PLATELET FUNCTION IN PLASMA AT PHYSIOLOGICAL CALCIUM CONCENTRATION. THE USE OF VAC AS AN ANTICOAGULANT

We have purified from human placenta an anticoagulatory protein (VAC, Mr= 32,000), which inhibits phospholipid dependent procoagulant reactions through a calcium dependent high affinity binding to procoagulant phospholipids..When tested in citrated platelet rich plasma (_cPRP), VAC does not affect platelet aggregation and secretion in reponse to ADP, collagen and thrombin. Purified VAC (0.05 μM, final concentration; f.c.) was used as an anticoagulant to prepare PRP (VAC-PRP). Platelet aggregation (optical density method) and release of newly absorbed ¹⁴C-sero-tonin (5HT) in response to adenosine diphospate (ADP) were measured and compared with platelet responses in_cPRP obtained simultaneously fromthe same donor.

The response of ADP stimulated platelets in VAC-PRP differs strikingly from that in cPRP. In the latter, platelets react with a dose-dependent primary aggregation, followed by a thrombin (Ila)-independent second wave of aggregation associated with 5HT-secre-tion.Platelets in VAC-PRP, however, demonstratean increased primary aggregation in responseto ADP, which is followed by a IIa-mediated second wave of aggregation and 5HT-secretion.Increasing the VAC concentration does not affect the primary aggregation response, but delayed the IIa-dependent secondary events in a dose-dependent way. At 0.5 μ M VAC, platelets react to ADP (10 μM f.c.) with reversible aggregation only. No matter this high ADP-dose, secretion reaction does not occur. At this VAC concentration, epinephrine (5 μM f.c.) does not cause aggregation and 5HT-release at all, whereas in_cPRP both reactions occur quite readily. Although in VAC-PRP, epinephrine retains its synergistic effect on ADP to aggregate platelets, no 5HT release was ever observed and the resulting aggregation was alwaysreversible It is concluded that VAC is a suitable anticoagulant to investigate platelet function in the presence of physiological calcium concentration. Since platelet aggregation and release appear very different from results obtained in the usual way (cPRP, low calcium concentration) the physiological meaning of this latter method needs re-evaluation. Finally, our results cast severe doubt on epinephrine as an important platelet stimulant under physiological conditions.