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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 (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.

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.

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.

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. Objectives To 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.

Blood cell production is regulated by peripheral nerve fibers that innervate the bone marrow. However, little is known about the development or maintenance of hematopoietic innervation. Schwann cells (SCs) are the primary axon 'support cells' of the peripheral nervous system (PNS), and abnormal SC development is sufficient to impair peripheral nerve function. SCs are also the primary repair cell for the PNS which makes them an attractive therapeutic target for normalization of drug or malignancy-induced 'hematopoietic neuropathy'. We hypothesized that neural regulation of hematopoiesis is dependent on SC development. To test this hypothesis, we used the Myelin Protein Zero-Cre (MP0-Cre); Lamc1fl/fl mouse line in which laminin-γ1 expression is deleted from SC precursors and their progeny1. Early SC maturation is dependent on autocrine SC precursor-derived molecules such as laminin-γ1. SC differentiation arrests prior to axon sorting and ensheathment in MP0-Cre; Lamc1fl/fl mice, and causes a global peripheral neuropathy that persists throughout the lifetime of the animal. Preliminary hematopoietic analysis of 'steady state' MP0-Cre; Lamc1fl/fl and littermate control mice has shown the following: (1) MP0-Cre; Lamc1fl/fl bone marrow is innervated, and Cre-mediated gene recombination occurs in cells immunophenotypically consistent with SCs throughout the peripheral nervous system, including those in the bone marrow; (2) MP0-Cre; Lamc1fl/fl mice are lymphopenic but not neutropenic; (3) MP0-Cre; Lamc1fl/fl mice have significantly reduced spleen size and cellularity; and (4) MP0-Cre; Lamc1fl/fl bone marrow has an ~50% reduction in Lin-Sca-1+Kit+(LSK) cells (measured as a percentage of the Lin- compartment of the bone marrow). These results are consistent with earlier work by our groups in which we found that global Lamc1 gene deletion in adult mice induced peripheral blood lymphopenia, reduced spleen size, and a niche-dependent reduction of lymphoid progenitor and precursor cells that was secondary to increased lymphoid precursor cell apoptosis and reduced proliferation (UBC-CreERT2; Lamc1fl/fl mouse line). As with the SC-specific laminin-γ1 deficient mice, myelopoiesis was preserved in the UBC-CreERT2; Lamc1fl/fl mice. Based on results from MP0-Cre; Lamc1fl/fl and UBC-CreERT2; Lamc1fl/fl mice, we conclude that early lymphoid but not myeloid development requires laminin-γ1 expression by MP0-Cre-targetted niche cells, i.e. Schwann Cells. Our results are consistent with reports from other labs that hematopoietic sympathetic neuropathy promotes aberrant myeloid expansion at the expense of lymphopoiesis2. Going forward, we will determine whether lymphopoietic development is dependent on global versus laminin-specific SC-derived cues, and whether these signals are transmitted directly between SCs and lymphoid biased HSPCs or indirectly via other components of the hematopoietic niche. We anticipate that this line of investigation will provide molecular insights and pharmacologic targets for prevention and or normalization of the 'hematopoietic neuropathy' induced by diabetes, aging, neurotoxic chemotherapies and myeloid malignancies. REFERENCES: 1 Yu, W. M., Feltri, M. L., Wrabetz, L., Strickland, S. & Chen, Z. L. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci25, 4463-4472, doi:10.1523/JNEUROSCI.5032-04.2005 (2005). 2 Maryanovich, M. et al. Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche. Nat Med24, 782-791, doi:10.1038/s41591-018-0030-x (2018). Disclosures No relevant conflicts of interest to declare.

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.

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

HSCs lacking the guanosine nucleotide-binding protein Gimap5, which stabilizes expression of the Mcl-1 Bcl2 family protein, exhibit impaired survival and long-term repopulation capacity., 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.

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.

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.

