IL-17A increases ADP-induced platelet aggregation

The increased risk of thromboembolism and higher incidence of cardiovascular disorders are among the most common causes of morbidity in patients suffering from autoimmune diseases. In this study we tested the hypothesis that IL-17A, a key pro-inflammatory cytokine involved in the development of autoimmune diseases, exerts pro-aggregant effects on both human and mouse platelets. Human or murine platelets were incubated with IL-17A for 2 min at 37 °C prior the addition of the stimuli. Aggregation was monitored in a light transmission aggregometer measuring changes in turbidity with continuous observation over a 5-min interval after the addition of the stimuli. IL-17RA, CD42b and CD62P expression as well as fibrinogen bindings were measured by FACS while Erk-2 phosphorylation was analyzed by western blot using phospho-specific antibodies. Pre-incubation with IL-17A increased ADP-, but not collagen-induced platelet aggregation and accelerated CD62P expression and exposure of fibrinogen binding sites. These effects were associated with a faster kinetic of ADP-induced Erk-2 phosphorylation and were lost in platelets deficient in the IL-17 receptor. Together these results unveil a novel aspect of the inflammatory nature of IL-17A suggesting, at the same time, that therapeutic strategies targeting this cytokine might provide further benefit for the treatment of autoimmune diseases by reducing the risk of cardiovascular-related pathologies.     

Oxygen-radical-mediated lipid peroxidation and inhibition of ADP-induced platelet aggregation

The effects of lipid peroxidation on ADP-induced aggregation of washed rat platelets were examined using a oxygen-radical-generating system consisting of H2O2 and ferrous ion. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS). Incubation of the platelets with various concentrations of H2O2 (2-10 mM) in the presence of 10 microM Fe2+ resulted in a decrease of the aggregating capacity and an increase of TBARS value, depending on the concentrations of H2O2. Addition of catalase (0.1 mg/ml) to the incubation medium containing 10 microM Fe2+ and 10 mM H2O2 effectively protected the aggregating capacity, but superoxide dismutase (0.1 mg/ml) did not protect H2O2/Fe(2+)-induced inhibition of the platelet aggregation. The results of kinetic studies on the platelet aggregation with varying ADP and Ca2+ concentrations suggested that treatment of the platelets with H2O2/Fe2+ causes decreases in the binding affinities of ADP and Ca2+ for the platelets. On the basis of these results, change in the aggregating capacity of the platelets by treatment with H2O2/Fe2+ is discussed in relation to lipid peroxidation.     

Inhibition of platelet aggregation by acyl-CoA thioesters

The aggregation of platelets induced by ADP, collagen, or epinephrine in human platelet-rich plasma is inhibited by long chain acyl-CoA thioesters. Palmityl-CoA exerts a concentration dependent inhibition of collagen-induced aggregation of the primary and secondary waves of ADP-induced aggregation. Palmitlyl-CoA also inhibits the secondary wave of epinephrine-induced aggregation but has no effect on the primary wave. The inhibitory effect of palmityl-CoA can be reversed by addition of excess ADP and cannot be attributed to a detergent action.     

Inhibition of ADP-induced aggregation of human platelets by beta, gamma-methylene-ATP.

Like ATP the analogue beta, gamma-methylene-ATP (AMP-PCP) is shown to be an inhibitor of both ADP-induced shape change and aggregation of human platelets. The effect of AMP-PCP on aggregation is not dependent on its conversion to adenosine, though in the presence of plasma adenosine is produced and the inhibitory effect is enhanced. Since AMP-PCP cannot be enzymatically cleaved at the beta, gamma-position the inhibitory effect cannot be attributed to utilisation of the analogue by a surface-located ATPase as has been suggested for ATP. Alternative explanations for the effect are considered with respect to some current theories of ADP-induced platelet aggregation.     

