The effect of D 2O and chlortetracycline on ADP-induced platelet shape change and aggregation

Abstract

The role of intracellular Ca 2+ in platelet shape change (SC) and aggregation was investigated by exchanging platelet H 2O for D 2O. Human platelets were isolated by D 2O-albumin density gradient centrifugation and resuspended in D 2O Tyrode's (37°C). Such treatment inhibited SC and aggregation in response to 10 −4 or 10 −5 M ADP, but did not inhibit the extent of aggregation induced by 10 −5 or 10 −6 M epinephrine. When platelets were incubated (37°C) with 500 μM chlortetracycline (CT), a divalent cation chelator, SC and aggregation in response to 10 −5 M ADP were inhibited. This inhibition could not be reversed by the addition of 5 mM CaCl 2. CT incubation also resulted in inhibition of 10 −5 M epinephrine-induced aggregation, but, in this case, addition of CaCl 2 (5 mM) completely restored the epinephrine response to control levels. The above results are consistent with the contention that ADP initiates SC and aggregation by triggering an increase in cytoplasmic Ca 2+.