Summary Background The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coagulation factor V (FV) has been speculated to reflect positive selection during evolution. Heterozygous Leiden carriers enrolled in the placebo arm of the PROWESS sepsis trial and heterozygous Leiden mice challenged with endotoxin both showed reduced mortality, whereas homozygous Leiden mice were not protected from lethal endotoxemia. Follow-up analyses of clinical outcomes and of mouse models of infection with various pathogens remained inconclusive. Objective To establish whether activated protein C resistance of FV Leiden modifies the outcome of bacterial infection in murine sepsis models. Methods Homozygous and heterozygous FV Leiden mice were subjected to gram-positive (S. aureus) or gram-negative (Y. pestis; E. coli) septic peritonitis or polymicrobial, focal septic peritonitis induced by cecal ligation and puncture. The effect of FV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared with the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 [protease activated receptor 4] deficiency, endothelial protein C receptor [ProcR/EPCR] deficiency). Results Heterozygous, but not homozygous, Leiden mice were protected from lethal infection with highly virulent S. aureus and Y. pestis strains. FV Leiden did not affect the outcome of sepsis induced by cecal ligation and puncture, staphylokinase-deficient S. aureus, Pla-deficient Y. pestis, or E. coli. Thrombocytopenia, deficiency of PAR1 or PAR4 did not affect S. aureus sepsis survival, whereas hemophilia A increased mortality. ProcR deficiency selectively abolished the survival advantage of heterozygous Leiden mice. Conclusions In mice, heterozygous FV Leiden carriers are protected from sepsis mortality after infection with clinically relevant human bacterial pathogens.

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.

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.

BACKGROUND AND APPROACH: Thrombomodulin (Thbd) is a multifunctional transmembrane molecule expressed on endothelial, hematopoietic, placental trophoblast, and various other cells. Thbd facilitates protein C and TAFI activation by thrombin, but also exerts thrombin- and protein C-independent functions to regulate complement activity and inflammatory signaling. Complete Thbd deficiency causes embryonic death through as yet unclear mechanisms, while ablation of the lectin-like Thbd domain, reduction of Thbd cofactor activity for protein C (pC) activation, or truncation of the cytoplasmic domain do not impair embryonic development1-3. Here we applied in vivo structure-function and genetic complementation analyses to differentiate between pC-dependent and -independent Thbd functions. RESULTS: Three mouse strains with various function-selective Thbd mutations were generated, i.e. ThbdPro/ΔCS, ThbdEGF4-6, and ThbdLT (Table 1). Of these, only the latter mutant with defective thrombin-binding reproduced the embryonic lethal phenotype of complete Thbd deficiency. To examine the developmental role of Thbd expression in cell types other than placental trophoblast, Thbd LoxP mice carrying a conditional Thbd allele were crossed with mice expressing Cre recombinase under control of the endogenous Meox2 gene promotor, resulting in selective ablation of Thbd in the embryo proper, but preservation in extra-embryonic placental tissue. This yielded a normal number of Thbd-deficient embryos at term pregnancy. Preservation of Thbd function in the embryonic placenta therefore was sufficient to prevent intrauterine lethality until birth. Immunohistochemistry of term embryos and adult mice (week 10-16) confirmed persistent absence of Thbd expression. Half of Thbd-null mice were lost prior to weaning (3 weeks). Surviving mice were smaller, yet exhibited normal weight gain thereafter. By 4 weeks of age, 19 of 28 Thbd-deficient animals developed grossly observable lesions, i.e. purple discoloration and swelling of tail segments, followed by, edematic swelling and discoloration of the tail and hind limb digits, distal tail necrosis and/or uni- or bilateral eye degeneration. More than half of the Thbd-null mice expired within the first 10 weeks of life, and only 2 of 28 mice survived to week 40. Neither gross anatomical inspection nor histological surveys of internal organs revealed evidence of severe thrombotic organ damage, and except for elevated IL-6, cytokine plasma levels were normal. Tamoxifen-induced Thbd LoxP gene ablation in adult mice (10-12 week old) reproduced the phenotype of Meox2Cre-Thbd-null mice. Genetic supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen variant (“hyperactivatable” protein C4), prevented perinatal lethality, partially restored normal birth weight, and largely prevented the phenotype of Thbd-null mice. However, Thbd-null females expressing the pC transgene exhibited pregnancy-induced morbidity and mortality with complete penetrance. When mated to wild type males (embryonic placenta expresses Thbd), 8 of 8 pregnancies ended in death or severe distress during gestation or postpartum. CONCLUSIONS: These findings indicate that the lack of protein C activation by Thbd-bound thrombin is the sole cause of lethality of embryonic and adult Thbd-null mice; and that Thbd expression in extra-embryonic placental tissue is sufficient to prevent intra-uterine lethality. The mortality of pregnant Thbd-null females expressing the protein C transgene constitutes a novel model for pregnancy-specific complications associated with defects in the function of natural anticoagulant pathways. REFERENCES: 1. Isermann B et al. Nat Med. 2003;9(3):331-337. 2. Conway EM et al. Blood. 1999;93(10):3442-3450. 3. Conway EM et al. J Exp Med. 2002;196(5):565-577. 4. Isermann B et al. Nat Med. 2007;13(11):1349-1358. Figure. Survival of Thbd-null mice with and without the hyperactivatable PC transgene (hmPC). Figure. Survival of Thbd-null mice with and without the hyperactivatable PC transgene (hmPC). Disclosures No relevant conflicts of interest to declare.

