Rapid Aequorin Loading into Platelets in the Presence of DMSO - Characteristics of the Responses (Changes in Light Transmission and in Calcium) to Various Agonists

Summary

We have looked at different parameters which could modify platelet behaviour during and after aequorin loading in the presence of DMSO. There is a decreased platelet reactivity in response to ADP, PAF and A-23,187 which appears to be mainly due to the exposure to EGTA during washing and loading and the 1 ml volume of the test suspension. All the studied agonists (including PMA) which elicit aggregation are able to induce an intracellular Ca²⁺ change detected with the aequorin probe. By contrast, epinephrine alone induces neither aggregation nor Ca²⁺ rise, but potentiates the responses to ADP. Different consecutive phases in Ca²⁺ changes after stimulation with ADP, PMA and A-23,187 can be evidenced. In the presence of external Ca²⁺, the second component of the Ca²⁺ change evoked with ADP is dependent on aggregation and the subsequent TXA₂ synthesis. When the external medium is Ca²⁺ depleted, the two Ca²⁺ peaks induced by ADP disappear whereas a Ca²⁺ rise persists (endogenous mobilization) with the other agonists, being independent of TXA₂ and ADP release. Ca²⁺ mobilization parallels activation with A-23,187 but not with low concentrations of thrombin.

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