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)

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII. 762 Jan 60

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.
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PMID:Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma. 770 88

Protein S is a plasma factor essential for prevention of thrombosis, partly due to its activity as a cofactor for the plasma anticoagulant protease-activated protein C. To expand knowledge about structure-function relationships in homologous protein S molecules, studies of protein S from different species have been performed. Protein S anti-coagulant activity in human, monkey, bovine, and porcine plasma has been inactivated by purified human C4b binding protein (C4BP) with dose-dependence, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active. Purified porcine protein S has a 10-fold higher Kd for human C4BP than has human protein S. Protein S residues 420-434 provide an essential binding site for the negative regulator C4BP. cDNA sequences show that protein S residues 420-434 are highly conserved in all four species with the notable exception of Lys-429-Ile in porcine protein S. Differences between porcine and human protein S, e.g. Lys-429-Ile, Lys-43-Ala, Ser-197-Leu, Ser 199-Phe, Glu-463-Gly, Lys-571-Glu, Asn-602-Ile, Gln-607-Pro, may contribute to the decreased affinity of porcine protein S for human C4BP. Moreover, the species specificity of cofactor activities of various species of protein S is determined for human versus bovine-activated protein C, and these results, combined with sequence comparisons, agree with previous evidence that the thrombin-sensitive region and the first epidermal growth factor domain of protein S, i.e. residues 47-116, are responsible for recognition of activated protein C.
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PMID:Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C. 783 52

Increased frequency of thromboembolic events has been recently observed in patients with thalassemia major (TM), causing hypoxemia and cor pulmonale. Autopsy findings demonstrated "old" and recent pulmonary and renal infarcts as well as premature atherosclerosis. Studies to determine hypercoagulability showed: impaired platelet aggregation, increased circulating platelet aggregates, shortened platelet survival, enhanced excretion of urinary metabolites of thromboxane A2 (TXA2) and prostacyclin and decreased plasma levels of Protein C, Protein S or anti-thrombin III. Erythrocytes from TM patients enhanced thrombin formation in a "prothrombinase" assay (using a chromogenic substrate). Chronic anti-thrombotic therapy may be indicated in thalassemic patients to prevent the cardiac and pulmonary complications.
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PMID:A chronic hypercoagulable state and life-long platelet activation in beta thalassemia major. 788 16

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa. Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment. All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Construction and characterization of thrombin-resistant variants of recombinant human protein S. 790 76

Protein S (PS) and protein C (PC) anticoagulant activities and thrombin-antithrombin complex (TAT) were measured in 20 patients with AIS, 25 patients with chronic stable angina (CSA) and a control group (C). Although plasma levels of TAT were significantly elevated in patients with CSA (p < 0.01 vs C), they were much higher in patients with AIS (p < 0.001 vs CSA). PC anticoagulant activity was similar in patients and controls. At variance, PS anticoagulant activity was lower in patients with AIS than in those with CSA and controls (p < 0.05), reflecting differences in total PS and C4B-binding protein (C4B-BP) antigen possibly resulting from involvement in the mechanisms of inflammation, complement activation and acute-phase response. The ratios of anticoagulant PS and PC to procoagulant vitamin K-dependent factors IX and II were reduced in AIS patients (0.05 > p > 0.005 vs C). In addition, the ratios of anticoagulant PC and PS to factor IX were lower in patients with AIS than in those with CSA (p < 0.05). These results indicate that in patients with acute ischemic cardiac syndromes the markedly increased in vivo thrombin generation is associated with an unbalance between coagulant and anticoagulant vitamin K-dependent factors.
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PMID:Protein S and protein C anticoagulant activity in acute and chronic cardiac ischemic syndromes. Relationship to inflammation, complement activation and in vivo thrombin activity. 797 87

