Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: inhibition by a selective inhibitor of phospholipase C.
We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner. The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer. A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity. MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619. Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using [32P]-phosphate and [14C]-arachidonic acid labeled platelets. PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol. Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes. MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner. An increase in phosphatidic acid was also observed in PLC-stimulated platelets. Up to 100 microM MDBA did not inhibit production of phosphatidic acid. PLC-treated platelets did not produce any TXB2. In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with [14C]-casein. PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of [14C]-casein, whereas more than 30% of [14C]-casein was hydrolyzed by trypsin. PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP. In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner. The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner. These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D010447:Peptide Hydrolases / Q000502:physiology; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010740:Phospholipases / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects*; D013929:Thromboxane B2 / Q000096:biosynthesis; D014213:Tretoquinol / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology*
D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D013929:Thromboxane B2; C024531:1-(2',4',5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; D000255:Adenosine Triphosphate; D010740:Phospholipases; D010738:Type C Phospholipases; D010447:Peptide Hydrolases; D014213:Tretoquinol; D000241:Adenosine; D002118:Calcium