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Query: UMLS:C0155339 (Brown)
 12,436 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Coagulation factor Xa is a plasma serine protease that catalyzes prothrombin to thrombin conversion, which, in turn, leads to the generation of the fibrin clot. Of the several parameters that govern the plasma level of factor Xa, control of its catabolism is of crucial importance. However, little is known regarding the mechanisms by which factor Xa is catabolized. In the present study we examine the cellular basis for the uptake and degradation of factor Xa. 125I-Factor Xa was degraded by hepatoma cells and embryonic fibroblasts via a process which required cell surface-bound tissue factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa. Uptake and degradation of cell surface-bound 125I-TFPI was also markedly stimulated in response to factor Xa binding. The intracellular kinetics of 125I-factor Xa and cell surface-bound 125I-TFPI display a strikingly similar pattern, suggesting that factor Xa and cell surface-bound TFPI are taken up as a bimolecular complex. Using cell lines either deficient in low density lipoprotein receptor-related protein, an endocytic receptor that mediates the degradation of uncomplexed TFPI (Warshawsky, I., Broze, G.J., Jr., and Schwartz, A.L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668), or deficient in tissue factor (TF), an integral membrane protein capable of forming quarternary complexes with factor Xa, TFPI, and factor VIIa, we demonstrated that the receptor that mediates the uptake and degradation of factor Xa-TFPI complex was neither low density lipoprotein receptor-related protein nor TF. As the vascular endothelial cell surface retains a substantial pool of TFPI (Sandset, P.M., Alildgaard, U., and Larsen, M.L. (1988) Thromb. Res. 50, 803-813; Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., and Broze, G.J., Jr. (1991) Blood 78, 387-393), our data suggest that endothelial cell surface TFPI may be actively involved in the clearance of factor Xa from the circulation via mediated uptake and degradation.
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PMID:Receptor-mediated endocytosis of coagulation factor Xa requires cell surface-bound tissue factor pathway inhibitor. 862 21

Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI-(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.
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PMID:Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor. 863 47

Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p<0.0001), Hoplocephalus stephensii (Stephen's-banded snake; p<0.0001) and Notechis scutatus (tiger snake; p=0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy.
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PMID:Endogenous thrombin potential as a novel method for the characterization of procoagulant snake venoms and the efficacy of antivenom. 2033 89

The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.
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PMID:Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis. 2413 80

Thrombin is generated from prothrombin through cleavage at two sites by the prothrombinase complex. Prothrombinase is composed of a protease, factor (f) Xa, and a cofactor, fVa, which interact on negatively charged phospholipid surfaces and cleave prothrombin into thrombin 300 000 times faster than fXa alone. The balance between bleeding and thrombosis depends on the amount of thrombin produced, and this in turn depends on the function of the prothrombinase complex. How fXa and fVa interact and how improved prothrombin processing is conferred are of critical importance for understanding healthy and pathological blood clotting. Until recently, little structural information was available, and molecular models were built on partial structures with assembly guided by biochemical data. Last year our group published a crystal structure of a prothrombinase complex from the venom of the Australian Eastern Brown snake (known as Pseutarin C). Here we use the crystal structure of Pseutarin C as a starting point for homology modelling and assembly of the full human prothrombinase complex. The interface is complementary in shape and charge, and is consistent with much of the published biochemical data. The model of human prothrombinase presented here provides a powerful resource for contextualizing previous data and for designing future experiments.
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PMID:Homology model of human prothrombinase based on the crystal structure of Pseutarin C. 2515 92