Mechanisms of Reduced Protein Phosphorylation Induced by Thrombin in SHRSP Platelets: Thrombin-induced Diacylglycerol Formation

Aggregation and secretion of washed platelets from stroke-prone spontaneously hypertensive rats (SHRSP) were greatly reduced by the development of the hypertension compared with those of platelets from age-matched normotensive Wistar-Kyoto rats (WKY). Concomitantly, thrombin-induced phosphorylation of the 47kDa protein in SHRSP platelets was significantly decreased. However, TPA-induced aggregation, secretion and 47kDa protein phosphorylation in SHRSP platelets were similar to those in WKY platelets. Thus, it was suggested that protein kinase C activity and its substrate are normally present in SHRSP platelets, and defects are in the receptor-mediated activation of protein kinase C (Umegaki et al. FEBS 196, 139, 1986). Since diacylglycerols (DG) and Ca²⁺ reportedly play important roles in 47kDa protein phosphorylation in human platelets, the mass content of phosphatidylinositol (PI) and thrombin-induced DG formation were investigated in 12-16 weeks old SHRSP platelets in comparison with age-matched WKY platelets.<br>Phospholipids were extracted from platelets by CHCI₃: CH₃OH (1:2) and resolved by HPTLC using CHCI₃: McOH: CH₃NH₂ (63:35:10). Each phospholipid was determined either by fluoro-metrically or by phosphorus assay. The content of PI in SHRSP platelets was approximately 10% lower than the content of PI in WKY platelets; there is no difference in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents. However, incorporation of ³H-arachidonic acid (AA) into PC was significantly higher in SHRSP platelets while those into PI and PE were lower in comparison with WKY platelets.<br>Aggregation responses of SHRSP platelets to lower doses of thrombin were much smaller than those in WKY platelets. With these concentrations of thrombin no significant formation of DG was observed in both platelets. In supramaximal doses of thrombin there was no difference in aggregation of SHRSP and WKY platelets; DG formation was apparently observed and it was lower in SHRSP platelets than in WKY platelets. The maximal formation of DG occurred 15sec after a thrombin stimulation.<br>It is concluded that reduced DG formation from PI is not responsible for attenuated responses of aggregation, secretion, and protein phosphorylation to thrombin in SHRSP platelets. With other evidence, defective Ca²⁺ functions in stimulus-response coupling appear to be an underlying mechanism for the hypofunctions in SHRSP platelets.