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis. METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined. RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations. CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. Disclosures No relevant conflicts of interest to declare.

Introduction: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two anticoagulant proteins directly bound to endothelium. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and early forms of prothrombinase. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. An in vitro model system of purified coagulation proteins has shown that TFPI and thrombomodulin act synergistically to quench tissue factor mediated thrombin generation via neutralization of prothrombinase activity. However, it is unclear how these two proteins cooperate within different vascular beds in vivo, particularly in the brain that has large amounts of tissue factor procoagulant activity. We have previously shown that mice with decreased thrombomodulin function (TMpro) do not have cerebral fibrin deposition even following LPS injection, while partial TFPI deficiency does induce intravascular fibrin deposition in the brain of the TMpro mice, suggesting that these two anticoagulant proteins have distinct functions within the brain vasculature. TFPI null mice die in utero with brain fibrin deposition. A hyperactivatable murine protein C (hMPC) can be produced by altering two acidic amino acids near the thrombin cleavage site. hMPC is activated 30-fold more efficiently by thrombin than wild type protein C and its activation does not require TM. We have used murine model systems to investigate how hMPC expression alters the (patho)physiology of mice with TFPI deficiency. Methods: Transgenic mice producing hMPC under control of the transthyretin promoter were produced. hMPC was bred into TFPI+/- mice, which were then characterized to define the anticoagulant activity of the transgene. Offspring from these initial matings were interbred to determine if hMPC expression would rescue the embryonic lethality of TFPI null mice. Results: hMPC expression elevated plasma protein C 2-fold and plasma APC 3-fold producing a potent anticoagulant effect. In thrombin generation assays, plasma from TFPI+/-/hMPC+ mice had peak thrombin generation of 44.9±7.5 nM vs. 63.0±3.5 nM in TFPI+/-/hMPC- mice (p Conclusions: Expression of the hMPC transgene produces a potent anticoagulant effect that partially rescues TFPI null embryonic lethality. Surviving TFPI-/-/hMPC+ pups are susceptible to death from severe hydrocephalus associated with hemorrhage and vascular abnormalities in the brain as they mature. These findings demonstrate that TFPI produces physiologically important anticoagulant activity within murine brain vasculature that is not fully compensated by over-expression of activated protein C. Disclosures Mast: Novo Nordisk: Honoraria, Research Funding.

