TY - JOUR
T1 - A method to prepare degranulated human platelets
T2 - Use for studies of platelet aggregation and ca2+ mobilization
AU - Pulcinelli, F. M.
AU - Daniel, J. L.
AU - Riondino, S.
AU - Gazzaniga, P. P.
AU - Russo, M. A.
AU - Salganicoff, L.
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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.
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U2 - 10.3109/09537109309013220
DO - 10.3109/09537109309013220
M3 - Article
AN - SCOPUS:0027194255
VL - 4
SP - 212
EP - 218
JO - Platelets
JF - Platelets
SN - 0953-7104
IS - 4
ER -