Elevated cytosolic Ca2+ activates phospholipase D in human platelets.
We have examined the activation of phospholipase D in human platelets treated with alpha-thrombin. When incubated with 1-O-[9,10-3H2]hexadecyl-2-lysophosphatidylcholine (PtdCho) and 1-alkyl-[32P]lysoPtdCho for 2 h, platelets formed 3H/32P-labeled PtdCho in a ratio of 11:1. After incubation of such labeled platelets with alpha-thrombin for 5 min, increased accumulation of 3H/32P-labeled phosphatidic acid (PtdOH) was detected in the same ratio, indicating the action of phospholipase D. The Ca2+ ionophore A23187 and alpha-thrombin each stimulated the formation of labeled PtdOH as above in a time- and concentration-dependent manner, with only minor changes in labeled diglyceride. A23187 was able to cause increases in labeled PtdOH comparable to those observed with alpha-thrombin. beta-Phorbol 12,13-dibutyrate, an activator of protein kinase C, only slightly stimulated the accumulation of labeled PtOH. The protein kinase C inhibitor, staurosporine, totally blocked these changes but only slightly inhibited the increases in labeled PtdOH promoted by alpha-thrombin. These results suggest that an increase in intracellular Ca2+, rather than protein kinase C activity, is a major factor regulating phospholipase D in platelets exposed to alpha-thrombin. We have also examined the relative contributions of phospholipase D and diglyceride kinase (following phospholipase C action) to PtdOH accumulation in [32P]Pi-labeled platelets by comparing the 32P-specific radioactivities of PtdOH, PtdCho, and metabolic gamma-ATP in control and alpha-thrombin-exposed platelets. Based on these determinations, we conclude that 13 and 87% of incremental PtdOH in human platelets exposed to alpha-thrombin arises via phospholipase D acting on PtdCho and phospholipase C/diglyceride kinase, respectively.
Huang
R
R
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Kucera
G L
GL
Rittenhouse
S E
SE
D000470:Alkaloids / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D015539:Platelet Activation; D019311:Staurosporine; D013917:Thrombin / Q000378:metabolism
D000470:Alkaloids; D004075:Diglycerides; D010712:Phosphatidic Acids; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D010739:Phospholipase D; D013917:Thrombin; D019311:Staurosporine; D002118:Calcium