Inhibitory effect of glyburide on thrombin-induced platelet aggregation and phosphoinositide metabolism in normal human platelets

We previously reported the effects of diet, sulphonylureas or insulin on thrombin-induced platelet aggregation, phosphoinositide metabolism and protein phosphorylation in non-insulin-dependent diabetes mellitus (NIDDM) patients. To clarify the mechanism of glyburide and insulin on platelet function, here we studied the in vitro effects of glyburide and insulin on thrombin-induced metabolic changes using normal human platelets. Platelet aggregation stimulated with <0.5 U/ml thrombin, 0.75-3 microM adenosine diphosphate (ADP) or 1 microg/ml collagen was significantly lower in glyburide-treated platelets, but not in insulin-treated platelets, than in untreated ones (control). Thrombin-induced incorporation of 32P radioactivity into phosphatidic acid (PA) in glyburide-treated platelets was lower than that in control but not in insulin-treated platelets. Phosphorylated proteins of platelets induced by thrombin and 12- O -tetradecanoylphorbol 13-acetate (TPA) in glyburide-treated platelets were suppressed, but not in insulin-treated platelets, compared with control. These results suggest that glyburide induces suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP(2) and production of PA, and subsequently inhibits platelet aggregation.     

Thrombin-induced platelet aggregation,phosphoinositide metabolism and protein phosphorylation in NIDDM patients treated by diet,sulphonylurea or insulin

Summary We studied thrombin-induced metabolism of phosphoinositide, protein phosphorylation and platelet aggregation in platelets from 32 NIDDM patients and 12 control subjects. To clarify the effect of diet, sulphonylureas, or insulin treatment, the subjects were divided into three groups based on the type of treatment. Thrombin-induced platelet aggregation was measured with an aggregometer. Low-dose thrombin (0.25 U/ml)-stimulated platelet aggregation in diabetic patients was significantly increased compared with the control subjects. Platelet aggregation in the sulphonylurea and insulin groups was significantly lower than in the diet group. On the other hand, in platelets incubated with [³²P]orthophosphate, thrombin-induced incorporation of ³²P radioactivity into phosphatidic acid (PA) was significantly lower in the sulphonylurea and insulin groups than in the diet group. Thrombin-induced incorporation of [³²P] radioactivity into phosphatidylinositol (PIP) for 10 s was significantly higher in the sulphonylurea group than in the diet group. There were no differences in thrombin-induced 47 kDa protein phosphorylation between platelets from the diet, sulphonylurea, or insulin groups. These results suggest that sulphonylureas and insulin induce suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP₂ and production of PA, which leads to inhibition of platelet aggregation.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - IDDM insulin-dependent diabetes mellitus - PA phosphatidic acid - PI phosphatidylinositol - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - TPA 12-O-tetradecanoylphorbol-13-actate - IP₃ inositol 1,4,5-trisphophate - E/I expiration/inspiration - HPLTC high performance thin layer chromatography     

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.     

Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).     

Changes in Phosphoinositides in Rabbit Platelets during Clot Formation. Comparison of Platelets Stimulated by ADP or by Thrombin in the Presence of Polymerising Fibrin

