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

Abstract

Several laboratories have reported that diacylglycerol levels in human platelets (≈100 pmol/109 platelets) increased severalfold in response to 0.5–1 U/ml thrombin. We report here fluctuations in diacylglycerol mass in control platelets, the magnitude of which were 60–90% of that measured in platelets treated with 0.2–0.5 U/ml of thrombin. These control platelets were not activated by such criteria as absence of aggregation, secretion, phosphatidic acid production and phosphorylation of the protein kinase C substrate, pleckstrin. Thrombin treatment evoked all of the above responses. Analysis of the diacylglycerol molecular species by reverse‐phase HPLC of the dimethylated, phosphorylated derivatives showed that all of the molecular species that were present in control platelets were also present in thrombin‐treated platelets. Most of the species appeared to fluctuate at random in control platelets with the exception of 1‐stearoyl‐2‐arachidonoyl‐sn ‐glycerol which was more or less stable and increased severalfold over control values only upon thrombin treatment. Furthermore, only this species accumulated as [32P]phosphorylated PtdOH in thrombin‐treated platelets prelabelled with [32P]Pi. Our findings show that, in platelets, elevation of diacylglycerol molecular species other than the 1‐stearoyl‐2‐arachidonoy1 species occurs, but these changes are not necessarily linked to activation of protein kinase C as measured by pleckstrin phosphorylation which was observed only upon elevation of 1‐stearoyl‐2‐arachidonoyl‐sn ‐glycerol.

Metabolic characterization of sciadonic acid (5c,11c,14c‐eicosatrienoic acid) as an effective substitute for arachidonate of phosphatidylinositol

“…In this study, we investigated the metabolism of sciadonic acid by fatty acid desaturase. Under our assay conditions, where D9, D6 and D5 desaturase exerts its activity, substantial amounts of 20:4 were not formed from [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]polymethylene-interrupted 20:3. Although our data is limited for the liver microsome, several investigations [31,32] have reported evidence that fatty acid desaturases in rat liver and testis could not desaturate sciadonic acid to arachidonic acid under in vivo conditions using [ 14 C]sciadonic acid [32].…”

“…Therefore, we used this mixture of polymethylene-interrupted 20:3 for fatty acid desaturase assay. The yields of [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]polymethylene-interrupted 20:3 from [1-14 C]arachidonic acid under our system was 6.5%. D9, D6 and D5 desaturase activities of rat liver microsomes were investigated using radiolabeled stearic acid, linoleic acid and dihomo-g-linolenic acid, respectively.…”

“…Because the membrane-permeable diacylglycerols, such as 1-oleoyl-2-acetyl glycerol and 1,2-dioctanoyl glycerol can activate PKC, the requirement of an arachidonic acid residue of diacylglycerol as activator of PKC is questionable. But evidence has been reported that activation of PKC is dependent on the composition of diacylglycerol molecular species [10,11] and correlates with the transient accumulation of diacylglycerol deriverd from inositolphospholipid [12] such as 1-stearoyl-2-arachidonoyl glycerol in intact cells [13]. Pettitt et al reported that a mixture of diacylglycerols containing polyunsaturated fatty acids, such as arachidonic acid and mead acid, was a more potent activator of PKC in Swiss 3T3 fibroblasts even in a cell free system [14].…”

Physical Properties of the Transmembrane Signal Molecule, sn-1-Stearoyl 2-Arachidonoylglycerol

“…This SAG is rapidly phosphorylated to sn-1-stearoyl-2-arachidonolyl phosphatidic acid by DAG kinase and thus exists only transiently in the membrane (22). However, the tendency of DAGs in general to form microdomains in membranes (30,32) suggests that PLC-generated SAG also may become transiently concentrated in microdomains in the cell membranes.…”

“…Moreover, upon thrombin stimulation of platelets many molecular species of DAG are formed, but the concomitant PKC-induced phosphorylation of pleckstrin only correlates with the transient, large accumulation of SAG, showing that at least in intact platelets the SAG species specifically activates PKC (22). Furthermore, DAG lipases from many sources use SAG as the preferred DAG species (8 -10).…”