Inhibition of ADP-induced platelet shape change and aggregation by o-phthalaldehyde: evidence for covalent modification of cysteine and lysine residues

Platelets play a major role in the hemostatic process following vascular injury. Chemical modification of cysteine and/or lysine residues in platelet proteins has been shown to cause loss of platelet aggregation induced by diverse agonists; however, these investigations have not addressed the identity of the specific proteins affected. o-Phthalaldehyde (OPTH) is a unique chemical modification reagent that forms and permits the identification of fluorescent isoindole derivatives with proteins by covalently and simultaneously modifying closely spaced cysteine and lysine residues. We found that OPTH inhibited platelet aggregation induced by ADP, collagen, and U46619 (an analog of prostaglandin H2), but had minimal effect on platelet aggregation induced by thrombin, plasmin, chymotrypsin, A23187 (a calcium ionophore), PMA (phorbol 12-myristate 13-acetate), and PMA + A23187. Since platelet aggregation induced by ADP, collagen, and U46619 has been shown to involve binding of endogenous or exogenous ADP to the platelet receptor, our further studies focused on platelet aggregation induced by ADP. OPTH inhibited ADP-induced shape change and aggregation in a concentration-dependent manner. The second-order rate constant for the inhibition of ADP-induced platelet shape change (Ksc = 1.0 X 10(3) M-1 s-1) was lower than that for aggregation (Kagg = 5.4 X 10(3) M-1 s-1). Fluorescence excitation and emission spectra of OPTH-platelet adduct exhibited maxima at 346 and 437 nm, respectively, consistent with the formation of an isoindole derivative(s). The nonpenetrating thiol-specific reagent, p-chloromercuribenzenesulfonate (pCMBS) (0.8 mM), is known to block the inhibition of stimulated adenylate cyclase induced by ADP but not the ADP-induced platelet shape change. The inhibition of ADP-induced platelet shape change (Ksc = 1.5 X 10(3) M-1 s-1) by OPTH was not affected by pCMBS. OPTH, at concentrations (15-50 microM) that inhibited ADP-induced platelet aggregation and shape change did not raise the intracellular levels of adenosine cyclic 3',5'-monophosphate (cAMP) in platelets nor did it impair the ability of iloprost (a stable analog of prostaglandin I2) to raise the platelet cAMP level. Thus, OPTH under these conditions did not interact with platelet adenylate cyclase. 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been previously shown to inhibit ADP-induced platelet shape change and aggregation by covalently modifying aggregin (Mr = 100 kDa), a putative ADP receptor on platelet surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blockade of ADP-induced Ca2+-signal and platelet aggregation by lipoxygenase inhibitors

Stimulation of platelets results in the liberation of arachidonic acid (AA) which is further metabolized via the cyclooxygenase or lipoxygenase (LPG) pathway. We have examined the effect of inhibition of LPG on (i) the ADP-induced increase of cytoplasmic Ca2+ concentration and (ii) platelet aggregation. Lipoxygenase inhibitors, nordigidroguaiaretic acid (NDGA) and BW-755C, both suppressed ADP-induced Ca2+-signals and aggregation in a dose-dependent manner, with an IC50 value of 1 2 microM for NDGA. Qualitatively the same effect was obtained with 4-bromophenylacyl bromide, the inhibitor of phospholipases A2 and C. By contrast, cyclooxygenase inhibitor indomethacin had only a negligible effect on Ca2+-signals and suppressed only the second phase of ADP-induced aggregation. It is concluded that the LPG pathway of AA metabolism in platelets might play a crucial role in ADP-induced Ca2+-signal generation and platelet aggregation.     

