Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Abstract

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta- thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.

The P2 Receptors in Platelet Function

“…52 The P2Y 12 receptor is also involved in potentiation of platelet secretion independently of TXA 2 generation and macroaggregate formation. 76,87,88 The P2Y 12 receptor also mediates the stabilization of platelet aggregates induced by thrombin 1,89,90 or TXA 2 . 58 The P2Y 12 receptor is coupled to inhibition of adenylyl cyclase activity through activation of a Ga i2 G protein subtype.…”

“…116 These pathways are involved in the long recognized role of ADP as a stabilizing factor of thrombin-induced platelet aggregates. 1 Interestingly, although in many cases adrenaline is able to rescue the P2Y 12 -deficient function, its ability to restore the stabilization of a thrombus was minimal in human platelets 1 or only partial in the P2Y 12 knockout mice. 78 These observations reinforce the unique role of the P2Y 12 receptor in the stabilization of the thrombus.…”

“…Adenine nucleotides play a crucial role in hemostasis and thrombosis. For proof, ADP by itself is an aggregating agent and a necessary cofactor for normal activation of platelets by strong agonists such as thrombin and collagen [1][2][3] or weak agonists such as serotonin, 4 adrenaline, 5 and chemokines. 6,7 Inhibitors of ADPinduced platelet aggregation are effective antithrombotic drugs.…”

Platelet Aggregation and Adenosine Diphosphate/Adenosine Triphosphate Receptors: A Historical Perspective

“…ADP also stabilizes aggregates formed by strong agonists such as thrombin. 19,20 Trumel et al 20 have attributed this effect to late activation by released ADP of phosphoinositol 3-kinase.…”

“…3,16,17 This synergistic effect of ADP is also apparent with low concentrations of stronger agonists, such as collagen, thrombin, arachidonic acid, and thromboxane A 2 , which in higher concentrations can cause aggregation in the absence of releasable ADP. 11,18 Stabilization of platelet aggregates by released ADP, 19,20 as well as the recognition of storage pool disease (lack of dense granule contents) as a cause of some bleeding abnormalities, 21 emphasized the importance of ADP in hemostasis and the contribution ADP makes to arterial thrombosis.…”