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.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

HEPARIN IS NOT AN EFFICIENT INHIBITOR OF THE FACTOR Xa-DEPENDENT ACTIVATION OF FACTOR V AND FACTOR VIII

1987 ◽  
Author(s):  
F A Ofosu ◽  
G J Modi ◽  
M R Buchanan ◽  
J Hirsh ◽  
M A Blajchman
Keyword(s):  
Factor Viii ◽  
Antithrombin Iii ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Inhibitory Effects ◽  
Efficient Inhibitor ◽  
System 1 ◽  
Prothrombin Activation

We have previously proposed that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent activation of factor V and factor VIII. This observation was based on the demonstration that therapeutic concentrations of heparin or 1μM of the thrombin specific inhibitor, phe-pro-arg CH2Cl (PPACK) completely inhibited the activation of prothrombin when contact-activated plasma (CAP) was recalcified for up to 1 min. Under similar conditions, heparin and PPACK only partially inhibited the activation of factor X. Moreover, the addition of thrombin (lOnM) to CAP 1 min before that of heparin or PPACK reversed their inhibitory effects. We now provide further support for our hypothesis by showing that when the activity of thrombin is suppressed by heparin or PPACK, efficient activation of radiolabelled prothrombin occurs only when the factor Xa then present activates factor V and factor VIII. We compared the effects of HEP of PPACK on the following four systems for initiating the activation of prothrombin: (1) CAP; (2) CAP + lOnM thrombin; (3) CAP + InM Xa and (4) unactivated plasma + InM Xa + InM Va + coagulant phospholipids. In each system, the enzymes were added 1 min before the heparin or PPACK. In the absence of heparin or PPACK, all four systems generated the same amount of thrombin activity in 45s. Complete inhibition of prothrombin activation by heparin and PPACK was observed only in system 1 which did not contain exogenous thrombin or factor Xa. No inhibition by heparin or PPACK was observed when thrombin or factor Xa was added to CAP in systems (2) and (3). Only partial inhibition was observed in system (4) which contained exogenous prothrombi-nase complex. Factor Xa thus provides an effective by-pass mechanism for the activation of factor VIII and factor V in plasma containing therapeutic concentrations of heparin. Our data provide further evidence that the heparin-antithrombin III system is not effective in inactivating factor Xa. These results support the hypothesis that in unactivated normal plasma, the primary anticoagulant effect of heparin is the inhibition of the thrombin-dependent activation of factor V and factor VIII.

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 Characterization of an inducible endothelial cell prothrombin activator

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2989-2994 ◽  
Author(s):  
L Liu ◽  
GM Rodgers
Keyword(s):  
Thrombin Generation ◽  
Interleukin 1 ◽  
Factor Xa ◽  
Apparent Molecular Weight ◽  
Factor V ◽  
Factor X ◽  
Exogenous Factor ◽  
Km Value ◽  
Prothrombin Activation

In vivo prothrombin activation is thought to occur via a factor Xa/factor V-dependent mechanism. We investigated whether human venous endothelial cells (EC) could be induced to express a prothrombin activator. EC treated with lipopolysaccharide (LPS) or interleukin-1 activated prothrombin in the absence of exogenous factors Xa and V. This activity resided in the membrane fraction of EC and was not inhibited by an antibody to factor V. The apparent Km value was 3.3 +/- 0.3 mumol/L. Comparative studies of thrombin generation using a model system of phospholipid and factors Xa/V versus LPS-treated EC were performed to quantitate the effects of known inhibitors to factor Xa. The factor Xa inhibitor DEGR-chloromethyl ketone and an antibody to factor X inhibited prothrombin activation. However, the EC activator did not hydrolyze a factor Xa chromogenic substrate, and recombinant tick anticoagulant peptide did not suppress activity of the prothrombin activator. The apparent molecular weight of the EC activator was approximately 30 kD. Exogenous factor V enhanced the activity of the EC activator, such that in the presence of factor V, the apparent K(m) value was 1.28 +/- 0.10 mumol/L. Additionally, LPS-treated EC activated exogenous factor V. This activator has several characteristics of a previously described inducible murine monocyte prothrombin activator and may contribute to thrombin generation associated with pathologic stimuli.

The Role of Phospholipids and Factor Va in the Mechanism of Prothrombin Activation

1979 ◽  
Author(s):  
J. Rosing ◽  
G. Tans ◽  
J.W.P. Govers-Riemslag ◽  
R.F.A. Zwaal ◽  
H.C. Hemker
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Clotting Factor ◽  
Factor V ◽  
Factor X ◽  
Factor Va ◽  
Prothrombin Activation ◽  
Thrombin Formation

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

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.

scholarly journals The Factor V Activation Paradox

2004 ◽  
Vol 279 (19) ◽  
pp. 19580-19591 ◽  
Author(s):  
Thomas Orfeo ◽  
Nicole Brufatto ◽  
Michael E. Nesheim ◽  
Hung Xu ◽  
Saulius Butenas ◽  
...  
Keyword(s):  
Rate Constants ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Reaction Pathways ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activation Products ◽  
Initial Activation ◽  
Prothrombin Activation

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. α-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.

Studies On The Interaction Between Factor Va And Factor Xa

1981 ◽  
Author(s):  
M Lindhout ◽  
J Govers-Riemslag ◽  
J Rosing ◽  
H Hemker
Keyword(s):  
Factor Xa ◽  
Factor V ◽  
Molecular Weights ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
A Value ◽  
Wide Range ◽  
Carboxyglutamic Acid ◽  
Polypeptide Chains ◽  
Prothrombin Activation

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.

Characterization of Clotting Factors Associated with Blood Platelets after Gel Filtration

1973 ◽  
Vol 30 (02) ◽  
pp. 289-298
Author(s):  
Oddvar Tangen ◽  
Eva B. Lestrup ◽  
Herbert J. Berman
Keyword(s):  
Factor Viii ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Factor Viii Activity ◽  
Clotting Factors ◽  
Platelet Surface ◽  
Filtration Factor

SummaryAggregation of human and rabbit gel filtered platelets (GFP) has been studied in presence of Ca2+, activated factor X (Xa) and different plasma preparations. It was found that factor Xa by itself is not a platelet aggregating agent. However, the platelets aggregated immediately when platelet poor plasma (PPP) was added to a mixture of GFP, Ca2+ and factor Xa. Aggregation also occurred immediately when factor V-deficient plasma was substituted for PPP, but not when factor II-deficient plasma was used. In the absence of factor Xa, aggregation occurred on addition of factor V- or VIII-deficient plasma, but only after some delay. The platelet aggregation experiments and experiments with centrifugations and resuspensions of the platelets, clotting experiments, and gel filtration of platelet free plasma (PEP) led to the following conclusions : Factors II and X are totally removed from the platelets by gel filtration, factor V is closely associated with the platelet surface, and part of the factor VIII-activity in the plasma is eluted together with the GFP without being associated with the platelets. This factor VIII-activity belonged to an extremely large molecule or molecular complex with a Mw in the order of 2 - 5 · 107.

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.

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