Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions. To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276 could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.
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PMID:Molecular recognition sites on factor Xa which participate in the prothrombinase complex. 160 96

The inactivation of factor Va by the natural anticoagulant, activated protein C (APC) is subject to a number of regulatory mechanisms. This report examines the efficacy of APC in plasma and evaluates the role of the APC cofactor protein S in this milieu. The ability of protein S to enhance the anticoagulant effects of activated protein C was demonstrated using a factor Xa recalcification time of Al(OH)3 adsorbed plasma. At a set concentration of APC, increasing concentrations of protein S resulted in a linear and saturable potentiation of the activity of APC. This result was not reflected in a purified component assay, where the extent of factor Va inactivation by APC was only marginally augmented by protein S. The efficacy of the cofactor was not affected by variations in the concentration of factor Va. Similarly, increasing the protein S:APC molar ratio of 200:1 resulted in only a trivial enhancement of APC activity. To directly examine the proteolysis of factor Va by APC in plasma, a novel assay system containing Al(OH)3 adsorbed, factor V deficient plasma supplemented with purified human factor Va was developed. The addition of APC in varying concentrations to this system consistently yielded factor Va inactivation rates inferior to those seen in a purified component assay. This finding is consistent with the presence of APC inhibitory activity in plasma. When protein S was added to the plasma system, factor Va inactivation by APC was restored to a similar level to that observed in the purified system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein S negates the activated protein C inhibitory activity of plasma. 164 2

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.
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PMID:Platelet-derived microparticles express high affinity receptors for factor VIII. 165 28

Human coagulation factor V is an integral component of the prothrombinase complex. Rapid activation of prothrombin is dependent on the interactions of this nonenzymatic cofactor with factor Xa and prothrombin in the presence of calcium ions and a phospholipid or platelet surface. Factor V is similar structurally and functionally to the homologous cofactor, factor VIII, which interacts with factor IXa to accelerate factor X activation in the presence of calcium and phospholipids. Both of these cofactors, when activated, possess homologous heavy and light chains. Binding to anionic phospholipids is mediated by the light chains of these two cofactors. In bovine factor Va, a phosphatidylserine-specific binding site has been localized to the amino-terminal A3 domain of the light chain. In human factor VIII, on the other hand, a region within the carboxyl-terminal C2 domain of the light chain has been shown to interact with anionic phospholipids. We have constructed a series of recombinant deletion mutants lacking domain-size fragments of the light chain of human factor V (rHFV). These mutants are expressed and secreted as single-chain proteins by COS cells. Thrombin and the factor V activator from Russell's viper venom process these deletion mutants as expected. The light chain deletion mutants possess essentially no procoagulant activity, nor are they activated by treatment with factor V activator from Russell's viper venom. Deletion of the second C-type domain results in essentially complete loss of phosphatidylserine-specific binding whereas the presence of the C2 domain alone (rHFV des-A3C1, which lacks the A3 and C1 domains of the light chain) results in significant phosphatidylserine-specific binding. The presence of the A3 domain alone (rHFV des-C1C2) does not mediate binding to immobilized phosphatidylserine. Increasing calcium ion concentrations result in decreased binding of recombinant human factor V and the mutant rHFV des-A3C1 to phosphatidylserine, similar to previous studies with purified plasma factor V and phospholipid vesicles. These results indicate that human factor V, similar to human factor VIII, possesses a phosphatidylserine-specific binding site within the C2 domain of the light chain.
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PMID:Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain. 174 Apr 60

A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1-10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.
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PMID:Simulation model for thrombin generation in plasma. 179 46

A simple model of the initiation of thrombin formation in plasma as a response to factor Xa generation was constructed. In this model factor Xa is considered as an input with a constant concentration. Substrate depletion and inactivation by activated protein C are neglected. The resulting linear model allows a closed form solution by standard methods. With values of the reaction rate constants, as determined in purified systems, this model predicts a highly explosive and complete activation of factor V and prothrombin as a response to any given (steady state) factor Xa concentration even in situations where prothrombinase and(/or) thrombin are rapidly inactivated. However, the time delay to rapid thrombin production becomes longer at lower factor Xa concentrations. Analysis of this time delay as a function of the factor Xa concentration indicates that the gain of the feedback loop of factor V activation by thrombin is so high that the contribution of factor V activation by factor Xa is relatively unimportant for factor Xa concentrations in the nanomolar range. It appears that the time lag is mainly determined by the gain of this feedback loop: similar proportional reductions of each of these reaction rates causes a similar effect. The effects of moderately enhanced inhibition rates of thrombin and prothrombinase on the time delay depend strongly on factor Xa concentration. Only a minor prolongation of the delay is predicted for factor Xa concentrations in the nanomolar range, but for factor Xa concentrations in the 1-10 pM range, the enhanced decay will cause considerable delays. Simultaneous reduction of the turnover rate of prothrombinase results in much larger delays for the entire range of factor Xa concentrations.
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PMID:Linearized model for the initiation of factor Va, and thrombin generation. 179 50

