A thrombin-PAR1/2 feedback loop amplifies thromboinflammatory endothelial responses to the viral RNA analogue poly(I:C).

Activation of blood coagulation and endothelial inflammation are hallmarks of respiratory infections with RNA viruses that contribute significantly to the morbidity and mortality of patients with severe disease. We investigated how signaling by coagulation proteases affects the quality and extent of the response to the TLR3-ligand poly(I:C) in human endothelial cells. Genome-wide RNA profiling documented additive and synergistic effects of thrombin and poly(I:C) on the expression level of many genes. The most significantly active genes exhibiting synergistic induction by costimulation with thrombin and poly(I:C) included the key mediators of 2 critical biological mechanisms known to promote endothelial thromboinflammatory functions: the initiation of blood coagulation by tissue factor and the control of leukocyte trafficking by the endothelial-leukocyte adhesion receptors E-selectin (gene symbol, SELE) and VCAM1, and the cytokines and chemokines CXCL8, IL-6, CXCL2, and CCL20. Mechanistic studies have indicated that synergistic costimulation with thrombin and poly(I:C) requires proteolytic activation of protease-activated receptor 1 (PAR1) by thrombin and transactivation of PAR2 by the PAR1-tethered ligand. Accordingly, a small-molecule PAR2 inhibitor suppressed poly(I:C)/thrombin-induced leukocyte-endothelial adhesion, cytokine production, and endothelial tissue factor expression. In summary, this study describes a positive feedback mechanism by which thrombin sustains and amplifies the prothrombotic and proinflammatory function of endothelial cells exposed to the viral RNA analogue, poly(I:C) via activation of PAR1/2.

Tissue factor pathway inhibitor is required for cerebrovascular development in mice.

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi-/- and Tfpi-/-F5-/- mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi-/- embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi-/- embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9.

BACKGROUND: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species-specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF-/-) rat by excision of all 52 exons of the VWF locus. METHODS: The entire VWF gene was eliminated in Sprague-Dawley (Crl:SD) rats via CRISPR/Cas9-mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF-/- rats were infused with FVIII-/- rat platelet poor plasma (PPP) to determine response of plasma FVIII. RESULTS: Breeding of VWF ± rats yielded VWF-/- offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF-/- rats. VWF-/- rats bled longer and more than VWF+/- and VWF+/+ rats when challenged. Transfusion of FVIII-deficient platelet-poor plasma induced a rapid rise in endogenous FVIII:C in VWF-/- rats. CONCLUSION: This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Role of thrombomodulin expression on hematopoietic stem cells.

BACKGROUND: Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated protein C (aPC) differentially regulate the quiescence and bone marrow (BM) retention of hematopoietic stem cells (HSC). Murine HSC co-express THBD, PAR1, and endothelial protein C receptor (EPCR), suggesting that HSC sustain quiescence in a quasi-cell autonomous manner due to the binding of thrombin present in the microenvironment to THBD, activation of EPCR-bound protein C by the thrombin-THBD-complex, and subsequent activation of PAR1 by the aPC-EPCR complex. OBJECTIVE: To determine the role of THBD expression on HSC for sustaining stem cell quiescence and BM retention under homeostatic conditions. METHODS: Hematopoietic stem cell function was analyzed in mice with constitutive or temporally controlled complete THBD-deficiency by flow cytometry, functional assays, and single cell RNA profiling. RESULTS: THBD was expressed in mouse, but not human, HSC, progenitors, and immature B cells. Expression in vascular endothelium was conserved in humans' BM. Mice with constitutive THBD deficiency had a normal peripheral blood profile, altered BM morphology, reduced numbers of progenitors and immature B cells, pronounced extramedullary hematopoiesis, increased HSC frequency, and marginally altered transcriptionally defined HSC stemness. Transplantation experiments indicated near normal engraftment and repopulating ability of THBD-deficient HSC. Transgenic aPC supplementation normalized BM histopathology and HSC abundance, and partially restored transcriptional stemness, but had no effect on B cell progenitors and extramedullary hematopoiesis. Temporally controlled THBD gene ablation in adult mice did not cause the above abnormalities. CONCLUSION: THBD expression on HSPC has minor effects on homeostatic hematopoiesis in mice, and is not conserved in humans.

The Adult Murine Intestine is Dependent on Constitutive Laminin-γ1 Synthesis.

