Elevated cytosolic Ca2+ activates phospholipase D in human platelets

• Published in: The Journal of biological chemistry 1991-01, Vol.266 (3), p.1652-1655
• Main Authors: RUSONG HUANG, KUCERA, G. L, RITTENHOUSE, S. E
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood Platelets - enzymology; Calcimycin - pharmacology; Calcium - physiology; Diglycerides - metabolism; Enzyme Activation; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; In Vitro Techniques; Phorbol 12,13-Dibutyrate - pharmacology; Phosphatidic Acids - metabolism; Phospholipase D - metabolism; Platelet Activation; Staurosporine; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52344-9

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.