BACKGROUND: Pharmacologic supplementation of protein C pathway function by infusion of recombinant Thbd or activated protein C supports recovery of hematopoietic function from lethal radiation injury in mice [Geiger et al., Nature Medicine, 2012]. Partial Thbd deficiency in hematopoietic stem and progenitor cells (HSPC) or bone marrow endothelium results in augmented sensitivity towards radiation injury [Geiger et al., Nature Medicine, 2012]. The underlying cellular and molecular mechanisms of Thbd function in hematopoiesis are not yet characterized. The objective of the current study was to determine the expression pattern and functional role of Thbd in HSPC. RESULTS: Flow cytometric analysis was employed to detect Thbd expression in defined subsets of murine HSPC. Thbd was co-expressed with the endothelial protein C receptor (Procr/EPCR) in the majority of bona fide stem cells with long-term-repopulating capacity (LT-HSC), and was also expressed on EPCR-negative stem cells with short-term repopulating capacity (ST-HSC), multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP). In contrast, only a subset of megakaryocyte-erythrocyte progenitors (MEP) expressed low levels of Thbd. In the bone marrow, Thbd was also expressed by B cells in early stages of maturation (from progenitor B cell stage till immature stage). In this lineage, the fraction of Thbd-positive cells was inversely correlated with the stage of B cell maturation. In addition, Thbd was detected in three distinct subsets of bone marrow-resident myeloid cells (CD11b+CD115+, CD11b+CD11c+ and CD11b-CD115+). Thbd expression outside the bone marrow was limited to a small fraction of hematopoietic cells (2-5% in the peripheral blood and spleen). These cells included myeloid cells (macrophage/monocyte and dendritic cells). Approximately 1-2% of all B cells in the peripheral blood and the spleen expressed Thbd, possibly reflecting recent bone marrow emigrants. Thbd expression was largely absent from splenic follicular and marginal zone B cells. Adult mice with complete, ubiquitous ablation of Thbd gene function (Meox2Cre-ThbdloxP -mice; "Thbd-null") were generated to analyze the functional role of Thbd in hematopoiesis. Thbd-null mice exhibited low birth weight, but only a mild prothrombotic diathesis, reflected in occasional peripheral vascular occlusion limited to the tail vein. Flow cytometric analyses revealed increased frequency of LT- and ST-HSC, a trend towards reduced CLP frequency, but normal relative abundance of MPP, CMP, GMP, and MEP. Thbd deficiency was also associated with a significant increase in the absolute number of LT-HSC and a reduction in the absolute number of CLP in the bone marrow. No such derangements were observed in mice lacking EPCR. In functional assays, bone marrow from wildtype and mutant mice yielded comparable numbers of CFU-GM. In contrast, the number of CFU-GM was increased in the spleen and peripheral blood of Thbd-null animals. The absolute number and frequency of all B cell precursors, as well as mature B cells in the bone marrow was reduced by ~50%. In the spleen, the absolute number of B cells was increased, whereas other hematopoietic populations in peripheral organs of Thbd-null mice were identical to that of wildtype controls. CONCLUSION: Thbd is expressed in the majority of hematopoietic progenitor cells in the bone marrow, including LT- and ST-HSC, and B cell precursors. Despite abundant Thbd expression in bone marrow resident cells and a modest prothrombotic phenotype, complete Thbd deficiency had only mild effects on steady-state hematopoiesis. Hematopoietic derangements were limited to the B cell compartment, and an Increased presence of CFU-GM in the spleen and peripheral blood of Thbd-null mice, possibly reflecting stimaulation of extramedullary hematopoiesis and/or altered bone marrow retention of precursors. Disclosures No relevant conflicts of interest to declare.

Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical role in placentation and the maintenance of pregnancy. Severe Protein C deficiency in the mother results in pregnancy failure in early gestation. Thrombomodulin (Thbd) or the Endothelial Protein C Receptor (EPCR/ProcR) gene deletions result in embryonic death, secondary to developmental and functional abnormalities of the placenta. These molecules play multiple roles in coagulation and inflammation. The mechanisms governing their role in placental development and maintenance of placental function remain to be fully understood. The objective of this work is to identify the critical functions of EPCR and Thbd that are required for placental development. Both Thbd and EPCR augment activated protein C generation, albeit to different extents. We have examined if reduced activated Protein C generation mediates placental abnormalities of EPCR- and Thbd-null mice. Activation of thrombin receptors expressed on platelets and trophoblast cells can also contribute to placental failure. We examined the role of thrombin receptor Par4 in placental failure of EPCR-null mice. Methods: To assess the role of a PC generation in placental phenotype of Thbd- and EPCR-null mice, we used a transgene to express a hyperactivatable form of murine protein C (hMPC) under the control of transthyretin promoter. Thrombin cleaves this mutant form of Protein C 30-fold more efficiently than wild type protein C, without requiring the cofactor function of thrombomodulin. Wild type mice expressing hMPC show 2-fold increase in PC and 3-fold increase in aPC levels. hMPC expression in PC-null mice restores their ability to carry pregnancies. Breeding strategies were used to generate hMPCtg ProcR+/- or hMPCtg Thbd+/- female mice. These were mated to ProcR+/- or Thbd+/- males, respectively, and survival of ProcR-/- and Thbd-/- embryos was analyzed. Similar genetic strategy was used to analyze the role of thrombin receptor Par4 in the demise of EPCR-null embryos. Placental phenotypes and embryonic survival was compared with experiments in which the mother was continuously infused with LMWH using a subcutaneous osmotic pump. Results: As previously reported, EPCR-null mice die before 10.5 days post coitum (dpc) (ProcR+/- intercrosses, out of 41 live embryos none were ProcR-/-, 10 were expected, 21 aborted not genotyped, 7 pregnancies analyzed at 11.5 dpc) and none are found at wean (out of 30 live pups none were ProcR-/-, 8 were expected, 5 litters analyzed). Transgenic expression of hMPC in the mother resulted in some live ProcR-/- embryos at 11.5 dpc (4 ProcR-/- out of 41 live embryos, 10 were expected, 15 aborted not genotyped, 7 pregnancies at 11.5 dpc) and pups at wean (2 ProcR-/- out of 28 live, 7 litters analyzed). Despite transgenic hMPC expression ProcR-/- embryos and pups were underrepresented (P=0.007, chi square GOF test). Surviving ProcR-/- embryos showed normal placental histology grossly comparable to littermate controls. Expression of hMPC in the mother did not ameliorate fetal death of Thbd-null mice (out of 38 live embryos none were Thbd-/-, 10 expected, 16 aborted not genotyped, 7 pregnancies at 9.5 dpc). Continuous infusion of LMWH also resulted in some live ProcR-/- embryos at 11.5 dpc (3 ProcR-/- out of 19 live embryos, 5 expected, 11 aborted not genotyped, 3 pregnancies analyzed), but two were growth retarded and all 3 placentae showed markedly reduced placental labyrinth formation. In contrast to transgenic expression of hMPC and treatment with LMWH, when Par4-/- ProcR+/- animals were intercrossed, ProcR-/- animals were born at an expected Mendelian frequency (7 ProcR-/- out of 35 live pups, 9 expected, 7 litters analyzed). Conclusions: Our results show that transgenic expression of hMPC allows normal placental development and rescues a fraction of EPCR-null embryos. Thus, placental defect of EPCR-null mice is in part mediated by reduced generation of aPC on placental cells. In contrast to the transgenic expression of hMPC and LMWH treatment, genetic absence of Par4 completely overcame the placental defect and allowed development of EPCR-null embryos. Further studies will clarify contributions of maternal versus fetal Par4 in this phenomenon. Disclosures No relevant conflicts of interest to declare.

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-γ 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.

Studies of mice with genetic deficits in specific coagulation factors have shown that many, but not all, components of the hemostatic system serve an essential role in mouse embryogenesis and pregnancy. Although the developmental failures observed in these mice are typically associated with severe hemorrhage, it is uncertain whether the role of coagulation factors in embryogenesis and reproduction is specifically tied to their function in thrombus formation and prevention of blood loss (i.e. hemostasis). Here, we show (i) that a complete loss of fibrinogen- and platelet-dependent hemostatic capacity does not reproduce the developmental defects occurring in mice with either deficits in thrombin generation or unfettered thrombin consumption; (ii) that the essential role of fibrinogen in the maintenance of pregnancy does not involve interaction with platelets; and (iii) that the previously described in utero growth retardation of gene-targeted mice lacking NF-E2, a transcription factor critical for megakaryopoieis, is not caused by a loss of platelet-dependent hemostatic function. In addition, we demonstrate (iv) that fibrinogen can confer physiologically relevant hemostatic function in the absence of platelets, but that a complete loss of hemostatic capacity results if a combined absence of these components is genetically imposed. These findings support the notion that the function of coagulation factors for in utero development and pregnancy is mechanistically distinct from their ability to mediate the formation of hemostatic platelet-fibrin(ogen) aggregates.

