Platelet Prothrombin-converting Activity Research Articles

Synergistic effect of antithrombin III, activated protein C and heparin on the inhibition of the tissue thromboplastin-mediated coagulation.

The synergistic effect of antithrombin III (ATIII), activated protein C (APC) and heparin on the tissue thromboplastin (TP)-mediated coagulation cascade was studied. APC prolonged the activated partial thromboplastin time of human plasma with increasing APC concentration but affected the prothrombin time only slightly. Neither ATIII nor heparin prolonged the prothrombin time by itself, while a mixture of APC and heparin strongly inhibited the coagulation. When the effects of APC, heparin and ATIII on the TP-mediated coagulation were examined with a solution consisting of prothrombin, factor V, factor VII, factor X and fibrinogen at physiological concentrations, the coagulation time was prolonged only slightly by the APC-heparin or ATIII-heparin mixture. However, the coagulation time was prolonged markedly by simultaneous addition of APC, ATIII and heparin to the solution. Inhibition of thrombin activity by the ATIII-APC-heparin mixture was weak as compared with that by the ATIII-heparin mixture after a 1-min incubation, but after a 2-min incubation the inhibitory activities were equal. Suppression of thrombin activity by the ATIII-APC-heparin mixture was supposed to be due to the inhibition of the interaction between ATIII and heparin on thrombin by APC because the APC-ATIII-heparin complex was detected by crossed immuno-electrophoresis but not by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. When the inhibitory effect of APC alone or the APC-heparin mixture on the platelet prothrombin converting activity (PPCA) was examined, heparin accelerated the PPCA inhibition by APC.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators

Purified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombin-converting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet prothrombin converting activity in hereditary disorders of platelet function.

Prothrombinase activities of platelets have been measured in diluted platelet-rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense-body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and alpha-granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard-Soulier syndrome, however, have approximately 10-fold higher prothrombinase activities in the non-stimulated form than normal non-stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non-stimulated Bernard-Soulier platelets, earlier described by Perret et al (1983).

Two separable steps in the development of platelet prothrombin-converting activity

Platelets catalyze conversion of prothrombin to thrombin in the presence of coagulation Factors Va and Xa. Bovine Factor Va was found to bind to human and bovine platelets washed by centrifugal pelleting. Factor Va was bound to sites varying in affinities but only those sites saturated at approximately 1 mM are active. Platelets in citrate or benzamidine-anticoagulated plasma or those washed by gel filtration bound substantially less Factor Va with these affinities, indicating that the binding sites increased in number or affinity or both during the centrifugal pelleting and redispersal. Thrombin production catalyzed by platelets with developed binding sites started at a low initial rate but after a lag period rapidly reached a maximum rate. Addition of 0.25 mM dinitrophenol increased the lag period and decreased the maximum rate obtained concomitantly with the reduction in ATP levels. Neither dinitrophenol nor its induced depletion of the ATP levels reduced Factor Va binding. Therefore, the activation of platelet pro thrombin-converting activity was separated into two steps; one being the development of the high-affinity binding sites and the other a step partially dependent on sufficient levels of ATP within the platelet.

Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III

We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC 50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute to the action of this drug in blood coagulation.

Collagen derived octapeptide inhibits platelet procoagulant activity induced by the combined action of collagen and thrombin

Platelet prothrombin converting activity was measured in a system using washed human platelets and purified coagulation factors Xa, Va and prothrombin. Exposure of platelet prothrombin converting activity evoked by collagen or the combined action of collagen and thrombin was effectively inhibited when a collagen derived octapeptide was added prior to platelet activation. Half maximal inhibition of prothrombinase activity of platelets stimulated by collagen plus thrombin- or collagen alone was obtained at 0.9 mM and 0.5 mM octapeptide, respectively. This suggests a modifying effect of thrombin on the platelet-collagen interaction. Octapeptide either alone or in combination with thrombin was unable to enhance platelet procoagulant activity. The increased prothrombin converting activity seen upon treatment of platelets with ionophore A23187 was not affected by octapeptide, added either before or after treatment with ionophore. It is concluded that octapeptide specifically interferes with the platelet-collagen interaction required to generate a procoagulant surface which enhances the rate of thrombin formation.

Evidence for independent pathways for the induction of platelet prothrombin-converting activity by thrombin and collagen

Evidence for independent pathways for the induction of platelet prothrombin-converting activity by thrombin and collagen

Enhanced prothrombin-converting activity and factor Xa binding of platelets activated by the alternative complement pathway

Platelet prothrombin-converting activity and factor Xa binding were studied after exposure of human platelet rich plasma (PRP) to various conditions leading to platelet activation. Zymosan resulted in increased platelet-bound C3, enhanced prothrombin-converting activity and increased factor Xa binding. Similar findings were observed with normal platelets resuspended in factor XII-deficient plasma. The combined use of zymosan and thrombin to activate platelets resulted in synergistic prothrombin-converting activity and factor Xa binding. In contrast, no synergism was obtained with the concomitant use of zymosan and collagen, suggesting that collagen and zymosan share the same pathway for platelet activation. Heterologous antibody to factor V completely inhibited the platelet prothrombin-converting activity for all modes of platelet activation, indicating that this activity is mediated by factor V.

