Severe coagulation factor V deficiency associated with an interstitial deletion of chromosome 1q
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Prothrombinase
Factor V
Activated protein C (APC)-resistance, the most common risk factor for venous thrombosis described so far, is due to a single point mutation in the factor V gene. As a result, inactivation of factor-activated factor V by APC is impaired, which leads to a hypercoagulable state and a lifelong increased risk of thrombosis. The importance of protein S as an anticoagulant protein is illustrated by the association between protein S deficiency and venous thrombosis. The objective of this article is to examine the most recent advances on the role of factor V and protein S as cofactors to activated protein C.The material examined in the present review includes several personal papers in this field, and articles and abstracts published in journals covered by the Science Citation Index.Factors V and VIII are homologous, high molecular weight glycoproteins with similar functional properties. Factors Va and VIIIa bind to negatively charged phospholipid and function as high affinity receptors/cofactors for factors Xa and IXa, respectively. Factors Va and VIIIa account for at least a 10(3) increase in the rate of activation of prothrombin and factor Xa, respectively. The potent anticoagulant activity of APC is mediated by the degradation of factors VIIIa and Va, resulting in inhibition of both Xase and prothrombinase activities. APC specifically degrades the membrane-bound activated forms of factors V and VIII, whereas the unactivated factors V and VIII are poor substrates for APC. Mature human protein S is a single chain glycoprotein composed of multiple domains, including a thrombin-sensitive region. Protein S acts as a cofactor to activated protein C. Thus function of protein S is lost upon thrombin cleavage, suggesting that the thrombin-sensitive region interacts with APC on the phospholid surface.Recent data suggest that factor V and protein S work in synergy as phospholipid-bound cofactors to APC in the degradation of factor VIIIa and that factor VIIIa is preferred over factor Va as APC-substrate. Thus complicated multimolecular complexes, which are the result of protein-protein as well as protein-phospholipid interactions, appear to form the basis for efficient cleavage and inhibition of factors VIIIa and Va.
Prothrombinase
Factor V
Factor IXa
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Purified coagulation factors and specific antibodies to factor V and factor X were used to investigate the action of thrombin on factor V and the mechanism by which thrombin-treated factor V influences prothrombin activation. The formation of a complex or complexes between phospholipid, factor V, factor Xa and calcium was demonstrated by column chromatography on Sephadex gel, and by immunological analysis of the column fractions including the use of solid-phase antibodies. Kinetic experiments demonstrated that generation of thrombin from purified prothrombin was accomplished by this complex. Pre-treatment of factor V with trace quantities of thrombin resulted in increased yield and rate of thrombin generation. It was shown that phospholipid became saturated when incubated with increasing concentrations of factor V and that the initial saturating concentration of the latter was reduced by pre-treatment with thrombin. The findings confirm that optimum conversion of prothrombin to thrombin is accomplished by a complex or complexes of phospholipid, factor V, factor Xa and calcium and it is suggested that thrombin plays an autocatalytic role in these reactions.
Prothrombinase
Factor V
Sephadex
Thromboplastin
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Factor V
Cleavage (geology)
Factor IXa
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We have demonstrated the presence of a saturable, reversible, and Ca(2+)-dependent binding site for 125I-labeled factor X ([125I]factor X) on human platelets (16000 +/- 2000 sites per platelet, Kd = 320 +/- 40 nM, n = 12) activated with either thrombin or the thrombin receptor agonist peptide, SFLLRN-amide, but not with ADP. Bound [125I]factor X could be completely removed by the addition of a Ca2+ chelator or an excess of unlabeled factor X. Antibodies that inhibit binding of factor X to the MAC-1 integrin receptor of monocytes and those directed against human factor V, failed to disrupt [125I]factor X binding to platelets. Prothrombin, but neither factor VII, factor IX, protein C, nor protein S, was an effective competitor of [125I]factor X binding with a K1 approximately Kd. [125I]Prothrombin also binds to activated (but not unactivated) platelets in a saturable, reversible, and Ca(2+)-dependent manner (20500 +/- 1500 sites, Kd = 470 +/- 110 nM, n = 3). Annexin V potently inhibited the binding of both [125I]factor X and [125I]prothrombin (IC50 approximately 3 nM). Factor X, prothrombin, and prothrombin fragment 1 (residues 1-155) were equipotent inhibitors of [125I]prothrombin and [125I]factor X binding, whereas Gla-domain-less factor X was unable to compete with [125I]factor X for platelet binding sites. Thus, it is the Gla-domains of factor X and prothrombin that appear to contain the regions necessary for platelet binding. The results of studies utilizing artificial phospholipid surfaces have led to the hypothesis that the substrates (FX and prothrombin) for the intrinsic pathway FXase and prothrombinase complexes are bound to the phospholipid surface. The factor X/prothrombin binding site we have described on the surface of activated platelets permits the utilization of surface-bound substrates by these complexes when they are assembled on a physiologic surface.
