Collagen increases cytoplasmic free calcium in human platelets

The role of changes in cytoplasmic free calcium in response to collagen was studied in human platelets loaded with the fluorescent calcium indicator, quin2. In the presence of 1mM external calcium, collagen caused a biphasic increase in cytoplasmic free calcium. In the absence of external calcium, there was a much smaller increase in cytoplasmic free calcium. These findings suggest that collagen increases cytoplasmic free calcium, partly by discharge of internal calcium, but mainly by stimulating calcium influx. Inhibition of cyclooxygenase by aspirin markedly reduced the second phase of the calcium response. Removal of ADP with apyrase resulted in complete inhibition of the second phase of the calcium response. The combination of apyrase and aspirin completely inhibited aggregation and the shape change caused by collagen. The calcium-entry blocking agent, verapamil, also inhibited the second phase of the calcium response to collagen. The increase in cytoplasmic free calcium is fast enough to be involved in the platelet response to collagen and these findings suggest that ADP and metabolites of arachidonic acid mediate the second phase of the calcium response to collagen.