The role of the phosphatidylinositol turnover in 12-hydroxyeicosatetraenoic acid generation from human platelets by Escherichia coli alpha-haemolysin, thrombin and fluoride.

Activation of human platelets with either the Escherichia coli alpha-haemolysin or thrombin, or with pharmacological agonists such as sodium fluoride (NaF), Ca-ionophore A23187 or phorbol myristate acetate (PMA), induced a similar pattern of serotonin release, unlike 12-hydroxyeicosatetraenoic acid (12-HETE) generation. In the presence of neomycin (0.1, 1, 10 mM), an inhibitor of the phosphatidylinositol-specific phospholipase C (PIP2-PLC), the E. coli alpha-haemolysin-induced 12-HETE generation was enhanced up to threefold in a dose-dependent manner. 12-HETE generation by NaF and thrombin was slightly inhibited at high neomycin concentrations (10 mM). Treatment of human platelets with E. coli alpha-haemolysin induced a different activation pattern of PIP2-PLC and phosphatidylinositol4-kinase (PI4-kinase), compared to NaF and thrombin. Haemolysin treatment resulted in a down-regulation of PIP2-PLC and PI4-kinase enzymatic activities by 20 +/- 9/40 +/- 8% compared to unstimulated cells; the decrease in enzymatic activities was observed within 2 min of stimulation and was still apparent after 60 min of stimulation. In NaF- and thrombin-stimulated platelets, dose- and time-dependent increases in PIP2-PLC (by up to 21 +/- 10%, 34 +/- 11%, respectively) and PI4-kinase (by up to 71 +/- 18, 54 +/- 14) activities were measured. Maximal enzymatic activities were reached after 5-20 min of stimulation (NaF, thrombin) followed by a decline to baseline levels (thrombin) or below baseline levels (NaF). Our results indicate that the phosphatidylinositolphosphate metabolism plays an important role in the regulation of 12-HETE release from human platelets by E. coli alpha-haemolysin.