Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.
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PMID:Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII. 69 98

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.
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PMID:Improved procedures for the purification of selected vitamin K-dependent proteins. 78 72

Treatment of human platelets with purified bovine Factor VIII caused three types of aggregation: (a) primary agglutination; (b) secondary aggregation involving the platelet release reaction; and (c) super-aggregation, in which the platelets were gathered into only a few large clumps. Removal of calcium ions or treatment with p-hydroxymercuiriphenyl sulfonate blocked the release reaction, but not primary agglutination or super-aggregation. Platelets treated with formalin were not aggregated by ADP, thrombin, or collagen, but were agglutinated by bovine Factor VIII, although they did not show super-aggregation. For malin-treated platelets were agglutinated by phytohemagglutinin P less extensively and less rapidly than by bovine Factor VIII. Treatment of platelets and Factor VIII with neuraminidase released 60 and 53%, respectively, of the sialic acid residues without affecting the agglutination reaction or the procoagulant activity of the Factor VIII. Agglutination was inhibited by high salt concentrations, dextran sulfate, and heparin. During agglutination, both the procoagulant and platelet-agglutinating activities of Factor VIII became bound to the platelet surface.
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PMID:The interaction of bovine factor VIII with human platelets. 80

Factor VIII is present in plasma in a precursor or inactive form. When bovine factor VIII that has been purified approximately 10,000-fold is incubated with thrombin, an activated product is formed which participates in the conversion of factor X to factor Xa in the presence of factor IXa, calcium ions, and phospholipid. This activated product, which has been tentatively identified as activated factor XIII, was stable when formed in the presence of 0.25M CaCl2 but was rapidly inactivated in the absence of CaCl2. It was inhibited by diisopropyl phosphorofluoridate and antithrombin III, suggesting that it is a serine enzyme. The exact role of this serine enzyme in the intrinsic pathway of coagulation remains to be established.
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PMID:Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor) and thrombin. 87 60

Possible increased activation of the coagulation pathway was measured in a group of patients with neoplastic diseases. In addition to standard tests, the thromboplastin generation test, thrombin generation test and immunologic and coagulant activities of both Factor VIII and antithrombin III were utilized in the evaluation. The correlation between immuno-Factor VIII (VIII-Ag) and its clotting activity (VIII-C1) was good (r = 0.83). In contrast, this was not the situation for antithrombin III-Ag and its clotting activity. Thromboplastin generation was accelerated in 60% and thrombin generation was accelerated in 40% of the patients. Fibrinogen was elevated in half the cases: in most of these patients, thrombin times were slightly prolonged. These results indicate that some patients who have cancer have abnormal clotting patterns and are often in a potentially hypercoagulable state that is reflected by the thromboplastin generation test, thrombin generation test, and high levels of Factor VIII (both VIII-Ag and VIII-C1).
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PMID:Antithrombin III and Factor VIII in patients with neoplasms. 87

Factor VIII is a large glycoprotein which can be separated into a high molecular weight component which retains factor VIII-related antigens and supports ristocetin-induced platelet agglutination, and a low molecular weight component which has procoagulant activity. It has been suggested that this separation, observed in high ionic strength buffers, might be the consequence of proteolysis by plasma proteins. To consider this possibility, we have examined the interaction of the proteolytic enzyme thrombin with factor VIII. This study, carried out with highly purified materials, has used thrombin-mediated factor VIII proagulant activation as an indicator of this interaction. Several proteolytic inhibitors have been studied to determine their ability to inhibit factor VIII activation by thrombin. Under the current experimental conditions, diisopropylfluorophosphate (DFP) and hirudin inhibited the reaction, while heparin was an effective inhibitor only when plasma proteins were added. Benzamidine inhibited factor VIII activation when used at high concentrations, and phenylmethyl sulphonylfluoride and soybean trypsin inhibitor were found to be ineffective. These results show that DFP and hirudin prevent thrombin alteration of factor VIII and will be useful in purification and characterization studies of the factor VIII molecule.
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PMID:Thrombin activation of factor VIII: the effect of inhibitors. 88 21

