Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbß3.

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet-bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbß3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5'-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria.

Inhibition of destructive autoimmune arthritis in FcgammaRIIa transgenic mice by small chemical entities.

The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.

Platelet-derived thrombospondin-1 is necessary for the vitamin D-binding protein (Gc-globulin) to function as a chemotactic cofactor for C5a.

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D-binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56-kDa plasma protein that binds and transports several diverse ligands. The objective of this study was to investigate the mechanisms by which DBP functions as a chemotactic cofactor for C5a using neutrophils and U937 cells transfected with the C5aR (U937-C5aR cells). The results demonstrate that U937-C5aR cells show C5a chemotactic enhancement only to DBP in serum, but, unlike mature neutrophils, this cell line cannot respond to DBP in plasma or to purified DBP. Analysis by SDS-PAGE and isoelectric focusing revealed no structural difference between DBP in serum compared with DBP in plasma. However, plasma supplemented with either serum, DBP-depleted serum, or activated platelet releasate provides a required factor and permits DBP to function as a chemotactic cofactor for C5a. Fractionation of activated platelet releasate revealed that the additional factor possessed the properties of thrombospondin-1 (TSP-1). Finally, purified TSP-1 alone could reproduce the effect of serum or platelet releasate, whereas Abs to TSP-1 could block these effects. These results provide clear evidence that TSP-1 is needed for DBP to function as a chemotactic cofactor for C5a.

Effects of Saiko-ka-ryukotsu-borei-to in patients with hyperlipidemia.

We measured and compared levels of platelet-derived microparticles (PMPs), monocyte-derived microparticles (MMPs), CD62P on activated platelets, soluble E-selectin (sE-selectin), and anti-oxidized low density lipoprotein (LDL) antibody in hyperlipidemia patients and control subjects. Binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies to platelets was not significantly different between hyperlipidemia patients and controls. However, expression of CD62P on platelets and levels of PMPs were higher for hyperlipidemia patients than in controls, although the difference between groups in CD62P expression was not significant (PMPs: 534 +/- 63 vs. 388 +/- 47, p < 0.05; CD62P: 9.1% +/- 1.45 vs. 7.3% +/- 1.15, N.S.). Although there were no differences in expression of CD36 and CD40 by monocytes between the two groups, levels of MMPs were higher in hyperlipidemia patients than in controls (MMPs: 147 +/- 21 vs. 59 +/- 8, respectively, p < 0.01). Levels of anti-oxidized LDL antibody and sE-selectin were also higher in hyperlipidemia patients. We studied the effects of Saiko-ka-ryukotsu-borei-to on levels of these factors in patients with elevated triglyceride levels. After Saiko-ka-ryukotsu-borei-to treatment, levels of CD62P, PMPs, sE-selectin, and anti-oxidized LDL antibody were reduced significantly. Levels of triglycerides, total cholesterol and MMPs also decreased, but the changes were not significant. These findings suggest that Saiko-ka-ryukotsu-borei-to prevents the development of vascular complications in hyperlipidemia patients.

Platelet activation in the lung after antigen challenge in a model of allergic asthma.

The purpose of this study was to investigate whether platelets are activated and release their products in the human lung after antigen challenge. Using subsegmental antigen challenge as a model of asthma, bronchoalveolar lavage fluids from ragweed-allergic asthmatic subjects were assayed for the alpha granule products, platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), prior to challenge (baseline) and at 5 min and 19 h after challenge with ragweed antigen. Airway segments challenged with normal saline were used as controls. Five minutes after antigen challenge, levels of platelet products in BAL fluid were not elevated from baseline or normal saline control levels. However, 19 h after antigen challenge, a 10-fold increase in platelet products in BAL fluids was found. The mean PF4 levels increased from baseline and saline control values of less than 1.0 to 7.2 ng/ml (p less than 0.05) 19 h after antigen challenge. beta-TG increased from baseline and control levels of less than 1.0 to 6.6 ng/ml (p less than 0.05). Elevations in PF4 and beta-TG were highly correlated with each other (r = 0.98, p less than 0.0001). Levels of platelet products during the 19-h response correlated with albumin, with kinins, with the prostaglandins 6-keto-PGF1 alpha, PGE2, and PGF2 alpha, and with the eosinophil-derived proteins, eosinophil-derived neurotoxin and eosinophil peroxidase. We conclude that platelet activation in the lung is a feature of the late inflammatory response to antigen challenge and that platelets may play an important role in allergic inflammation and asthma.