Thrombomodulin Regulates PTEN/AKT Signaling Axis in Endothelial Cells.

BACKGROUND: We recently demonstrated that deletion of thrombomodulin gene from endothelial cells results in upregulation of proinflammatory phenotype. In this study, we investigated the molecular basis for the altered phenotype in thrombomodulin-deficient (TM-/-) cells. METHODS: Different constructs containing deletions or mutations in the cytoplasmic domain of thrombomodulin were prepared and introduced to TM-/- cells. The phenotype of cells expressing different derivatives of thrombomodulin and tissue samples of thrombomodulin-knockout mice were analyzed for expression of distinct regulatory genes in established signaling assays. RESULTS: The phosphatase and tensin homolog were phosphorylated and its recruitment to the plasma membrane was impaired in TM-/- cells, leading to hyperactivation of AKT (protein kinase B) and phosphorylation-dependent nuclear exclusion of the transcription factor, forkhead box O1. The proliferative/migratory properties of TM-/- cells were enhanced, and cells exhibited hypersensitivity to stimulation by angiopoietin 1 and vascular endothelial growth factor. Reexpression of wild-type thrombomodulin in TM-/- cells normalized the cellular phenotype; however, thrombomodulin lacking its cytoplasmic domain failed to restore the normal phenotype in TM-/- cells. Increased basal permeability and loss of VE-cadherin were restored to normal levels by reexpression of wild-type thrombomodulin but not by a thrombomodulin construct lacking its cytoplasmic domain. A thrombomodulin cytoplasmic domain deletion mutant containing 3-membrane-proximal Arg-Lys-Lys residues restored the barrier-permeability function of TM-/- cells. Enhanced phosphatase and tensin homolog phosphorylation and activation of AKT and mTORC1 (mammalian target of rapamycin complex 1) were also observed in the liver of thrombomodulin-KO mice. CONCLUSIONS: These results suggest that the cytoplasmic domain of thrombomodulin interacts with the actin cytoskeleton and plays a crucial role in regulation of phosphatase and tensin homolog/AKT signaling in endothelial cells.

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.

Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging.

BACKGROUND: APC (activated protein C) is a plasma serine protease with anticoagulant and anti-inflammatory activities. EPCR (Endothelial protein C receptor) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. METHODS: Young (3-4 months) and aged (24-26 months) wild-type C57BL/6J mice, as well as EPCR point mutation (EPCRR84A/R84A) knockin C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild-type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. RESULTS: The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCRR84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMPK (AMP-activated protein kinase) mediates acute adaptive response while AKT (protein kinase B) is involved in chronic metabolic programming in the hearts with APC treatment. CONCLUSIONS: I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells.

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM-/- cells that resulted in enhanced adhesion of leukocytes to TM-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM-/- cells. In a flow chamber assay, isolated platelets adhered to TM-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Extracellular Histones Bind Vascular Glycosaminoglycans and Inhibit the Anti-Inflammatory Function of Antithrombin.

BACKGROUND/AIMS: Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. METHODS: We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. RESULTS: Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. CONCLUSION: We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.

PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells.

OBJECTIVE: Native and latent conformers of AT (antithrombin) induce anti-inflammatory and proapoptotic signaling activities, respectively, in vascular endothelial cells by unknown mechanisms. Synd-4 (syndecan-4) has been identified as a receptor that is involved in transmitting signaling activities of AT in endothelial cells. Approach and Results: In this study, we used flow cytometry, signaling assays, immunoblotting and confocal immunofluorescence microscopy to investigate the mechanism of the paradoxical signaling activities of high-affinity heparin (native) and low-affinity heparin (latent) conformers of AT in endothelial cells. We discovered that native AT binds to glycosaminoglycans on vascular endothelial cells via its heparin-binding D-helix to induce anti-inflammatory signaling responses by recruiting PKC (protein kinase C)-δ to the plasma membrane and promoting phosphorylation of the Synd-4 cytoplasmic domain at Ser179. By contrast, the binding of latent AT to endothelial cells to a site(s), which is not competed by the native AT, induces a proapoptotic effect by localizing PKC-δ to the perinuclear/nuclear compartment in endothelial cells. Overexpression of a dominant-negative form of PKC-δ resulted in inhibition of anti-inflammatory and proapoptotic signaling activities of both native and latent AT. CONCLUSIONS: These results indicate that the native and latent conformers of AT may exert their distinct intracellular signaling effects through differentially modulating the subcellular localization of PKC-δ in endothelial cells.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis.

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.

