The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 Pphosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA 2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-␣ or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stressactivated protein kinase (p38 mapk ), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38 mapk inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA 2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA 2 expressed in a baculovirus/insect cell system to cPLA 2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA 2 in different mammalian cells stimulated with multiple agonists.Cytosolic phospholipase A 2 (cPLA 2 ) 1 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids (1, 2). The 85-kDa enzyme is present in many mammalian cells (3), and strong evidence is accumulating for the role of cPLA 2 in the generation of tissue mediators that are metabolites of arachidonic acid, such as prostaglandins, leukotrienes, and thromboxanes. In contrast to the small molecular weight phospholipase A 2 s that are secreted and are active on the outside of cells, cPLA 2 is regulated by intracellular signals that are propagated from surface receptors. One important regulatory mechanism appears to be a rise in the intracellular Ca 2ϩ concentration which causes translocation of cPLA 2 from the cytosol to internal membranes (4 -7) where it binds through a Ca 2ϩ -dependent lipid-binding domain (8). A second, well established mechanism of the regulation of cPLA 2 activity is by phosphorylation on Ser-505 through a mitogen-activated protein kinase (MAPK) (9) which modestly increases the intrinsic activity of the lipase measured in vitro (3, 10, 11). Phosphorylation of cPLA 2 together with release of arachidonic acid has been observed in a variety of cells (11)(12)(13)(14)(15)(16).A thorough characterization of the phosphorylation sites of human cPLA 2 heterologously expressed in Spodoptera frugiperda (Sf9) cells by high performance liquid chromatography (HPLC), mass spectrometry, and protein sequencing has revealed four sites of phosphorylation: Ser-...
BACKGROUND The calibrated automated thrombogram (CAT) assay in plasma is a versatile tool to investigate patients with hypo- or hypercoagulable phenotypes. The objective was to make this method applicable for whole blood measurements. METHODS Thin-layer technology and the use of a rhodamine 110–based thrombin substrate appear to be essential for a reliable thrombin generation (TG) assay in whole blood. Using this knowledge we developed a whole blood CAT-based assay. RESULTS We demonstrated that the whole blood CAT-based assay is a sensitive and rapid screening test to assess function of the hemostatic system under more nearly physiological conditions than the TG assay in plasma. Under conditions of low tissue factor concentration (0.5 pmol/L) and 50% diluted blood, the intraassay CV of the thrombogram parameters, endogenous thrombin potential and thrombin peak height, were 6.7% and 6.5%, respectively. The respective interassay CVs were 12% and 11%. The mean interindividual variation (SD) of 40 healthy volunteers was 633 (146) nmol · min/L for the endogenous thrombin potential and 128 (23) nmol/L for the thrombin peak. Surprisingly, erythrocytes contributed more than platelets to the procoagulant blood cell membranes necessary for optimal TG. Statistically significant (P < 0.001) and potentially clinically significant correlations were observed between circulating factor-VIII concentrations in blood of hemophilia A patients and endogenous thrombin potential (r = 0.62) and thrombin peak height (r = 0.58). CONCLUSIONS We have developed a reliable method to measure TG in whole blood. The assay can be performed with a drop of blood and may provide a useful measurement of TG under more physiological conditions than plasma.
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