When purified antihemophilic factor (Factor VIII) was rechromatographed on 4% agarose in 0.15 M NaCl or 1.0 M NaCl, a single protein peak, containing both procoagulant activity and von Willebrand factor activity, as defined by ristocetin-induced platelet aggregation, was eluted in the void volume. Purified Factor VIII immediately lost about 30% of its procoagulant activity when dissolved in 0.25 M CaCl2, and when rechromatographed on 4% agarose in 0.25 M CaCl2, the protein peak and von Willebrand factor activity remained coincident in the void volume; however, most of the remaining procoagulant activity was eluted after the void volume. The elution position of Factor VIII procoagulant activity from 4% agarose in 0.25 M CaCl2, and hence its apparent molecular weight, varied with the protein concentration applied to the column; at low protein concentrations it was eluted close to the inner volume. Yet on Sephadex G-200 in 0.25 M CaCl2, the protein and procoagulant activity were eluted together in the void volume. These observations suggested that the Factor VIII procoagulant activity was not eluting according to size or shape, but was adsorbing to some extent to the agarose. Isolated activity peak material from the 0.25 M CaCl2 columns contained protein and had a typical ultraviolet spectrum. Even at high concentrations, the protein contained no thrombin, Factors IX, X, or Xa activity, or detectable phospholipid. In addition to Factor VIII procoagulant activity, which could be inactivated by a human antibody to Factor VIII, the activity peak protein also contained von Willebrand factor activity. Like native Factor VIII and the void volume protein, the activity peak contained protein that did not enter a sodium dodecyl sulfate 5% polyacrylamide gel in the absence of reducing reagent. After reduction of disulfide bonds, several subunits ranging from 195,000 to 30,000 daltons were observed. These results indicate that the protein in the shifted Factor VIII procoagulant activity peak is large and that its anomalous elution pattern from 4% agarose in 0.25 M CaCl2 results from interaction with the agarose. The Factor VIII-like properties of the activity peak protein and its electrophoretic pattern on sodium dodecyl sulfate gels suggest that it is a species of Factor VIII modified by proteolytic cleavage. These results allow an interpretation that is different from the recently proposed "carrier protein-small active subunit" hypotheses for the structure-function relationships of the Factor VIII molecule.
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PMID:Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure. 108 90

Two groups of specifically presensitized Macaca speciosa monkeys received renal allografts via anastomosis to an indwelling arteriovenous (A-V) shunt. One group was pretreated with heparin (2 mg/kg) intravenously and the other was also treated with constant heparin infusion (1 mg/kg/hr) directly into the renal artery. These studies were performed to evaluate the effects of heparin within the kidney on total and compartmental blood flow, complement (C3) levels, sequestration of formed elements, and activation of the coagulation, fibrinolytic, and kinin-forming systems during the initial 3 hours of hyperacute rejection. The results are compared with those previously reported in unmodified control allografts. Heparin prolonged blood clotting time to infinity, markedly prolonged total renal venous blood flow, and normalized compartmental distribution in both groups despite antibody deposition similar to that in controls. With heparin pretreatment only, initial morphologic injury was much reduced but then progressed rapidly. Marked initial cortical cyanosis with mottling appeared to change constantly and was associated with fluctuations in renal turgor, total blood flow, and sequestration of formed elements, all of which suggested repetitive local cortical arterial spasm and incremental destruction of the grafts. Activation of coagulation Factors II and XII was also revealed and marked net Factor VIII activity was observed in the venous effluent. The latter reflects either formation and release of this factor by the injured kidney, or provides in vivo documentation of the "hyperactivation" of Factor VIII by thrombin known to occur in vitro. The addition of intrarenal artery heparin infusion resulted in greater improvement in early total blood flow rates and more uniformly progressive cyanosis and loss of turgor, but the diffuse initial morphologic injury suggested more uniform perfusion of injured areas. Intrarenal consumption of C3 and sequestration of formed elements was similar to that in controls. Paradoxically, prompt activation and consumption of all coagulation factors, plasminogen, and prekallikrein were observed, but formed fibrin was sparse. The exess amount of Factor XIIa present during heparin blockade may have been diverted to production of plasminogen activator and kallikrein formation. The enormous numbers of neutrophils observed within vessels of grafts which showed the greatest kallikrein activation provide the probable in vivo demonstration of the chemotactic properties of kallikrein noted by others in vitro. Heparin-induced platelet aggregation may have played an important role in the failure of these grafts. These studies elucidate the intrarenal effects of heparin during hyperacute rejection and again suggest that vasoconstriction is the most important early determinant of graft failure, as blood flow appeared unrelated to the degree of vascular injury and apparent obstruction. Also, heparin may exer a beneficial effect on blood flow by other than its known action on coagulation.
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PMID:Hyperacute renal allograft rejection in the primate. Intrarenal effects of heparin and associated net release of factor VIII activity and kallikrein activation. 109 75