Laminin-γ1 is required for early embryonic development; however, the need for laminin-γ1 synthesis in adulthood is unknown. A global and inducible mouse model of laminin-γ1 deficiency was generated to address this question. Genetic ablation of the Lamc1 gene in adult mice was rapidly lethal. Despite global Lamc1 gene deletion in tamoxifen-induced mutant mice, there was minimal change in total cardiac, pulmonary, hepatic or renal laminin protein. In contrast, laminin-γ1 was significantly depleted in the small intestines, which showed crypt hyperplasia and dissociation of villous epithelium from adjacent mesenchyme. We conclude that the physiologic requirement for laminin-γ1 synthesis in adult mice is dependent on a tissue-specific basal rate of laminin-γ1 turnover that results in rapid depletion of laminin-γ1 in the intestine.

Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation.

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd-/-) mice to term. Half of the Thbd-/- offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd-/- animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd-/- mice. However, Thbd-/- females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy.

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.

A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development.

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.

EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice.

Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow-derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

Critical role for Gimap5 in the survival of mouse hematopoietic stem and progenitor cells.

Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1-which is essential for HSC survival-and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.

Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice.

Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor-1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1- dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-gamma production by natural killer-like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.

Maternal Par4 and platelets contribute to defective placenta formation in mouse embryos lacking thrombomodulin.

Absence of the blood coagulation inhibitor thrombomodulin (Thbd) from trophoblast cells of the mouse placenta causes a fatal arrest of placental morphogenesis. The pathogenesis of placental failure requires tissue factor, yet is not associated with increased thrombosis and persists in the absence of fibrinogen. Here, we examine the role of alternative targets of coagulation that might contribute to the placental failure and death of Thbd(-/-) embryos. We demonstrate that genetic deficiency of the protease-activated receptors, Par1 or Par2, in the embryo and trophoblast cells does not prevent the death of Thbd(-/-) embryos. Similarly, genetic ablation of the complement pathway or of maternal immune cell function does not decrease fetal loss. In contrast, Par4 deficiency of the mother, or the absence of maternal platelets, restores normal development in one-third of Thbd-null embryos. This finding generates new evidence implicating increased procoagulant activity and thrombin generation in the demise of thrombomodulin-null embryos, and suggests that platelets play a more prominent role in placental malfunction associated with the absence of thrombomodulin than fibrin formation. Our findings demonstrate that fetal prothrombotic mutations can cause localized activation of maternal platelets at the feto-maternal interface in a mother with normal hemostatic function.

Fetal gene defects precipitate platelet-mediated pregnancy failure in factor V Leiden mothers.

We describe a mouse model of fetal loss in factor V Leiden (FvL) mothers in which fetal loss is triggered when the maternal prothrombotic state coincides with fetal gene defects that reduce activation of the protein C anticoagulant pathway within the placenta. Fetal loss is caused by disruption of placental morphogenesis at the stage of labyrinth layer formation and occurs in the absence of overt placental thrombosis, infarction, or perfusion defects. Platelet depletion or elimination of protease-activated receptor 4 (Par4) from the mother allows normal placentation and prevents fetal loss. These findings establish a cause-effect relationship for the observed epidemiologic association between maternal FvL status and fetal loss and identify fetal gene defects as risk modifiers of pregnancy failure in prothrombotic mothers. Pregnancy failure is mediated by Par4-dependent activation of maternal platelets at the fetomaternal interface and likely involves a pathogenic pathway independent of occlusive thrombosis. Our results further demonstrate that the interaction of two given thrombosis risk factors produces markedly disparate consequences on disease manifestation (i.e., thrombosis or pregnancy loss), depending on the vascular bed in which this interaction occurs.

Cause-effect relation between hyperfibrinogenemia and vascular disease.

Elevated plasma levels of fibrinogen are associated with the presence of cardiovascular disease, but it is controversial whether elevated fibrinogen causally imparts an increased risk, and as such is a true modifier of cardiovascular disease, or is merely associated with disease. By investigating a transgenic mouse model of hyperfibrinogenemia, we show that elevated plasma fibrinogen concentration (1) elicits augmented fibrin deposition in specific organs, (2) interacts with an independent modifier of hemostatic activity to regulate fibrin turnover/deposition, (3) exacerbates neointimal hyperplasia in an experimental model of stasis-induced vascular remodeling, yet (4) may suppress thrombin generation in response to a procoagulant challenge. These findings provide direct experimental evidence that hyperfibrinogenemia is more than a by-product of cardiovascular disease and may function independently or interactively to modulate the severity and/or progression of vascular disease.

The thrombomodulin-protein C system is essential for the maintenance of pregnancy.

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.