Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist

1. Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C-type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of alpha- (16.5 kDa) and beta- (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS - PAGE and mass spectrometry. 2. In vitro, agkistin concentration-dependently inhibited ristocetin-induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u x ml(-1)). 3. 125I-agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223+/-10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I-agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I-agkistin binding reaction. 4. Agkistin (250 microg x kg(-1)) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein-treated mice in vivo. 5. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.     

Antiplatelet mechanisms of TA-993 and its metabolite MB3 in ADP-induced platelet aggregation

We investigated the antiplatelet mechanisms of TA-993 [(-)-cis-3-acetoxy-5-(2-(dimethylamino)ethyl)-2, 3-dihydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one maleate] and its metabolite MB3 (deacetyl and N-monomethyl TA-993) in human platelets stimulated by ADP in vitro. TA-993 and MB3 concentration-dependently inhibited fibrinogen binding to the ADP-stimulated platelets as well as inhibiting platelet aggregation. The antiplatelet effect of MB3 was about 300 times more potent than those of TA-993 and a glycoprotein IIb/IIIa receptor antagonist, Arg-Gly-Asp-Ser (RGDS). Aggregation of ADP-treated fixed platelets caused by the addition of fibrinogen was inhibited by RGDS but not by TA-993 and MB3. TA-993 and MB3 inhibited ADP-induced polymerization of actin filaments. Neither TA-993 nor MB3 affected cyclic AMP and cyclic GMP levels in resting platelets, and nor suppressed the increase in intracellular Ca(2+) concentration induced by ADP. These results suggest that the antiplatelet mechanisms of TA-993 and MB3 may involve inactivation of glycoprotein IIb/IIIa receptors via inhibition of the polymerization of actin.     

Discovery of an orally active non-peptide fibrinogen receptor antagonist based on the hydantoin scaffold

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a promising new class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible manner, human and dog platelet aggregation as well as 125I-fibrinogen binding to ADP-activated human gel filtered platelets and isolated GP IIb/IIIa with K(i) values of 9 nM and 0.17 nM, respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural features which are important for binding. Hydrophobic substitutions both at the hydantoin nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which is a promising drug candidate for the treatment of thrombotic diseases in humans.     

Aminopeptidase resistant Arg-Gly-Asp analogs are stable in plasma and inhibit platelet aggregation

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin α_11bβ₃) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH₂ were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH₂ with an acetylated or deleted α-amino group were prepared. Both analogs were stable when incubated in plasma, blocked ¹²⁵I-fibrinogen binding to activated platelets (IC₅₀= 10–30μm ) and inhibited ADP induced platelet aggregation (IC₅₀= 10–30μm ). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.     

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb.

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.     

Comparative studies of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody,YM337, and abciximab on in vitro antiplatelet effect and binding properties

The effects of YM337, the Fab fragment of a humanized anti-glycoprotein IIb/IIIa (GPIIb/IIIa) monoclonal antibody C4G1, on in vitro platelet function and binding properties were compared with those of abciximab, the Fab fragment of the human/murine chimeric anti-GPIIb/IIIa monoclonal antibody 7E3. Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin. Further, both inhibited human platelet adhesion to von Willebrand factor, fibrinogen, fibronectin and subendothelial matrix with similar potency. Fibrinogen binding to washed platelets was dose-dependently inhibited by both agents. In binding assay using 125I-YM337 and 125I-abciximab, Kd values determined with platelet-rich plasma were 6.74 +/- 0.56 nM for YM337 and 6.65 +/- 1.45 nM for abciximab, and the number of binding sites were 42,700 +/- 3,000 for YM337 and 76,000 +/- 5,400 for abciximab. GPIIb/IIIa was precipitated from the solubilized fraction of platelets by both agents. In contrast, integrin alphavbeta3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337. Fibrinogen binding to purified GPIIb/IIIa was dose-dependently inhibited by both agents. In contrast, vitronectin binding to purified integrin alphavbeta3 was dose-dependently inhibited by abciximab but not by YM337, supporting the idea that abciximab reacts to integrin alphavbeta3. Therefore, YM337 was suggested to bind to a different epitope of GPIIb/IIIa from abciximab. These results suggest that YM337 specifically acts on platelet GPIIb/IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab.     

Cyclic RGD peptide analogues as antiplatelet antithrombotics.

