased toward anticoagulant and cytoprotective activity as a therapeutic strategy and perhaps an effective alternative to high-dose methylprednisolone for the management of acute relapsing MS attacks.NEW & NOTEWORTHY There are limited treatment options for mitigating acute relapsing attacks for patients with multiple sclerosis. We tested the hypothesis that harnessing the cytoprotective activity of the blood coagulation enzyme, thrombin, would provide benefit and protection against relapsing disease in a mouse model of MS. Our results provide rationale for considering engineered forms of thrombin biased toward cytoprotective activity as a therapeutic strategy and perhaps an alternative to steroids for the management of relapsing MS attacks.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

An siRNA library screen identifies CYLD and USP34 as deubiquitinases that regulate GPCR-p38 MAPK signaling and distinct inflammatory responses.

G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-ß-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.

Blockade of CD47 function attenuates restenosis by promoting smooth muscle cell efferocytosis and inhibiting their migration and proliferation.

Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cß3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin ß3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia.

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity.

Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10-fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho-Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine-succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo-hirudin and desulfo-hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho-hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo-hirudin; succinylated hirudin was also prospective. Phospho-hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho-hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.