Inorganic Polyphosphate Amplifies High Mobility Group Box 1-Mediated Von Willebrand Factor Release and Platelet String Formation on Endothelial Cells.

Objective- Inorganic polyphosphate (polyP) is known to modulate coagulation, inflammation, and metabolic pathways. It also amplifies inflammatory responses of HMGB1 (high mobility group box 1) in endothelial cells. The objective of this study was to evaluate the effect of polyP on von Willebrand factor (VWF) release from endothelial cells with or without HMGB1. Approach and Results- EA.hy926 endothelial cells were treated with different concentrations of polyP70 alone or in combination with different concentrations of HMGB1. VWF release was measured by an ELISA assay in the absence or presence of pharmacological inhibitors of the receptor for advanced glycation end products, P2Y1, and Ca2+. A flow chamber assay was used to monitor polyP70-mediated platelet recruitment and VWF-platelet string formation. PolyP70 and HMGB1 induced VWF release from endothelial cells by a concentration-dependent manner. PolyP70 amplified HMGB1-mediated VWF release from endothelial cells. This was also true if boiled platelet releasate was used as the source of polyP. Gene silencing or pharmacological inhibitors of receptor for advanced glycation end products, P2Y1, and Ca2+ significantly inhibited VWF release. PolyP70 and HMGB1 synergistically promoted VWF-platelet string formation in the flow chamber assay, which was inhibited by the anti-GPIbα (glycoprotein Ib alpha) antibody. VWF release by polyP70-HMGB1 complex required phosphorylation of Src and phospholipase C because inhibitors of Src, phospholipase C, and Ca2+ signaling significantly decreased VWF secretion. The polyP70-HMGB1 complex also increased angiopoietin-2 release, indicating that Weibel-Palade body exocytosis is involved in the VWF release. Conclusions- PolyP70 can promote thrombotic and inflammatory pathways by inducing VWF release and platelet string formation on endothelial cells.

Activated Protein C Attenuates Severe Inflammation by Targeting VLA-3high Neutrophil Subpopulation in Mice.

The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1highCD11bhighVLA-3high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities.

The Cardioprotective Signaling Activity of Activated Protein C in Heart Failure and Ischemic Heart Diseases.

Activated protein C (APC) is a vitamin-K dependent plasma serine protease, which functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. APC also modulates cellular homeostasis by exhibiting potent cytoprotective and anti-inflammatory signaling activities. The beneficial cytoprotective effects of APC have been extensively studied and confirmed in a number of preclinical disease and injury models including sepsis, type-1 diabetes and various ischemia/reperfusion diseases. It is now well-known that APC modulates downstream cell signaling networks and transcriptome profiles when it binds to the endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) on various cell types. However, despite much progress, details of the downstream signaling mechanism of APC and its crosstalk with other signaling networks are far from being fully understood. In this review, we focus on the cardioprotective properties of APC in ischemic heart disease and heart failure with a special emphasis on recent discoveries related to the modulatory effect of APC on AMP-activated protein kinase (AMPK), PI3K/AKT, and mTORC1 signaling pathways. The cytoprotective properties of APC might provide a novel strategy for future therapies in cardiac diseases.

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.

Activated protein C protects against pressure overload-induced hypertrophy through AMPK signaling.

We found that the anticoagulant plasma protease, activated protein C (APC), stimulates the energy sensor kinase, AMPK, in the stressed heart by activating protease-activated receptor 1 (PAR1) on cardiomyocytes. Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. The results demonstrated that while no phenotypic differences can be observed between WT and AMPK-KD mice under normal physiological conditions, AMPK-KD mice exhibit significantly larger hearts after 4 weeks of TAC surgery. Analysis by echocardiography suggested that the impairment in the cardiac function of AMPK-KD hearts is significantly greater than that of WT hearts. Immunohistochemical staining revealed increased macrophage infiltration and ROS generation in AMPK-KD hearts after 4 weeks of TAC surgery. Immunoblotting results demonstrated that the redox markers, pShc66, 4-hydroxynonenal and ERK, were all up-regulated at a higher extent in AMPK-KD hearts after 4 weeks of TAC surgery. Administration of APC-WT and the signaling selective APC-2Cys mutant, but not the anticoagulant selective APC-E170A mutant, significantly attenuated pressure overload-induced hypertrophy and fibrosis. Macrophage infiltration and pShc66 activation caused by pressure overload were also inhibited by APC and APC-2Cys but not by APC-E170A. Therefore, the cardiac AMPK protects against pressure overload-induced hypertrophy and the signaling selective APC-2Cys may have therapeutic potential for treating hypertension-related hypertrophy without increasing the risk of bleeding.

