Human platelet activation by bacterial phospholipase C: mechanism of inhibition by flurazepam.
We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes platelet activation possibly by inducing turnover of phosphoinositides and phosphorylation of a 47,000 Dalton protein (P47). Moreover, only 15 microM and 11 microM flurazepam inhibits PLC-induced platelet aggregation and serotonin secretion by 50% respectively. This study was conducted to better understand the mechanism of platelet activation by PLC and its inhibition by flurazepam. Incubation of (14C)-arachidonic acid labelled platelets with PLC produced diacylglycerol in a time- and concentration-dependent manner. Flurazepam did not inhibit diacylglycerol production by PLC. Paranitrophenolphosphorylcholine and prostaglandin E1 inhibited diacylglycerol production by 75% and 20% respectively. In a platelet-free system PLC hydrolyzed 14C-choline-phosphatidylcholine (14C-PC) in a time- and calcium ions-dependent manner. Flurazepam had no effect on PLC-induced hydrolysis of 14C-PC. Platelet cytosolic fraction (PCF), containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed (3H-inositol)-phosphatidylinositol (3H-PI) in a platelet-free system. Flurazepam did not inhibit hydrolysis of 3H-PI by PCF. Phospholipase C caused phosphorylation of P47 in 32P-labelled platelets. Flurazepam did not block phosphorylation of P47 in the first three minutes and had very little inhibitory effect by five minutes. However, flurazepam completely blocked phosphorylation of P47 by seven minutes. Platelet aggregation induced by ionomycin, a calcium ionophore, was completely inhibited by 100 microM flurazepam whereas platelet aggregation induced by 12-O-Tetradecanoylphorbol-13-acetate (TPA), which mimics the action of diacylglycerol, was partially inhibited by 300 microM flurazepam. These findings suggest that PLC induced platelet activation depends, at least in part, on diacylglycerol production and phosphorylation of P47. These data also suggest that flurazepam does not inhibit PLC-induced platelet activation by inhibiting: (a) the production of diacylglycerol from phosphatidylcholine; and (b) the action of PI-PLC on phosphatidylinositol. The ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that flurazepam is able to block platelet activation by inhibiting the increase in free cytosolic calcium ions in platelets or by inhibiting a step subsequent to the rise in intraplatelet calcium ions.
D000527:Alprostadil / Q000494:pharmacology; D001696:Biomechanical Phenomena; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology; D001798:Blood Proteins / Q000378:metabolism; D003016:Clostridium perfringens / Q000032:analysis*; D004075:Diglycerides / Q000096:biosynthesis; D004987:Ethers / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005479:Flurazepam / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D015759:Ionomycin; D010713:Phosphatidylcholines / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D001798:Blood Proteins; D004075:Diglycerides; D004987:Ethers; D010713:Phosphatidylcholines; D010750:Phosphoproteins; C039077:platelet protein P47; D015759:Ionomycin; D010738:Type C Phospholipases; D000527:Alprostadil; D005479:Flurazepam; D013755:Tetradecanoylphorbol Acetate