Role of P-selectin in platelet sequestration in pulmonary capillaries during endotoxemia

Background: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. Methods: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. Results: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. Conclusion: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction. Copyright (c) 2006 S. Karger AG, Basel

Src-family kinases mediate an outside-in signal necessary for β2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin

An introduction to chemokines and their roles in transfusion medicine

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74808/1/j.1423-0410.2008.01127.x.pd

Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling.

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1-2.5 μg/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter- epidermal growth factor synthetic peptide L83FTRKL88(G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) but not by D- NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55-3.1 ng/ml) in a reaction inhibited by L- NAME and by the inter-epidermal growth factor peptide Leu83-Leu88 but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor-pathway inhibitor. These data suggest that factor Xa- induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo