Platelet Retention in Glass Bead Columns: Further Evidence for the Importance of ADP

Plasma of normal heparinized blood contained 0.284 µM ± SD 0.097 (ADP + ATP) with an ATP:ADP ratio of 2.5:1. Plasma from thrombocytopenic blood contained only 0.106 µM ± 0.073 (ADP + ATP). Blood with normal platelet retention released 0.234 µM ± 0.187 (ADP + ATP) during passage through a glass bead column, with an ATP:ADP ratio of 1.6:1. Significantly less was released in blood with low retention, i.e., samples from patients with von Willebrand’s disease, thrombasthenia, or thrombocytopenia, and some samples from normal individuals. Thus, nucleotides in the plasma of pre- and postcolumn blood appear to be derived from platelets; their release within glass bead columns is closely associated with normal platelet retention. Since release occurred at room temperature and was not prevented by acetylsalicylic acid or accompanied by measurable release of ¹⁴C-serotonin, the classic release reaction may not have been responsible. The low retention in platelet-rich plasma was variably increased by adding 0.5 µM ADP, an increase at least partly due to trapping of preformed aggregates. Retention in undisturbed blood was markedly inhibited by creatine phosphokinase with creatine phosphate (CPK-CP) and moderately inhibited by apyrase I (ATPase:ADPase 0.8:1) at an ADP-removing activity between 1 and 5 U/ml, indicating that ADP is essential for retention. At less than 1 U/ml, both apyrase I and II (ATPase: ADPase 2.8:1) enhanced retention in undisturbed blood, but CPK-CP was still inhibitory. These results suggest that enhancement is due to conversion of released ATP to ADP, as shown to occur in studies of platelet aggregation with ATP and ADP. At less than 1 U/ml, all three enzymes protected against the inhibitory effect of disturbance; this protection was marked with apyrase II, moderate with apyrase I and slight with CPK-CP. These observations provide additional evidence that ADP is responsible for the low retention caused by disturbance of the blood.