Platelet phosphoinositide metabolism was examined during platelet-fibrin clot formation stimulated by ADP (10 μM) plus reptilase, or by thrombin (1 U/ml), for 120 s in the presence of fibrinogen, to determine which changes are specifically associated with this process. Stirring at 200 rpm was used to minimise the contribution of aggregation to the platelet changes. Under these conditions, thrombin caused extensive release of the contents of platelet granules; ADP plus reptilase did not. The presence of fibrinogen decreased the amount of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) by 46.4±5.5% when thrombin was the stimulus, and by 47.4±5.5% when the platelets were stimulated by ADP plus reptilase. Fibrinogen did not decrease the extraction of other phospholipids. The amount of phosphatidylinositol 4-phosphate (PIP) increased when platelets were stimulated in either the presence or absence of fibrinogen. These increases were greater in the presence of fibrinogen and the thrombin-induced increase was smaller than the increase induced by ADP plus reptilase; with ADP plus reptilase, the increase in PIP more than accounted for the loss of extractable PIP(2). In platelets prelabelled with [(3)H]inositol, the decrease in PIP(2) labelling induced by fibrinogen with ADP plus reptilase as the stimulus was accounted for by the increase in PIP labelling; the decrease induced by fibrinogen with thrombin as the stimulus was not. With thrombin, 46.5% of the decrease in PIP(2) labelling, caused by fibrinogen, was accounted for by label that remained with the interfacial protein after lipid extraction; with ADP plus reptilase, the amount of label with this protein was the same with or without fibrinogen. Only thrombin increased the amount of label in inositol trisphosphate (IP(3)) and the amount of phosphatidic acid (PA); these changes were not increased by fibrinogen. Thus, the results with ADP plus reptilase indicate that clot formation is not dependent on release of granule contents, formation of detectable IP(3) or PA (and hence does not require activation of phospholipase C) or association of [(3)H]inositol-labelled compounds with protein. Clot formation is associated with a shift in the PIP(2)-PIP equilibrium toward PIP.     

Plasmin inhibition of thrombin-induced platelet aggregation.

The effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10-20 muM, PLASMIN (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin's inhibition of aggregation. Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction. Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin-treated platelets, upon centrifugation and treatment with SBTI. With the use of a "cold initiation" technique, the release by thrombin of 46.7 plus or minus 6.7 (mean plus or minus SEM) mu-g of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by STBI) aggregated normally upon addition of as little as 10 mu-g human plasma fibrinogen per mg platelet protein. It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.     

Thrombin interaction with human platelets. Potentiation of thrombin-induced aggregation and release by inactivated thrombin

The possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombin-induced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.     

Carbon monoxide released by CORM-3 inhibits human platelets by a mechanism independent of soluble guanylate cyclase

OBJECTIVE: Carbon monoxide (CO) modulates several physiological functions through activation of a cGMP-dependent pathway similar to that of nitric oxide (NO). Here we investigated the possible involvement of soluble guanylate cyclase in the anti-aggregatory effect of micromolar concentrations of CO released by a novel, water-soluble, CO releasing molecule (CORM) in human platelets. METHODS: Human platelet aggregation was induced by collagen or thrombin, and the effects of CO releasing molecule (CORM-3) and an NO donor on platelet aggregation were compared. RESULTS: CORM-3 liberated CO in a time- and concentration-dependent manner as evidenced by the formation of carbon monoxy myoglobin (MbCO) using a spectrophotometric assay. When added to washed platelets, CORM-3 (10-300 microM) inhibited collagen- and thrombin-induced aggregation in a concentration-dependent manner. The anti-aggregatory effect of CORM-3 was reversed by deoxy-Mb (50 microM). Interestingly, in the presence of an inhibitor of guanylate cyclase (ODQ, 5 microM), inhibition of collagen-induced aggregation by CORM-3 was not blocked but potentiated. Under the same experimental conditions, inhibition of platelet aggregation by an NO donor (SNAP, 1 microM) was prevented by ODQ. In collagen-induced or thrombin-induced platelet aggregation, a stimulator of guanylate cyclase (YC-1, 0.3 microM) did not alter the effect of CORM-3, whereas it markedly potentiated the inhibition of platelet aggregation mediated by SNAP. Notably, CORM-3-induced inhibition of platelet aggregation was of similar degree when platelets were activated by a low (20 mU/ml) or by high concentration of thrombin (100-200 mU/ml), whereas NO donors (SNP and SNAP)- or carbaprostacylin (cPGI(2))-induced effects were considerably attenuated when platelets were activated by high concentrations of thrombin. CONCLUSIONS: Inhibition of platelet aggregation by CO released by a novel, water-soluble CORM is not mediated by activation of soluble guanylate cyclase. In contrast to NO and PGI(2), CO effectively inhibits platelets even when cells are activated excessively. We suggest that despite the fact that CO is not a potent inhibitor of platelet activation, it may gain importance when NO and PGI(2) alone are insufficient to overcome excessive platelet activation.     

Original Article: Intracellular Ca(2+) Mobilization and Aggregatory Response to ADP and Thrombin in Aged Rat Platelets

We previously reported that thrombin-induced Ca(2+) mobilization was enhanced in aged rat platelets. Since Ca(2+) mobilization in platelets is believed to be closely associated with platelet activation, we examined Ca(2+) mobilization and the aggregatory response to ADP and thrombin in young (3 months) and aged (24 months) rat platelets. Blood levels of fibrinogen and Ca(2+) in aged rats were higher than those in young rats. ADP-induced platelet aggregation in aged rats was significantly enhanced in platelet rich plasma and in washed platelet suspension, suggesting that age-associated hyperaggregability to ADP is attributable to changes in platelets themselves. On the other hand, thrombin (0.03-0.3 unit/ml)-induced aggregation in washed platelet suspension from aged rats was comparable to that from young rats. But, thrombin (0.3 unit/ml)-induced intracellular Ca(2+)mobilization was enhanced in aged rat platelets in the presence of extracellular Ca(2+). Likewise, ADP-induced Ca(2+) mobilization was enhanced in aged rat platelets. These results suggest that enhanced Ca(2+) mobilization in aged rat platelets is associated with hyperaggregability to ADP but not to thrombin.     

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.     

Platelet Aggregation and Release by ADP and Thrombin: Evidence for Two Separate Effects of ADP on Platelets, Involvement of Fibrinogen in Release, and Mechanism of Inhibitory Action of Acetylsalicylic Acid

S ummary . The previously demonstrated roles of thrombin and fibrinogen in platelet interaction (Ardlie & Han, 1974) suggested a possible relationship between platelet aggregation and the release reaction. The present results show that a product of the action of thrombin on fibrinogen induced the release of platelet constituents. This activity was not inhibited by heparin. The thrombin-fibrinogen reaction which occurs when platelets come into contact could be the cause of the subsequent release reaction. ADP was shown to be essential for the thrombin-induced aggregation, but not the alteration in platelet morphology. Evidence of involvement of ADP in cell contact was obtained and it is possible that its role may be to facilitate aggregation through an effect on platelet surface charge. Thus, ADP induces aggregation through two distinct actions: (1) alteration of platelet morphology; and (2) reduction in net negative surface charge of platelets. Incubation of thrombin with aspirin inhibited its action on platelets and it is suggested that acetylation of thrombin (or prothrombin) by aspirin may account for the action of this compound on platelets. The significance of these observations and a possible mechanism for the release reaction is discussed.     

Original Article: Intracellular Ca2+ Mobilization and Aggregatory Response to ADP and Thrombin in Aged Rat Platelets

We previously reported that thrombin-induced Ca²⁺ mobilization was enhanced in aged rat platelets. Since Ca²⁺ mobilization in platelets is believed to be closely associated with platelet activation, we examined Ca²⁺ mobilization and the aggregatory response to ADP and thrombin in young (3 months) and aged (24 months) rat platelets. Blood levels of fibrinogen and Ca²⁺ in aged rats were higher than those in young rats. ADP-induced platelet aggregation in aged rats was significantly enhanced in platelet rich plasma and in washed platelet suspension, suggesting that age-associated hyperaggregability to ADP is attributable to changes in platelets themselves. On the other hand, thrombin (0.03-0.3 unit/ml)-induced aggregation in washed platelet suspension from aged rats was comparable to that from young rats. But, thrombin (0.3 unit/ml)-induced intracellular Ca²⁺mobilization was enhanced in aged rat platelets in the presence of extracellular Ca²⁺. Likewise, ADP-induced Ca²⁺ mobilization was enhanced in aged rat platelets. These results suggest that enhanced Ca²⁺ mobilization in aged rat platelets is associated with hyperaggregability to ADP but not to thrombin.     

Proteoglycan obtained from bovine aorta suppress thrombin-induced platelet aggregation.

