Thrombin Induces Activation of p38 MAP Kinase in Human Platelets

Abstract

Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.We have recently shown that thrombin stimulates the activity of the MAP 1 kinases ERK1 and ERK2 but also activates another proline-directed kinase that is distinguishable from ERK1/2 based on its strong binding to anion exchange resin and the lack of reactivity with anti-ERK1/2 antibodies (1). We further noted that this kinase readily phosphorylates cPLA 2 but not the S505A mutant of cPLA 2 . This observation indicated that the serine residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser 505 -Pro) is the target phosphorylation site for the kinase. Significantly, the thrombin receptor agonist peptide SFLLRN also activated this proline-directed kinase but completely failed to stimulate ERK1/2. Nonetheless SFLLRN, like thrombin, mediated activation of cPLA 2 by phosphorylation, and we reasoned that this unidentified kinase could play a role in the signal transduction pathways activated through the thrombin receptor. We therefore further characterized the kinase with the goal to determine its identity and define its role in the thrombin-induced signaling events during platelet activation. EXPERIMENTAL PROCEDURESPlatelet Isolation and Incubation-Fresh human platelets were prepared from platelet-rich plasma of drug-free volunteers in the presence of prostacyclin (10 Ϫ8 M) and apyrase (0.5 units/ml) as described previously (2), suspended at 1.25 ϫ 10 9 /ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl 2 , 2.2 mM CaCl 2 , 5.5 mM glucose, 0.2 mM EGTA, 10 mM Hepes, pH 7.4, containing 30 M cyclo(S,S)-Mpr(Har)-GDWP-Pen-NH 2 (where Mpr is mercaptopropionyl, Har is homoarginine, and Pen is penicillamine) (3) (kindly provided by Dr. Robert Scarborough, COR Therapeutics), and incubated at 37°C with 5 units/ml ␣-thrombin (ϳ3500 NIH units/mg, Enzyme Research Laboratories). Reactions were terminated by adding (final concentrations) 1% Triton X-100, 5 mM EGTA, 1 mM DTT, 0.2 mM Na 3 VO 4 , 100 nM microcystin (Life Technologies, Inc.), 100 M leupeptin, 0.2 mg/ml aprotinin, 10 M pepstatin A, 1 mM Pefabloc (Centerchem), and 50 mM ␤-glycerophosphate, pH 7.5. The suspension was then briefly sonicated, centrifuged for 30 min at 100,000 ϫ g using a Sorvall RC M120EX microcentrifuge, and diluted with MonoQ buffer as indicated.Partial Purification of p38 and ERK Kinases by MonoQ Chromatography-High speed supernatants were subjected to chromatography on a MonoQ HR 5/5 column (Pharmacia Biotech Inc.) at a flow rate of 1.5 ml/min ...

TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

“…Several authors have suggested that TRAP is only a partial agonist because it induces less expression of activated GPIIb-IIIa, fails to induce platelet prothrombinase activity, and causes weaker responses in terms of Ca ϩϩ mobilization, arachidonate production, and serotonin release. 8,9,28,29 Our studies do not fully support these concepts. In our hands, concentrations of thrombin and TRAP that induced similar aggregation responses in washed platelets resulted in nearly equivalent levels of phosphorylation at tyrosine residues of proteins present in whole platelet lysates.…”

Phosphorylation and Activation of Cytosolic Phospholipase A2 by 38‐kDa Mitogen‐Activated Protein Kinase in Collagen‐Stimulated Human Platelets

“…In contrast, column fractions from thrombin-stimulated platelet lysates containing a prolinedirected kinase different from p42Ip44 MAP kinase phosphorylated recombinant cytosolic PLA, but not [Ala505]cytosolic PLA, [ll]. These fractions were analysed and contained p38 MAP kinase [15]. Recently, we reported that SB 203580 partially inhibits phosphorylation of cytosolic PLA, by thrombin [36].…”

“…[ 15] showed activation of the 38-kDa MAP kinase in thrombinstimulated platelets and suggested that this kinase may phosphorylate cytosolic PLA,.…”

Not so strange bedfellows: G-protein-coupled receptors and Src family kinases

“…Early experiments demonstrated that Src, Fyn or Yes could be activated by a host of GPCRs, including the lysophosphatidic acid (LPA), a2A adrenergic, M1 muscarinic, M2 muscarinic, a-thrombin, endothelin, angiotensin II, bombesin, bradykinin, and V2 vasopressin receptors (Simonson and Herman, 1993;Chen et al, 1994;Ishida et al, 1995;Kramer et al, 1995;Linseman et al, 1995;Ptasznik et al, 1995;Luttrell et al, 1996;Rodriguez-Fernandez and Rozengurt, 1996;Schieffer et al, 1996;Simonson et al, 1996). From the outset, there was evidence for heterogeneity in the mechanisms underlying GPCR control of Src activity.…”