Unexpected effects of aurin tricarboxylic acid on human platelets.

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of rabbit platelet aggregation by 1,4-naphthoquinones

The effects of four 1,4-naphthoquinone derivatives on the aggregation of rabbit platelets were examined. All the four 1,4-naphthoquinone derivatives inhibited the platelet aggregation of washed rabbit platelets induced by thrombin (0.1 U/ml) and the IC50 is: 2-chloro-3-methyl-1,4-naphthoquinone (CMN), 5 micrograms/ml; 3-methyl-5,8-dihydroxy-1,4-naphthoquinone, 13 micrograms/ml; 5,8-dihydroxy-1,4-naphthoquinone, 18 micrograms/ml; 3-methyl-1,4-naphthoquinone (vitamin K3), 53 micrograms/ml. CMN was the most potent in inhibiting the aggregation and release reaction induced by ADP, arachidonic acid, PAF, ionophore A23187, collagen and thrombin in a dose-dependent manner in washed platelets, platelet-rich-plasma and whole blood. The thromboxane B2 formation caused by collagen and ionophore A23187 was inhibited by CMN. However, the thromboxane B2 formation by arachidonic acid was markedly increased. The platelet inhibitory effect of CMN could not be antagonized either by raising the concentrations of extracellular Ca++ or by wash out. The phosphoinositides breakdown induced by thrombin was inhibited by CMN. Phospholipids (PE, PC, PI) could slightly antagonize the antiplatelet effect of CMN. It is concluded that the inhibitory effect of CMN on rabbit platelet aggregation may be due to the inhibition of phosphoinositides breakdown caused by the inducers.     

Functional involvement of thrombospondin in platelet aggregation induced by low versus high concentrations of thrombin

Thrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (less than or equal to 0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (greater than or equal to 60 micrograms/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (greater than or equal to 60 micrograms/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of G619, a new platelet aggregation inhibitor.

G619, a 4-OH-isophthalic acid derivative, was studied for its capacity to inhibit platelet aggregation. G619 dose-dependently inhibited U46619, collagen, ADP, PAF, thrombin and epinephrine-induced platelet aggregation in vitro. The IC50 values for inhibition of U46619-induced human and rabbit platelet aggregation were 39 and 43 microM, respectively. G619, at 100 microM, inhibited high concentration collagen (10 micrograms/ml)-induced aggregation of rabbit platelets pretreated with indomethacin and increased the level of cAMP in washed rabbit platelets by 30% (p less than 0.01 vs basal). However, G619, did not inhibit fibrinogen binding to GPIIb/IIIa receptor, phosphodiesterase, U46619-induced contractile responses on canine saphenous vein or rabbit aorta, calcium-induced vasoconstriction and thrombin or PAF-induced elevation of [Ca++]i in platelets in vitro. In vivo, the U46619-induced maximal thrombocytopenia in rats was reduced from 40% (vehicle) to 22% and 18% by 10 and 30 mg/kg of G619 i.v., respectively. G619 (30 mg/kg) had no effect on the U46619-induced vasopressor response or sudden death in rats, and had no effect on TxB2 formation. Our results indicate that G619 is a broad-spectrum platelet aggregation inhibitor and may have its effect on a common mechanism for platelet aggregation besides an effect on the thromboxane A2 receptor.     

Antiplatelet effect of capsaicin

Capsaicin was found to be a potent inhibitor of platelet aggregation and release reaction. It inhibited the aggregation of rat platelet induced by collagen and thrombin, but only slightly reduced those of AA and A23187. The IC₅₀ on collagen-induced platelet aggregation was about 85 μg/ml. Less inhibition was observed in the aggregation of platelet-rich plasma. Increase of the calcium concentration could not overcome the inhibitory effect. Washing of the capsaicin-pretreated platelets only partially reversed the inhibition. Capsaicin also inhibited ATP release induced by thrombin and A23187 in the presence of EDTA. MDA and TXB₂ formation were markedly inhibited by capsaicin in platelets challenged by collagen, thrombin and A23187. In AA-stimulated platelets, MDA formation was slightly decreased and TXB₂ formation was not affected. Capsaicin showed more marked inhibition in the presence of CP/CPK, indomethacin or a combination of both. Capsaicin reduced the hemolysis of RBCs induced by hydrogen peroxide or hypotonicity. It was concluded that capsaicin had some membrane stabilizing property and this might lead to the interferance of the activation of phospholipase A₂.     