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Prothrombinase
Factor V
Factor IXa
Protein A/G
Factor IX
Intrinsic factor
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Summary In this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogen-ic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM). Addition of 0.2 nM APC and 20 μM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 rnin. Under the same conditions, APC inactivated ∼ 60% and ∼20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg506⟶Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = {factor Va+APC/factor Va−APC} × 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p <0.0001). In all cases our test gave the same result as the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.
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Factor V
Thromboplastin
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Human plasma factor V is heterogeneous and yields two forms of activated factor V that bind with low (factor Va1) and high affinity (factor Va2) to phospholipids. The properties of factor Va1 and factor Va2 in the anticoagulant and procoagulant pathways were evaluated by comparing their sensitivity for inactivation by APC and their ability to act as cofactor in prothrombin activation. At low phospholipid concentrations and on membranes containing low amounts of phosphatidylserine (PS), factor Va1 was inactivated by APC at 15-fold lower rates than factor Va2, both in the absence and in the presence of protein S. At high phospholipid concentrations and on membranes with more than 15 mol % PS, factor Va1 and factor Va2 were inactivated with equal efficiency. Differences between cofactor activities of factor Va1 and factor Va2 in prothrombin activation were only observed on membranes with less than 7.5 mol % PS. Due to the different phospholipid requirements of APC-catalyzed factor Va inactivation and of expression of factor Va cofactor activity in prothrombin activation, the thrombin-forming capacity of factor V1 was 7-fold higher than that of factor V2 in a reaction system containing factor Xa, prothrombin, APC, protein S, vesicles with a phospholipid composition resembling that of activated platelets, and traces of thrombin to initiate prothrombin activation. This shows that in the process of generation, expression, and down-regulation of factor Va cofactor activity on physiological membranes, the overall procoagulant activity of factor V1 can considerably exceed that of factor V2.
Prothrombinase
Factor V
Thromboplastin
Factor IXa
Factor VII
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Prothrombinase
Factor V
Factor IXa
Factor IX
Protein A/G
Intrinsic factor
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Thrombin activated bovine factor V is composed of two polypeptide chains with molecular weights 94,000 and 80,000. The two polypeptide chains are complexed via Ca2+ions. Factor Va enhances the rate of thrombin formation by drastically increasing the Vmax of the prothrombin activation. We have undertaken a study of the interactions of factor Va with the different components of the prothrombinase complex (e.g. factor Xa and prothrombin), in order to get more insight in the mode of action of factor Va. Our kinetic experiments in solution show that the functional enzyme in the prothrombinase complex is a equimolar complex of factor Va and factor Xa. The dissociationconstant, as determined over a wide range of prothrombin concentrations, has a value of 3×10-9M. For the stimulating effect of factor Va on the prothrombin activation by factor Xa in solution, the presence of Ca2+ions is required. The dissociationconstant of the Va-Xa complex was found to be independent of the Ca2+ concentration. In order to reveal whether an interaction between Ca2+ and γ- carboxyglutamic acid residues is responsible for the observed Ca2+ requirement, identical experiments were carried out with decarboxyfactor Xa and decarboxyprothrombin. The isolated polypeptide chains of factor Va have, in the presence or absence of factor Va, no effect on the kinetic parameters of the prothrombin activation. This let us conclude that there is no interaction between factor Xa and the separate polypeptide chains of factor Va. The affinity of factor Xa for negatively charged phospholipid or stimulated bloodplatelets is greatly enhanced by the presence of factor Va. Our Kd value measured for the Xa-Va complex in combination with reported dissociationconstants of factor Xa-phospholipid and Factor Va-phospholipid complexes give a quantitative explanation for the above mentioned effect of factor Va on the binding of factor Xa to phospholipid membranes.
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Factor V
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