Background Fibrin deposition in cerebral vessels contributes to the pathogenesis of stroke and vascular dementia. Under quiescent conditions fibrin deposition is prevented or limited by natural anticoagulants of the endothelium. Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two major endothelial anticoagulants and act on different portions of the clotting cascade. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and FXa. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. Both proteins are reported to have low expression in the brain and their function in prevention of cerebral vascular fibrin deposition is unclear. This is emphasized by findings from murine models of decreased TFPI and TM (TMpro/pro) activity. TFPI null embryos have fibrin deposition in the brain and liver, while TMpro/pro mice have fibrin deposition in the lungs, heart, spleen and liver, but the brain is protected from fibrin deposition even following lethal LPS doses. Interestingly, TFPI heterozygosity combined with TMpro/pro results in cerebral vascular fibrin. Here, we report a detailed analysis of TFPI expression in the brain vasculature, the consequences of vascular TFPI deficiency in adult mice, and the capacity of APC to compensate for TFPI deficiency. Methods and Results Real time PCR studies demonstrated that TFPI expression in total brain is lower than in other tissues in comparison to the RPL-19 housekeeping gene. However, when the endothelial-specific genes, VE-cadherin or CD31 were used as housekeeping genes, brain TFPI expression was higher than that of lung endothelium (TFPIa: ∼9 fold higher; TFPIb: ∼5-fold higher). In situ hybridization studies revealed that TFPI and TM are expressed on endothelium throughout the brain parenchyma. In addition, TFPI was expressed within the granule layer of the cerebellum. TFPI null embryos succumb to embryonic lethality with necrotic lesions and fibrin deposition in the brain. To determine if increased TM-APC anticoagulant activity would rescue TFPI null embryos, a transgene over expressing human activated protein C (hHPC) was bred into heterozygous TFPI mice, which were then bred to determine if TFPI null mice expressing hHPC survive embryogenesis. However, the hHPC transgene did not rescue TFPI null embryos. Furthermore, TFPI null embryos had similar brain lesions and brain fibrin deposition regardless of the presence of the hHPC transgene. A hematopoietic cell transplant model system was used to examine the consequences of TFPI deficiency in the adult mouse brain. Mice transplanted with hematopoietic cells lacking TFPI had cerebral vascular fibrin deposition that was not observed in mice transplanted with hematopoietic cells producing normal amounts of TFPI. Interestingly, the fibrin was often found at sites of endothelial injury as demonstrated by co-localization with anti-C1qA, which binds to phosphatidylserine on damaged endothelium. Conclusions TFPI prevents necrotic brain lesions and cerebral fibrin deposition in developing mouse embryos and over expression of hHPC is not sufficient to compensate for the absence of TFPI. Adult mice lacking TFPI in hematopoietic cells have increased amounts of fibrin deposited at sites of cerebral vascular injury despite having normal endothelial TM. These data indicate that the distinct function of TFPI in the cerebral vasculature is not compensated by the anticoagulant activity of TM in regulating cerebral vascular thrombosis. Disclosures: Mast: Novo Nordisk: Honoraria, Research Funding.

Background Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor that dampens tissue factor (TF) initiated blood coagulation. It is produced by endothelial cells and megakaryocytes. Mice lacking TFPI activity die in utero from yolk sac hemorrhage or a presumptive consumptive coagulopathy. The embryonic lethal phenotype of TFPI null mice can be rescued by TF or factor VII deficiency. Our laboratory has shown that platelet TFPI limits platelet accumulation, but not fibrin formation, in an in vivo vascular injury model. Interestingly, mice lacking PAR4, the major thrombin receptor in mouse platelets, have decreased platelet accumulation but not fibrin formation following vascular injury. Therefore, we hypothesized that PAR-4 deficiency would counterbalance the absence of TFPI in platelets and rescue the embryonic lethal phenotype of TFPI null mice. Methods/Results TFPI heterozygous mice (TFPI+/-; C57/Bl6) were bred with PAR4 null mice (PAR4-/-; C57/Bl6) to generate TFPI+/-/PAR4-/- mice. These mice were bred to assess rescue of TFPI lethality. Of 76 pups closely monitored following birth, 12 (15.8%) were TFPI-/-/PAR4-/-, with 8 (10.5%) surviving to wean. TFPI-/-/PAR4-/- male mice were bred with TFPI+/-/PAR4-/- females for subsequent studies. Of 447 pups generated, 101 TFPI-/-/PAR4-/- mice (22.6%) were produced. Thus, TFPI-/-/PAR4-/- pups survive to wean at approximately one-half the expected frequency. TFPI-/-/PAR4-/- mice surviving to wean appeared healthy with no overt signs of thrombosis, and have apparently normal survival to over 12 months of age. Compared to TFPI+/-/PAR4-/- littermates, TFPI-/-/PAR4-/- have reduced body weight at 8 weeks. Female TFPI-/-/PAR4-/- mice can reproduce successfully. Approximately one-third of the TFPI-/-/PAR4-/- mice have short or totally absent tails. The short tail phenotype develops in utero and changes in tail:body proportion do not occur following birth. There was no difference in blood loss following tail clip between TFPI+/+/PAR4-/-, TFPI+/-/PAR4-/- or TFPI-/-/PAR4-/- mice. There also was no difference in fibrin or platelet accumulation between TFPI+/+/PAR4-/- or TFPI-/-/PAR4-/-mice following venous electrolytic injury. Conclusions This is the first reported rescue of the TFPI null embryonic lethal phenotype by a platelet activation defect. Almost one-half of the TFPI-/-/PAR4-/- mice survive through adulthood despite having normal tissue factor function not regulated by TFPI. This, and our inability, thus far, to identify a procoagulant state in the adult mice, suggests that an essential TFPI anticoagulant function is to modulate PAR-4 mediated platelet function; likely by limiting thrombin generation. TFPI may do this through inhibition of TF-fVIIa, or via inhibition of early forms of prothrombinase on the platelet surface, a very recently recognized inhibitory function of TFPI. The long term survival of these mice also demonstrates that unfettered TF-fVlla-fXa mediated signaling events through PAR-2 are not solely responsible for the embryonic lethality of TFPI null mice. However, the short or absent tails that arise during development of TFPI deficient mice suggests that TF and/or TFPI may have roles in notochord development that is separate from the coagulation system. Disclosures: Mast: Novo Nordisk: Honoraria, Research Funding.

