Blood coagulation is initiated when tissue factor binds to coagulation factor VIIa to give an enzymatically active complex which then activates factors IX and X, leading to thrombin generation and clot formation. We have determined the crystal structure at 2.0-A degrees resolution of active-site-inhibited factor VIIa complexed with the cleaved extracellular domain of tissue factor. In the complex, factor VIIa adopts an extended conformation. This structure provides a basis for understanding many molecular aspects of the initiation of coagulation.

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH 2 ) was determined to be 266 ؎ 8, 320 ؎ 22, and 467 ؎ 47 nM for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGFdependent signaling requires proteolysis from an inactive single-chain precursor into an active a/b-heterodimer. We show that the serine protease-like HGF b-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed b-propeller, where the bottom face of blades 2 and 3 binds to the HGF b-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF b binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF bchains brings two HGF b:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibodyinduced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury IntroductionThrombosis and inflammation are linked in many clinical conditions. 1 Tissue factor (TF), the major cellular initiator of the coagulation protease cascade, plays important roles in both thrombosis and inflammation. 2 The coagulation cascade is initiated by the complex of TF and factor VIIa (FVIIa). The TF:FVIIa complex activates its substrates factor X and factor IX by limited proteolysis. Activated FX (FXa) then converts prothrombin to thrombin, which cleaves fibrinogen and activates platelets leading to the formation of a hemostatic plug. TF also contributes to inflammation. TF complexes (TF:FVIIa and TF:FVIIa:FXa) induce the expression of TNF-␣, interleukins, and adhesion molecules by cleaving protease activated receptors (PARs). [3][4][5] Monocytes from patients with antiphospholipid (aPL) antibodies express TF and in vitro experiments showed that monocytes incubated with aPL antibodies express TF. 6,7 A variety of inflammatory stimuli, including mitogens, bacterial cell products, components of the complement system, and cytokines, is known to induce the expression of TF on the surface of endothelial cells, monocytes, and neutrophils. 8,9 TF expression on these cells is a characteristic feature of acute and chronic inflammation in conditions such as sepsis, atherosclerosis, Crohn disease, systemic lupus erythematosus, and transplant rejection reactions. 10-14 TF on monocytes and synovial cells promotes leukocyte adhesion and transendothelial migration, potentiating inflammation in joints, 15 while decreased TF activity abrogates the systemic expression of inflammatory mediators in several animal models. 16,17 The antiphospholipid syndrome (APS) is considered a thrombophilic disorder. However, animal studies from our laboratory have shown the importance of inflammation in the pathogenesis of aPL-induced pregnancy loss, a common complication in APS. 18,19 Using a mouse model of APS, we demonstrated that complement activation, through the action of anaphylotoxin C5a, promotes neutrop...

Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gramnegative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3-induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1 -/-mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1 +/+ mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1 -/-mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1 +/+ mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.

Summary We report an unexpected role for protease signaling in neural tube closure and formation of the central nervous system. Mouse embryos lacking protease-activated receptor 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, Par2 selectively along the line of closure. Ablation of Gi/z and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often co-expressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

IntroductionThe link between cancer and venous thromboembolism (VTE) is referred to as Trousseau syndrome. Interestingly, different cancer types are associated with different rates of VTE, with pancreatic cancer having one of the highest rates. 1,2 A VTE risk-scoring model has been developed that stratifies ambulatory cancer patients undergoing chemotherapy into 3 VTE risk categories based on 5 parameters: (1) the site of the primary tumor, (2) prechemotherapy leukocyte count, (3) platelet count, (4) hemoglobin level, and (5) body mass index. 3 Recently, this model was expanded to include the biomarkers D-dimer and soluble P-selectin. 4 Another potential circulating biomarker of VTE risk in pancreatic cancer patients is microparticle (MP) tissue factor (TF). [5][6][7][8][9] Full-length TF (flTF) is a transmembrane protein that activates the coagulation cascade. 10 In addition, an alternatively spliced form of TF (asTF) has been identified that lacks a membrane anchor and therefore can be released as a soluble protein. 11 Increased TF expression is correlated with poor prognosis in pancreatic cancer. [12][13][14] Cultured human pancreatic tumor lines express variable levels of both flTF and asTF and release TF-positive MPs containing flTF into the culture medium. [14][15][16][17][18][19] In some patients with pancreatic cancer, high levels of TF-positive MPs are found in the circulation and, in a small pilot study, were predictive of VTE. [5][6][7]9,20 In a mouse model of human colorectal tumors, human TF protein is released into the circulation. 21 In nude mice bearing orthotopic human pancreatic tumors (L3.6pl) plasma levels of human TF protein were correlated with the levels of thrombin-antithrombin (TAT) complex, a marker of the activation of coagulation. 22 Further, plasma from these tumor-bearing mice was found to enhance thrombin generation in vitro in a human TF-dependent manner. 22 Another study found that human (SOJ-4) and mouse (PANC02) pancreatic cell lines expressed TF, and the investigators observed an accumulation of tumor-derived MPs at the site of thrombosis and increased thrombosis in a microvascular model. 18 The objective of the present study was to determine the role of tumor-derived TF in the activation of coagulation and thrombosis in a xenograft mouse model of human pancreatic tumors. We found that only TF-positive tumors activated coagulation and that this activation was abolished by inhibition of human TF. Two TF-positive pancreatic tumor cell lines activated coagulation, but only one had detectable levels of circulating TF-positive MPs, which suggested that activation of coagulation was due to TF expression by the tumor itself rather than to TF on the MPs. Mice with elevated levels of TF-positive MPs exhibited increased thrombosis in a saphenous vein model, but not in an inferior vena cava (IVC) stenosis model. Methods Cell linesHuman pancreatic (MIAPaCa-2 [CRL-1420] Abs and proteinsPE-labeled mouse IgG control (#555574), mouse anti-human TF (#550312) and FITC-conjugated anti-human MUC...