PLATELET-FIBRIN CLOTS FORMED BY THROMBIN SELECTIVELY RETAIN PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIF2)

Abstract

It is established that stimulation of human platelets with thrombin for 60 s in the absence of fibrinogen increases the amount of PIPp compared with unstimulated controls (4.7 ± 0.24 nmol/109 plat, vs 3.83 ± 0.14 nmol/109 plat., p<0.01, n=8). However, stimulation with thrombin for 60 s in the presence of fibrinogen causes a large decrease in the amount of PIP2 that can be extracted with acidified chloroform/methanol compared with unstimulated controls (1.62 ± 0.39 nmol/109 plat, vs 3.84 ± 0.44 nmol/109 plat., p<0.001, n=6). Stimulation of rabbit platelets with thrombin in the presence of fibrinogen also decreases the amount of extractable PIP2 (60% at 60 s, p<0.001, n=8). Similar decreases in amount can not be demonstrated for phosphatidylinositol 4-phosphate, phosphatidylinositol, phosphatidic acid or phosphatidylcholine under the same conditions, indicating that the decrease is specific for PIP2. With rabbit platelets, polymerized fibrin formed by reptilase, which does not stimulate platelets or induce clot retraction, does not cause the decrease in extractable PIP, (3.06 ± 0.05 nmol/109 plat, were extracted compared with 3.182 ± 0.07 nmol/109 plat, without reptilase). However, stimulation of rabbit platelets with ADP in the presence of polymerizing fibrin formed by reptilase causes | larger decrease in extractable PIP2 (to 2.54 ± 0.19 nmol/109 plat., p<0.05, n=4) than is caused Dy ADP and fibrinogen alone (to 2.87 ± 0.06 nmol/10 plat., p<0.05, n=4). Inhibition by glycyl-L-prolyl-L-arginyl-L-proline of polymerization of fibrin formed by the action of thrombin prevents the large.decrease in the amount of extractable PIP2 (4.37 ± 0.30 nmol/10 plat, were extracted) from human platelets. These results indicate that the interaction of polymerizing fibrin with stimulated platelets is required for the decrease in PIP2. The decrease in extractable PIP2 seen with polymerizing fibrin can not be explained by increased degradation of PIP2 to IP3 or PIP. Thus, when human or rabbit platelets are stimulated with thrombin in the presence of fibrinogen, an association of polymerizing fibrin with the stimulated platelets occurs that leads to decreased extractability of PIP2. This may mean that PIP2 forms a specific association with pratelet proteins that are involved in clot retraction.