Proteoglycan (PG), isolated and purified from bovine aorta (intima-media), consisted of 68.6% chondroitin 4/6-sulfate (CS 4/6-S), 30% dermatan sulfate (DS), 1.4% heparan sulfate (HS), and a trace of hyaluronic acid (HA). PG did not affect platelet aggregation induced by ADP, collagen, and epinephrine, but inhibited that induced by thrombin. Of the standard GAGs investigated, hyaluronic acid (HA) and CS-4/6-S slightly inhibited only thrombin-induced platelet aggregation. However, PG and standard GAGs did not affect the thrombin induced aggregation of washed platelets. The effect of PG after papain digestion on thrombin-induced platelet aggregation was less potent than that before. It is suggested by the results of this study that PG in the aorta inactivates plasma thrombin, probably by inhibiting thrombin activators or potentiating substances which inactivate thrombin and that these effect of PG would be mainly due to PG-DS and partly due to PG-HS.     

Anti-platelet effects of different phenolic compounds from Yucca schidigera Roezl. bark

Resveratrol (3,4',5-trihydroxystilbene) has been reported to have a variety of anti-inflammatory, anti-carcinogenic, anti-fungal and anti-platelet effects. It occurs naturally in different medicinal plants. Recently, resveratrol and other related phenolic compounds including trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene and yuccaols A and C were isolated from the bark of Yucca schidigera. The aim of the present study was to evaluate in vitro the effects of these compounds on platelet aggregation induced by thrombin and ADP. Pretreatment of platelets with resveratrol or other tested phenolics (1-25 microg/ml) slightly reduced platelet aggregation stimulated by 5 microM ADP (P < 0.05) or 10 microM ADP (P < 0.005). The comparison of the inhibitory effects of tested compound in thrombin-induced platelet aggregation revealed that phenolic showed even stronger antiplatelet actions than resveratrol. These compounds also had an inhibitory effect on the thrombin-induced enzymatic platelet lipid peroxidation determined as the level of thiobarbituric acid reactive substances.     

Nitric Oxide (NO)-Platelet Interactions: Inhibition is Independent of the Prostanoid and ADP Pathways

Effects of nitric oxide (NO) on thrombin-induced responses in gel-filtered, [(32)P] Pi-(pre) labeled platelets (GFP) were examined. NO did not alter the levels of (32)P-labeled polyphosphoinositides in unstimulated platelets and did not inhibit the forskolin-induced elevation of [(32)P]PIP (phosphatidylinositol 4-phosphate), which indicates that NO does not concomitantly increase the level of cAMP in resting human platelets. In aspirinated platelets NO inhibited thrombin (0.05 U/ml)-induced formation of [(32)P]phosphatidic acid (PA), secretion of ATP + ADP from the dense granules and secretion of acid glycosidases in a dose-dependent manner. At 0.2 U/ml of thrombin NO still inhibited these responses, although to a lesser degree. In aspirinated platelets in the presence of creatine phosphate/creatine phosphokinase (CP/CPK) to remove secreted ADP, increasing concentrations of NO still produced strong inhibition of [(32)P] PA-formation and secretory responses.     

Inhibitory mechanism of human platelet aggregation by nafamostat mesilate

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myrisate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 mu M. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 mu M, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 mu M, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 mu M. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 mu M NM and was completely inhibited by 50 mu M NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.     

Thrombin-, Epinephrine- and Collagen-Induced Platelet Aggregation Inhibited by α1-Acid Glycoprotein

α₁-acid glycoprotein (α₁-acid GP) isolated from human plasma was found to inhibit thrombin-induced aggregation of washed human platelets (final thrombin concentration 0.05 NIH U/ml), and inhibition was complete with physiological concentrations of the glycoprotein (1.0–1.5 g/l final conc.). The inhibitory effect seemed to occur immediately on thrombin addition, thus being similar to the effect of heparin previously observed. As opposed to heparin, however, α₁-acid GP did not affect spontaneous platelet aggregation. Furthermore, α₁-acid GP (in optimal concentrations) reduced the combined inhibitory effect of heparin and antithrombin III on thrombin-induced platelet aggregation, thus being consistent with the previous findings using heparin thrombin clotting time. Snyder & Coodley (1976) found α₁-acid GP to inhibit platelet aggregation induced by epinephrine and adenosine diphosphate in platelet-rich plasma. As we also found α₁-acid GP to inhibit collagen-induced platelet aggregation, this acid glycoprotein may possibly act as an inhibitor of the release reaction although fairly high concentrations (10 mg/ml final conc.) were needed for complete inhibition.     

