The differences in Ca(2+)-sensitivity of protein kinase C in platelets from Wistar Kyoto rat and stroke-prone spontaneously hypertensive rat.

Thrombin-induced phosphorylation of 47 kDa protein (P47) in platelets, a substrate of protein kinase C (PKC), was defective in stroke-prone spontaneously hypertensive rats (SHRSP) (Hypertens. 14, 304-315, 1989). Platelet PKC from SHRSP and Wistar Kyoto rats (WKY) was partially purified and Ca(2+)-sensitivity of PKC activity was examined to understand the defect in the protein phosphorylation. When the platelets from SHRSP and WKY were homogenized in a buffer containing 10 mM EGTA and 2 mM EDTA, approx. 80% of PKC was in the cytosol fraction. PKC in this fraction was purified by DE52 and hydroxyapatite column chromatography. Both platelet PKC preparations contained only PKC-alpha, and there was no significant difference in the Ca(2+)-dependency of activity between them. When the platelets from SHRSP and WKY were homogenized in a buffer containing 10 microM CaCl2, 90% of PKC was found to be bound to the membrane. PKC was extracted from the membrane with a buffer containing 2.5 mM EGTA and 2.5 mM EDTA, and purified by DE52 column chromatography. PKC from WKY platelets eluted as a single peak whereas that from SHRSP platelets eluted as two peaks (peak 1 and peak 2). Ca(2+)-sensitivity of peak 1 PKC was much lower than that of WKY PKC. In contrast, the Ca(2+)-sensitivity of peak 2 PKC appeared to be slightly higher than that of WKY PKC. The specific activity of peak 2 PKC was 4% to 5% of that of peak 1 and WKY PKC. These results suggest that there are two different types of PKC, normal and low Ca(2+)-sensitive in SHRSP platelets. Defective P47 phosphorylation in SHRSP platelets might be attributable to the occurrence of this low Ca(2+)-sensitive PKC.