Tissue Factor/ FVIIa prevents the extrinsic pathway of apoptosis by regulation of the tumor suppressor Death-Associated Protein Kinase 1 (DAPK1).

INTRODUCTION: This study determines the impact of tissue factor (TF)-signaling on the extrinsic pathway of apoptosis in cancer cells and propose death associated protein kinase-1 (DAPK1) as a novel key regulator. MATERIALS AND METHODS: In MDA-MB-231 breast and PC3 prostate cancer cells, mRNA levels were analyzed by real-time PCR and protein expressions were assessed by flow cytometry or western blot. Caspase-8 and -3 levels, cell size, and changes in nuclear morphology were recorded using the ArrayScan microscope and 84 apoptosis-related genes were screened with the RT2 Profiler™ PCR Array. RESULTS: In serum starved MDA-MB-231 cells, a TF/FVIIa-sensitive upregulation of apoptosis markers was recorded. Similarly, TRAIL-induced apoptosis was negatively regulated by TF/FVIIa (10 and 100 nM) and TF/FVIIa/FXa but not by active-site inhibited FVIIa. FVIIa, moreover, decreased the transcription of DAPK1 and thereby diminished the association between DAPK1 and FADD in the caspase-8 activating death-inducing signaling complex (DISC). TF/FVIIa regulation of caspase-8 and DAPK1 was dependent on PI3-kinase/AKT and independent of the protease activated receptors (PAR) 1 and 2. Despite of receptor expression and functional signaling, both PAR-agonist treatment and PAR-blocking antibodies in combination with FVIIa failed to influence the anti-apoptotic signal. CONCLUSIONS: We hereby report that TF/FVIIa-induced signaling governs the extrinsic pathway of apoptosis by reducing the levels of DAPK1 in the DISC independently of PAR1 and PAR2. This implies the conceivable involvement of cell surface components other than the PARs and entails the search for TF-dependent regulators of DAPK1 transcription.

Tissue factor and IL8 production by P-selectin-dependent platelet-monocyte aggregates in whole blood involves phosphorylation of Lyn and is inhibited by IL10.

BACKGROUND: P-selectin and CD40L expressed by activated platelets induce tissue factor (TF) and inflammatory cytokines in monocytes, but little is known of the cellular signaling pathways involved. The anti-inflammatory cytokine IL10 reduces atherosclerotic plaque formation. OBJECTIVES: To evaluate the importance of P-selectin upon platelet-monocyte aggregate (PMA) formation in thrombin receptor activator peptide (TRAP) stimulated whole blood, the P-selectin-P-selectin glycoprotein ligand (PSGL)-1-induced cellular signaling pathway, and the effects of IL10 on these functions. METHODS: TF, IL8, and monocyte chemotactic protein-1 (MCP-1) production, PMAs and phosphorylation of Lyn were analyzed in whole blood, purified monocytes, and vitamin D(3)-differentiated U-937 cells stimulated with TRAP or P-selectin with or without IL10. Anti-P-selectin or anti-CD40L antibodies (Abs), Src-kinases inhibitors, SU6656 or PP2, were added in some experiments. RESULTS: TRAP and P-selectin increased TF, IL8, and MCP-1 mRNA in whole blood and purified monocytes. Anti-P-selectin Ab reduced TRAP-induced PMA formation by 80 +/- 2% (P = 0.001) and production of TF (P = 0.04) and IL8 (P = 0.01). IL10 and SU6656 had no effect on PMA formation, although both significantly reduced TF (P = 0.002 and P = 0.02) and IL8 (P = 0.009 and P = 0.001) mRNA upon TRAP and P-selectin stimulation. Induced Lyn phosphorylation in monocytes was diminished by SU6656 (P = 0.02), anti-P-selectin Ab (P = 0.02), and IL10 (P = 0.03) upon TRAP or P-selectin stimulation. These results were confirmed in the vitamin D(3)-differentiated U-937 cells. CONCLUSIONS: The formation of PMAs in whole blood was P-selectin-dependent in the long term. P-selectin-PSGL-1-induced TF and IL8 expression through Lyn phosphorylation, and part of the inhibitory effect of IL10 depends on reduced phosphorylation.

The influence of different heparin surface concentrations and antithrombin-binding capacity on inflammation and coagulation.

