scholarly journals Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1015-1022 ◽  
Author(s):  
JP Miletich ◽  
WH Kane ◽  
SL Hofmann ◽  
N Stanford ◽  
PW Majerus
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Severe Bleeding ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Factor Va ◽  
Number Of Binding Sites ◽  
Stimulation Of

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.

scholarly journals Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1015-1022 ◽  
Author(s):  
JP Miletich ◽  
WH Kane ◽  
SL Hofmann ◽  
N Stanford ◽  
PW Majerus
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Severe Bleeding ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Factor Va ◽  
Number Of Binding Sites ◽  
Stimulation Of

Abstract Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.

Human Platelets Contain Forms of Factor V in Disulfide-Linkage with Multimerin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1933-1933
Author(s):  
Catherine P.M. Hayward ◽  
Nola Fuller ◽  
Shilun Zheng ◽  
Frederic Adam ◽  
Samira Jeimy ◽  
...  
Keyword(s):  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Disulfide Linkage ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Plasma Factor ◽  
Large Factor ◽  
V Antigen

Abstract Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

scholarly journals Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 875-880 ◽  
Author(s):  
T Wiedmer ◽  
CT Esmon ◽  
PJ Sims
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Exogenous Factors

Abstract The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

Interaction of Factor V and Factor Va with Platelets

1979 ◽  
Author(s):  
P.B. Tracy ◽  
J. M. Peterson ◽  
M.E. Nesheim ◽  
F.C. McDuffie ◽  
K. G. Mann
Keyword(s):  
Binding Sites ◽  
Intrinsic Factor ◽  
Factor V ◽  
Single Chain ◽  
Apparent Dissociation Constant ◽  
Platelet Factor ◽  
High Affinity ◽  
Factor Va ◽  
Thrombin Activation ◽  
Triton X

We have used homogeneous single chain bovine factor V to examine the binding of both factor V and factor Va to bovine platelets, as well as to develop a double-antibody radioimmunoassay (RIA) to measure intrinsic platelet factor V. Reaction of the protein with 125I Bolton-Hunter reagent produced a labelled product which retained 90% of its cofactor activity and gave products indistinguishable from native factor V following thrombin activation. When incubated separately with washed bovine platelets, both 125I-factor V and Va underwent saturable and exchangeable binding. There are high affinity binding sites to which 500-900 V(Va) molecules are bound per platelet with an apparent dissociation constant of 3 χ 10-10 M, as well as binding sites of slightly lower affinity (Kd = 3 χ 10-9M) to which as many as 3500 V (Va) molecules are bound per platelet. Thrombin pretreatment of the platelets was not required for the binding of either factor V or Va. The RIA data for Triton X-100 lysed, washed bovine platelets revealed that 400-1000 intrinsic factor V molecules were present per platelet. Factor V clotting assays produced results consistent with the RIA data. These studies suggest that the factor V molecules intrinsic to the platelet are equivalent to the number of high affinity factor V (Va) binding sites present on the platelet memhrane surface. (Supported by Grant HL-17430 and the hayo Foundation).

scholarly journals Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 875-880 ◽  
Author(s):  
T Wiedmer ◽  
CT Esmon ◽  
PJ Sims
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Exogenous Factors

The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

The Influence of Purified Factor V, Factor Va, and Activated Platelets on the Factor Xa Catalyzed Activation of Prothrombin

1979 ◽  
Author(s):  
M. E. Nesheim ◽  
K.G. Mann
Keyword(s):  
Factor Xa ◽  
Maximum Velocity ◽  
Fold Increase ◽  
Factor V ◽  
Platelet Factor ◽  
Factor Va ◽  
Radioimmune Assay ◽  
I Factor ◽  
Measurable Activity ◽  
Kinetics Of

The kinetics of prothrombin activation by factor Xa and Ca++ plus purified factor V and phospholipid or activated bovine platelets were monitored continuously with dansylarginine-4-ethylpiperidine amide which specifically inhibits thrombin and displays enhanced fluorescence upon binding. With purified components and saturating amounts of factor Va a 500,000 fold increase in activity occurred compared to that with factor Xa alone, with phospholipid and Ca++ present, the rate increase with factor Va was 24,000 fold. The reaction rate with factor V was only 0.5 to 1.0% of that with factor Va, although saturating concentrations of either form of the cofactor were approximately equivalent. At fixed levels of factor Xa and Ca++, the process was saturable with platelets; it was also saturable with factor Xa at fixed platelet levels. Saturation occurred at levels consistent with the notion that platelet factor Va (measured by bioassay and radioimmune assay) was limiting in both cases. The maximum velocity with platelets was about 1100 moles thrombin min-1/mole factor Xa. These results suggest that I) factor Va influences prothrombinase activity to a greater degree than previously recognized: 2) unactivated factor V has low but measurable activity; and 3) in terms of maximum catalytic capacity the synthetic prothrombinase utilizing phospholipid and purified factor Va is about equivalent to that utilizing platelets. (Supported by NHLBI Grants HL-07069 and HL-17430D).

