Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Underlying disorders of the coagulation system such as inhibitor deficiencies or decreased fibrinolysis are common in patients suffering from venous thrombosis. They may lead to the necessity of a lifelong prophylaxis. Prompt diagnosis is obviously to the patients benefit. We investigated 22 patients suffering from venous thromboses for the inhibitors antithrombin III (ATIII), protein C, and protein S during the first 8 to 12 days after admission to hospital and in addition after withdrawal from anticoagulant treatment after several months. At the day of admission ATIII and protein C levels were comparable to those several months later, but after 2 days they shifted downward or upward, respectively. Protein S did not shift during the period of hospitalisation, but was initially slightly lower than several months later. For inhibitors the day of admission to hospital is most suitable to take the samples. About 50% of the patients still had elevated activation markers (prothrombin fragments F1+2, thrombin-antithrombin complex TAT, and D-dimers) after several months.
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PMID:Parameters of haemostasis during acute venous thrombosis. 858 90

Factor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chromogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell's viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.
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PMID:Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the factor VR506Q mutation. 886 33

The effect of the Arg506 --> Gln mutation in factor VLEIDEN on thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pM factor VIIa.TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 microM thrombin. The factor VLEIDEN mutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nM TFPI, 65 nM protein C, and 10 nM recombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nM thrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDEN increased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nM, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDEN suggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI. Markedly increased thrombin generation in the presence of both 1.25 nM TFPI and factor VLEIDEN was also observed when antithrombin-III was added to the system to complete the natural set of coagulation inhibitors. Protein S (300 nM) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with either normal factor V or factor VLEIDEN. Protein S also failed to significantly potentiate the action of the protein C pathway in the presence of antithrombin-III in reactions employing normal factor V or factor VLEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in the reaction. Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis when combined with the Arg506 --> Gln mutation in factor VLEIDEN.
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PMID:Increased tissue factor-initiated prothrombin activation as a result of the Arg506 --> Gln mutation in factor VLEIDEN. 925 93

Protein S (PS), which functions as a species-specific anticoagulant cofactor to activated protein C (APC), is a mosaic protein that interacts with the phospholipid membrane via its gamma-carboxyglutamate-rich (Gla) module. This module is followed by the thrombin-sensitive region (TSR), sensitive to thrombin cleavage, four epidermal growth factor (EGF)-like modules and a last region referred to as the sex hormone binding globulin (SHBG) domain. Of these, the TSR and the first EGF-like regions have been shown to be important for the species-specific interaction with APC. Difficulties in crystallising PS have so far hindered its study at the atomic level. Here, we report theoretical models for the Gla and EGF-1 modules of human PS constructed using prothrombin and factor X experimental structures. The TSR was built interactively. Analysis of the model linked with the large body of biochemical literature on PS and related proteins leads to suggestions that (i) the TSR stabilises the calcium-loaded Gla module through hydrophobic and ionic interactions and its conformation depends on the presence of the Gla module; (ii) the TSR does not form a calcium binding site but is protected from thrombin cleavage in the calcium-loaded form owing to short secondary structure elements and close contact with the Gla module; (iii) the PS missense mutations in this region are consistent with the structural data, except in one case which needs further investigation; and (iv) the two PS 'faces' involving regions of residues Arg49-Gln52-Lys97 (TSR-EGF-1) and Thr103-Pro106 (EGF-1) may be involved in species-specific interactions with APC as they are richer in nonconservative substitution when comparing human and bovine protein S. This preliminary model helps to plan future experiments and the resulting data will be used to further validate and optimise the present structure.
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PMID:A theoretical model for the Gla-TSR-EGF-1 region of the anticoagulant cofactor protein S: from biostructural pathology to species-specific cofactor activity. 926 56

Danazol, a synthetic attenuated anabolic steroid, has been administered for 36 months to a 32 year old male with hereditary Protein S (PS) deficiency who had become non-compliant for warfarin therapy. The patient has an eleven year history of venous thrombosis. Since danazol therapy was initiated, the patient has not experienced a thrombotic event or adverse side-effects. Levels of PS, other inhibitors, fibrinolytic components, and markers for thrombin and platelet activation were measured prior and subsequent to therapy. Following danazol administration, marked and sustained increases were noted in Free Protein S, Antithrombin, and Protein C. Platelet CD62 (P-selectin) positivity which was elevated before therapy, decreased to assay threshold limits within five weeks. Both Prothrombin Fragment 1.2 and thrombin-antithrombin complexes were elevated post danazol therapy indicating continued clearance of generated thrombin. These data suggest that the protective effect provided by danazol in this patient with hereditary PS deficiency, may in large part be due to suppression of platelet activation by thrombin inhibition than simply through elevation of PS.
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PMID:Efficacy of danazol in a patient with congenital protein-S deficiency: paradoxical evidence for decreased platelet activation with increased thrombin generation. 930 21