Inhibition of platelet aggregation by anthrax edema toxin

Edema toxin is a key virulence determinant in anthrax pathogenesis that causes augmentation of cAMP inside host cells. This exotoxin has been implicated in facilitating bacterial invasion by impairing host defenses. Here, we report for the first time that edema toxin plays an important role in suppression of platelet aggregation; an effect that could be of vital significance in anthrax afflicted subjects. It was found that edema toxin induces a dose dependent and time dependent increase in cAMP inside rabbit platelets. Elevation of cAMP led to suppression of platelet aggregation as demonstrated by in vitro aggregation assays. A 95% suppression of platelet aggregation in response to thrombin and a complete suppression in response to ADP, at toxin concentrations of 7 and 2.2 nM, respectively, were observed. Antibody neutralized wild type edema factor and non-toxic mutants of this binary toxin failed to show any alteration in the normal aggregation pattern. Edema toxin caused the activation of cAMP dependent protein kinase A inside platelets, a phenomenon that could be speculated to initiate the cascade of events responsible for suppressing platelet aggregation. Furthermore, in vivo bleeding time registered a sharp increase in response to edema toxin. These findings can explicate the systemic occurrence of hemorrhage, which is a prominent symptom of anthrax. This study exemplifies how Bacillus anthracis has evolved the ability to use host's physiological processes by mimicking the eukaryotic signal transduction machinery, thus inflicting persistent infection.     

Inhibition of platelet aggregation by acyl-CoA thioesters.

The aggregation of platelets induced by ADP, collagen, or epinephrine in human platelet-rich plasma is inhibited by long chain acyl-CoA thioesters. Palmityl-CoA exerts a concentration dependent inhibition of collagen-induced aggregation and of the primary and secondary waves of ADP-induced aggregation. Palmityl-CoA also inhibits the secondary wave of epinephrine-induced aggregation but has no effect on the primary wave. This inhibitory effect of palmityl-CoA can be reversed by addition of excess ADP and cannot be attributed to a detergent action.     

Inhibition of blood platelet aggregation by dioxo-ene compounds

Compounds with a conjugated oxo-ene-oxo system were tested for inhibition of blood platelet aggregation. All compounds with this structure in trans configuration were effective inhibitors of aggregation induced by thrombin and by arachidonic acid. While the oxo-trans-ene-oxo system is prerequisite for such activity, other structural features of the compounds may be varied without loss of activity. Inhibition is exemplified by 9,12-dioxo-trans-10- and 10,13-dioxo-trans-11-octadecenoic acids and their methyl esters, by 11,14-dioxo-trans-12- eicosenoic acid, by 4,7-dioxo-trans-5- decene and by trans- dibenzoylethylene . The half-inhibition concentrations are in the order of 2-6 microM, with complete inhibition at 8-20 microM. According to experiments with the inhibiting 9,12-dioxo-trans-10-octadecenoic acid, the normal oxygenation of exogenous arachidonic acid by platelets is not affected but the thrombin-induced internal release of this acid seems to be abolished by the inhibitor. The inhibition of aggregation in the presence of exogenous arachidonic acid and its products suggests that the inhibitor also interferes with other events leading to aggregation. By implication from other properties of the oxo-trans-ene-oxo system, reaction with SH groups may be a mechanism for inhibition.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate,and its mechanism

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate, and its mechanism.

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VIII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content of dextran sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Inhibition of ADP-triggered blood platelet aggregation by diadenosine polyphosphate analogues

The synthesis and biological evaluation of new diadenosine polyphosphate analogues on blood platelet aggregation are reported. The most active are compounds with a sulfur atom replacing one or both non-bridging oxygens at phosphorus bound to adenosyl residues and hydroxymethyl groups of bis(hydroxymethyl)phosphinic acid.     

Inhibition of human blood coagulation factor Xa by alpha 2-macroglobulin

The inactivation of activated factor X (factor Xa) by alpha 2-macroglobulin (alpha 2M) was studied. The second-order rate constant for the reaction was 1.4 X 10(3) M-1 s-1. The binding ratio was found to be 2 mol of factor Xa/mol of alpha 2M. Interaction of factor Xa with alpha 2M resulted in the appearance of four thiol groups per molecule of alpha 2M. The apparent second-order rate constants for the appearance of thiol groups were dependent on the factor Xa concentration. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and factor Xa. Under nonreducing conditions, four factor Xa-alpha 2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225,000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of alpha 2M, while the second complex (Mr 400,000) consisted of the heavy chain of factor Xa molecule and two subunits of alpha 2M. Factor Xa was able to form a bridge between two subunits of alpha 2M, either within one molecule of alpha 2M or by linking two molecules of alpha 2M. Complexes involving more than two molecules of alpha 2M were not formed.     