A possible association between plasma coagulant activity and the presence of vascular complications in patients with diabetes mellitus was studied by measuring the generation of thrombin in plasma of 20 control subjects and 50 diabetic patients classified according to the presence or absence of microvascular complications. Thrombin production was determined in defibrinated plasma using a semi-automated technique with measurement of thrombin activity using chromogenic peptide S2238. Values determined were the lag time to appearance of thrombin activity and time taken to generate 50% maximal thrombin activity. Thrombin activity was related to concentrations of coagulant factor VIII activity and fibrinopeptide A and these were correlated with HbA1C levels. The median time to generate 50% maximal thrombin activity was not significantly reduced in diabetic patients compared with control subjects (53 vs 54 s, p = 0.076) and there were no significant differences between patients with and without microvascular complications. There were no differences in median fibrinopeptide A concentrations between the diabetic and control subjects (1.5 vs 2.2 nmol/l, p = 0.169). Time to 50% maximal thrombin activity correlated inversely with factor VIII:C concentrations in diabetic patients (r = -0.344, p = 0.015, n = 50) and both this and lag time correlated with factor VIII:C in diabetic patients and control subjects combined (r = -0.395, p less than 0.01; r = -0.327, p = 0.006, n = 70). Factor VIII:C concentrations increased with age of the subject and with HbA1C concentrations. The results failed to show enhancement of coagulation in contact-activated diabetic plasma compared with control plasma and suggest that a relationship between high levels of factor VIII:C in diabetes and the development of mcirovascular complications is unlikely to be mediated through procoagulant activity in plasma.
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PMID:Generation of thrombin activity in relation to factor VIII:C concentrations and vascular complications in type 1 (insulin-dependent) diabetes mellitus. 139 82

Triflavin, an Arg-Gly-Asp (RGD)-containing peptide, purified from snake venom of Trimeresurus flavoviridis, inhibits human platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex. In this report, we examined the effect of triflavin on tumor cells (human hepatoma J-5)-induced platelet aggregation (TCIPA) of heparinized platelet-rich plasma (PRP). ADP-scavenger agents, apyrase (10 U/ml) and creatine phosphate (5 mM)/creatine phosphokinase (5 U/ml) did not inhibit TCIPA while hirudin (5 U/ml) completely inhibited it. J-5 cells initially induced platelet aggregation, then blood coagulation occurred. J-5 cells concentration-dependently shortened the recalcification time of normal as well as Factor VIII, IX-deficient human plasmas, while it was inactive at shortening the recalcification time of Factor VII-deficient plasma, suggesting J-5 cells induced platelet aggregation through activation of extrinsic pathway, leading to thrombin formation as evidenced by the amidolytic activity on s-2238 by expressing tissue factor-like activity. Triflavin inhibited TCIPA in a dose-dependent manner (IC50, 0.02 microM). When compared on molar ratio, triflavin was approximately 30,000 times more potent than GRGDS (IC50, 0.58 mM). On the other hand, GRGES showed no significant effect on TCIPA, even its concentration was raised to 4 mM. Additionally, the monoclonal antibodies, raised against glycoprotein IIb/IIIa complex (i.e., 7E3 and 10 E5) inhibited J-5 TCIPA. In conclusion, we suggest the inhibitory effect of triflavin on J-5 TCIPA may be chiefly mediated by the binding of triflavin to the fibrinogen receptor associated with glycoprotein IIb/IIIa complex on platelet surface membrane.
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PMID:Triflavin, an Arg-Gly-Asp containing snake venom peptide, inhibits aggregation of human platelets induced by human hepatoma cell line. 151 27


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