Ligand induced activation of the beta‐receptor for platelet‐derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta‐receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF‐BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild‐type beta‐receptors were attenuated by inhibition of phosphatidylinositol 3′‐kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF‐BB‐induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild‐type beta‐receptor was less affected. Moreover, the PDGF‐BB‐stimulated tyrosine phosphorylation of phospholipase C‐gamma was increased in the mutant receptor cells compared with wild‐type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C‐gamma, and shifts the response to increased mitogenicity.

The Corline Heparin Surface used in cardiopulmonary bypass proved to be more biocompatible than an uncoated surface when using a standard systemic heparin dose. The low dose of systemic heparin might not be sufficient to maintain the antithrombotic activity, and the high dose resulted in direct cell activation rather than a further anti-inflammatory and anticoagulatory effect.

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Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF β-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4,5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.

Background-We have previously reported the potentiation of PDGF-BB-induced chemotaxis of fibroblasts, vascular smooth muscle cells, and endothelial cells by FVIIa. Here we studied the role of TF/FVIIa and the induced signaling pathways in regulation of chemotaxis of human monocytes, fibroblasts, and porcine aorta endothelial cells. Methods and Results-Human monocytes were obtained by using Ficoll-Paque gradient and the MACS system (for highly purified population), fibroblasts and PAE cells have been characterized previously. Inhibitors of selected signaling intermediates were used, and the effect of TF/FVIIa on the migratory response of all cells to chemotactic agents was analyzed.The induced signaling was studied by immunoprecipitation and Western blotting. TF/FVIIa complex selectively enhanced PDGF-BB-induced chemotaxis in a Src-family, PLC, and PAR-2-dependent manner. Using PAE cells we identified c-Src and c-Yes as the Src-family members activated by TF/FVIIa. We report for the first time the PAR-2 and Src family-dependent transactivation of PDGFR␤ by TF/FVIIa involving phosphorylation of a subset of PDGFR␤ tyrosines. Conclusions-The

We have previously shown that porcine aortic endothelial cells expressing the Y934F platelet-derived growth factor (PDGF) ␤-receptor mutant respond to PDGF-BB in a chemotaxis assay at about 100-fold lower concentration than do wild-type PDGF ␤-receptor-expressing cells (Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engströ m, U., Wernstedt, C., Heldin, C.-H., and Rö nnstrand, L. (1996) EMBO J. 15, 5299 -5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-␥1 (PLC-␥1), measured as inositol-1,4,5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-␥1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-␥1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-␥1. To assess whether increased activation of PLC-␥1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wildtype or a catalytically compromised version of PLC-␥1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-␥1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-␥1 mutant had no effect on PDGF-BBinduced chemotaxis. Furthermore, in cells expressing normal levels of PLC-␥1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-␥1 was not inhibited by the PI3-kinase inhibitor LY294002. These observations suggest the existence of two different pathways which mediate PDGF-induced chemotaxis; depending on the cellular context, the PI3-kinase pathway or the PLC-␥1 pathway may dominate.

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This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.