Release of Choline Metabolites from Human Platelets: Evidence for Activation of Phospholipase D and of Phosphatidylcholine-specific Phospholipase C

Abstract

In aspirin-treated platelets labelled by preincubation with [(3)H]-choline, enhanced release of both [(3)H]-choline and [(3)H]-choline phosphate resulted from stimulation by collagen or thrombin. No such release accompanied stimulation by ADP, platelet-activating factor or adrenaline. Release of [(3)H]-choline phosphate was entirely dependent on aggregate formation whereas release of [(3)H]-choline was reduced but not eliminated, if aggregation was prevented. The properties of [(3)H]-choline and [(3)H]-choline phosphate release indicated that both collagen and thrombin induced activation of phospholipase D in the absence of aggregate formation. Such activation was augmented if aggregate formation occurred. Aggregation induced by these two agonists also caused activation of phosphatidylcholine-specific phospholipase C. These effects were prevented in the presence of staurosporine and could also be induced by addition of a synthetic 1,2-diacylglycerol indicating a role for protein kinase C.

Diacylglycerol Elevations in Control Platelets are Unaccompanied by Pleckstrin Phosphorylation. Implications for the Role of Diacylglycerol in Platelet Activation

“…They observed a monophasic [25], biphasic [24,271 or multiphasic [26] increase in diacylglycerol levels which were 2-4-fold greater than the stable control levels. We had embarked on an analysis of the molecular species of diacylglycerol formed to determine the precursor(s) and our findings are in agreement with those Petty et al [29]. However, Petty and Scrutton [29] found increases in choline and choline phosphate with platelet stimulation.…”

“…Several groups have reported on the kinetics of diacylglycerol formation associ-ated with thrombin [24-271 and arachidonic acid stimulation [28]. However, Petty and Scrutton [29] found increases in choline and choline phosphate with platelet stimulation. Nakashima et al [24] concluded from their analysis of the possible phospholipase C products, inositol and choline derivatives, that the inositol phospholipids were the only source of diacylglycerol in platelets.…”

Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins

“…We therefore investigated the activation of PLD in stimulated platelets labeled with [ 3 H]arachidonic acid. In contrast to the situation in neutrophils, PAF did not lead to PLD activation in either patient or control platelets (data not shown), a ®nding also made by others (22). For this reason, we chose to stimulate platelets with thrombin, which is known to activate PLD in platelets (19) and, like PAF, also has been shown to induce a decreased aggregatory response in PV platelets (25).…”

Diacylglycerol Elevations in Control Platelets are Unaccompanied by Pleckstrin Phosphorylation. Implications for the Role of Diacylglycerol in Platelet Activation

“…Nakashima et al [24] concluded from their analysis of the possible phospholipase C products, inositol and choline derivatives, that the inositol phospholipids were the only source of diacylglycerol in platelets. However, Petty and Scrutton [29] found increases in choline and choline phosphate with platelet stimulation. We had embarked on an analysis of the molecular species of diacylglycerol formed to determine the precursor(s) and our findings are in agreement with those Petty et al [29].…”

“…However, Petty and Scrutton [29] found increases in choline and choline phosphate with platelet stimulation. We had embarked on an analysis of the molecular species of diacylglycerol formed to determine the precursor(s) and our findings are in agreement with those Petty et al [29]. We also found unexpectedly that, in our hands, the mass levels of diacylglycerol in control platelets varied widely, occasionally as much as in thrombintreated platelets.…”