Hartmut Weiler - Academia.edu (original) (raw)

Papers by Hartmut Weiler

PLOS ONE, 2021

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure... more Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart...

Blood Advances, 2021

Activation of blood coagulation and endothelial inflammation are hallmarks of respiratory infecti... more 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. Mechan...

Blood, 2021

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that l... more 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, f...

Blood, 2005

Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory dise... more Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory disease associated with multi-organ failure. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects. The contribution of these distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. The aim of the study is to delineate the mechanism underlying the protective effect of APC in mouse endotoxemia. We first establish an experimental mouse model to demonstrate that recombinant murine APC reduces 6 day mortality of mice subjected to LPS-induced endotoxemia. APC treatment did not alter the extent of inflammatory cytokine release. Recombinant human APC did not exhibit therapeutic efficacy in this model. In contrast, recombinant human and mouse APC reduced to a similar extent experimentally induced arterial thrombus formation. The therapeutic efficacy of wild type recombinant murine APC was abolished in genetically engineered mice with reduced expre...

Blood, 2006

Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis a... more Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis and multi-organ failure. APC can exert both anticoagulant activity and direct cytoprotective effects on cells (anti-inflammatory, anti-apoptotic, endothelial barrier stabilization, etc.). The contribution of distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. Lethal mouse endotoxemia (i.p. LPS administration) and bacterial sepsis (i.p. Staphylococcus aureus) models were used to clarify mechanisms for APC’s beneficial mortality reduction effects and to distinguish the relative importance of APC anticoagulant effects vs. APC direct effects on cells. Murine rec wtAPC (APC) was administered as bolus plus i.v. infusion (over < 2 hr) in total doses ranging from 0.2 to 0.04 mg/kg and was given coincident with or at times up to 3 hr after challenge. Following induction of LPS-mediated septicemia in normal mice, APC markedly reduced mortality (eg., from 50...

Journal of Thrombosis and Haemostasis, 2019

BACKGROUND-Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated pro... more 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 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-HSC 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.

Blood, 2012

3360 The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most pre... more 3360 The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most prevalent genetic risk factor for venous thrombosis. We have now shown that heterozygous carriers, but not homozygous carriers, exhibit a robust survival advantage in murine models of lethal infection with gram-positive and gram-negative bacterial pathogens. FV Leiden augments the thrombin-mediated formation of activated protein C (aPC) and thereby enables the aPC-mediated inhibition of a specific component of the overall inflammatory response of myeloid immune cells. This specific, aPC-inhibited inflammatory response was mediated by the induction of tissue factor (TF) expression, assembly of the ternary TF-VIIa-Xa complex, and the EPCR-dependent activation of Protease Activated Receptor 2 (PAR2) by the ternary TF complex. The inhibition of inflammation-induced, PAR2-dependent gene expression by APC required factor V, protein S, and Protease Activated Receptor 3. This anti-inflammatory bioa...

Blood, 2013

Background Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhi... more 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...

Blood, 2015

Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical rol... more 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 e...

Blood, 2015

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels... more 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 o...

Blood, 2012

SCI-43 The phase 3 PROWESS clinical trial in 2001 resulted in approval of recombinant human activ... more SCI-43 The phase 3 PROWESS clinical trial in 2001 resulted in approval of recombinant human activated protein C (APC) using low-dose, 96 hour infusion therapy to reduce mortality for adult severe sepsis linked to bacterial infection (1). In that trial, APC reduced mortality from 30.8% to 24.7%, an absolute mortality reduction of 6.1% (19.4% relative risk reduction), but this therapy carried a risk of serious bleeding (4.0 vs. 1.5%, p=0.06). Trials of APC therapy for less than severe adult or pediatric sepsis failed to show benefit but confirmed increased risk for serious bleeding. Subsequently, 10 years after the PROWESS trial, the similarly designed large trial (PROWESS SHOCK) (2) failed to document therapeutic efficacy in adult severe sepsis, reporting mortalities of 26.4% vs. 24.2% and, curiously, no increased risk of serious bleeding (1.2 vs. 1.0%, p=0.81). Recombinant APC was thus withdrawn from the market in late 2011. A simple comparison of PROWESS to PROWESS-SHOCK is confoun...