The corline heparin surface (CHS) used in the extracorporeal circuit during coronary artery bypass grafting is shown to decrease the activation of inflammation and coagulation. Synchrotron radiation studies have shown that a single layer of the CHS may not completely cover the substrate surface. However, a double layer of CHS results in a uniform surface. We investigated the effect of surfaces with different surface concentrations of heparin on cell activation and coagulation compared to an uncoated surface. The CHS is prepared by a conditioning layer of polymeric amine onto which a macromolecular heparin conjugate is attached. We used PVC tubing, uncoated or modified with a single or double layer of the CHS, and circulated fresh whole blood from healthy volunteers in a loop model system at 37 degrees C up to 4 h. Blood was drawn from the loops at different times and activation of inflammation and coagulation was studied by real-time PCR, flow cytometry and ELISA. The activation of leukocytes and platelets and formation of leukocyte-platelet aggregates were reduced by use of the single-layered CHS compared to the uncoated surface. Use of double-layered CHS resulted in significantly reduced cell activation and thrombin generation. Development of the CHS obtained by the double layer of the coating has improved the biocompatibility of the surface.

Cell adhesion and tissue factor upregulation in oxygenators used during coronary artery bypass grafting are modified by the Corline Heparin Surface.

OBJECTIVE: Cardiopulmonary bypass (CPB) is associated with inflammatory response and activation of coagulation. We investigated the influence of a new heparin surface on the activation of cells retrieved from oxygenators used during coronary artery bypass grafting (CABG). DESIGN: Sixty patients undergoing CABG with CPB were randomly assigned to either uncoated or completely Corline Heparin Surface (CHS)-coated circuits with one of three different levels of systemic heparin: standard, high or low. At end of surgery adhered cells were retrieved from the oxygenators and cell count, tissue factor (TF)- and CD11b-expression on monocytes and monocytic TFmRNA were analysed. RESULTS: The heparin coating of the oxygenator prevented adhesion of granulocytes, monocytes and platelets. TF-expression on monocytes from the oxygenators was significantly higher than on circulating cells in all groups. Monocytes from the uncoated oxygenators showed low levels of TF-expression with high levels of TFmRNA. The coated group with high level of heparin showed higher surface-expression of TF with low levels of TFmRNA. CONCLUSION: The CHS was most biocompatible with the standard level of heparin used during CABG whereas elevation of systemic heparin rather increased the activation and TF upregulation in monocytes from oxygenators.

Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB-stimulated chemotaxis.

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 beta-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.

Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis.

We have previously shown that porcine aortic endothelial cells expressing the Y934F platelet-derived growth factor (PDGF) beta-receptor mutant respond to PDGF-BB in a chemotaxis assay at about 100-fold lower concentration than do wild-type PDGF beta-receptor-expressing cells (Hansen, K., Johnell, M., Siegbahn, A. , Rorsman, C., Engström, U., Wernstedt, C., Heldin, C.-H., and Rönnstrand, L. (1996) EMBO J. 15, 5299-5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma1 (PLC-gamma1), measured as inositol-1,4, 5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-gamma1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-gamma1. To assess whether increased activation of PLC-gamma1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wild-type or a catalytically compromised version of PLC-gamma1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-gamma1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-gamma1 mutant had no effect on PDGF-BB-induced chemotaxis. Furthermore, in cells expressing normal levels of PLC-gamma1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-gamma1 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-gamma1 pathway may dominate.

Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.

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.

C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway.

Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved are undefined. Since most natural leukocyte secretagogues also induce cell migration, and since the anaphylatoxins C3a and C5a are mast cell secretagogues, we hypothesized that both C3a and C5a are also mast cell chemotaxins. Here we report that C3a and C5a are, in fact, potent chemotaxins for the human mast cell line HMC-1. The optimal concentrations, half-maximal effective concentrations (a measure of agonist potency) and the efficacy (response at the optimal concentration) compared with medium control were, for C3a: 10 nM, 0.5 nM, and 256%, respectively; for C5a: 1 nM, 10 pM and 145%. Chemotaxis of HMC-1 cells to both C3a and C5a was blocked by pertussis toxin, suggesting that Gi-coupled receptors are involved in signal transduction. C3a and C5a also induced transient pertussis toxin-inhibitable increases in [Ca2+]i (ED50 = 1 nM for both) that could be homologously but not heterologously desensitized, suggesting that the receptors for C3a and C5a are distinct. These results make C3a the most effective mast cell chemotaxin identified to date. The chemotactic potency described here for C3a is also 100- to 1000-fold greater than for all of its previously described cellular actions. Direct chemoattraction of mast cells by C3a and C5a may help explain the rapid accumulation of mast cells at sites of inflammation.