Stimulation of platelets activates GPIIbIIIa, the heterodimeric integrin receptor, to bind fibrinogen (Fg), which results in platelet aggregation. GPIIbIIIa/Fg binding inhibitors are potentially suitable for acute use during and after thrombolytic therapy as antithrombotic agents. Incorporation of the tripeptide sequence Arg-Gly-Asp (RGD), a common structural element of many integrin ligands, into cyclic peptides produced a series of peptides of the general structure BrAc-(AA1)-RGD-Cys-OH, which were prepared by solid-phase peptide synthesis. Cyclization was accomplished by reaction of the N-terminal bromoacetyl group with the cysteine sulfhydryl at pH 8 at high dilution, resulting in thioether-bridged cyclic peptides [cyclo-S-Ac-(AA1)-RGD-Cys-OH]. Use of alpha-substituted bromoacetyl groups gave rise to an analogous series of acetyl-substituted thioether-bridged cyclic peptides. Oxidation of the thioethers produced separable diastereomeric sulfoxide-bridged cyclic peptides. After thorough evaluation in a GPIIbIIIa ELISA assay and a platelet aggregation assay, G-4120 (70A; AA1 = D-Tyr; sulfoxide bridge) was selected for further investigation as an antithrombotic agent. G-4120 was equipotent in the platelet aggregation assay to kistrin, a highly potent inhibitor of fibrinogen-mediated platelet aggregation isolated from snake venom (IC50 = 0.15 microM).     

Ex-vivo and In-vivo Antithrombotic Effect of Triflavin,an RGD-containing Peptide

Abstract— Triflavin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation through the blockade of fibrinogen binding to the activated platelets. It binds to fibrinogen receptors associated with the glycoprotein IIb/IIIa complex with a K_d value of 7 × 10^?8 m. In this study, we found that ¹²⁵I-triflavin reached the maximal binding to human platelets within 5 min at 25°C. In addition, when triflavin was intravenously administered at 1·0 mg kg^?1 to rabbits, it reversibly impaired the platelet aggregation of platelet-rich plasma caused by ADP (20 μm) ex-vivo over 30 min. The platelet counts of the experimental rabbits remained unchanged. Triflavin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at a dose of 2 μg g^?1. Therefore, triflavin was proven to be an effective antithrombotic agent in preventing ADP-induced acute pulmonary thromboembolism in mice and impairing reversibly the platelet function of rabbits when given intravenously.     

Characterization of a novel protein from Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist.

Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.     

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist

The pharmacological properties of YM-57029 [4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino]acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 microg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.     

The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man

The thienopyridines, ticlopidine and PCR 4099, inhibit ex vivo aggregation in response to ADP and other agonists. It has been shown that ticlopidine induces a functional defect in the binding of fibrinogen to its platelet membrane receptor. We have studied the effects on platelet functions of PCR 4099 in rat and in man. The aim of the study was to check the possibility of a direct modification of the fibrinogen binding site on the GP IIb-IIIa complex. Washed platelet suspensions were used for aggregation and fibrinogen binding studies. Platelet lysates were submitted to SDS-polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and immunoprecipitation. We found that administration of PCR 4099 inhibited selectively and irreversibly ADP-induced aggregation. Although the effect of ADP on aggregation was blocked, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin on platelet aggregation were inhibited. Fibrinogen binding was dramatically inhibited in rat and in man when platelets were stimulated with ADP and low concentrations of thrombin. At high concentration of thrombin there still remained a part of fibrinogen binding inhibition although aggregation was not impaired. Electrophoretic and immunoelectrophoretic studies showed no difference before and after treatment by PCR 4099. In particular, the GP IIb-IIIa-complex was not dissociated, its electrophoretic mobility was not changed and three monoclonal anticomplex antibodies recognized it in the same manner before and after treatment. We conclude that PCR 4099 selectively inhibits the ADP aggregation pathway and that the inhibition of fibrinogen binding is probably not due to a direct modification of the GP IIb-IIIa complex.     

Snake venom probes of platelet adhesion receptors and their ligands

Snake venom proteins that modulate platelet adhesive interactions are chiefly from either of two main structural families: the C-type lectin-like family, or the metalloproteinase-disintegrins. Snake venom probes from both families selectively target platelet adhesion receptors, including glycoprotein (GP) Ib-IX-V, GP VI, 2β1 and IIbβ3 (GP IIb-IIIa). These receptors act together to mediate platelet adhesion, activation and aggregation (thrombus formation) under hydrodynamic shear stress in flowing blood. The receptors are members of the leucine-rich repeat family (GP Ib-IX-V), the immunoglobulin superfamily (GP VI), or integrins (2β1, IIbβ3). In addition, adhesive glycoproteins in matrix and/or plasma such as von Willebrand factor (that binds GP Ib and IIbβ3), collagen (that binds GP V, GP VI and 2β1), or fibrinogen (that binds IIbβ3), are also targeted by C-type lectin family or metalloproteinase-disintegrin snake venom proteins. Emerging structural and functional evidence is beginning to explain how interactions between the conserved structural module-domains that make up these mammalian and snake proteins are regulated. Whether homologous adhesion/counter-receptors on platelets and other vascular cells are also potential snake venom targets is as yet largely unexplored.     

Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to guinea-pig platelets

The binding of [125I]9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxypheny l)-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 [( 125I]PTA-OH), a thromboxane A2/prostaglandin H2 receptor antagonist, to washed guinea-pig platelets was studied. [125I]PTA-OH bound to guinea-pig platelets in a saturable and displaceable manner. The Kd for [125I]PTA-OH was 14.5 +/- 2 nM (n = 4) and the Bmax was 32 +/- 7 fmol/10(7) platelets or 1,927 +/- 422 binding sites/platelet. The IC50 value for a series of 13-azapinane TXA2 analogs to antagonize the TXA2/PGH2 mimetic U46619-induced platelet aggregation and displace [125I]PTA-OH from its binding site was determined. The IC50 values for the series of five antagonists were highly correlated (r = 0.99) in the binding assays and aggregation studies. The ability of a series of five agonists to displace [125I]PTA-OH from its binding site was compared to their ability to induce platelet aggregation. All the agonists completely displaced the ligand from its binding site but their rank order did not correlate well with their ability to induce aggregation (r = 0.37). Collectively, the data are consistent with the notion that [125I]PTA-OH binds to a putative TXA2/PGH2 receptor in guinea-pig platelets.     

Pharmacodynamics and pharmacokinetics of YM128, a GPIIb/IIIa antagonist prodrug

We examined the biochemical properties of YM‐57029 ({4‐[4‐(4‐Carbamimidoylphenyl)‐3‐oxopiperazin‐1‐yl]piperidino}acetic acid monohydrochloride trihydrate) and the pharmacodynamics and pharmacokinetics of its prodrug, YM128 (Ethyl (Z)‐(4‐{4‐[4‐(N²‐hydroxycarbamimidoyl)phenyl]‐3‐oxopiperazin‐1‐yl}piperidino)acetate), an orally‐active glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist. YM‐57029 strongly inhibited aggregation of human platelets induced by various agonists, with IC50 values ranging from 3.6 to 51 nM. YM‐57029 specifically inhibited fibrinogen binding to purified GPIIb/IIIa about 1,000‐fold more potently than Arg‐Gly‐Asp‐Ser (RGDS). Moreover, YM‐57029 effectively inhibited an Arg‐Gly‐Asp (RGD) peptide binding to platelets, suggesting that YM‐57029 competed with the RGD sequence of ligand. YM‐57029 or YM128 dose‐dependently inhibited ex vivo platelet aggregation after iv bolus injection or oral administration to beagle dogs and cynomolgus monkeys. However, YM128 exerted more potent and prolonged inhibitory effects on platelet aggregation than YM‐57029 after oral administration to cynomolgus monkeys. Furthermore, YM‐57029 prolonged template bleeding time at a dose that inhibited ex vivo platelet aggregation during cumulative iv infusion to cynomolgus monkeys. Metabolic and pharmacokinetic studies showed that YM128 effectively converted into YM‐57029 in liver microsomes from humans as well as dogs and monkeys, and that bioavailabilities of YM128 in dogs and monkeys were 32.3 and 22.2%, respectively. These results suggest that YM128, a prodrug of YM‐57029, may be a valuable GPIIb/IIIa antagonist with good bioavailability in humans. Drug Dev. Res. 55:149–161, 2002. © 2002 Wiley‐Liss, Inc.     

Snake venoms and the hemostatic system

Snake venoms are complex mixtures containing many different biologically active proteins and peptides. A number of these proteins interact with components of the human hemostatic system. This review is focused on those venom constituents which affect the blood coagulation pathway, endothelial cells, and platelets. Only highly purified and well characterized snake venom proteins will be discussed in this review. Hemostatically active components are distributed widely in the venom of many different snake species, particularly from pit viper, viper and elapid venoms. The venom components can be grouped into a number of different categories depending on their hemostatic action. The following groups are discussed in this review: (i) enzymes that clot fibrinogen; (ii) enzymes that degrade fibrin(ogen); (iii) plasminogen activators; (iv) prothrombin activators; (v) factor V activators; (vi) factor X activators; (vii) anticoagulant activities including inhibitors of prothrombinase complex formation, inhibitors of thrombin, phospholipases, and protein C activators; (viii) enzymes with hemorrhagic activity; (ix) enzymes that degrade plasma serine proteinase inhibitors; (x) platelet aggregation inducers including direct acting enzymes, direct acting non-enzymatic components, and agents that require a cofactor; (xi) platelet aggregation inhibitors including: -fibrinogenases, 5′-nucleotidases, phospholipases, and disintegrins. Although many snake venoms contain a number of hemostatically active components, it is safe to say that no single venom contains all the hemostatically active components described here. Several venom enzymes have been used clinically as anticoagulants and other venom components are being used in pre-clinical research to examine their possible therapeutic potential. The disintegrins are an interesting group of peptides that contain a cell adhesion recognition motif, Arg–Gly–Asp (RGD), in the carboxy-terminal half of their amino acid sequence. These agents act as fibrinogen receptor (integrin GPIIb/IIIa) antagonists. Since this integrin is believed to serve as the final common pathway leading to the formation of platelet–platelet bridges and platelet aggregation, blockage of this integrin leads to inhibition of platelet aggregation regardless of the stimulating agent. Clinical trials suggest that platelet GPIIb/IIIa blockade is an effective therapy for the thrombotic events and restenosis frequently accompanying cardiovascular and cerebrovascular disease. Therefore, because of their clinical potential, a large number of disintegrins have been isolated and characterized.     