Research and practice in thrombosis and haemostasis, 2018

This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel... more This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advance...

Biology of Reproduction, 2007

Blood advances, Jan 13, 2016

Platelets are a rich source of many cytokines and chemokines including transforming growth factor... more Platelets are a rich source of many cytokines and chemokines including transforming growth factor β 1 (TGF-β1). TGF-β1 is required to convert conventional CD4(+) T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. Whether platelet contents will affect Treg cell properties has not been explored. In this study, we show that unfractionated platelet lysates (pltLys) containing TGF-β1 efficiently induced Foxp3 expression in Tconv cells. The common Treg cell surface phenotype and in vitro suppressive activity of unfractionated pltLys-iTreg cells were similar to those of iTreg cells generated using purified TGF-β1 (purTGFβ-iTreg) cells. However, there were substantial differences in gene expression between pltLys-iTreg and purTGFβ-iTreg cells, especially in granzyme B, interferon γ, and interleukin-2 (a 30.99-, 29.18-, and 17.94-fold difference, respectively) as determined by gene microarray analysis. In line with these gene signatures, we f...

Blood, Jan 2, 2015

Infection and inflammation are invariably associated with activation of the blood coagulation mec... more 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 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 mRNAs 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, ...

Hämostaseologie, 2011

The central effector protease of the protein C pathway, activated protein C (APC), interacts with... more The central effector protease of the protein C pathway, activated protein C (APC), interacts with the endothelial cell protein C receptor, with protease activated receptors (PAR), the apolipoprotein E2 receptor, and integrins to exert multiple effects on haemostasis and immune cell function. Such receptor interactions modify the activation of PC and determine the biological response to endogenous and therapeutically administered APC. This review summarizes the current knowledge about interactions of APC with cell surface-associated receptors, novel substrates such as histones and tissue factor pathway inhibitor, and their implications for the biologic function of APC in the control of coagulation and inflammation.

Journal of Thrombosis and Haemostasis, 2015

Background-The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coa... more 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 aPC-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 (CLP); and the effect of fV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared to the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 deficiency, 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 CLP, 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.

PLOS ONE, 2021

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure... more Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart...

Blood Advances, 2021

Activation of blood coagulation and endothelial inflammation are hallmarks of respiratory infecti... more 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. Mechan...

Blood, 2021

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that l... more 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, f...

Blood, 2005

Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory dise... more Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory disease associated with multi-organ failure. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects. The contribution of these distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. The aim of the study is to delineate the mechanism underlying the protective effect of APC in mouse endotoxemia. We first establish an experimental mouse model to demonstrate that recombinant murine APC reduces 6 day mortality of mice subjected to LPS-induced endotoxemia. APC treatment did not alter the extent of inflammatory cytokine release. Recombinant human APC did not exhibit therapeutic efficacy in this model. In contrast, recombinant human and mouse APC reduced to a similar extent experimentally induced arterial thrombus formation. The therapeutic efficacy of wild type recombinant murine APC was abolished in genetically engineered mice with reduced expre...

Blood, 2006

Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis a... more Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis and multi-organ failure. APC can exert both anticoagulant activity and direct cytoprotective effects on cells (anti-inflammatory, anti-apoptotic, endothelial barrier stabilization, etc.). The contribution of distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. Lethal mouse endotoxemia (i.p. LPS administration) and bacterial sepsis (i.p. Staphylococcus aureus) models were used to clarify mechanisms for APC’s beneficial mortality reduction effects and to distinguish the relative importance of APC anticoagulant effects vs. APC direct effects on cells. Murine rec wtAPC (APC) was administered as bolus plus i.v. infusion (over < 2 hr) in total doses ranging from 0.2 to 0.04 mg/kg and was given coincident with or at times up to 3 hr after challenge. Following induction of LPS-mediated septicemia in normal mice, APC markedly reduced mortality (eg., from 50...

Journal of Thrombosis and Haemostasis, 2019

BACKGROUND-Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated pro... more 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 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-HSC 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.

