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

Stephen S. Navran, Karl Romstedt, Jane Chang, Duane Miller, Dennis R. Feller, Huzoor-Akbar

Research output: Contribution to journalArticle

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Abstract

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 μM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 μM 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 μM) 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 μM 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 μM 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 PLCinduced 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.

Original languageEnglish (US)
Pages (from-to)499-510
Number of pages12
JournalThrombosis Research
Volume33
Issue number5
DOIs
StatePublished - Mar 1 1984
Externally publishedYes

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Phosphatidic Acids
Platelet Activation
Type C Phospholipases
Phosphatidylinositols
Hydrolysis
Blood Platelets
Thromboxane B2
Caseins
Platelet Aggregation
Arachidonic Acid
Adenosine
Adenosine Diphosphate
Tretoquinol
Peptide Hydrolases
Adenosine Triphosphate
Calcium
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Clostridium perfringens
Thrombin
Trypsin

All Science Journal Classification (ASJC) codes

  • Hematology

Cite this

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. / Navran, Stephen S.; Romstedt, Karl; Chang, Jane; Miller, Duane; Feller, Dennis R.; Huzoor-Akbar.

In: Thrombosis Research, Vol. 33, No. 5, 01.03.1984, p. 499-510.

Research output: Contribution to journalArticle

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