GTP gamma S-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate soluble phospholipase C from human platelets requires soluble GTP-binding protein.

GTP-binding activity was fractionated into two peaks (GI and GII) by chromatography on heparin-agarose. GTP-dependent PLC activity eluted as a single peak, which co-chromatographed with GTP-binding peak GII. Rechromatography of peak GII on heparin-agarose, in the presence of 0.5% sodium cholate, resulted in separation of PLC and GTP-binding activities, and loss of GTP-dependent PLC activity. Recombining fractions containing PLC and GTP-binding activities restored GTP-dependent PLC activity. A specific GTP-binding protein of 29,000 daltons was identified in peak GII by Western blotting of column fractions with [alpha-32P]GTP. These results demonstrate that the soluble phospholipase C from human platelets is regulated by GTP S-binding protein (G29).

Inhibition of fibrinogen receptor expression and serotonin release by leupeptin and antipain.

Human platelet agonists such as thrombin, ADP, and collagen stimulate the rapid expression of fibrinogen receptors. In other cell types, calcium-activated proteases have been suggested to participate in the mechanism of expression of cell surface receptors (Lynch, G., and Baudry, M. (1984) Science 224, 1057-1063). In platelets the majority of the neutral protease activity is calcium-activated protease. We examined the effects of leupeptin and antipain, two calcium-activated protease inhibitors, on the expression of platelet fibrinogen receptors. These inhibitors abolished thrombin and ADP-induced fibrinogen binding. This inhibition required the addition of leupeptin or antipain prior to the agonist and was not due to displacement of fibrinogen from its receptor or inhibition of agonist binding to platelets. Leupeptin and antipain also inhibited fibrinogen-independent thrombin-stimulated release of serotonin. These results are discussed in relation to the involvement of calcium-activated protease in early events of platelet activation.

Studies on the nature of the human platelet alloantigen, PlA1: localization to a 17,000-dalton polypeptide.

We have examined the biochemical properties of the human platelet alloantigen system, PlA, using a preparation enriched in plasma membrane glycoproteins (GPs) IIb and IIIa as the starting material. After confirming that GPIIIa contains the PlA epitope, attempts were made to distinguish the two allelic forms of PlA (A1 and A2) using electrophoretic techniques. Whereas no difference could be discerned in the mol. wt of GPIIIa extracted from A1/A1, A1/A2 or A2/A2 platelets by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis revealed a reproducible difference in the isoelectric point of GPIIIa derived from A2/A2 individuals. Treatment of GPIIIa with a combination of exo- and endoglycosidases resulted in apparently complete deglycosylation of the molecule, as indicated by its co-migration with chemically deglycosylated GPIIIa in SDS-PAGE. The enzymatically deglycosylated protein retained its full ability to react with anti-PlA1 antibodies. Tryptic digestion of GPIIIa resulted in the generation of a number of smaller polypeptides, including one of 17,000 daltons, that contained the PlA1 determinant. These results suggest that the carbohydrate moieties of GPIIIa are unimportant to the expression of the PlA system, and that the charge difference between the two allelic forms of GPIIIa may reflect a subtle amino acid difference(s) within a 17K polypeptide region of the GP.

Reconstruction of platelet proteins into phospholipid vesicles. Functional proteoliposomes.

Platelet membrane glycoproteins IIb and IIIa were reconstituted into liposomes containing phosphatidylcholine. The reconstituted vesicles bound antiplatelet antibodies and showed specific binding to thrombin-activated platelets. Prostacyclin, a known inhibitor of thrombin-activated platelet aggregation, inhibited the binding of the proteoliposomes to thrombin-activated platelets. The reconstituted vesicles also specifically bound 125I-labeled fibrinogen. This binding was insensitive to ADP but dependent on calcium ions. These data indicate that platelet glycoproteins IIb and IIIa have been successfully reconstituted into phospholipid vesicles such that their behavior is similar to that in intact platelets.