Phospholipase D in human platelets: Presence of isoenzymes and participation of autocrine stimulation during thrombin activation

Abstract

Phospholipase D (PLD), which hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline, is present in human platelets. Thrombin and other agonists have been shown to activate PLD but the precise mechanisms of activation and PLDs role in platelet activation remains unclear. We measured thrombin-stimulated PLD activity in platelets as formation of phosphatidylethanol. Since no specific PLD inhibitors exist, we investigated possible roles for PLD in platelets by correlating PLD activity with platelet responses such as thrombin-mediated secretion and F-actin formation (part of platelet shape change). Extracellular Ca2+ potentiated thrombin-stimulated PLD, but did not stimulate PLD in the absence of thrombin. Thrombin-induced PLD activity was enhanced by secreted ADP and binding of fibrinogen to its receptors. In contrast to others, we also found a basal PLD activity. Comparison of time courses and dose responses of platelets with PLD showed many points of correlation between PLD activation and lysosomal secretion and F-actin formation. The finding of different PLD activities suggested that different PLD isoenzymes exist in platelets as reported for other cells. Here we present evidence for the presence of both PLD1 and PLD2 in platelets by use of specific antibodies with immunoblotting and immunohistochemistry. Both isoforms were randomly localized in resting platelets, but became rapidly translocated to the proximity of the plasma membrane upon thrombin stimulation, thus indicating a role for PLD in platelet activation.

Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances

“…This is in accordance with experiments performed by Vorland and Holmsen who found no effect on lysosomal secretion when using the specific TXA2 inhibitor SQ 29.548 in thrombin-stimulated platelets [27] and Chronos et al who compared the exposure of CD63 and P-selectin in healthy subjects before and after ingestion of ASA [31]. McKenzie et al reported that the spontaneous expression of LAMP-1, LIMP and P-selectin was reduced in platelets treated with ASA in vitro [32].…”

“…A reduction in NAG release has previously been found upon pre-incubation of platelets with the ADP-removing system, creatine phosphate/creatine phosphokinase [27]. Moreover, we observed that thrombin-induced aggregation was not reduced by cangrelor.…”

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

“…While PLD1 has a low basal activity and is readily activated by PKC and small GTPases of the adenosine diphosphate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Elvers et al PLD as negative regulator of platelets 5 marginally induced by a variety of activators. In platelets, both PLD isoforms are present [23].…”

Pharmacological Inhibition of Phospholipase D Protects Mice From Occlusive Thrombus Formation and Ischemic Stroke—Brief Report

“…8,10 In platelets, both PLD isoforms are present and become activated upon stimulation with various agonists. 9,11 Recently, we demonstrated that PLD1 contributes to GPIb-dependent platelet integrin activation, whereas absence of both PLD isoforms leads to a defect in α-granule release in addition to the defects observed in PLD1-deficient platelets. 12 Moreover, Pld1 −/− /Pld2 −/− mice are protected from arterial thrombosis and ischemic stroke but do not exhibit hemostatic defects.…”

“…The 2 mammalian PLD isoforms, PLD1 and PLD2, are widely expressed and are 50% identical in amino acid sequence. 8,9 PLD1 and PLD2 locate to distinct intracellular membrane compartments and have been implicated in many important signaling pathways and cell biological processes. 8,10 In platelets, both PLD isoforms are present and become activated upon stimulation with various agonists.…”