Inhibition of platelet factor XIIIa-catalyzed reactions by calmodulin

Calmodulin was found to exhibit an inhibitory effect on platelet factor XIIIa-catalyzed incorporation of pseudodonor amines into dimethylcasein, platelet actin and myosin. The inhibitory action of calmodulin on the calcium-dependent enzyme reactions was analogous to the effects of EGTA and parvalbumin on these reactions. The extent of inhibition of factor XIIIa activity was a function of calmodulin concentration when factor XIII and Ca2+ concentrations were held constant. These results indicate that calmodulin inhibits platelet factor XIIIa-catalyzed reactions by sequestering calcium.     

Inhibition of platelet factor 3 availability by prostacyclin

Preincubation of human platelet rich plasma with PGI₂ in a concentration preventing collagen induced platelet aggregation abolished also platelet factor 3 availability brought about by collagen. Following PGI₂ pretreatment no second wave aggregation could be elicited by ristocetin. However, primary aggregation as well as platelet factor 3 activity were only partially inhibited in this case and the inhibitory action of PGI₂ was not increased by raising its concentration. Similarly, marked but not complete inhibition of platelet factor 3 availability was obtained when kaolin was used as activating agent.     

Inhibition of platelet aggregation and arachidonate metabolism in platelets by procyanidins

The effects of procyanidins on platelet aggregation and arachidonate metabolism in platelets were studied. Nine procyanidins were used in this investigation. Procyanidins B-2-S, EEC and C-1 significantly induced the inhibition of platelet aggregation, and the potency of inhibition was comparable with aspirin. Procyanidin B-2-S was used as a representative of procyanidins for further studies on the effect on arachidonate metabolism. In arachidonate metabolism by fatty acid cyclooxygenase pathway, B-2-S inhibited TXB2 and HHT formation by intact platelets treated with exogenous arachidonic acid. It also inhibited TXB2 formation measured by a specific radioimmunoassay when the cells were challenged with calcium ionophore A23187. In cell-free system, B-2-S inhibited both TXB2 and 12-HETE bioxynthesis in platelet microsome and cytosol, respectively. The inhibitory effect on thromboxane biosynthesis might explain the inhibitory effect of procyanidins on platelet aggregation.     

Dietary soya lecithin decreases plasma triglyceride levels and inhibits collagen- and ADP-induced platelet aggregation

Elevated plasma lipid concentrations and increased platelet activation are risk factors in the development of atherosclerosis. Nine patients with type IIa hyperlipoproteinemia and nine patients with type IV hyperlipoproteinemia were given soya lecithin, 12 g/day, for 3 months. Plasma cholesterol and triglycerides were reduced by 15 and 23%, respectively, and HDL-cholesterol increased by 16% in the hypercholesterolemic patients. Platelet function was unchanged. In the hypertriglyceridemic patients, total cholesterol fell by 18%, triglycerides by 36%, and HDL-cholesterol increased by 14%. There was a 27% reduction in platelet aggregation (P less than 0.01). Seventeen hypertriglyceridemic patients then received increasing doses of soya lecithin for 1-month periods (6, 12, and 18 g/day). The optimal lipoprotein-lowering effect was achieved with a daily dose of 12 g soya lecithin per day. Both low-density lipoprotein and very-low-density lipoprotein levels were reduced, and HDL-cholesterol and apolipoprotein levels were reduced, and HDL-cholesterol and apolipoprotein A-I concentrations were increased. Platelet aggregation in response to collagen and ADP was significantly reduced, parallel with the reduction in triglyceride level. Soya lecithin supplementing the diet may be useful in the management of the hypertriglyceridemic patient.