Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen.     

Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization

The effect of ethanol on signal generation in collagen-stimulated human platelets was evaluated. Incubation of washed human platelets with physiologically relevant concentrations of ethanol (25-150 mM) resulted in a dose-dependent inhibition of aggregation and secretion in response to collagen (0.5-10 micrograms/ml), but did not inhibit shape change. In platelets labeled with [3H]arachidonic acid, ethanol significantly inhibited the release of arachidonic acid from phospholipids, in both the presence and the absence of indomethacin. Thromboxane B2 formation was also inhibited in proportion to the reduction in free arachidonic acid. There was a close correlation between the extent of inhibition of arachidonic acid release and secretion. The inhibition of platelet aggregation and secretion by ethanol was partially overcome by the addition of exogenous arachidonic acid. In the presence of indomethacin, ethanol had no effect on the activation of phospholipase C by collagen as determined by the formation of inositol phosphates and phosphatidic acid. Moreover, ethanol had no effect on the mobilization of intracellular calcium by collagen and only minimally inhibited the early phases of the phosphorylation of myosin light chain (20 kDa) and a 47-kDa protein, a known substrate for protein kinase C. Arachidonic acid formation was also inhibited by ethanol in response to ionomycin under conditions where phospholipase C activation was prevented. The results suggest that the functional effects of ethanol on collagen-stimulated platelets are due, at least in part, to an inhibition of phospholipase A2.     

Inhibition of fractalkine ameliorates murine collagen-induced arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-gamma by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.     

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 platelet secretion of ATP by phalloidin

The involvement of actin in the secretion of ATP by platelets was studied using two stimulants, ADP and A23187, and two actin-mediating reagents, cytochalasin B and phalloidin. The degree of actin polymerization was determined using DNase I. Preincubation of platelets with cytochalasin B suppressed the polymerization of actin and ATP secretion induced by stimulants. In the absence of the stimulant, phalloidin-treated platelets exhibited time-dependent actin polymerization and the maximum level was reached at 5 min. No secretion of ATP was observed. The polymerization was enhanced by phalloidin when the platelets were preincubated for 3 to 5 min with the stimulants, but little ATP was secreted. After a 30-min preincubation, the amount of polymerized actin was lower than that after a 5-min incubation, and no ATP was secreted.     

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.     

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 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.     

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein (or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.     

Inhibition of ADP-induced platelet aggregation by reduced factor Xa

Treatment of blood coagulation factor Xa with insolubilized hexyl-agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes. Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.     

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.     

L—精氨酸L—门冬氨酸盐对血小板功能的抑制

用血小板聚集、粘附、释放实验和出血时间测定观察L-精氨酸*L-门冬氨酸盐(DR)对血小板功能的作用。实验结果显示DR15mg/kg静脉给药,可明显抑制腺苷二磷酸(ADP)诱导的大鼠血小板聚集(P<0.01);15mg/kg单次口服给药可明显抑制ADP诱导的家兔血小板聚集;其药效可持续8h以上(P<0.01);DR7.5、15、30mg/kg灌胃给药(Bid×3.5d),可明显抑制ADP、胶原或凝血酶诱导的大鼠血小板聚集(P<0.01),并延长出血时间(P<0.05)。DR30mg/kg可明显抑制大鼠血小板粘附,并促进血管内皮释放前列环素(PGI2),但对活化的血小板释放血拴素(TXA2)无明显影响。本研究发现,DR可抑制血小板聚集和粘附功能,其作用机制不同于阿司匹林。这些作用部分是由于DR增加了血管内皮PGI2的释放。此结果为血小板功能的调节提供了新线索。     

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.     

Effect of gender difference on platelet reactivity

Background

Previous studies have suggested that women do not accrue equal therapeutic benefit from antiplatelet medication as compared with men. The physiological mechanism and clinical implications behind this gender disparity have yet to be established.

Methods

On-treatment platelet reactivity was determined in 717 men and 234 women on dual antiplatelet therapy, undergoing elective coronary stent implantation. Platelet function testing was performed using arachidonic acid and adenosine diphosphate-induced light transmittance aggregometry (LTA) and the VerifyNow P2Y12 and Aspirin assays. Also the incidence of all-cause death, non-fatal acute myocardial infarction, stent thrombosis and ischaemic stroke was evaluated.

Results

Women had higher baseline platelet counts than men. Women exhibited a higher magnitude of on-aspirin platelet reactivity using LTA, but not using the VerifyNow Aspirin assay. The magnitude of on-clopidogrel platelet reactivity was significantly higher in women as compared with men with both tests used. The cut-off value to identify patients at risk as well as the incidence of clinical endpoints was similar between women and men (16/234[6.8%] vs. 62/717[8.6%], p = 0.38).

Conclusion

Although the magnitude of platelet reactivity was higher in women, the absolute difference between genders was small and both the cut-off value to identify patients at risk and the incidence of the composite endpoint were similar between genders. Thus, it is unlikely that the difference in platelet reactivity accounts for a worse prognosis in women.