Protein S is a vitamin K-dependent non-enzymatic coagulation factor involved in the regulation of activated protein C (APC). In this paper we report an APC-independent anticoagulant function of protein S. We observed an inhibition of prothrombinase activity on endothelial cells and platelets which was half-maximal at physiological concentrations of free protein S in plasma. On endothelial cells, thrombin-cleaved protein S (PSt) as well as protein S in complex with C4b-binding protein (C4BP) inhibited prothrombinase activity to the same extent as protein S did. In solid-phase binding assays, direct binding of protein S and PSt to factor V and factor Va were observed. Protein S-C4BP complex did not bind to factor V or factor Va, implicating that the factor V(a) binding site on protein S is lost by the interaction with C4BP. A direct inhibition of factor Xa activity by protein S was also observed. Incubation of factor Xa with protein S revealed a noncompetitive inhibition of factor Xa by protein S with a Ki of (4.9 +/- 0.8) x 10(-7) M. Both protein S and protein S-C4BP complex were able to inhibit factor Xa to the same extent, whereas PSt had lost its inhibitory activity. This suggests that the conformational change induced by cleavage of the amino-terminal thrombin-sensitive loop results in a loss of a factor Xa binding site on protein S. The inhibitory effect of protein S involves interactions with both factor Va and factor Xa. The interaction of protein S with factor Xa is influenced by cleavage of the thrombin-sensitive loop, whereas C4BP blocks the interaction of protein S with factor Va. Inhibition of the prothrombinase complex by either forms of protein S might be an important mechanism in regulating thrombin generation in blood coagulation. The importance of the APC-independent anticoagulant action of protein S was emphasized by experiments in which the addition of protein S to normal plasma induced a prolongation of clotting time in a dilute activated partial thromboplastin time (dAPTT) assay. Furthermore, inhibition of endogenous protein S by the addition of monoclonal antibodies against protein S to normal plasma, induced a shortening of the clotting time in a dAPTT assay.
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PMID:Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factors Va and Xa. 806 24

Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investigated possible interaction of protein S with human factor Xa. Factor Xa, diisopropylphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of approximately 19 nM. In fluid phase, protein S bound to factor Xa with a Kd of approximately 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition of prothrombin conversion to thrombin by factor Xa was phospholipid-independent and was 1.6 times stimulated by Ca2+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at approximately 8 nM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mechanisms of anticoagulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.
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PMID:Protein S binds to and inhibits factor Xa. 814 82

Proteins C and S are vitamin K-dependent proteins with an essential anti-coagulant function. Protein C exists in an inactive form and is activated by a thrombin-thrombomodulin complex. Protein S combines with protein C and forms a stoichiometric complex which regulates coagulation in the presence of calcium. As patients with sickle cell disease (SCD) bear a high risk of developing thrombo-embolic disorders, we studied the coagulation derangement in 100 patients and 40 normal age- and sex-matched controls. The patients were clinically assessed and classified into sickle cell homozygotes (Hb SS), Hb S heterozygotes (Hb AS) and double heterozygotes for Hb S/beta 0-thalassaemia based on haematological parameters, red cell indices, Hb A2 and F levels and genetic studies. The proteins C and S were estimated and related to the type of the gene defect. The results showed significantly reduced levels of proteins C and S in SCD patients with the highest prevalence of deficiency in patients with a severe disease and frequent episodes of crisis. However, no significant differences were encountered in the level of proteins C and S in the same patients during the steady state and during episodes of crisis. It was concluded that the lower protein C and S levels in SCD is either due to decreased production or increased consumption though this reduction does not seem to play a role in producing thrombo-embolic disorders.
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PMID:Blood proteins C and S in sickle cell disease. 829 68

Since plasma protein S serves an anticoagulant function by mechanisms which are not completely understood, its possible interaction with Factor Va was investigated. Human protein S bound to immobilized human Factor Va in a calcium-dependent, saturable, and reversible manner and Factor Va bound similarly to immobilized protein S. Binding of protein S to immobilized Factor V was greatly enhanced by pretreatment of the surface-bound Factor V with increasing doses of thrombin up to 1 unit/ml. Binding of protein S to Factor Va was also demonstrated in fluid phase with a Kd of 33 +/- 9 nM. Biotin-labeled heavy chain of Factor Va bound to immobilized protein S, and this binding was reversed by a 17-fold molar excess of intact unlabeled Factor Va. Protein S competed efficiently with prothrombin for binding to immobilized Factor Va. The prothrombinase activity in a reaction mixture of purified clotting factors was inhibited by protein S and exhibited a pattern of mixed inhibition. The concentration of protein S needed for 50% inhibition of the prothrombinase activity of a mixture containing 1 nM Factor Xa, 20 pM Factor Va, and 50 microM phospholipids was about 16 nM. Since not all protein S preparations exhibited this degree of prothrombinase inhibitory activity, extensive control experiments were performed to verify that the inhibitory activity was associated with protein S during immunoaffinity chromatography and was not caused by traces of activated protein C in the protein S preparations. These data show that protein S has an anticoagulant function which is independent of activated protein C and, at least in part, that this is because of its competition with prothrombin for direct binding to Factor Va.
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PMID:Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. 842 62


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