Ticlopidine selectively inhibits human platelet responses to adenosine diphosphate.

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.     

Antithrombogenic effects of calcium channel blockers: synergism with prostacyclin and thromboxane synthase inhibitors

Four calcium channel blockers (nimodipine, nifedipine, verapamil and diltiazem) of three chemical classes were tested in vitro for inhibition of platelet aggregation using heparinized human platelet rich plasma. Both ADP- and thrombin-induced aggregation were inhibited as was the biosynthesis of thromboxane A2 in response to ADP or thrombin. However, the IC50's for the calcium channel blockers were greater than or equal to 110 microM. Nimodipine was also tested in combination with prostacyclin, the potent platelet antiaggregatory agent, or with a thromboxane synthase inhibitor, U63557A. At concentrations at which neither nimodipine or prostacyclin inhibited platelet aggregation greater than or equal to 10%, the two compounds is combination synergistically inhibited both ADP- and thrombin-induced platelet aggregation. U63557A inhibited biosynthesis of thromboxane A2 by platelets in response to ADP or thrombin, but did not inhibit either ADP- or thrombin-induced platelet aggregation. However, U63557A in combination with a threshold inhibitory concentration of nimodipine resulted in a synergistic inhibition of platelet aggregation induced by ADP or thrombin. These results suggest that calcium channel blockers may be of therapeutic value as a new class of antithrombogenic agents when used in combination with agents that inhibit either platelet aggregation or synthesis of platelet thromboxane A2.     

Inhibition of platelet thromboxane formation and phosphoinositides breakdown by osthole from Angelica pubescens

Osthole, isolated from Chinese herb Angelica pubescens, inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin in washed rabbit platelets. It showed a weak activity in platelet-rich plasma. Osthole inhibited the thromboxane B2 formation caused by arachidonic acid, collagen, ionophore A23187 and thrombin in washed platelets, and also the thromboxane B2 formation caused by the incubation of lysed platelet homogenate with arachidonic acid. The generation of inositol phosphates in washed platelets caused by collagen, PAF and thrombin was suppressed by osthole. These data indicate that the inhibitory effect of osthole on platelet aggregation and release reaction was due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Thrombin receptor occupancy modulates aggregation efficiency and platelet surface expression of vWF and thrombospondin at low thrombin concentrations.

Previous studies evaluating requirements for occupancy of thrombin receptors in normal platelet secretion and aggregation, using the thrombin antagonists hirudin and PPACK (D-Phe-Pro-Arg-chloromethylketone), have suggested that at low thrombin activating concentrations (0.025-0.13 U/ml), occupancy was required only in the first 45-60 s following activation. In our study, we differentiate between thrombin receptor occupancy requirements for surface expression of secreted adhesive proteins, for activation of GPIIb-IIIa receptors, and for aggregation of washed platelets (WP) in laminar shear flow. Platelets activated with 0.05 U/ml thrombin for 10 min to allow maximal secretion (hereafter referred to as "pre-activated platelets"), then sheared, showed a 50-70% decrease in platelet counts after 60 s of shear. Treatment of pre-activated platelets with hirudin or PPACK produced a 65% reduction of capture efficiencies, alphaG (reflecting experimental/theoretical initial rates of aggregation), as well as a 30-40% decrease in the surface expression of von Willebrand factor (vWF) and thrombospondin (TSP). However, alpha-granule membrane P-selectin expression and numbers of activated GPIIb-IIIa receptors were comparable for treated and non-treated platelets. No significant difference in any of the parameters tested was observed when platelets were similarly pre-activated with 0.2 U/ml thrombin, due to treatment with thrombin antagonists. Binding of soluble FITC-vWF (GRGDSP-sensitive) to pre-activated, thrombin antagonist treated platelets, was greatly reduced (> or =80%). Soluble Fg was shown to bind to antagonist-treated pre-activated platelets, but could not significantly enhance platelet aggregation. Although occupancy of thrombin receptors by catalytically active thrombin is required transiently for secretion and activation of platelets, there is a further requirement for thrombin occupancy at low thrombin concentrations, for optimizing initial rates of platelet aggregation, surface expression of vWF and TSP, and activated GPIIb-IIIa ligand recognition.     

