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Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins “activation reporter.” When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP-or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.

Bleeding time, blood loss and platelet retention by glass beads, measured by standardized techniques, were significantly altered in a group of 30 non-thrombocytopenic patients with chronic renal failure undergoing maintenance haemodialysis. Bleeding time or blood loss did not correlate with platelet retention either before or after haemodialysis. No correlation could be found between the above tests and a number of biochemical parameters characterizing the uraemic condition. Haemodialysis only partially corrected the abnormal bleeding time, blood loss and platelet retention. These tests were still significantly different after haemodialysis from those of 30 normal subjects. It is suggested that some non-dialyzable material could play an important role in the aetiology of uraemic bleeding.

The platelet-polymorphonuclear (PMN) leukocyte interaction may occur in vivo, as observed in animal models of inflammatory and thrombotic diseases. Mixed masses of PMN and platelets have been observed in the lungs of experimental animals subjected to hemorrhagic shock (1) or anaphylatoxin instillation (2) or following intradermal or intravenous infusion of n-formyl-Methionyl-Leucyl-Phenylalanine (fMLP) (3,4), or in rat glomeruli inamodel o f immune complex nephritis (5). Plateletaccumulation in experimental myocardial infarction was depressed by PMN depletion (6). Leukocyte accumulation at the site of platelet thrombus was reported (7) as well as platelet-leukocyte interaction after injection of oxidized lipoproteins in vivo (8). Thepathogenesis of the adult respiratory distress syndrome also appears to involve activation o f PMN and platelets (9). The broad range o f interactions between PMN and platelets in vitro includes reciprocal modulatory effects (10-13). In view of epidemiological evidence of a positive correlation between the number o f PMN and the development of ischemic disease (14), the observation that activated PMN induce platelet activation may be of great physiopathological relevance. We have focussed our attention on the stimulating effects ofPM N on platelets. Different pathways and mediators o f this cell-cell interaction have been suggested: several PMN-derived products, such as myeloperoxidase, hydrogen peroxide (H20 2) and superoxide anion (O2), may induce platelet activation (15-17). PAF, a lipid mediator produced by PMN, also has platelet stimulating activity. Though it seems to play a role in rabbitPMN-platelet cooperation (18), this mediator seems not to be implicated in PMN-dependent platelet activation with human cells (19). A direct role for PMN-derived proteinases was shown by the enhancement o f platelet serotonin release induced by thrombin or immune com plexes, using chym otrypsin-like cationic proteins (20). Bykowska et al. (21) provided evidence that a neutral-chymotrypsin-like-serine proteinase purified from human neutrophils, stimulated platelet function. Marked platelet activation, involving cytoplasmic Ca2+ increase, was also reported when mixed human platelet-PMN suspensions were challenged with fMLP (or other PMN agonists) in the presence of cytochalasin B, which allow s azurophilic granule discharge. Cell-free supernatants o f fMLP-activated PMN also induced platelet activation, suggesting a soluble factor was involved (19,22). Electron microscopic observation o f aggregates from mixed

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