Novel nucleotide variations in the thrombomodulin (THBD) gene involved in coagulation pathways can increase the risk of recurrent pregnancy loss (RPL).

Recurrent pregnancy loss (RPL) is a common but complex complication in fertility conditions, affecting about 15-20% of couples. Although several causes have been proposed for RPL, it occurs in about 35-60% of cases without a known explanation. A strong assumption is that genetic factors play a role in the etiology and pathophysiology of PRL. Therefore, several genes are proposed as candidates in the pathogenesis of RPL. The current study aimed to investigate the effects of nucleotide changes in the THBD (thrombomodulin) gene as an RPL-related candidate gene. This gene encodes a cell receptor for thrombin and is involved in reproductive loss in RPL cases. Its involvement in the natural anticoagulant system has been extensively studied. By genetic screening of the entire coding and noncoding regions of the THBD gene, we found twenty-seven heterozygous and homozygous nucleotide changes. Ten of them led to amino acid substitutions, seven variants were identified in the promoter region, and eight of them occurred in 3'UTR. Potentially, the pathogenicity effects of these variations on THBD protein were evaluated by several prediction tools. The numerous genomic variations prompted noticeable modifications of the protein's structural and functional properties. Furthermore, in-silico scores were consistent with deleterious effects for these mutations. The results of this study provide genetic information that will be useful in the future for clinicians, scientists, and students to understand the unknown causes of RPL better. It may also pave the way for developing diagnostic/prognostic approaches to help treat PRL patients.

VhH anti-thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow.

BACKGROUND: Thrombomodulin on endothelial cells can form a complex with thrombin. This complex has both anticoagulant properties, by activating protein C, and clot-protective properties, by activating thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI (TAFIa) inhibits plasmin-mediated fibrinolysis. OBJECTIVES: TAFIa inhibition is considered a potential antithrombotic strategy. So far, this goal has been pursued by developing compounds that directly inhibit TAFIa. In contrast, we here describe variable domain of heavy-chain-only antibody (VhH) clone 1 that inhibits TAFI activation by targeting human thrombomodulin. METHODS: Two llamas (Lama Glama) were immunized, and phage display was used to select VhH anti-thrombomodulin (TM) clone 1. Affinity was determined with surface plasmon resonance and binding to native TM was confirmed with flow cytometry. Clone 1 was functionally assessed by competition, clot lysis, and thrombin generation assays. Last, the effect of clone 1 on tPA-mediated fibrinolysis in human whole blood was investigated in a microfluidic fibrinolysis model. RESULTS: VhH anti-TM clone 1 bound recombinant TM with a binding affinity of 1.7 ± 0.4 nM and showed binding to native TM. Clone 1 competed with thrombin for binding to TM and attenuated TAFI activation in clot lysis assays and protein C activation in thrombin generation experiments. In a microfluidic fibrinolysis model, inhibition of TM with clone 1 fully prevented TAFI activation. DISCUSSION: We have developed VhH anti-TM clone 1, which inhibits TAFI activation and enhances tPA-mediated fibrinolysis under flow. Different from agents that directly target TAFIa, our strategy should preserve direct TAFI activation via thrombin.

Recombinant soluble thrombomodulin accelerates epithelial stem cell proliferation in mouse intestinal organoids and promotes the mucosal healing in colitis.

BACKGROUND AND AIM: Epithelial regeneration, a critical step for the mucosal healing in inflammatory bowel disease, is tightly regulated by stem cells. Therefore, identification of the specific factors that induce stem cell proliferation could contribute to the development of effective strategies for treating inflammatory bowel disease. Recombinant soluble thrombomodulin (rsTM) has previously been shown to promote cell proliferation in skin and corneal wound healing in murine models, but its effects on intestinal epithelial cell proliferation remains unclear. METHODS: Mouse intestinal organoids and dextran sulfate sodium (DSS)-induced colitis mouse model were used to assess the effects of rsTM on proliferation of intestinal epithelial cells. The size and budding morphologies of organoids were studied by confocal microscopy. The gene expression levels were analyzed by quantitative real-time polymerase chain reaction and immunofluorescence analysis. The effects of rsTM on DSS-induced colitis were investigated by evaluating body weight changes, colon length, histological score, and survival rate. RESULTS: The rsTM markedly stimulated the growth of intestinal organoids, thereby increasing the surface areas and budding phenotypes of the organoids. rsTM also significantly upregulated the gene expression of intestinal stem cell-specific and epithelial cell-specific markers in a dose-dependent manner. Furthermore, the treatment with high concentrations of rsTM significantly improved the recovery of body weight, histological outcomes, colon length shortening, and prolonged the survival of mice with colitis. CONCLUSIONS: The rsTM promotes intestinal stem cell proliferation in intestinal organoids and enhances the mucosal healing during recovery phase in DSS-induced colitis.

Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex.

Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-ß-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced µs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased µs-ms dynamics in a ß-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two ß-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

Recombinant Human Soluble Thrombomodulin Suppresses Monocyte Adhesion by Reducing Lipopolysaccharide-Induced Endothelial Cellular Stiffening.

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells regulate leukocyte adhesion in a substrate- and cellular stiffness-dependent manner. Our result show that LPS-induced cellular stiffening enhances monocytic THP-1 cell line adhesion, whereas rsTM suppresses THP-1 cell adhesion to inflamed endothelial cells by reducing cellular stiffness. Endothelial cells increase cellular stiffness in reaction to inflammation, thereby promoting monocyte adhesion. Treatment of rsTM reduced LPS-induced cellular stiffening and suppressed monocyte adhesion in a cellular stiffness-dependent manner.

Recombinant human soluble thrombomodulin is associated with attenuation of sepsis-induced renal impairment by inhibition of extracellular histone release.

Multiple organ dysfunction induced by sepsis often involves kidney injury. Extracellular histones released in response to damage-associated molecular patterns are known to facilitate sepsis-induced organ dysfunction. Recombinant human soluble thrombomodulin (rhTM) and its lectin-like domain (D1) exert anti-inflammatory effects and neutralize damage-associated molecular patterns. However, the effects of rhTM and D1 on extracellular histone H3 levels and kidney injury remain poorly understood. Our purpose was to investigate the association between extracellular histone H3 levels and kidney injury, and to clarify the effects of rhTM and D1 on extracellular histone H3 levels, kidney injury, and survival in sepsis-induced rats. Rats in whom sepsis was induced via cecal ligation and puncture were used in this study. Histone H3 levels, histopathology of the kidneys, and the survival rate of rats at 24 h after cecal ligation and puncture were investigated. Histone H3 levels increased over time following cecal ligation and puncture. Histopathological analyses indicated that the distribution of degeneration foci among tubular epithelial cells of the kidney and levels of histone H3 increased simultaneously. Administration of rhTM and D1 significantly reduced histone H3 levels compared with that in the vehicle-treated group and improved kidney injury. The survival rates of rats in rhTM- and D1-treated groups were significantly higher than that in the vehicle-treated group. The results of this study indicated that rhTM and its D1 similarly reduce elevated histone H3 levels, thereby reducing acute kidney injury. Our findings also proposed that rhTM and D1 show potential as new treatment strategies for sepsis combined with acute kidney injury.

End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity.

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. The 4, 5, 6 EGF-like domain of TM (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 is a promising anticoagulant candidate but has a very short half-life. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM456 modification. Recombinant TM456 with a C-terminal LPETG tag (rTM456-LPETG) was expressed in Escherichia coli for its end-point modification with NH2-diglycine-PEG5000-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific PEGylation of rTM456via CFCC. Both PEGylated rTM456 conjugates retained protein C activation activity as that of rTM456. Also, they were more stable than rTM456 in Trypsin digestion assay. Further, both PEGylated rTM456 conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.

Thrombomodulin Regulation of Mitogen-Activated Protein Kinases.

The multifaceted role of mitogen-activated protein kinases (MAPKs) in modulating signal transduction pathways in inflammatory conditions such as infection, cardiovascular disease, and cancer has been well established. Recently, coagulation factors have also emerged as key players in regulating intracellular signaling pathways during inflammation. Among coagulation factors, thrombomodulin, as a high affinity receptor for thrombin on vascular endothelial cells, has been discovered to be a potent anti-inflammatory and anti-tumorigenic signaling molecule. The protective signaling function of thrombomodulin is separate from its well-recognized role in the clotting cascade, which is to function as an anti-coagulant receptor in order to switch the specificity of thrombin from a procoagulant to an anti-coagulant protease. The underlying protective signaling mechanism of thrombomodulin remains largely unknown, though a few published reports link the receptor to the regulation of MAPKs under different (patho)physiological conditions. The goal of this review is to summarize what is known about the regulatory relationship between thrombomodulin and MAPKs.

Anti-adhesive effects of human soluble thrombomodulin and its domains.