Enhanced prothrombin-converting activity and factor Xa binding of platelets activated by the alternative complement pathway.

Platelet prothrombin-converting activity and factor Xa binding were studied after exposure of human platelet rich plasma (PRP) to various conditions leading to platelet activation. Zymosan resulted in increased platelet-bound C3, enhanced prothrombin-converting activity and increased factor Xa binding. Similar findings were observed with normal platelets resuspended in factor XII-deficient plasma. The combined use of zymosan and thrombin to activate platelets resulted in synergistic prothrombin-converting activity and factor Xa binding. In contrast, no synergism was obtained with the concomitant use of zymosan and collagen, suggesting that collagen and zymosan share the same pathway for platelet activation. Heterologous antibody to factor V completely inhibited the platelet prothrombin-converting activity for all modes of platelet activation, indicating that this activity is mediated by factor V.

The nature of the binding for prothrombinase at the platelet surface as revealed by lipolytic enzymes.

The nature of the receptor for the prothrombinase complex at the surface of non-activated platelets was investigated by measuring the platelet prothrombin-converting activity wih a chromogenic substrate assay, after treatment of the platelets with various phospholipases or three different proteolytic enzymes. Platelet prothrombin-converting activity only decreased after treatment with those phospholipases which are able to hydrolyse phospholipids in the intact platelet and also have the ability to degrade negatively charged phospholipids, phosphatidylserine and phosphatidylinositol. Those phospholipases which do hydrolyse phospholipids in the intact platelet but have no activity towards phosphatidylserine (and phosphatidylinositol) produce an increase in the platelet prothrombin-converting activity. Proteolytic treatment of platelets with trypsin, chymotrypsin or papain did not result in a decrease of prothrombin-converting activity. It is concluded that negatively charged phosphatidylserine and possibly phosphatidylinositol are involved in the prothrombin-converting activity of non-activated platelets. We could not demonstrate the involvement of platelet membrane proteins in a receptor for the components of the prothrombinase complex.

Activated protein C inhibits platelet prothrombin-converting activity

Bovine platelets that have been activated by thrombin facilitate the conversion of prothrombin to thrombin in the presence of calcium ions and factor Xa. Activated protein C, a vitamin-K-dependent plasma protein, inhibits this platelet prothrombin-converting activity. The inhibition is time dependent and is not reversed by increasing concentrations of factor Xa. However, factor Xa is able to protect the platelet prothrombin-converting activity from inactivation by activated protein C. The activated protein C causes a parallel loss of factor Xa receptor sites and platelet prothrombin-converting activity. Activated protein C may contribute to the regulation of clotting through inactivation of the platelet prothrombin-converting activity.

Activated Protein C Inhibits Platelet Prothrombin-Converting Activity

Bovine platelets that have been activated by thrombin facilitate the conversion of prothrombin to thrombin in the presence of calcium ions and factor Xa. Activated protein C, a vitamin-K-dependent plasma protein, inhibits this platelet prothrombin-converting activity. The inhibition is time dependent and is not reversed by increasing concentrations of factor Xa. However, factor Xa is able to protect the platelet prothrombin-converting activity from inactivation by activated protein C. The activated protein C causes a parallel loss of factor Xa receptor sites and platelet prothrombin-converting activity. Activated protein C may contribute to the regulation of clotting through inactivation of the platelet prothrombin-converting activity.

The Effect of Aggregation and Release on Platelet Prothrombin-Converting Activity

AbstractPlatelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostasis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel-filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytryptamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125l-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

The effect of aggregation and release on platelet prothrombin- converting activity

Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel- filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

Effect of oral contraceptive use on platelet prothrombin converting (platelet factor 3) activity

Platelets obtained from women using estrogen-progesterone oral contraceptives are evaluated for activity as cofactor for Factor Xa-catalyzed prothrombin activation. Donor platelet-rich plasma is diluted into a standard platelet-poor plasma to minimize variations in plasma Factor X and prothrombin concentrations. Activation is initiated with Russell's viper venom, arrested at intervals with EDTA, and residual prothrombin is measured. Compared with controls, platelets from women using oral contraceptives promote an increase in both rate and extent of prothrombin activation, with control and test groups falling into non-overlapping populations. The increased prothrombin activation correlates with an increase in antithrombin III consumption. Enhanced antithrombin III consumption explains the decreased serum antithrombin III levels among women using oral contraceptives.