Blood, 2012

3360 The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most pre... more 3360 The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most prevalent genetic risk factor for venous thrombosis. We have now shown that heterozygous carriers, but not homozygous carriers, exhibit a robust survival advantage in murine models of lethal infection with gram-positive and gram-negative bacterial pathogens. FV Leiden augments the thrombin-mediated formation of activated protein C (aPC) and thereby enables the aPC-mediated inhibition of a specific component of the overall inflammatory response of myeloid immune cells. This specific, aPC-inhibited inflammatory response was mediated by the induction of tissue factor (TF) expression, assembly of the ternary TF-VIIa-Xa complex, and the EPCR-dependent activation of Protease Activated Receptor 2 (PAR2) by the ternary TF complex. The inhibition of inflammation-induced, PAR2-dependent gene expression by APC required factor V, protein S, and Protease Activated Receptor 3. This anti-inflammatory bioa...

Blood, 2013

Background Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhi... more 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...

Blood, 2015

Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical rol... more 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 e...

Blood, 2015

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels... more 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 o...

Blood, 2012

SCI-43 The phase 3 PROWESS clinical trial in 2001 resulted in approval of recombinant human activ... more SCI-43 The phase 3 PROWESS clinical trial in 2001 resulted in approval of recombinant human activated protein C (APC) using low-dose, 96 hour infusion therapy to reduce mortality for adult severe sepsis linked to bacterial infection (1). In that trial, APC reduced mortality from 30.8% to 24.7%, an absolute mortality reduction of 6.1% (19.4% relative risk reduction), but this therapy carried a risk of serious bleeding (4.0 vs. 1.5%, p=0.06). Trials of APC therapy for less than severe adult or pediatric sepsis failed to show benefit but confirmed increased risk for serious bleeding. Subsequently, 10 years after the PROWESS trial, the similarly designed large trial (PROWESS SHOCK) (2) failed to document therapeutic efficacy in adult severe sepsis, reporting mortalities of 26.4% vs. 24.2% and, curiously, no increased risk of serious bleeding (1.2 vs. 1.0%, p=0.81). Recombinant APC was thus withdrawn from the market in late 2011. A simple comparison of PROWESS to PROWESS-SHOCK is confoun...

Research and practice in thrombosis and haemostasis, 2018

This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel... more This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advance...

Biology of Reproduction, 2007

Blood advances, Jan 13, 2016

Platelets are a rich source of many cytokines and chemokines including transforming growth factor... more Platelets are a rich source of many cytokines and chemokines including transforming growth factor β 1 (TGF-β1). TGF-β1 is required to convert conventional CD4(+) T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. Whether platelet contents will affect Treg cell properties has not been explored. In this study, we show that unfractionated platelet lysates (pltLys) containing TGF-β1 efficiently induced Foxp3 expression in Tconv cells. The common Treg cell surface phenotype and in vitro suppressive activity of unfractionated pltLys-iTreg cells were similar to those of iTreg cells generated using purified TGF-β1 (purTGFβ-iTreg) cells. However, there were substantial differences in gene expression between pltLys-iTreg and purTGFβ-iTreg cells, especially in granzyme B, interferon γ, and interleukin-2 (a 30.99-, 29.18-, and 17.94-fold difference, respectively) as determined by gene microarray analysis. In line with these gene signatures, we f...

Blood, Jan 2, 2015

Infection and inflammation are invariably associated with activation of the blood coagulation mec... more 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 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 mRNAs 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, ...

Hämostaseologie, 2011

The central effector protease of the protein C pathway, activated protein C (APC), interacts with... more The central effector protease of the protein C pathway, activated protein C (APC), interacts with the endothelial cell protein C receptor, with protease activated receptors (PAR), the apolipoprotein E2 receptor, and integrins to exert multiple effects on haemostasis and immune cell function. Such receptor interactions modify the activation of PC and determine the biological response to endogenous and therapeutically administered APC. This review summarizes the current knowledge about interactions of APC with cell surface-associated receptors, novel substrates such as histones and tissue factor pathway inhibitor, and their implications for the biologic function of APC in the control of coagulation and inflammation.

Journal of Thrombosis and Haemostasis, 2015

Background-The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coa... more 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 aPC-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 (CLP); and the effect of fV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared to the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 deficiency, 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 CLP, 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.