The common pathway for alpha- and gamma-thrombin-induced platelet activation is independent of GPIb: a study of Bernard-Soulier platelets

The responses to alpha- and gamma-thrombin were studied in normal and Bernard-Soulier platelets labelled with [32P]phosphate, to investigate the relationship between thrombin binding to the platelet membrane glycoprotein Ib (GPIb) and thrombin-induced platelet activation. For this purpose we conducted parallel studies of the kinetics of platelet aggregation, granule secretion, hydrolysis of polyphosphoinositides, formation of phosphatidic acid, phosphorylation of the myosin light chain (p20) and of the 43 kDa protein (p43), and thromboxane B2 formation. Like alpha-thrombin, gamma-thrombin activated control platelets via all the above metabolic responses, but only after a prolonged lag. In Bernard-Soulier platelets, alpha-thrombin induced polyphosphoinositide hydrolysis and phosphatidic acid formation, p20 and p43 phosphorylation, thromboxane B2 formation, secretion and to a lesser extent aggregation, but only after a prolonged lag. The metabolic responses of Bernard-Soulier platelets to gamma-thrombin were very similar to those of control platelets. We have previously showed that GPIb which is not present in Bernard-Soulier platelets binds alpha- but not gamma-thrombin. The present results indicate that thrombin binding to GPIb is not directly coupled either with the activation of phospholipase C specific to polyphosphoinositides, or with the activation of protein kinase C and phospholipase A2. However, thrombin binding to GPIb appears to promote an early mechanism which accelerates all the platelet responses.     

Role of homocysteine and folic acid on the altered calcium homeostasis of platelets from rats with biliary cirrhosis

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state. Different studies indicate that cholestatic diseases are associated with hyperhomocysteinemia; thus, we hypothetized that it could contribute to those platelet alterations. In the present study, we have investigated the role of homocysteine (HCY) in platelet aggregation and calcium signaling in the BDL model. The effect of chronic folic acid treatment was also analyzed. Acute treatment with HCY increased the aggregation response to ADP and calcium responses to thrombin in platelets of control and BDL rats. Capacitative calcium entry was not altered by HCY. Chronic treatment with folic acid decreased platelet aggregation in control and BDL rats, but this decrease was greater in BDL rats. In folic acid-treated rats, thrombin-induced calcium entry and release were decreased in platelet of control rats but unaltered in BDL rats; however, capacitative calcium entry was decreased in platelets of control and BDL rats treated with folic acid. Reactive oxygen species were produced at higher levels by BDL platelets after stimulation with HCY or thrombin and folic acid normalized these responses. HCY plays a role in the enhanced platelet aggregation response of BDL rats, probably through an enhanced formation of ROS. Folic acid pretreatment normalizes many of the platelet alterations shown by BDL rats.     

Anti-platelet effects of different phenolic compounds from Yucca schidigera Roezl. Bark

Resveratrol (3,4',5-trihydroxystilbene) has been reported to have a variety of anti-inflammatory, anti-carcinogenic, anti-fungal and anti-platelet effects. It occurs naturally in different medicinal plants. Recently, resveratrol and other related phenolic compounds including trans -3,3',5,5'-tetrahydroxy-4'-methoxystilbene and yuccaols A and C were isolated from the bark of Yucca schidigera . The aim of the present study was to evaluate in vitro the effects of these compounds on platelet aggregation induced by thrombin and ADP. Pretreatment of platelets with resveratrol or other tested phenolics (1-25 w g/ml) slightly reduced platelet aggregation stimulated by 5 w M ADP ( P < 0.05) or 10 w M ADP ( P < 0.005). The comparison of the inhibitory effects of tested compound in thrombin-induced platelet aggregation revealed that phenolic showed even stronger antiplatelet actions than resveratrol. These compounds also had an inhibitory effect on the thrombin-induced enzymatic platelet lipid peroxidation determined as the level of thiobarbituric acid reactive substances.