Evidence that the rat is not an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation

Abnormal platelet aggregation seen in experimentally induced diabetic, hypercholesterolemic and spontaneously hypertensive rats (SHR) has been linked with increased prostaglandin synthesis. The present study was conducted to examine the role of prostaglandins in rat platelet activation using normal Wistar Kyoto (WKY) and SHR rats. Up to 30 microM ADP did not induce secondary phase of platelet aggregation in rat PRP and up to 30 microM epinephrine did not produce any response in rat PRP. In other experiments ADP (1.0 microM) and epinephrine (2.0 microM) induced typical biphasic aggregation responses in human PRP. Up to 20 microM U46619, a stable analog of prostaglandin H2, did not induce platelet aggregation in rat PRP or washed rat platelets. In contrast 2.0 microM U46619 caused maximal aggregation in human PRP and washed human platelets. Arachidonic acid (1.5-2.0 mM) induced aggregation in washed rat platelets. However, this was associated with excessive (67% and 94%) loss of cytoplasmic LDH. The low concentrations of thrombin (0.04 and 0.05 U/ml), induced two to three-fold increase in aggregation response in SHR platelets as compared to WKY platelets. Higher concentrations of thrombin (0.1 and 0.3 U/ml) induced similar aggregation responses in SHR and WKY platelets. Thrombin (0.04-0.3 U/ml) induced serotonin secretion in a concentration dependent manner. The extent of secretion was the same in SHR and WKY platelets at all concentrations. Thrombin-induced synthesis of thromboxane A2 (TXA2) in WKY and SHR platelets was quantified using a radioimmunoassay for TXB2. Thrombin (0.04-0.3 U/ml) produced TXB2 in WKY and SHR platelets in a concentration dependent manner. The SHR platelets produced significantly larger amounts of TXB2 as compared to WKY platelets. In other experiments aspirin (500 microM) inhibited thrombin (0.05 U/ml) induced TXB2 synthesis by 75% in both WKY and SHR platelets but failed to inhibit aggregation or secretion in either WKY or SHR platelets. Based on these data it is suggested that: (a) rat platelets inspite of their ability to synthesize TXA2 do not require TXA2 for aggregation; and (b) the rat may not be an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation.     

Aptamer inhibits degradation of platelet proteolytically activatable receptor, PAR-1, by thrombin.

We investigated the in vitro effects of the site-directed thrombin inhibitor-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTTGG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and platelet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin-dependent degradation of proteolytically activatable receptor, PAR-1, exposed on platelet surface membrane at micromolar concentrations. The incubation of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up to fourfold, P<.0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46+/-20% inhibition at 0.15 U/ml thrombin, P<.001), as well as thrombin-mediated platelet aggregation in PRP (7+/-10% inhibition at 1 U/ml thrombin, P<.05). Furthermore, aptamer inhibited the thrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17%, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 nM (P<.025 by randomised block analysis; P(regression slope)<.0001). We conclude that aptamer is able to considerably attenuate thrombin proteolytic activity regardless of the molecular size of thrombin substrates. Our observations directly proved that aptamer may be successfully used for the inhibition of thrombin activity towards various physiological targets: one related to fibrin generation in the final stage of coagulation cascade, and another concerning the interaction of thrombin with its surface membrane receptor, PAR-1, in blood platelets.     

Low molecular weight heparin as an anticoagulant for in vitro platelet function studies

We evaluated the suitability of low molecular weight (LMW) heparin as an anticoagulant for in vitro platelet function tests in 11 normal volunteers. Results with citrated platelets were considered as the standard. Spontaneous platelet aggregation and the aggregation responses to ADP, epinephrine, collagen, ristocetin and thrombin were measured turbidimetrically in an aggregometer. Dose-response and dose-rate curves were constructed for ADP- and epinephrine-induced aggregation. The maximum aggregation response (EDmax) and rate (EDRmax), and the estimated dose of agonist to induce 50% of the maximum response (ED50) and rate (EDR50) were calculated from these curves. The inhibition of ADP-induced aggregation with PGI2 was expressed as per cent inhibition. The release of ATP and TxA2, from platelets aggregated with collagen was measured. No spontaneous aggregation occurred with either anticoagulant. The ED50 and the EDR50 for heparinized platelets were significantly lower for ADP induced aggregation (0.8 +/- 0.3 microM vs 2.1 +/- 1.0 microM [p = 0.001] and 0.4 +/- 0.1 microM vs 0.8 +/- 0.3 microM [p = 0.003]). The EDRmax with ADP was significantly higher (p = 0.004) for heparinized platelets (64.6 +/- 17.0 units/ml vs 50.4 +/- 7.6 units/ml). The heparinized platelets aggregated slightly, but significantly, less in response to ristocetin than the citrated platelets. The response of washed heparinized and citrated platelets to thrombin was not significantly different. The per cent inhibition of ADP aggregation with PGI2, was significantly lower with heparinized platelets. The release of TxA2 and ATP was similar for both anticoagulants. These results indicate that LMW heparin is a satisfactory anticoagulant for platelet aggregation tests.     

