Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN- platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.

No 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.

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the aminoand carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process.We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for a-helix barely initiated a turbidity increase at conditions favoring selfassembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.