We reported previously that leukocyte ß2 integrins (LFA-1 and Mac-1) bind to the serine/threonine-rich domain of thrombomodulin (TM) expressed on vascular endothelial cells (VECs). Recombinant human soluble TM (rhsTM, TMD123) has been approved as a therapeutic drug for septic disseminated intravascular coagulation. However, the roles of TMD123 on the adhesion of leukocyte integrins to VECs remain unclear. In the current study, we have revealed that an integrin-dependent binding between human peripheral blood mononuclear cells (PBMCs) and VECs was inhibited by TMD123. Next, using mutant proteins composed of isolated TM extracellular domains, we examined the structural characteristics responsible for the anti-adhesion properties of TMD123. Namely, we investigated whether the effects of the binding of TM and leukocytes was inhibited by the administration of TMD123. In fact, we confirmed that TMD123, TMD1, and TMD3 inhibited the binding of PBMCs to the immobilized recombinant proteins TMD123 and TMD3. These results indicate that TMD123 inhibited the adhesion of leukocytes to endothelial cells via ß2 integrins and endothelial TM. Moreover, since TMD1 might bind to leukocytes via other adhesion receptors than integrins, TMD1 and TMD3 appear to inhibit leukocyte binding to TM on VECs via different mechanisms. In summary, TMD123 (rhsTM), TMD1 or TMD3 is a promising treatment option for sepsis that attenuates integrin-dependent binding of leukocytes to VECs, and may inhibit the undesirable adhesion and migration of leukocytes to VECs in sepsis.

Cell and tissue responses at the interface with a chitosan hydrogel intended for vascular applications: in vitro and in vivo exploration.

AIMS: The need for small caliber vessels to treat cardiovascular diseases has grown. However, synthetic polymers perform poorly in small-diameter applications. Chitosan hydrogels can provide a novel biological scaffold for vascular engineering. The goal of this study was to explore host cell and tissue behavior at the interface with chitosan-based scaffolds in vitro and in vivo. METHODS AND RESULTS: in vitro, we assessed the ability of endothelial cells lining chitosan hydrogels to produce tissue factor (TF), thrombomodulin (TM) and nitric oxide. We showed that endothelial cells behave as a native endothelium since under stimulation, TF and TM expression increased and decreased, respectively. Endothelial cells seeded on chitosan produced nitric oxide, but no change was observed under stimulation. After in vivo subcutaneous implantation of chitosan hydrogels in rats, macrophage activation phenotypes, playing a crucial role in biomaterial/tissue, were explored by immunohistochemistry. Our results suggested a balance between pro- and anti-inflammatory signals since we observed an inflammatory response in favor of macrophage M2 phenotype. CONCLUSION: in vitro exploration of endothelial cell response at the interface with chitosan hydrogel showed a functional endothelium and in vivo exploration of tissue response revealed a biointegration of chitosan hydrogels.

Sustained release of recombinant thrombomodulin from cross-linked gelatin/hyaluronic acid hydrogels potentiate wound healing in diabetic mice.

Thrombomodulin (TM) is a type-I transmembrane glycoprotein expressed on the surfaces of endothelial cells and epidermal keratinocytes. It is known to regulate blood coagulation, inflammation, and cell-cell adhesion. A recombinant TM, which contains an epidermal growth factor-like domain and serine/threonine-riches domain, has been demonstrated to stimulate cell proliferation and migration of keratinocytes and wound healing. In this study, we developed the biodegradable hydrogels and evaluated the efficacy of sustained release of rhTM from the hydrogel for the treatment of diabetic wounds. The hydrogels were composed of gelatin with or without hyaluronic acid, and fabricated by chemical cross-linking followed by lyophilization. Gelatin-based hydrogels had porous structure, good swelling property, and were biodegradable with characteristics of slow rhTM release in a short term. The once every-3-day rhTM-loaded hydrogel (with hyaluronic acid) markedly promoted wound healing and were superior to rhTM solution, once daily rhTM hydrogel (without hyaluronic acid), hydrogel controls, and once every-3-day rhEGF hydrogel treatment groups. The rhTM hydrogels enhanced granulation tissue formation, re-epithelialization, collagen deposition, and angiogenesis in wound repair. The once every-3-day rhTM hydrogel was stable and drug release was maintained up to 11-month of storage at 4 °C. The developed rhTM hydrogels could meet the needs for clinical practice, and may have future medical applications for wound care in diabetic patients.

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Herein we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from Gpr15 knock-out (KO) mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal EC numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. The use of TME5C may be a promising treatment strategy to prevent or treat lethal complications, such as sinusoidal obstruction syndrome, whose pathogenesis is based on endothelial insults.

Exploring traditional and nontraditional roles for thrombomodulin.