TR47, a PAR1-based peptide, inhibits melanoma cell migration in vitro and metastasis in vivo.

Activated Protein C (APC) is a serine-protease that displays antithrombotic and anti-inflammatory properties. In addition, cleavage of protease-activated receptor 1 (PAR1) by APC exerts endothelial cytoprotective actions. The effects of APC on endothelial cells may be reproduced by TR47, a PAR1-based peptide that mimics the novel N-terminus of PAR1 generated upon cleavage at Arg-46 by APC. In this study we demonstrate that wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), display similar inhibitory effects towards the transendothelial migration of A375 human melanoma cells. Consistent with this observation, APC and APC-2Cys significantly reduced the in vivo metastatic potential of the B16F10 murine melanoma cells. TR47 recapitulated the in vitro and in vivo protective profiles of APC and APC-2Cys. Treatment of EA.hy926 endothelial cells with TR47 (20 µM) significantly decreased the A375 cell migration. In addition, treatment of C57/BL6 mice with a single TR47 dose (125 µg/animal) strongly reduced the metastatic burden of B16F10 cells. Together, our results suggest that protection of the endothelial barrier by APC/TR47-mediated signaling pathways might be a valuable therapeutic approach to prevent metastasis.

Occupancy of human EPCR by protein C induces ß-arrestin-2 biased PAR1 signaling by both APC and thrombin.

Activation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective ß-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the ß-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of ß-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via ß-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered ß-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates ß-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing ß-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model.

Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays.

Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree.

Intraperitoneal administration of activated protein C prevents postsurgical adhesion band formation.

Postsurgical peritoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of adhesion and compared the protective effect of activated protein C (APC) to that of the Food and Drug Administration-approved antiadhesion agent, sodium hyaluronate/carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7, and peritoneal fluid concentrations of tissue plasminogen activator (tPA), d-dimer, thrombin-antithrombin complex, and cytokines (IL-1ß, IL-6, interferon-γ, tumor necrosis factor-α, transforming growth factor-ß1) were evaluated. Inflammation scores were also measured based on histologic data obtained from peritoneal tissues. Relative to Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor (EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model. These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands.

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor.

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models. By using small interfering RNA knockdowns, pharmacologic inhibitors and extracellular domains of the receptors TLR2, TLR4, RAGE, and P2Y1 as competitive inhibitors, we demonstrate that polyP amplifies H4- and HMGB1-mediated inflammatory signaling in human umbilical vein endothelial cells specifically through interaction with the RAGE and P2Y1 receptors, thereby eliciting intracellular Ca(2+) release. Finally, we demonstrate that the natural anticoagulant protease, activated protein C, potently inhibits polyP-mediated proinflammatory effects of both nuclear proteins in cellular and in vivo systems.

Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients.

We recently identified two hemophilia B patients who carried Gly-317 to Arg (FIX-G317R) or Gly-317 to Glu (FIX-G317E) substitutions in their FIX gene. The former mutation caused severe and the latter moderate bleeding in afflicted patients. To understand the molecular basis for the variable clinical manifestation of Gly-317 mutations, we prepared recombinant G317R and G317E derivatives of FIX and compared their kinetic properties to those of recombinant wild-type FIX in appropriate assay systems. Both physiological activators, factor XIa and extrinsic Tenase (factor VIIa-tissue factor), activated both zymogen variants with an ∼1.5-fold elevated K(m); however, extrinsic Tenase activated FIX-G317E with an ∼2-fold improved k(cat). By contrast to zymogen activation, the catalytic activities of both FIXa-G317R and FIXa-G317E enzymes toward the natural substrate, factor X, were dramatically (>4 orders of magnitude) impaired, but their apparent affinity for interaction with factor VIIIa was only slightly (<2-fold) decreased. Further studies revealed that the reactivity of FIXa-G317R and FIXa-G317E with antithrombin has been impaired 10- and 13-fold, respectively, in the absence and 166- and 500-fold, respectively, in the presence of pentasaccharide. As expected, the clotting activities of FIX variants could not be measured by the aPTT assay. These results implicate a critical role for Gly-317 in maintaining normal catalytic function for FIX/FIXa in the clotting cascade. The results further suggest that improved k(cat) of FIX-G317E activation in the extrinsic pathway together with dramatically impaired reactivity of FIXa-G317E with antithrombin may account for the less severe bleeding phenotype of a hemophilia B patient carrying the FIX-G317E mutation.