ENHANCED THROMBIN-INDUCED AGGREGATION AND INOSITOL TRISPHOSPHATE FORMATION OF PLATELETS FROM SPONTANEOUSLY HYPERCHOLESTEROLEMIC RATS

Platelets from rats with diet-induced hypercholesterolemia are hypersensitive to thrombin through a pathway independent of released ADP or thromboxane A₂ (TXA₂) formation. We examined if platelets from rats with spontaneous hypercholesterolemia (HC) are similarly hypersensitive. HC rats (plasma cholesterol: 130±4 mg/dl, n=15) were compared with their normocholesterolemic genetic controls (NC) (87±4 mg/dl, p<0.001, n=16). Total cholesterol/109 platelets was not different between the groups (HC: 0.314±0.032 μmole, n=7; NC: 0.357±0.046 ymole, n=7). Washed platelets were prelabelled with ¹⁴c-serotonin. In the presence of aspirin (to inhibit TXA₂ formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (22±2%, n=ll) than NC platelets (10±4%, n=12, p<0.01) in response to thrombin (0.065 U/ml); ¹⁴C release was not different. Thrombin causes inositol mono-, bis-, and trisphosphate (IP, IP₂, IP₃) formation from phosphoinositides (PI, PIP, PIP₂ respectively) in rabbit platelets; IP3 may be involved in Ca⁺⁺ mobilization and the release of granule contents. We examined if enhanced inositol phosphate formation was associated with hypersensitivity of HC platelets. Platelets were prelabelled with 3H-inositol and stimulated with thrombin (0.057 U/ml) for 30 sec in the presence of aspirin and CP/CPK. Li⁺ (20 mM) was used to prevent degradation of inositol phosphates to inositol. ³H-IP, IP₂ and IP₃ were isolated by ion-exchange chromatography. The increase in radioactivity (dpm/10⁹ platelets) in IP₂ and IP₃ following thrombin stimulation was greater in HC platelets (IP₂: 2210±160, n=4; IP₃: 1430±180, n=4) than in NC platelets (IP₂: 660±150, n=4, p<0.001; IP₃: 490±100, n=4, p<0.01); IP was not different.

Thus platelets from spontaneously HC rats are hypersensitive to thrombin independently of released ADP or TXA₂ formation. This hypersensitivity is associated with only moderate increases in plasma cholesterol and no detectable increase in total platelet cholesterol. Enhanced labelling of IP₃ may indicate that enhanced activity of the pathways leading to IP₃ formation is associated with this hypersensitivity.