The majority of fatal acute myocardial infarctions occur in the elderly. Since these events are predominantly thrombotic, we studied the cross-sectional associations of the anticoagulant proteins Antithrombin, Protein C, Protein S. and Tissue Factor Pathway Inhibitor (TFPI) in a subgroup (n = 400) of the Cardiovascular Health Study (a study of healthy men and women > or = 65 years) free of clinical cardiovascular disease (CVD). We did not observe any strong age-associated trends, although Protein C was lower in older women (p < or = 0.001), and TFPI was higher in older men (p < or = 0.01). The inhibitors were highly intercorrelated, and were associated with increased levels of inflammation-sensitive proteins (e.g., fibrinogen. plasminogen), lipids (especially total and LDL-cholesterol), and coagulation factors, such as Factors VIIc, IXc, and Xc. None was associated with the procoagulant markers Prothrombin Fragment F1-2 or Fibrinopeptide A. Only TFPI was associated with subclinical atherosclerosis: ankle-arm index and internal carotid artery stenosis, p trend < or = 0.01; and carotid wall thickness, p trend < or = 0.05. In multivariate analysis the independent predictors of TFPI were levels of fibrinogen; the fibrinolytic marker plasmin-antiplasmin complex; LDL-cholesterol; and carotid wall thickness (R2 for the model = 0.35). In summary, the inhibitors did not appear to increase with age, and were predominantly associated with inflammation markers and lipids. Since markers of thrombin production do increase with age, we hypothesize that an age-related hemostatic imbalance may ensue, with associated increased thrombotic risk. Only TFPI was associated with subclinical CVD, suggesting that it may more closely reflect endothelial damage.
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PMID:Correlates of antithrombin, protein C, protein S, and TFPI in a healthy elderly cohort. 968 99

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.
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PMID:Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling. 984 74

Coagulation factor V is composed of domains A1-A2-B-A3-C1-C2 and is activated by thrombin through proteolytic cleavage at Arg 709, Arg 1018 and Arg 1545. Upon thrombin activation, the B-domain is released and the active factor Va is formed by the heavy (A1-A2) and light chains (A3-C1-C2). Factor Va functions as an essential cofactor to factor Xa in the conversion of prothrombin to thrombin during coagulation. Recently it was shown that coagulation factor V, apart from being a precursor form to the procoagulant factor Va, also has anticoagulant properties, as it functions as a cofactor to activated protein C (APC). APC is a member of the anticoagulant pathway and downregulates the coagulation process through proteolytic inactivation of factors VIII/VIIIa and factors V/Va. In a factor VIIIa degradation assay, the APC-mediated inactivation of factor VIIIa is potentiated by the synergistic cofactors protein S and factor V. Protein S alone has little cofactor activity, whereas in the presence of factor V it is dramatically enhanced. This study provides insights into the molecular mechanisms that regulate the anticoagulant activity of factor V. Thrombin cleavage of factor V occurs in a sequential order. The thrombin cleavage site Arg 1545 is kinetically less favored than the other two sites, and cleavage at this site is the last to occur during thrombin activation of factor V As a consequence of this, different activation intermediates exist that express different levels of procoagulant activity. The anticoagulant activities of these intermediates have now been studied. It was found that factor V could be cleaved by thrombin at both Arg 709 and Arg 1018 and still work fully as a cofactor to APC, whereas cleavage at Arg 1545 completely abolished the anticoagulant activity of factor V. This suggests that the APC cofactor function of factor V depends on the B-domain remaining attached to the A3 domain. This study further shows that APC converts coagulation factor V into a member of the anticoagulant pathway by cleaving factor V in the A2 domain at Arg 506. By cleavage of factor V, APC not only produces an anticoagulant cofactor, but at the same time eliminates the pool of procoagulant factor V, since APC cleaved factor V will have no future as a cofactor in the coagulation. The unique way by which APC and thrombin, through proteolytic cleavage, can convert factor V into either an anticoagulant or a procoagulant adds to the intriguing mechanisms that balance the procoagulant and anticoagulant forces.
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PMID:Mechanisms that regulate the anticoagulant function of coagulation factor V. 1009 86

The independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (approximately 0.2 microM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd approximately 10(-8) M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and prothrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 x 10(8)/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.
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PMID:Regulation of prothrombinase activity by protein S. 1045 58

The anticoagulant factor protein S is a secreted vitamin K-dependent gamma-carboxylated protein that is mainly made in the liver. Protein S is homologous to the growth arrest specific protein, Gas6, the expression of which is up-regulated in cultured fibroblasts upon serum withdrawal. We report here the synthesis and secretion of protein S by cultured human vascular smooth muscle cells (HVSMCs). Western blot analysis revealed that similar amounts of protein S are secreted by both growing and growth-arrested HVSMCs. HVSMC-derived protein S was found to be gamma-carboxylated as it was precipitated by barium citrate and was shown to possess protein C cofactor activity. Treatment with the vitamin K antagonist warfarin led to the accumulation of intracellular undercarboxylated protein S forms that were rapidly secreted upon the reintroduction of vitamin K. Northern blotting analysis showed that cultured HVSMCs express a protein S transcript. The expression of protein S messenger RNA was unaffected by either warfarin, growth arrest, or various VSMC mitogens, such as platelet-derived growth factor-BB, basic fibroblast growth factor, transforming growth factor-beta, or hepatocyte growth factor. Thrombin, however, induced an up-regulation of protein S expression at both messenger RNA and protein levels. The evidence we provide for protein S secretion by cultured HVSMCs and its up-regulation by thrombin, together with earlier reports showing that protein S acts as a mitogen for these cells, suggests that, in addition to its known role in regulating blood clotting, protein S may also be an important autocrine factor in the pathophysiology of the vasculature. (Blood. 2000;95:2008-2014)
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PMID:The anticoagulant factor, protein S, is produced by cultured human vascular smooth muscle cells and its expression is up-regulated by thrombin. 1070 68


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