Kinetics of thrombin-induced release and activation of platelet factor V

Abstract

The kinetics of thrombin-induced platelet factor V activation were studied in suspension of washed human platelets. The effect of thrombin in stimulating the release reaction could be separated from its effect on factor V activation by use of a potent inhibitor of the release reaction, the prostacyclin analogue ZK 36374. When platelets were incubated with ZK 36374 prior to stimulation with thrombin, the amount of ZK 36374 required to inhibit 50% of factor V, formation was 15 pM. ZK36374 at a final concentration of 1 nM was found to block instantaneously and completely the release of factor V,, whereas it has no effect neither on platelet factor V activation nor on the factor V, assay. By varying the time interval between the addition of thrombin (0.5 nM) and ZK36374 to suspensions of 4.6 x lo6 platelets/ml the rate of factor V release was found to be 12 pM factor V/min. In the absence of ZK36374 the total amount of factor V released was 8 pM, whereas Triton X-100-treated platelets gave 13 pM factor V. It appeared that the amount of factor V that could be released was dependent on the thrombin concentration. Maximum release was obtained at 1 nM thrombin. The rate of factor V release increased in proportion to the thrombin concentration. The rate of factor V activation was found to be proportional to the thrombin concentration as well as to the amount of released factor V. When 4.6 x lo6 platelets/ml were activated by 0.5 nM thrombin, the rates of factor V activation were found to be 0.3 pM and 1.2 pM factor V,/min at 20% and 90% completion of the release reaction. Therefore, the rate of factor V release was at least one order of magnitude faster than the rate of factor V activation. The kinetics of thrombin-induced platelet factor V activation were compared to those of plasma factor V activation in platelet-rich and platelet-free plasma. The results clearly demonstrate that platelets have no effect on the rate of factor V activation and that the kinetics of plasma factor V activation are identical to those of platelet factor V activation.Thrombin-activated platelets are essential to the normal hemostatic mechanism. Two types of procoagulant activity from activated platelets have been recognized. In the first place factor V is released and activated during stimulation by thrombin [l, 21 and secondly the altered membrane of platelets activated by thrombin plus collagen provides a procoagulant surface [3, 41. These two events enable factor X, to function in a platelet-bound complex of factor V, and factor X,, the so-called prothrombinase, thus causing a dramatic amplification of the thrombin-generating capacity of factor X, [5, 61. Factor V occurs as a plasma protein as well as a protein present in the alpha granules of platelets.The question of the physiological importance of platelet factor V over plasma factor V in affecting the efficiency of the hemostatic process has been recently addressed by Tracy et al. [7]. These authors described an inherited factor V disorder and the association of a significant bleeding...

Plasmin accelerates platelet‐dependent prothrombinase formation without activating the platelets

“…Additionally, neither 10 M hirudin nor 10 M r-TAP had any effect on the amidolytic activities of plasmin towards the three substrates. Thrombin translocates factor V from its storage granules to the surface of platelets where it is activated by thrombin to support prothrombinase formation on the platelets (Baruch et al, 1986;Wyschock et al, 1994). Plasmin did not directly activate prothrombin in platelet-rich plasmas in this study, in contrast to the proposal by Seitz et al (1993).…”

“…Activation of platelets by -thrombin results in the translocation of factor V from the -granules to the surface of platelets where it is activated by -thrombin (Baruch et al, 1986;Wyshock et al, 1994), and platelets are the principal source of factor V when prothrombinase assembles in platelet-rich plasma (Liu et al, 1994a). Whether plasmin increased the translocation of factor V from the -granules of thrombin-stimulated platelets to enhance prothrombinase assembly was then explored.…”

“…Platelet activation and platelet-dependent prothrombin activation are linked processes since thrombin activates platelets to provide factor Va and coagulant surfaces for prothrombinase to assemble on (Baruch et al, 1986;Tracy, 1988;Coller, 1990;Winters et al, 1991;Mann et al, 1992;Liu et al, 1994a). The present study determined whether plasmin directly activates platelets to increase prothrombin activation in recalcified platelet-rich plasmas.…”

Mode of Action of Unfractionated and Low Molecular Weight Heparins on the Generation of Thrombin in Plasma

“…The lat ter process consists of shedding of vesicles that exhibit phosphatidylserine at the out side and of flip-flop of the platelet mem brane [32], Factor V that is released is acti vated by the thrombin present. It has been shown that this activation is slow as com pared with the release reaction [29], Because of the presentation of factor Va and of procoagulant phospholipid, the con ditions for prothrombin conversion become much more favorable, and a burst of throm bin occurs. We could demonstrate that espe cially the availability of phospholipids is re sponsible for the thrombin burst, although platelet factor V does contribute [58].…”

“…The amount of factor V seques tered in the platelets is roughly 20% of the amount present in the plasma [28]. Throm bin, that causes the release reaction, will also activate the released factor V. It has been shown that this activation rather than the release reaction itself is the rate-limiting fac tor for the generation of factor V activity from triggered platelets [29], The concentra tion of factor V in platelet-poor plasma is about 25 nM whereas that of its partner, fac tor X, is around 200 nM. This may lead one to think that the contribution of platelet fac tor V may be important in vivo.…”