Ex situ hepatic surgery, in which a diseased liver is resected from outside the body, was first reported in 1988. This study investigates the hemostatic changes occurring during such surgery in two cases. During the anhepatic period of more than 5 hours, veno-venous bypass without heparin was performed. The tests included platelet count, prothrombin activity (PT), partial thromboplastin time (PTT), fibrinogen (Fbg), factor II (F.II), factor V (F.V), and thromboelastography (TEG). Three to 4 hours after entering the anhepatic phase, marked fibrinolysis and a fall in the values of PT, Fbg and F.V. were observed. Every parameter temporarily deteriorated immediately after revascularization of the graft, however, all returned to almost normal values within 1-2 hours after hepatic reperfusion except for F.V and platelets. In conclusion, the coagulopathy during ex situ hepatic surgery is caused by the marked fibrinolysis and depletion of hemostatic factors which develop 3-4 hours after the onset of the anhepatic phase.
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PMID:Hemostatic studies of ex situ hepatic surgery. 181 94

Activation of the extrinsic plasmatic clotting system was simulated in a computerized analysis. The results were compared with previously described experimental investigations in plasma with isolated clotting factor deficiency, which led to the conclusion that the sequence of patterns for constants of a function describing the extinction curves is related to the sequence of steps of clotting factor activation. The kinetics of activation resulting in extinction curves that correspond to the curves obtained from experimental measurements are described by sets of stiff coupled linear differential equations. The set of functions can be numerically solved without further approximations. As for experimental extinction curves the simulated extinction curves are characterized by an empirical function with three constants. The distribution patterns for the constants are qualitatively similar to experimental patterns, if the following assumptions are made: (1) feedback reaction occurs from factor IIa via factor V, and (2) the conversion of factor II by factor Xa occurs at a rate considerably slower than the conversion of factor II by the prothrombinase complex. A feedback reaction by factor Xa via factor VII accelerates the formation of factor X, although the distribution pattern remains similar to the distribution pattern for a mechanism without the feedback reaction, provided that the initial activation of factor X occurs at a fast rate. A feedback reaction by factor Xa via factor V in addition to the feedback reaction by factor IIa via factor V accelerates the activation, while the pattern distribution remains unchanged. The simultaneous inhibition of factor Xa and factor IIa by antithrombin III does not change the pattern distribution, while the formation of activated factor II is decelerated.
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PMID:Simulation of the extrinsic pathway of the plasmatic clotting system. 182 78

Thirty-six patients with chronic myeloproliferative disorders (CMPD) were studied as regards blood coagulation and fibrinolysis. These studies revealed various mild abnormalities: activated thromboplastin time (APTT) tended to prolong and the level of factor V decreased significantly. In several cases, the levels of D-dimer, thrombin-antithrombin III complex and plasmin-alpha 2-plasmin inhibitor complex were elevated compared to normal. These results suggest that abnormal coagulation system in the patients with CMPD is related to low grade disseminated intravascular coagulation. Many coagulation factors did not correlate with peripheral blood cell counts. Two patients with polycythemia vera were evaluated for several abnormalities of the coagulation system before and during treatment. Coagulation abnormalities persisted after hematologic control had been achieved. Our results suggest that patients with CMPD have a chronic state of abnormal blood coagulation system even after normalization of blood cell counts.
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PMID:[Abnormal blood coagulation and fibrinolysis in chronic myeloproliferative disorders]. 187 Feb 70

TF mediated initiation of coagulation appears to play a critical role in normal hemostasis and probably pathologic thrombosis as well. Although teleological considerations would seem to suggest that a specific regulator of this process should exist, and although the presence in plasma of such an inhibitor was documented many years ago, it was not until the past five years that the inhibitor was characterized and its mechanism of action defined. LACI produces factor Xa-dependent feedback initiation of the VIIa/TF catalytic complex. The mechanism of this feedback inhibition is novel. First, LACI, a multi-headed protease inhibitor, binds factor Xa, a product of VIIa/TF catalysis, at one of its inhibitory domains. The Xa-LACI complex, possibly acting as a pseudosubstrate, then is able to bind to VIIa/TF in an appropriate conformation such that a second inhibitory domain of LACI is positioned to interact with factor VIIa in the VIIa/TF complex. Whether such a unique means of eliciting feedback inhibition in a protease cascade is repeated in nature is unknown. The existence of LACI appears to help explain the clinical need for both "extrinsic" and "intrinsic" coagulation pathways. In addition, data to the present are consistent with the notion that, in normal hemostasis at least, TF is responsible for an initial burst of factor Xa generation which provides sufficient thrombin to induce the aggregation of platelets and the activation of the critical coagulation cofactors factor V and factor VIII. Ultimate and persistent hemostasis, however, appears to require the continued production of additional factor Xa through the action of factor IXa and factor VIII. The fact that patients with factor XI deficiency suffers a variable but usually mild bleeding diathesis suggests that under certain conditions the initial burst of factor IXa formed through the action of VIIa/TF is insufficient and supplemental factor IXa generated by factor XIa is needed for normal hemostasis. The mechanism by which this factor XIa is generated in vivo, however, has not been determined. We stress that the predicted in vivo role of LACI is simply that--a prediction based on its known in vitro properties. Documentation of its physiologic importance remains to be provided and is an area of active research. Further, although significant progress has been made over the past few years in the characterization of LACI, many questions remain unanswered. For example: What is the mechanism for LACI's association with lipoproteins in plasma? What function, if any, does the third Kunitz-type protease inhibitor domain in LACI serve? (ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The lipoprotein-associated coagulation inhibitor. 200 33


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