Inhibition of phospholipase D of human platelets by protein tyrosine kinase inhibitors.
Protein tyrosine kinases (PTKs) of the src family are thought to play an important role in platelet signal transduction, but little is known about the targets of these enzymes in platelets. We determined that exposure of human platelets to pervanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in the activity of phospholipase D (PLD), an enzyme that might be involved in signaling events leading to aggregation or secretion. To further investigate whether tyrosine phosphorylation is a step in the pathway of activation of PLD in response to thrombin, we tested the effects of a series of PTK inhibitors on the activity of platelet PLD. PLD was activated in response to 0.3 U/ml thrombin, and this activation was reduced by several of the PTK inhibitors, especially genistein, methyl 2,5-dihydroxycinnamate (MDHC), ST271, and the tyrphostins A25 and A47. In saponin-permeabilized platelets, we observed a marked inhibition of GTP-gamma S-stimulated PLD by many of the PTK inhibitors, consistent with the possibility that PTKs are involved in the regulation of PLD activity by a G-protein or small GTP-binding protein. MDHC did not affect PLD activity in permeabilized cells, which suggests that this compound might inhibit PLD in intact platelets via another pathway. The inhibitors were also tested for their effects on the phosphorylation of a peptide substrate of src-family PTKs in a platelet membrane preparation and in permeabilized platelets. Several of the compounds partially inhibited peptide phosphorylation in the membrane preparation and in permeabilized platelets, most notably ST271, ST638, and tyrphostin A25.(ABSTRACT TRUNCATED AT 250 WORDS)
Martinson
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EA
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Scheible
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Presek
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P
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002396:Catechols / Q000494:pharmacology; D002934:Cinnamates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D019833:Genistein; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D008969:Molecular Sequence Data; D009570:Nitriles / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000097:blood; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000097:blood; D015398:Signal Transduction; D013343:Styrenes / Q000494:pharmacology; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology; D020032:Tyrphostins; D014638:Vanadates / Q000494:pharmacology
D002396:Catechols; D002934:Cinnamates; D007529:Isoflavones; D009570:Nitriles; D010455:Peptides; D013343:Styrenes; D020032:Tyrphostins; C076715:pervanadate; C086531:ST 271; C060152:tyrphostin 47; D014638:Vanadates; C067472:methyl 2,5-dihydroxycinnamate; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D010739:Phospholipase D; D013917:Thrombin; D019204:GTP-Binding Proteins