Cathepsin G-dependent platelet stimulation by activated polymorphonuclear leukocytes and its inhibition by antiproteinases: role of P-selectin-mediated cell-cell adhesion

Abstract

Human PMN stimulated by fMLP are able to activate coincubated, autologous platelets. Cathepsin G, a neutral serine protease stored in the azurophilic granules of PMN, is the major platelet activator in this system. We previously proposed that shear-induced close PMN- platelet contact creates the conditions for which cathepsin G activity on platelets is protected against antiproteinases. The aim of this study was to investigate the adhesive mechanisms, possibly creating between PMN and platelet membranes the microenvironment in which cathepsin G, discharged from stimulated PMN onto adherent platelets, is protected against antiproteinases. Microscopic examination showed that under conditions of high shear, 71.3% +/- 6.1% of PMN were associated to platelets forming small clumps. This percentage decreased to 10% +/- 2% and 13% +/- 4%, respectively, in the presence of an inhibitory antibody to P-selectin or 20 mmol/L mannose-1-phosphate and to 10.8% +/- 3.7% when cells were not stirred. Similarly, PMN pretreatment with neuraminidase abolished PMN binding to platelets. These results indicate that P-selectin mediates PMN-platelet adhesion occurring before PMN stimulation. Prevention of PMN-platelet contact significantly potentiated the inhibitory effect of alpha 1-protease inhibitor on subsequent cathepsin G-induced platelet serotonin release. Because anti-P-selectin antibody, mannose-1-phosphate, and neuraminidase treatment of PMN did not modify PMN-induced platelet activation in the absence of antiproteinases, it is suggested that P- selectin-mediated PMN-platelet adhesion results in the formation of a sequestered microenvironment between cell membranes, in which higher amounts of antiproteinases are required to prevent the activity of released cathepsin G. These data add a new functional role to P- selectin-mediated PMN-platelet adhesion that could be important in vivo because of the presence of antiproteinases in plasma.

Characterization of blood components separated from donated whole blood after an overnight holding at room temperature with the buffy coat method

“…Granulocyte-mediated PLT activation requires close cellto-cell contact because proteinases released from granulocytes are quickly inactivated by plasma inhibitors. 25 PLT active factor remains mostly attached to the granulocytes' membrane. 26 The HSR of PLTs has been reported to correlate well with PLT viability and posttransfusion recovery of PLTs.…”

Circulating platelet–neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing

“…These ®ndings are indicative that neutrophils within platelet±neutrophil complexes represent an activated subset of cells. In addition, it can be inferred that the platelet component of PNCs must also represent an activated subpopulation of platelets, as CD62P expression is an essential prerequisite for PNC formation (de Bruijne Admiraal et al, 1992;Evangelista et al, 1993).…”

Adhesion of flowing leucocytes to immobilized platelets

“…We and others have previously shown that P-selectin mediates adhesion of flowing neutrophils to immobilized activated platelets Yeo et al, 1994), and that thrombin-activated platelets express P-selectin on their surface and bind neutrophils in suspension via this receptor (McEver, 1991;Evangelista et al 1993;Spangenberg et al, 1993). In the current study, platelet-coated microslides bound autologous neutrophils or lymphocytes.…”

“…The suspension was placed in a sterile plastic petri dish, and incubated for 30 min at 3 7°C in 5% C02 atmosphere to allow monocytes to sediment and attach to the surface. Lymphocytes were rinsed from the dish, centrifuged at 3008 for 5min and resuspended in PBS/5% plasma, counted by Coulter Counter (model ZF; Coulter Electronic Ltd, Luton) attached to a Pulse Width and Height Analyser (Fisher & Fisher, 1989), and diluted to 1 x lo6 cells/ml in the same medium. Pulse height analysis allowed assessment of the proportion of residual large Ieucocytes, outside the Iymphocyte volume peak, and this averaged N 10%.…”

“…and circulating leucocyte-platelet aggregates increased during cardiopulmonary bypass in humans (Rinder et al, 1992). Adhesion between leucocytes and platelets could recruit either cell type to the inflamed or damaged vessel wall, occlude vessels, and promote biochemical interaction by producing high local concentrations of released mediators and avoiding neutralization by plasma inhibitors (Bazzoni et al, 1991;Evangelista et al, 1993). In order to examine the ligands involved in leucocyte adhesion to P-selectin, and to test whether neutrophils alone are able to use this route to promote immobilization from flow, we have carried out a comparative study of the adhesion of flowing neutrophils, peripheral blood lymphocytes, and HL60 cells to immobilized platelets.…”