Integrin αIIbβ3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PI 3-kinase

AbstractActivation of the focal adhesion kinase pp125FAK correlates with its phosphorylation on tyrosine residues and is mediated by multiple receptor-ligand pairs. In platelets, pp125FAK phosphorylation is triggered by αIIbβ3 integrin or FcγRII receptor interaction with immobilized fibrinogen and IgG, respectively. In this study we used platelets as a model system to explore the role of PI 3-kinase relative to pp125FAK phosphorylation. Treatment of the platelets with two PI 3-kinase inhibitors, wortmannin and LY294002, inhibited in a dose-dependent manner αIIbβ3-mediated platelet spreading on fibrinogen having no effect on platelet spreading on IgG. Both inhibitors also completely abolished αIIbβ3-mediated pp125FAK phosphorylation but not pp72syk phosphorylation. Furthermore, FcγRII- and thrombin-induced pp125FAK phosphorylation were not affected by wortmannin and LY294002. Finally, the PI 3-kinase inhibitors’ effect on αIIbβ3-mediated spreading and pp125FAK phosphorylation was reversed by phorbol ester treatment. These results establish that the role of PI 3-kinase relative to pp125FAK phosphorylation in platelets is receptor type-specific yet essential for αIIbβ3-mediated cell spreading and pp125FAK phosphorylation

Protein kinase C-δ activation and tyrosine phosphorylation in platelets

AbstractSeveral protein kinase C (PKC) isoforms are expressed in human platelets. We report that PKC-δ is tyrosine phosphorylated within 30 s of platelet activation by thrombin. This correlated with a 2–3-fold increase in the kinase activity of PKC-δ relative to unstimulated platelets. The tyrosine phosphorylated PKC-δ isoform was associated with the platelet particulate (100 000×g insoluble) fraction. αIIbβ3 integrin mediated platelet adhesion to fibrinogen did not significantly affect PKC-δ activity. Tyrosine phosphorylation of PKC-δ was similarly not detected in fibrinogen adherent platelet lysates. Treatment of the platelets with mAb 7E3 prior to the addition of thrombin blocked aggregation having no effect on the thrombin induced PKC-δ activation. We conclude that PKC-δ is activated in platelets by an αIIbβ3 independent pathway

Neutrophil survival on biomaterials is determined by surface topography

AbstractPurpose: Cardiovascular device-centered infections are a major cause of hospital morbidity, mortality, and expense. Caused by opportunistic bacteria, this phenomenon is thought to arise because of a defect in neutrophil bacterial killing. We have shown that neutrophils that adhere to polystyrene remain viable, whereas neutrophils that adhere to the vascular biomaterials expanded polytetrafluoroethylene (ePTFE) and Dacron undergo a rapid nonapoptotic death. This study was designed to test the hypothesis that surface topography is a determinant of the nonapoptotic death response of neutrophils to biomaterials. Methods: We took advantage of the ease with which a polystyrene surface can be manipulated to examine the effect of surface topography on neutrophil viability. Neutrophils were exposed to smooth or roughened polystyrene surfaces both in vivo and in vitro. Changes in cell membrane permeability and production of reactive oxygen species by individual cells were monitored with fluorescent dyes. Results: Host cells and isolated human neutrophils died rapidly after adhesion to roughened polystyrene. Neutrophils adherent to roughened surfaces produced more reactive oxygen intermediates than those adherent to smooth surfaces and were first to die. The cell death response precipitated by expanded polytetrafluoroethylene, Dacron, or the roughened surfaces was significantly reduced with treatment of the neutrophils with catalase, diphenylene iodonium, or the src kinase inhibitor PP2 before adhesion. Conclusions: Neutrophil adhesion to roughened materials triggers rapid production of reactive oxygen species and precipitates a nonapoptotic cell death. Understanding the material properties that trigger these responses is essential to development of the next generation of implantable biomaterials. (J Vasc Surg 2003;37:1082-90.

Force-dependent integrin–cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2

As cells encounter new regions of the substrate, they develop bonds with new matrix molecules for migration, matrix remodeling and force generation. How cells orchestrate the assembly of adhesion sites is only partially understood. Here we show that fibroblasts deficient in the SH2 domain containing protein tyrosine phosphatase 2 (Shp2) have an increased number of immature focal complexes deficient in α-actinin. Inhibition of FAK restored α-actinin to focal complexes, whereas inhibition of RhoA did not. In correlation, adhesion site dynamics, measured by fluorescence recovery after photobleaching (FRAP) of GFP–paxillin and GFP–vinculin were dramatically increased in Shp2(–/–) cells and restored to normal by FAK inhibition. Shp2(–/–) cells failed to strengthen initial integrin–cytoskeleton linkages, as measured by optical tweezers and large bead assays, and were rescued by inhibition of FAK. In contrast, affinity modulation of adhesion receptors was unaffected. In addition, reinforcement correlated with α-actinin assembly through decreased dynamics. This shows for the first time that adhesion site dynamics are regulated during adhesion formation and that force-dependent strengthening of integrin–cytoskeleton linkages is correlated with α-actinin assembly and decreased adhesion site dynamics