1 Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2 Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more eective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the eects of trans-resveratrol (3,4',5-trihydroxytrans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4 trans-Resveratrol exerted a strong inhibitory eect on reactive oxygen species produced by PMN stimulated with 1 mM formyl methionyl leucyl phenylalamine (fMLP) (IC 50 1.3+0.13 mM, mean+ s.e.mean), as evaluated by luminol-ampli®ed chemiluminescence. 5 trans-Resveratrol prevented the release of elastase and b-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 mM, IC 50 18.4+1.8 and 31+1.8 mM), and C5a (0.1 mM, IC 50 41.6+3.5 and 42+8.3 mM), and also inhibited elastase and b-glucuronidase secretion (IC 50 37.7+7 and 25.4+2.2 mM) and production of 5-lipoxygenase metabolites leukotriene B 4 (LTB 4 ), 6-trans-LTB 4 and 12-trans-epi-LTB 4 (IC 50 48+7 mM) by PMN stimulated with the calcium ionophore A23187 (5 mM). 6 trans-Resveratrol signi®cantly reduced the expression and activation of the b 2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated ®xed platelets in a dynamic system were also prevented by transresveratrol. 7 These results, indicating that trans-resveratrol interferes with the release of in¯ammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective eect of red wine consumption against CHD.

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation.

Polymorphonuclear leukocyte (PMN)-platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of "outside-in" signaling by Srcfamily tyrosine kinases (SFKs) in the regulation of ␣M␤2-integrin-dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activationdependent epitopes in ␤2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from ␣M␤2 ؊/؊ , hck ؊/؊ fgr ؊/؊ , and hck ؊/؊ fgr ؊/؊ lyn ؊/؊ mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by

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.

Aspirin affords cardioprotection through the acetylation of serine 529 in human cyclooxygenase-1 (COX-1) of anucleated platelets, inducing a permanent defect in thromboxane A 2 (TXA 2 )-dependent platelet function. However, heterogeneity of COX-1 suppression by aspirin has been detected in cardiovascular disease and may contribute to failure to prevent clinical events. The recent recognized capacity of platelets to make proteins de novo paves the way to identify new mechanisms involved in the variable response to aspirin. We found that in washed human platelets, the complete suppression of TXA 2 biosynthesis by aspirin, in vitro, recovered in response to thrombin and fibrinogen in a time-dependent fashion (at 0.5 and 24 hours, TXB 2 averaged 0.1؎0.03 and 3؎0.8 ng/mL; in the presence of arachidonic acid [10 mol/L], it was 2؎0.7 and 25؎7 ng/mL, respectively), and it was blocked by translational inhibitors, by rapamycin, and by inhibitors of phosphatidylinositol 3-kinase. The results that COX-1 mRNA was readily detected in resting platelets and that [ 35 S]-methionine was incorporated into COX-1 protein after stimulation strongly support the occurrence of de novo COX-1 synthesis in platelets. This process may interfere with the complete and persistent suppression of TXA 2 biosynthesis by aspirin necessary for cardioprotection.A spirin affords cardioprotection inducing a complete and permanent defect in the capacity of platelets to generate thromboxane A 2 (TXA 2 ) through the acetylation of serine 529 of cyclooxygenase-1 (COX-1). 1,2 Because of a nonlinear relationship of inhibition of platelet TXA 2 generation with inhibition of TXA 2 -mediated platelet aggregation, an excess of 95% inhibition of COX-1 activity is required to influence platelet function. 3 In fact, even tiny concentrations of TXA 2 have been shown to cause platelet activation. Thus, 10 nmol/L of the TXA 2 mimetic U46619 induces platelet adhesion and shape change, 4 and in the presence of a subthreshold concentration of collagen, U46619 (0.5 to 10 nmol/L) causes platelet aggregation. 5 Recently, Maree et al 6 showed that many patients who are treated with low-dose enteric-coated aspirin (75 mg) for secondary prevention of cardiovascular events have persistent elevated serum TXB 2 levels (Ͼ2.2 ng/mL), which translates into a more frequent occurrence of arachidonic acid (AA)-induced platelet aggregation. Reduced bioavailability of aspirin 6 and genetic variants in COX-1 7 may participate in the intersubject variable response to aspirin. The recent recognized capacity of platelets to make proteins de novo 8 paves the way to identify new mechanisms involved in aspirin failure to cause complete and persistent suppression of platelet COX-1 activity in some individuals.Thus, in the present study, we assessed, in vitro, the hypothesis that de novo synthesis of COX-1 could account for TXA 2 biosynthesis in platelets in which the activity of preformed COX-1 was blocked by pretreatment with aspirin in vitro. Materials and MethodsHealthy volunteers (nϭ9 to 22...

Adhesion of polymorphonuclear leukocytes (PMNLs) to activated platelets requires a P-selectin-triggered, tyrosine kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of a 110-kd protein (P-110) in PMNLs. Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion to activated platelets or to P-selectin expressing Chinese hamster ovary

Inflammatory lung disease is a primary cause of morbidity and mortality in cystic fibrosis (CF). Mechanisms of unresolved acute inflammation in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in nonrespiratory cells is emerging. Here we examined CFTR expression and function in human platelets (PLTs) and found that they express a biologically active CFTR. CFTR blockade gave an ∼50% reduction in lipoxin A(4) (LXA(4)) formation during PLT/polymorphonuclear leukocytes (PMN) coincubations by inhibiting the lipoxin synthase activity of PLT 12-lipoxygenase. PLTs from CF patients generated ∼40% less LXA(4) compared to healthy subject PLTs. CFTR inhibition increased PLT-dependent PMN viability (33.0±5.7 vs. 61.2±8.2%; P=0.033), suppressed nitric oxide generation (0.23±0.04 vs. 0.11±0.002 pmol/10(8) PLTs; P=0.004), while reducing AKT (1.02±0.12 vs. 0.71±0.007 U; P=0.04), and increasing p38 MAPK phosphorylation (0.650±0.09 vs. 1.04±0.24 U; P=0.03). Taken together, these findings indicate that PLTs from CF patients are affected by the molecular defect of CFTR. Moreover, this CF PLT abnormality may explain the failure of resolution in CF.