A method to prepare degranulated human platelets: use for studies of platelet aggregation and ca(2+) mobilization

A method for the preparation of a suspension of thrombin-degranulated human platelets is described. Two peptides (RGDS and GPRP) are used to prevent fibrinogen binding and consequent aggregation, and to prevent fibrin polymerization during thrombin activation. A mixture of creatine phosphokinase and creatine phosphate is used to remove ADP. Hirudin and TAMe are used to neutralize thrombin after the platelets have been activated. [(14)C] Serotonin and PF4 release and electron microscopy demonstrate that the preparation is completely degranulated. After all inhibitors are removed and fibrinogen added, the preparation aggregates rapidly to a mixture of agonists composed of ADP, epinephrine and the synthetic analog of prostaglandin H(2)/thromboxane A(2), U46619. ADP and epinephrine when added individually are both able to induce a clearly detectable aggregation, while U46619 induces only a shape change. The preparation is also suitable for intracellular Ca(2+) studies and we find that the mixture of agonists produces an increase in the intracellular calcium concentration to about 1 µM.