A method to prepare degranulated human platelets: Use for studies of platelet aggregation and ca2+ mobilization

F. M. Pulcinelli, J. L. Daniel, S. Riondino, P. P. Gazzaniga, M. A. Russo, L. Salganicoff

Research output: Contribution to journalArticle

Abstract

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. [14C] 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 H2/thromboxane A2, 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 Ca2+ studies and we find that the mixture of agonists produces an increase in the intracellular calcium concentration to about 1 μM.

Original languageEnglish
Pages (from-to)212-218
Number of pages7
JournalPlatelets
Volume4
Issue number4
DOIs
Publication statusPublished - 1993

ASJC Scopus subject areas

  • Hematology
  • Cell Biology

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