HC, Lindhout T: Kinetics of thrombin induced release and activation of platelet factor V

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 instan-taneously 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 o