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Bidirectional signaling of integrin [[alpha].sub.[parallel]b][[beta].sub.3] requires the [[beta].sub.3] cytoplasmic domain. To determine the sequence in the [[beta].sup.3] cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein lb-IX, integrin [[alpha].sub.[parallel]b], and mutants of [[beta].sub.3] with truncations at sites COOH terminal to [T.sup.741], [Y.sup.747], [F.sup.754], and [Y.sup.759]. Truncation at [Y.sup.759] did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of [beta]3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at [F.sup.754], [Y.sup.747, or [T.sup.741] completely abolished integrin activation. A point mutation replacing [Y.sup.759 with alanine also abolished integrin activation. Thus, the [T.sup.755]NIT[Y.sup.759] sequence of [[beta].sub.3], containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at [Y.sup.759] in a population of [[beta].sub.3] during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin (alpha)(sub IIb)(beta)(sub 3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing (alpha)(sub IIb)(beta)(sub 3) adhere poorly to vWE Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin (alpha)(sub IIb)(beta)(sub 3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against (alpha)(sub IIb)(beta)(sub 3), vWF binding to GPIb-IX also activates soluble fibrinogen binding to (alpha)(sub IIb)(beta)(sub 3) indicating that GPIb-IX mediates a cellular signal leading to (alpha)(sub IIb)(beta)(sub 3) activation. Deletion of the 14-3-3-binding site in GPIb(alpha) inhibited GPIb-IX-mediated fibrinogen binding to (alpha)(sub IIb)(beta)(sub 3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIb(alpha) induced an endogenous integrin-dependent cell spreading on vWF without requiring (alpha)(sub IIb)(beta)(sub 3), but inhibited vWF-induced fibrinogen binding to (alpha)(sub IIb)(beta)(sub 3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of (alpha)(sub IIb)(beta)(sub 3) requires 14-3-3 interaction with GPIb(alpha).

Abstract Background Tissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways. Methods A plasmid containing TF promoter −2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells. Results We show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment. Conclusions This study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.

Research has been conducted on cGMP-dependent protein kinase. The role of this protein kinase in platelet activation has been investigated, and the results demonstrate that recombinant cGMP-dependent protein kinase's expression in reconstituted cell model can enhance von-Willebrand factor-induced platelet integrin activation.