Prothrombin Activation in the Presence of Platelets

1979 ◽  
Author(s):  
B.U. Dahlbäck ◽  
J. Stenflo
Keyword(s):  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Release Reaction ◽  
Prothrombinase Complex ◽  
Dependent Part ◽  
The One ◽  
Prothrombin Activation

The role of fragment 1, the vitamin K-dependent part of prothrombin containing γ-carboxyglutamic acid residues, and of fragment 2, the factor V-binding part of prothrombin for rapid prothrombin activation was studied. Activation rates of bovine prothrombin, acarboxyprothrombin and prethrombin 1 by factor Xa in the presence of platelets on the one hand and by the prothrombinase complex (factor Xa, factor V, phospholipid and Ca2+) on the other were compared. The conversion products of prothrombin in the presence of factor Xa and platelets were found to be the same as those seen when prothrombin was activated by the prothrombinase complex. The complete prothrombinase complex was more efficient even for activation of acarboxyprothrombin and prethrombin 1, which do not bind to phospholipid, than an abortive complex lacking the phospholipid. This was probably due to more effectively bound factor X . For rapid prothrombin activation by factor Xa in the presence of platelets both fragment 1 and fragment were found to be required. Acarboxyprothrombin and prethrombin 1 were slowly activated to thrombin by factor Xa in the presence of platelets but only after the platelet release reaction. The apparent KH of 0.6 uM prothrombin was 6 times lower than that of acarboxyprothrombin and the coefficient for proteolytic efficiency was approximately 50 times higher. The platelet surface ex posed upon the release reaction gradually lost its catalytic property during the prothrombin activation, probably due to destruction of the platelet factor Xa receptor by thrombin.

Prothrombin Activation on the platelet surface

1979 ◽  
Author(s):  
J.P. Miletich ◽  
W. Kane ◽  
P.W. Majerus ◽  
M.J. Lindhout ◽  
C.M. Jackson
Keyword(s):  
Factor Xa ◽  
Model System ◽  
Model Systems ◽  
Factor V ◽  
Platelet Surface ◽  
High Affinity ◽  
Normal Platelet ◽  
Factor Va ◽  
Modified Forms ◽  
Prothrombin Activation

Previously reported high affinity receptors for Factor Xa which exist on the platelet surface have been identified as Factor V (more accurately Factor Va). Approximately 200 Factor Xa receptors exist per normal platelet and the rate of prothrombin activation is correlated with the extent to which these receptors are occupied by Factor Xa. Platelets from congenitally Factor V deficient patients possess reduced numbers of Factor Xa receptors and proportionally reduced rates of prothrombin activation. The role ot phospholipid, in addition to that of Factor Va in mediating the association ot Factor Xa and prothrombin with the platelet surface has been assessed by employing structurally modified forms of Factor Xa and prothrombin. Possible explanations for the comparatively greater rate of prothrombin activation observed with platelets than with model system ca. 20-fold, will be discussed by comparing the platelet and model systems. (Supported by US NHLBI, SCOR in Thrombosis (HL14147) and HL12820).

The Human Prothrombin-Activating Enzyme

1969 ◽  
Vol 22 (01) ◽  
pp. 045-067 ◽  
Author(s):  
K Deggeller ◽  
J Vreeken
Keyword(s):  
Linear Relationship ◽  
Factor Xa ◽  
Enzyme Concentration ◽  
Factor V ◽  
Factor X ◽  
Active Centers ◽  
Factor Va

SummaryThe formation and action of human prothrombin-activating enzyme is described. The study of the formation of the enzyme leads to the following conclusions :1. The enzyme is formed from factor V, factor X and phospholipid in the presence of calcium. If one of the reagents is omitted no activity develops.2. Factor V and factor X need activation by thrombin and for instance Russell Viper Venom, respectively.3. A linear relationship exists between the inverse of factor Va concentration and the inverse of enzyme concentration.4. A linear relationship exists between the inverse of factor Xa concentration and the inverse of enzyme concentration.5. A linear relationship exists between the inverse of phospholipid concentration and the inverse of enzyme concentration at small phospholipid concentration.6. A linear relationship exists between the phospholipid concentration and the inverse of enzyme concentration at high phospholipid concentration.The study of the action of the enzyme leads to the conclusion that human prothrombin is substrate and an inhibitor if present in excess.The observed phenomena are best explained by the hypothesis that factor Va and factor Xa adsorb onto the phospholipid surface. When both factors are adsorbed close together they are active as an enzyme. This activity depends on two active centers, probably one derived from factor Va and one from factor Xa.

Sign in / Sign up

Export Citation Format

Share Document