Abstract
Mass changes in the various molecular species of phospholipids were determined after stimulation of human platelets with thrombin and collagen. Upon stimulation, every molecular species of phosphatidylinositol and phosphatidylserine was equally hydrolyzed, whereas the molecular species of phosphatidylcholine and diacyl- and alkenylacylphosphatidylethanolamine containing arachidonic acid were selectively hydrolyzed. At low Ca2+ concentrations, which result from mobilization of intracellular Ca2+ stores, phosphatidylinositol, phosphatidylcholine, and diacylphosphatidylethanolamine were hydrolyzed after stimulation with thrombin, whereas only phosphatidylinositol was hydrolyzed with production of thromboxane B2 after stimulation with collagen. At high Ca2+ concentrations, phosphatidylcholine and diacylphosphatidylethanolamine were hydrolyzed after stimulation with collagen, and phosphatidylserine and alkenylacylphosphatidylethanolamine were degraded after stimulation with both thrombin and collagen. [1-14C]Arachidonic acid was heterogeneously incorporated into the individual molecular species of the various phospholipid classes, indicating that the determination of mass is essential for an accurate picture of phospholipid hydrolysis. The data reported here indicate that the Ca2+ concentration affects the differential degradation of phospholipid molecular species in activated human platelets.
Highlights
Mass changes in the various molecular species of centrations of thrombin and collagen is controlled by thromphospholipids were determined after stimulation of boxane AP [8,9,10]
We report acomplete analysis of phospholipid breakdown during stimulation of human platelets with both graded after stimulation with both thrombin and col- thrombin and collagen by determining mass changes in the lagen. [l-14C]Arachidonic acidwas heterogeneously incorporated into the individual molecular species of the various phospholipid classes, indicating that the determination of mass is essential for an accurate picmolecular species of phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and diacyl- and alkenylacylphosphatidylethanolamine at different Ca2+concentrations
Each phospholipid class labeled with [1-'4C]204 prepared as described under "Experimental Procedures" and 18:0/180 phosphatidylcholine were hydrolyzed by phospholipase C reaction under the conditions described under "Experimental Procedures." Diradylglycerolswere extracted with ether andseparated by thin-layer chromatography with the solvent of hexane/diethyl ether/acetic acid (5050:l). 180/180 Diacylglycerol was determined as dinitrobenzoyldiacylglycerol by high-performance liquid chromatography as described under "Experimental Procedures." Approximately 40-160 nmol of radioactive phospholipids were used for the reaction
Summary
Each phospholipid class labeled with [1-'4C]204 prepared as described under "Experimental Procedures" and 18:0/180 phosphatidylcholine were hydrolyzed by phospholipase C reaction under the conditions described under "Experimental Procedures." Diradylglycerolswere extracted with ether andseparated by thin-layer chromatography with the solvent of hexane/diethyl ether/acetic acid (5050:l). 180/180 Diacylglycerol was determined as dinitrobenzoyldiacylglycerol by high-performance liquid chromatography as described under "Experimental Procedures." Approximately 40-160 nmol of radioactive phospholipids were used for the reaction. Data are given as themean values & S.E. ( n= 4)
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