Snake venom modulators of platelet adhesion receptors and their ligands.

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its adhesive ligand, von Willebrand factor, in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high hydrodynamic shear stress. This process closely resembles normal haemostasis at high shear, where GP Ib-IX-V-dependent platelet adhesion to von Willebrand factor in the injured blood vessel wall initiates platelet activation and integrin alphaIIb beta3 (GP IIb-IIIa)-dependent platelet aggregation. At low shear, other receptors such as those that bind collagen, the integrin alpha2beta1 (GP Ia-IIa) or GP VI, mediate platelet adhesion. Recently, snake venom proteins have been identified that selectively modulate platelet function, either promoting or inhibiting platelet aggregation by targeting GP Ib-IX-V, alpha2beta1, GP VI, alphaIIb beta3, or their respective ligands. Interestingly, these venom proteins typically belong to one of two major protein families, the C-type lectin family or the metalloproteinase-disintegrins. This review focuses on recent insights into structure-activity relationships of snake venom proteins that regulate platelet function, and the ways in which these novel probes have contributed in unexpected ways to our understanding of the molecular mechanisms underlying thrombosis.     

Advancing the Battle Against Acute Ischemic Syndromes: A Focus on the GP IIb-IIIa Inhibitors

Platelet aggregation and thrombus formation, resulting from disruption of a coronary artery plaque, play a critical role in the pathology of acute coronary syndromes. Currently, aspirin and heparin are administered to decrease platelet aggregation. The discovery of the platelet integrin receptor α_IIbβ₃, also known as the platelet glycoprotein (GP) IIb-IIIa receptor, is a breakthrough in antiplatelet therapy. The GP IIb-IIIa receptor is responsible for the crucial binding of fibrinogen to platelets, leading to cross-links between platelets and further platelet aggregation. Since the introduction of abciximab, the first GP IIb-IIIa-receptor antagonist, several other nonantibody agents have been studied for use in percutaneous transluminal coronary angioplasty and also in stent placement, treatment of unstable angina, and myocardial infarction.     

Effects of fibrinogen receptor antagonist GR144053F and aurintricarboxylic acid on platelet activation and degranulation.

Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100 enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response (IC(50) = 114.0 +/- 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma (IC(50) = 17.7 +/- 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 microM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.     

Practical applications of snake venom toxins in haemostasis

Snake venom toxins affecting haemostasis have facilitated extensively the routine assays of haemostatic parameters in the coagulation laboratory. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen/fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction. SVTLE are not inhibited by heparin and can thus can be used for assaying antithrombin III and other haemostatic variables in heparin-containing samples. Snake venoms are a rich source of prothrombin activators and these are utilised in prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin and non-enzymic forms of prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains toxins which have been used to assay blood clotting factors V, VII, X, platelet factor 3 and, importantly, lupus anticoagulants (LA). Other prothrombin activators (from the taipan, Australian brown snake and saw-scaled viper) have now been used to assay LA. Protein C and activated protein C resistance can be measured by means of RVV and Protac^®, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin^® from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing snake venom proteins, show potential for studying platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib. Snake venom toxins affecting haemostasis are also used in the therapeutic setting: Ancrod (from the Malayan pit viper, Calloselasma rhodostoma), in particular, has been used as an anticoagulant to achieve ‘therapeutic defibrination’. Other snake venom proteins show promise in the treatment of a range of haemostatic disorders.     

Non-peptide GPIIb/IIIa inhibitors. 20. Centrally constrained thienothiophene alpha-sulfonamides are potent, long acting in vivo inhibitors of platelet aggregation.

The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon = 3.3 x 10(6) M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 microg/kg [corrected], and an oral dose of 50-90 microg/kg [corrected] followed by low daily doses of 10 microg/kg [corrected] was sufficient to maintain approximately 80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 +/- 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.