Prothrombin-converting Activity Research Articles

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.

Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va.

The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a gamma-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another gamma-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.

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.

Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets.

In the present paper we compare prothrombin‐converting activities of platelets non‐activated, or activated by collagen, thrombin or collagen plus thrombin in the absence and presence of added factor Va. In all experiments described, the rate of thrombin formation for platelets activated by the combined action of collagen and thrombin is greater than that of platelets stimulated by collagen or thrombin alone. The presence of added factor Va enhanced the rate of thrombin formation in all cases, but the higher activity observed with platelets stimulated by collagen plus thrombin remains. When platelets are activated by collagen plus thrombin in the presence factor Xa and prothrombin, a lag period of approximately 10 min is observed before the rate of thrombin formation reaches a steady state. Addition of an excess of factor Va in this experiment reduces the lag time to 3 min. This lag period is interpreted as the time required to generate extra binding sites for the prothrombinase complex at the platelet surface. These extra sites explain the difference in thrombin formation rate between these platelets and platelets activated by either collagen or thrombin only.The exposure of phospholipids at the platelet outer surface was studied with phospholipases after various activations of the platelets. It is demonstrated that activation by collagen plus thrombin is accompanied by increased susceptibility of platelet phospholipids towards phospholipase A2. Among these degradable phospho‐ lipids are 25% of the phosphatidylserine and 30% of the phosphatidylethanolamine. On the other hand, little no phosphatidylserine is exposed at the membrane exterior of thrombin‐treated or control platelets. We propose that the exposure of phosphatidylserine at the outer surface of platelets activated with thrombin plus collagen essential for the rate enhancement of thrombin formation observed under these conditions. The possibility of transbilayer movement of phospholipids in the platelet membrane as a result of the activation process will be discussed.

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.

Effects of polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4 · 10 −6 M detergent. Efflux of [ 14C]serotonin, 45Ca 2+ and labile aorta contracting substance (thromboxane A 2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4 · 10 −5 M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca 2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [ 14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8 · 10 −4 to 5 · 10 −3 M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregation agents, while higher concentrations produce membrane destabilization and cell lysis.

Action of human thrombokinase on human prothrombin and [formula omitted] methyl ester

An enzyme, thrombokinase, has been partially purified from human plasma. Its defining property is the ability to convert prothrombin to thrombin in the absence of other cofactors. This prothrombin-converting activity is independent of residual thrombin and is inhibited by soybean trypsin inhibitor but not by DFP. Various cofactors (polylysine or calcium alone, or beef serum or phospholipid in the presence of calcium) accelerate the conversion of prothrombin by thrombokinase.

Formation of prothrombin converting activity.

After measurement of the conversion of prothrombin to thrombin in a purified system, a model has been constructed for the kinetics of the formation of prothrombin converting activity. In the model phospholipid provides a surface on to which coagulant factors bind.

The Effect of Hirudin, a Potent Antithrombin, on the Thromboplastin Generation Test

SummaryThe presence of hirudin at the time adsorbed plasma and serum were combined in TGT inhibited the formation of the prothrombin converting activity. Hirudin, a potent antithrombin, probably exerted its inhibitory effect by neutralizing any thrombin which might form. If adsorbed plasma and serum were incubated together for a period as short as 1 to 3 min before the addition of hirudin, calcium and lipid, hirudin no longer exerted the same inhibitory effect, but, in order to exert the same inhibitory effect, had to be present in higher concentration. Preincubation of factor VIII deficient plasma with serum and calcium was not effective in overcoming hirudin inhibition, but preincubation with factor IX deficient serum with adsorbed plasma and calcium was able to overcome inhibition due to hirudin equally as well as normal serum.Once again the importance of traces of thrombin in the formation of activity capable of rapidly clotting substrate plasma in TGT has been shown by using a specific thrombin inhibitor. A thrombin dependent interaction appears to take place between adsorbed plasma and serum and also between product I and phospholipid.