The transmembrane thrombin receptor, thrombomodulin (TM) suppresses excessive activation of blood coagulation. Complete deficiency of TM results in developmental growth arrest and embryonic lethality in mice. These embryos are resorbed by 8.5 day of development, and do not show “head turning” characteristic of the next developmental stage. The mechanism underlying this phenomenon has remained enigmatic. Growth arrest of TM null embryos is mediated by lack of TM expression in fetal-origin cells of the placenta, but is not associated with increased fibrin clots. TM null embryos cannot be rescued by anticoagulation therapy of the mother with heparin or by absence of maternal and fetal fibrinogen. Lethality of TM null embryos is, however, prevented by genetic reduction of embryonic tissue factor expression, suggesting the involvement of coagulation activation in its pathophysiology. We investigated into the role of platelets in this developmental defect. Using appropriate genetic crosses we generated TM null embryos in NF-E2 −/− mice and examined their fate during gestation. Deficiency of NF-E2 has been previously shown to be associated with severe reduction in platelet counts. We show that TM null embryos are rescued from developmental growth arrest in NF-E2 null mothers. We next wanted to determine whether rescue is specific to the absence of maternal platelets or to the absence of the transcription factor NF-E2. We injected pregnant mice from TM +/− intercrosses with platelet depleting antibodies, during midgestation. Antibody induced depletion of maternal platelets also restored the development of TM null embryos. These results demonstrate that in the absence of TM expression in fetal origin cells of the placenta, maternal platelets disrupt placental function resulting in developmental growth arrest of the embryo. To determine if thrombin-mediated platelet activation is required for the deleterious effect of maternal platelets on TM null embryos, we examined the fate of TM null embryos in Protease Activated Receptor 4 (Par4) null mice. Par4 signaling is essential for the activation of mouse platelets by thrombin. Our results show that the absence of maternal Par4 does not rescue TM null embryos. However, anticoagulation therapy of the mother with enoxaparin, in combination with the genetic absence of maternal Par4, results in “turned” emobryos past the developmental block of untreated embryos. Enoxaparin treatment alone is not sufficient to rescue the embryos. These observations implicate maternal platelets, thrombin and the Par4 receptor in mediating the developmental growth arrest of TM null embryos. Toxic effects of platelet mediated excessive thrombin generation, or chemokine secretion, induced by activation of the platelet Par4 receptor are candidate mechanisms. We have substantiated our observations on the role of maternal platelets in pregnancy outcome in another mouse model of fetal loss that we have generated. In this model, combining maternal thrombophilia due to Factor V Leiden polymorphism with embryonic TM Pro mutation results in fetal loss. Depleting platelets in maternal peripheral blood by antibody treatment of pregnant mice rescues the embryos from midgestational lethality. Enoxaparin treatment, on the other hand, is ineffective. Our results highlight an important role of maternal platelets in determining pregnancy outcome. Understanding this role may open avenues for prevention and treatment of pregnancy disorders in prothrombotic mothers through attenuation of platelet function.