Effect of clopidogrel on thrombin generation in platelet-rich plasma in the rat.

Clopidogrel (25 mg/kg, p.o.), a potent and selective inhibitor of ADP-induced platelet aggregation, significantly inhibited, in the presence of platelets, ex vivo thrombin generation triggered by low concentrations of tissue factor. Clopidogrel reduced the area under the curve (23%, p <0.05) and the thrombin peak concentration (35%, p <0.05) but did not affect the lag phase of thrombin generation. Under the same experimental conditions, heparin (100 microg/ml) inhibited thrombin generation mostly by delaying and by reducing the burst of thrombin. In a stasis-induced venous thrombosis model in rats under low thrombogenic challenge, clopidogrel inhibited thrombus formation (ED50 = 7.9+/-1.5 mg/kg, p.o. - n = 10), confirming the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation and venous thrombosis.     

Inhibitory effect of vitamin E (alpha-tocopherol) on spontaneous platelet aggregation in whole blood

Vitamin E (D-alpha-tocopherol) inhibited spontaneous human platelet aggregation in whole blood in the 20-200 micrograms/ml range. When alpha-tocopherol (20 micrograms/ml) and aspirin (0.5 mM), or alpha-tocopherol and the mixture of phosphocreatine (1.5 mM) and creatine phosphokinase (50 U/ml) (CP/CPK) were added to this reaction system, a synergic inhibitory effect on aggregation was observed. On the other hand, when both alpha-tocopherol and the specific inhibitor of platelet activating factor (CV-3988; 0.38 mM) were added to this system, the inhibition was the same as that caused by the addition of CV-3988 alone, suggesting there was no synergism, i.e., that the effect of alpha-tocopherol is related to the inhibition of platelet activating factor (PAF)-induced platelet aggregation in whole blood. However, alpha-tocopherol (20 or 50 micrograms/ml) did not inhibit PAF (10 nM) induced platelet aggregation in platelet rich plasma (PRP). These results suggest that the inhibition of platelet aggregation in whole blood by alpha-tocopherol is due to the inhibition of PAF synthesis, and is unrelated to adenosine diphosphate (ADP) or thromboxane A2.     

Antiplatelet effects of protopine isolated from Corydalis tubers

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.     

Superoxide dismutase cooperates with prostacyclin to inhibit platelet aggregation: a comparative study in washed platelets and platelet rich plasma.

The role of superoxide anions (O2-) in human platelet aggregation in Krebs' buffer or plasma was investigated. In indomethacin (10 microM)-treated washed platelets superoxide dismutase (SOD; 60 U/ml) or ferricytochrome c (FCC; 70 microM) inhibited platelet aggregation by thrombin but not that by collagen or ADP. In addition, in indomethacin (10 microM)-treated washed platelets, SOD significantly potentiated the anti-aggregatory activity of prostacyclin (PGI2) or iloprost when thrombin but not collagen was used as the aggregating agent. In platelet rich plasma, SOD (60 U/ml) did not inhibit platelet aggregation nor did it potentiate the anti-aggregatory activity of iloprost when ADP, collagen or thrombin were used as aggregating agents. Thus, O2- participate in the aggregatory activity of thrombin but not collagen or ADP and PGI2 or iloprost, by reducing the sensitivity of platelets to thrombin, co-operate with SOD to inhibit thrombin-induced platelet aggregation. The interpretation of the use of SOD in experiments involving endothelium-derived relaxing factor (NO) is discussed.     

Lack of stability of aggregates after thrombin-induced reaggregation of thrombin-degranulated platelets.

The stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2Cl was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 microM) and chymotrypsin (10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymotrypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of a calcium dependent protease inhibitor on platelet activation and secretion

Continuous proteolysis resulting in consumption of major cytoskeletal proteins may be essential for platelet activation and aggregation. In this study we evaluated the effect of a known protease inhibitor, Leupeptin, on agonist induced platelet aggregation and secretion. Platelets exposed to 10 ugs/ml of Leupeptin did not aggregate in response to the action of thrombin (0.2u/ml). However, a concentration of Leupeptin as high as 250 ugs/ml did not prevent arachidonate induced aggregation and secretion. Leupeptin (100 ugs/ml) effectively blocked thrombin (0.2 u/ml) induced elevation of cytosolic calcium, but did not affect arachidonate induced elevation of platelet intracellular calcium levels. At a concentration of 100 ug/ml, Leupeptin effectively blocked thrombin (0.5u/ml) induced clot formation of platelet poor plasma, suggesting that it can exert its effect on thrombin by preventing fibrinogen degradation. Effective K_i for the competitive inhibition of thrombin induced hydrolysis of a chromogenic substrate, S2238, by Leupeptin was 2.4 uM. Leupeptin inhibition of platelet function was reversible by washing platelets free of the polypeptide. Results of our study demonstrate that Leupeptin inhibits thrombin induced platelet activation, probably by interfering with its proteolytic activity on the platelet surface membrane. However, inhibition of platelet surface membrane associated proteases did not prevent activation of platelets by other agonists.     

Inhibition of thrombin- and collagen-induced phosphoinositides breakdown in rabbit platelets by a PAF antagonist--denudatin B, an isomer of kadsurenone

Denudatin B, an isomer of kadsurenone, was isolated from Magnolia fargesii. It inhibited the aggregation and ATP release of washed rabbit platelets caused by platelet-activating factor (PAF) in a concentration-dependent manner. The IC50 on PAF (2 ng/ml)-induced aggregation was about 10 micrograms/ml. High concentration of denudatin B (greater than 50 micrograms/ml) also inhibited the aggregation and ATP release of platelets caused by ADP, collagen, arachidonic acid and thrombin. However, shape change of platelets still existed. Prolongation of the incubation time with platelets could not cause further inhibition, and the aggregability of platelets could be restored after denudatin B was washed out from platelets. Thrombin-induced thromboxane B2 formation was almost completely suppressed. In the absence of extracellular calcium (EGTA 1 mM), ATP release caused by thrombin was inhibited. Thrombin-induced rise of the intracellular calcium concentration was suppressed by denudatin B, but not by BN52021 or kadsurenone. The generation of inositol phosphate in washed platelets caused by collagen, PAF and thrombin was also suppressed. The data indicate that PAF antagonist denudatin B has nonspecific antiplatelet action at high concentration by inhibiting phosphoinositides breakdown induced by collagen and thrombin.     

Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation

Systemic inflammation has been shown to be a contributing factor to the instability of atherosclerotic plaques in patients with acute coronary syndromes (ACS). VX-702, a novel p38 mitogen-activated protein kinase (MAPK) inhibitor, is currently under investigation in ACS patients with unstable angina to evaluate its safety and efficacy during percutaneous coronary intervention (PCI). The role of p38 MAPK in platelet aggregation of normal individuals was examined using the selective second generation p38 MAPK inhibitor VX-702. Treatment of platelets with thrombin (activates PAR1 and PAR4 thrombin receptors), SFLLRN (PAR1), AYPGKF (PAR4), collagen (alpha2beta1 and GPVI/FCgammaIIR receptors) and U46619 (TXA(2)) resulted in strong activation of p38 MAPK. Activation of the GPIb von Willebrand factor receptor with ristocetin did not stimulate p38 MAPK. Pre-treatment of platelets with 1 microM VX-702 completely inhibited activation of p38 MAPK by thrombin, SFLLRN, AYPGKF, U46619, and collagen.There was no effect of VX-702 on platelet aggregation induced by any of the agonists in the presence or absence of aspirin, heparin or apyrase. It has been postulated that a potential role of p38 MAPK is to activate phospholipase A(2) (cPLA(2)) which catalyses formation of arachidonic acid leading to production of thromboxane. Interestingly, we show contrasting effects of p38 MAPK inhibition as compared to aspirin inhibition on platelet aggregation in response to collagen. Blockade of TXA(2) production by aspirin results in significant inhibition of collagen activation. However,VX-702 has no effect on collagen-mediated platelet aggregation, suggesting that blocking p38 MAPK does not effect thromboxane production in human platelets.Therefore, unlike aspirin blockade of thromboxane production in platelets, p38 MAPK inhibitors such as VX-702 do not significantly affect platelet function and would not be expected to contribute to an elevated risk of bleeding side-effects in treated patients.