Thrombomodulin (TM) is an integral component of a multimolecular system, localized primarily to the vascular endothelium, that integrates crucial biological processes and biochemical pathways, including those related to coagulation, innate immunity, inflammation, and cell proliferation. These are designed to protect the host from injury and promote healing. The "traditional" role of TM in hemostasis was determined with its discovery in the 1980s as a ligand for thrombin and a critical cofactor for the major natural anticoagulant protein C system and subsequently for thrombin-mediated activation of the thrombin activatable fibrinolysis inhibitor (also known as procarboxypeptidase B2). Studies in the past 2 decades are redefining TM as a molecule with many properties, exhibited via its multiple domains, through its interacting partners, complex regulated expression, and synthesis by cells other than the endothelium. In this report, we review some of the recently reported diverse properties of TM and how these may impact on our understanding of the pathogenesis of several diseases.

Human soluble thrombomodulin-induced blockade of peripheral HMGB1-dependent allodynia in mice requires both the lectin-like and EGF-like domains.

Thrombomodulin (TM), an endothelial protein with anti-coagulant activity, is composed of 5 domains, D1-D5. Recombinant human soluble TM (TMα) consisting of D1-D3, which is generated in CHO cells, suppresses inflammatory and nociceptive signals by inactivating high mobility group box 1 (HMGB1), one of damage-associated molecular patterns. TMα sequesters HMGB1 with the lectin-like D1 and promotes its degradation by thrombin binding to the EGF-like D2. We prepared TM's D123, D1 and D2 by the protein expression system of yeast, and evaluated their effects on HMGB1 degradation in vitro and on the allodynia caused by HMGB1 in distinct redox forms in mice in vivo. TMα and TM's D123, but not D1, promoted the thrombin-dependent degradation of all-thiol (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), an effect mimicked by TM's D2, though to a lesser extent. Intraplantar administration of TMα and TM's D123, but not D1, D2 or D1 plus D2, strongly prevented the mechanical allodynia caused by intraplantar at-HMGB1, ds-HMGB1 or lipopolysaccharide in mice. Our data suggest that, apart from the role of D3, TMα and TM's D123 require both lectin-like D1 capable of sequestering HMGB1 and EGF-like D2 responsible for thrombin-dependent degradation of HMGB1, in abolishing the allodynia caused by exogenous or endogenous HMGB1.

Peptide GC31 inhibits chemokines and ICAM-1 expression in corneal fibroblasts exposed to LPS or poly(I:C) by blocking the NF-κB and MAPK pathways.

In keratitis, keratocytes play a vital role by releasing inflammatory cytokines and expressing intercellular cell adhesion molecule-1(ICAM-1). GC31 is a peptide derived from thrombomodulin, an endogenous protein with potential anti-inflammation properties. We evaluated the protective effect of GC31 in LPS- or poly(I:C)-induced corneal fibroblasts. Cultured keratocytes were treated with either LPS or poly(I:C); The mRNA and protein expressions of IL-6, IL-8, MCP-1, and IFN-γ were determined by real-time RT-PCR and ELISA. The expression level of ICAM-1 was estimated by real-time RT-PCR, immunofluorescence, and western blot. The underlying pathways were investigated by detecting NF-κB p65 translocation and phosphorylation of IκBα, p65, p38, JNK, and ERK. The MTS assay was used to measure cell viability of keratocytes after GC31 incubation. The elevation of IL-6, IL-8, MCP-1, and IFN-γ expression induced by LPS or poly(I:C) was significantly inhibited by GC31 in a dose-dependent manner at both mRNA and protein levels. GC31 also reduced the expression of ICAM-1 in keratocytes after LPS or poly(I:C) stimulation. LPS or poly(I:C) induced p65 translocation and phosphorylation of IκBα, p65, p38, and JNK were suppressed by GC31.GC31 is not only an effective inhibitor of LPS-induced inflammatory response, but it also inhibits poly(I:C)-induced release of inflammatory cytokines and ICAM-1 expression by blocking the NF-κB and MAPK (p38 and JNK) pathways. This suggested that GC31 may exert a protective effect in attenuating corneal inflammation by suppressing the immune response of the fibroblasts.

Recombinant and chemo-/bio-orthogonal synthesis of liposomal thrombomodulin and its antithrombotic activity.

Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C anticoagulant pathway. The EGF-like domains 4-6 of TM (TM456) are essential for PC activation. In this study, we proposed a liposomal recombinant TM conjugate to mimic the membrane TM structure and its anticoagulant activity. First, a DSPE-PEG2000-TM456 was successfully synthesized by site-specific conjugation of azido-TM456 with DSPE-PEG2000-DBCO via copper-free click chemistry quantitatively. Then, liposome-TM456 was fabricated via direct liposome formation with the DSPE-PEG2000-TM456 and other lipids. This liposomal formulation of TM456 retained protein C activation activity as that of TM456. Also, liposome-TM456 was much more stable and had a longer plasma half-life than TM456 and DSPE-PEG2000-TM456, respectively. Moreover, liposome-TM456 showed in vivo anticoagulant effect by decreasing the mortality from 80% to 20% in a thrombin-induced thromboembolism mouse model. The reported liposome-TM456 conjugate mimics the endothelial TM anticoagulation activity and may serve as an effective anticoagulant agent candidate for future development.

Exosite 2-Directed Ligands Attenuate Protein C Activation by the Thrombin-Thrombomodulin Complex.

Thrombin activity, inhibition, and localization are regulated by two exosites that flank the active site. Substrates, cofactors, and inhibitors bind to exosite 1 to promote active site access, whereas exosite 2 interactions hold thrombin on cells, platelets, and proteins. The exosites also serve allosteric roles, whereby ligand binding alters thrombin activity. Previously, we showed that ligands that bind exosite 2 attenuate the exosite 1-mediated interaction of thrombin with fibrin, demonstrating allosteric connection between the exosites. To determine the functional consequences of these inter-exosite interactions, we examined the effect of exosite 2 ligands on thrombin's interaction with thrombomodulin, a key cofactor that binds exosite 1 and redirects thrombin activity to the anticoagulant protein C pathway. Exosite 2-directed ligands, which included the HD22 aptamer, glycoprotein 1bα-derived peptide, and fibrinogen γ'-chain peptide, reduced the level of exosite 1-mediated thrombin binding to the thrombomodulin peptide consisting of the fourth, fifth, and sixth epidermal-like growth factor-like domains, decreasing affinity by >10-fold, and attenuated thrombomodulin-dependent activation of protein C by 60-80%. The ligands had similar effects on thrombin-mediated protein C activation with intact soluble thrombomodulin and with thrombomodulin on the surface of cultured endothelial cells. Their activity was exosite 2-specific because it was attenuated when RA-thrombin, a variant lacking exosite 2, was used in place of thrombin. These results indicate that additional reactions mediated by exosite 1 are amenable to regulation by exosite 2 ligation, providing further evidence of inter-exosite allosteric regulation of thrombin activity.

Rational Design of Protein C Activators.

In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.

The fifth epidermal growth factor like region of thrombomodulin alleviates LPS-induced sepsis through interacting with GPR15.

Thrombomodulin (TM) exerts cytoprotection via the fifth region of epidermal growth factor (EGF)-like domain of TM (TME5) by interacting with G-protein coupled receptor 15 (GPR15) expressed on cell surface of vascular endothelial cells. TM is also implied to mediate anti-inflammatory functions by unknown mechanism. By applying a lipopolysaccharide (LPS)-induced murine sepsis model, we assessed the role of TME5 in septic inflammation and coagulation. We found that TME5 treatment protected mice in association with ameliorating inflammation and coagulopathy in LPS-induced sepsis. Further study confirmed that TME5 bound GPR15 in vitro. Knock out of GPR15 abolished protective role of TME5 in sepsis model. GPR15 mediated anti-inflammatory function of TME5 through suppression of phosphorylation of IκBα, nuclear translocation of NF-κB and release of pro-inflammatory cytokines in macrophages (Macs). Knock out of GPR15 resulted in dysregulated immune response of Macs, characterised by excessive expression of pro-inflammatory genes and failing to limit immune response. This study indicates that TME5 exerts anti-inflammatory function through inhibition of NF-κB in a GPR15-dependent manner. The use of TME5 may be a potential therapeutic option for treatment of sepsis.

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ' as thrombomobulin II.

Fibrin is an important matrix protein that provides the backbone to the blood clot, promoting tissue repair and wound healing. Its precursor fibrinogen is one of the most heterogeneous proteins, with an estimated 1 million different forms due to alterations in glycosylation, oxidation, single nucleotide polymorphisms, splice variation and other variations. Furthermore, ligation by transglutaminase factor XIII (cross-linking) adds to the complexity of the fibrin network. The structure and function of the fibrin network is in part determined by this natural variation in the fibrinogen molecule, with major effects from splice variation and cross-linking. This mini-review will discuss the direct effects of fibrinogen αEC and fibrinogen γ' splice variation on clot structure and function and also discuss the additional role of fibrinogen γ' as thrombomodulin II. Furthermore, the effects of cross-linking on clot function will be described. Splice variation and cross-linking are major determinants of the structure and function of fibrin and may therefore impact on diseases affecting bleeding, thrombosis and tissue repair.