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.

Virulent Shigella flexneri Causes Damage to Mitochondria and Triggers Necrosis in Infected Human Monocyte-Derived Macrophages

Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon. The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate. In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes. Within 2 h of infection, 59% ± 6% of the HMDM and ≤4% of the monocytes were stained with propidium iodide. Treatment of the cells with the inhibitors of caspases YVAD and zVAD, the antioxidants N-acetyl-l-cysteine and butylated hydroxyanisole, or an inhibitor of NADPH oxidase, diphenyleniodonium, did not alter the infection outcome. Importantly, we found that virulent Shigella caused a rapid drop in the ATP level to about 50% in infected HMDM. Furthermore, using a combination of fluorescent vital dyes and mitochondrial membrane potential-sensitive dyes, we observed that cells that exhibited a permeable cell membrane were not stained by the mitochondrion-specific dyes, indicating that the mitochondrial membrane potential was lost in these cells. We also observed infected cells that were not stained with either type of dye, indicating that the loss of the mitochondrial membrane potential preceded the increase in cell membrane permeability. Taken together, our studies showed that virulent Shigella flexneri targets the host cell mitochondria for destruction. This activity may account for the necrotic cell death precipitated by these pathogens