Abstract. Thrombin stimulates cultured endothelial ceils (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester-but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8/zg/ml for 5-15 min at 37~ which allow the introduction of small extracellular molecules without the loss of large intraceUular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.

The tumor-promoting phorbol diester 4,8-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2' in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; ICse vs. thrombin = 2 ng/ml, 3.4 nM: >90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2' mobilization was evident within 30 sec of pretreatment with PMA and was essentially complete by 6-10 min at 10-20 ng of PMA per ml. Thrombin-induced secretion was initially accelerated in the presence of PMA, but after 1 min it was progressively inhibited when Ca2+ mobilization was depressed by >60%. PMA did not inhibit Ca2+ mobilization or secretion caused by A23187. Thrombininduced phosphatidylinositol 4,5-[32P]bisphosphate breakdown and [32Plphosphatidic acid production were also initially increased by PMA and then progressively depressed. Inhibition of thrombin-induced lipid metabolism required higher concentrations of PMA (IC5s = 10 ng/ml), and it was not overcome by A23187. 4a-Phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit any responses to thrombin. These results suggest that activation of protein kinase C, which initially fosters secretion and aggregation, may subsequently exert negative feedback on the receptor-mediated mobilization of intracellular Ca2+ and the hydrolysis of phosphatidylinositol 4,5-bisphosphate.The stimulation of platelets to secrete and aggregate by agonists, such as thrombin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether), is associated with a rapid increase of the cytoplasmic free Ca2l concentration ([Ca2+]i) (1,2) and the hydrolysis of the phosphodiester bond of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) (3, 4). The latter reaction is closely correlated with stimulusresponse coupling in many cell types, including platelets (5). The products of this reaction, 1,2-diacylglycerol (acyl2Gro) and myo-inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), appear to play important roles in various cellular activation processes.Acyl2Gro acts as a second messenger to stimulate Ca2+/ phosphatidylserine-dependent protein kinase C by increasing the affinity of the enzyme-lipid complex for Ca2+, so that the enzyme can be activated even at resting levels of [Ca2+]i (6).Acyl2Gro is subsequently metabolized by further hydrolysis to release arachidonic acid and by phosphorylation to form phosphatidic acid (PtdOH). The action ofacyl2Gro on protein kinase C can be duplicated by tumor-promoting phorbol esters (7). Ins-1,4,5-P3, on the other hand, has been impli- (17) is mediated by cyclic AMP. In this paper we report that PMA inhibits Ca2+ mobilization, breakdown of Ptdlns-4,5-P2, formation of PA, and secretion caused by thrombin. This suggests that the protein kinase C pathway can exert feedback inhibition on receptor-mediated platelet activation subsequent to its initial role in promoting secretion. METH...

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