The role of tissue factor/factor VIIa in the pathophysiology of acute thrombotic formation.

Tissue factor (TF) is the essential cofactor for the coagulation protease factor VIIa (FVIIa), initiating the coagulation cascade. The role of TF in thrombotic diseases is becoming increasingly evident. Recent findings suggest that inhibition of TF/FVIIa activity could be important in the prevention of clinical sequelae associated with plaque rupture or vessel damage that exposes TF to blood. Furthermore, selective inhibitors of TF/FVIIa may be associated with less bleeding risk than other antithrombotic agents. Several TF/FVIIa inhibitors are in development, including the protein-based inhibitors (such as NAPc2, Corsevin M, FFR-FVIIa, and Tifacogin). Research into the development of small molecule inhibitors is on-going, but is at a less advanced stage.

A radioreceptor binding assay for measurement of platelet-activating factor synthesis by human neutrophils.

A new convenient method for the measurement of platelet-activating factor produced in vitro has been developed. This method involves incubation of neutrophils with stimuli, lipid extraction, purification of lipid extracts by normal phase high performance liquid chromatography (HPLC) and quantification of platelet-activating factor (PAF) by a competitive radioreceptor binding assay. The recovery of PAF from the extraction and purification procedures is 94.5 +/- 0.9%. The sensitivity of the assay is 10 pg/tube. Human neutrophils stimulated with 0, 0.25, 0.5, 1 and 2 microM A 23187 will produce ND, ND, 720, 840 and 900 pg of PAF, respectively (ND = not detectable). The values obtained for PAF produced by human neutrophils in the present assay are comparable to those obtained with the rabbit platelet aggregation assay.

Potent in vitro and in vivo inhibitors of platelet aggregation based upon the Arg-Gly-Asp-Phe sequence of fibrinogen. A proposal on the nature of the binding interaction between the Arg-guanidine of RGDX mimetics and the platelet GP IIb-IIIa receptor.

Peptide mimetics of the RGDF sequence in which Arg-Gly has been replaced with 5-(4-amidinophenyl)pentanoyl mimetic has led to a 1000-fold increase in inhibitory potency over the natural RGDF ligand. The guanidine residue of the arginine may be involved in a reinforced ionic interaction with a carboxylate of the receptor which could explain the dramatic increase in potency upon replacement with benzamidine. This hypothesis is supported by the observation of low inhibitory potency of the corresponding benzylamine (18) and no activity with the corresponding imidazoline derivative (19); plus, ab initio calculations on the respective complexes suggest that the benzamidine-carboxylate is more favorable than the guanidine-carboxylate interaction. The ED50 for the inhibition of ex vivo collagen induced platelet aggregation in the dog for SC-52012 (1) was 0.32 microgram/kg/min by iv infusion with a pharmacodynamic half-life for recovery of approximately 40 min.

Effect of the stable endoperoxide analog U-46619 on prostacyclin production and cyclic AMP levels in bovine endothelial cells.

The effects of the stable endoperoxide analog U46619 (U) on the regulation of prostacyclin (PGI2) formation and cyclic adenosine monophosphate (cAMP) were investigated in cultured bovine aortic endothelial (BAE) cells. Incubation of U (0.3, 3.0 and 30 microM) with BAE cells for 5 min results in a dose-dependent increase in PGI2. Cyclic AMP levels were not changed at 0.3 and 3.0 microM but were stimulated at 30 microM U. When cells were exposed to U for a second and third 5 min period, PGI2 formation at 0.3 and 3.0 microM U remained stimulated while at 30 microM, PGI2 was not increased. Five min incubation of BAE cells with the cyclooxygenase inhibitor indomethacin blocked the stimulation of PGI2 at all concentrations of U and also prevented the increase of cAMP levels at 30 microM. In cells prelabeled with 3H-arachidonate, U stimulated release of labeled products at 0.3 and 3.0 microM but not at 30 microM U. In cells treated with bradykinin in the presence of U, PGI2 production was stimulated at 0.3 and 3.0 microM but not 30 microM U. When cells were exposed to U and stimulated with PGI2 (with and without phosphodiesterase inhibition), U caused significant increases in cAMP. We conclude that incubation of BAE cells with U results in an initial dose-dependent increase in PGI2 formation. Cyclic AMP levels are increased at high concentrations of U. This increase in cAMP is mediated by the initial stimulated PGI2 and results in decreased PGI2 on further exposure to U. Data suggest that U stimulates phospholipase activity and, at high concentrations, inhibits phosphodiesterase.

A new method for measurement of plasma concentration of orally active glycoprotein IIb/IIIa antagonists.

A bioassay for determining concentrations of antiplatelet compounds in plasma or aqueous solution has been developed. The method uses an aliquot of plasma from treated animals to inhibit collagen-induced platelet aggregation in pooled platelet-rich plasma (PRP) obtained from donor dogs. The concentration in plasma from treated animals was estimated using a standard curve of inhibition established using plasma from untreated animals which had been spiked with known amounts of compound. For independent validation, plasma concentrations of certain compounds were determined in identical dog plasma samples by both bioassay and HPLC. Results from the two methods were concordant. The bioassay provides an accurate and sensitive method for measuring antiplatelet activity without the need for extraction of plasma samples and may be used to measure activity in any solution which is compatible with PRP. This assay is routinely used to provide an estimate of absorption of prodrugs and systemic conversion to active compound after oral dosing. Some of the compounds of interest are ester-acid pairs with the inactive ester prodrug being cleaved to the active acid following administration. Compounds were administered orally (ester) or IV (acid) and blood samples were taken periodically for 24 hours. Plasma concentration of active moiety was determined for each time point and the area under the curve (AUC) of concentration vs. time was calculated. Comparing the AUCs for oral and IV routes of administration yielded the Oral Systemic Activity (OSA), a measure of active compound available after oral dosing.

In vitro and in vivo effects of a peptide mimetic (SC-47643) of RGD as an antiplatelet and antithrombotic agent.

Platelet aggregation requires binding of fibrinogen (fgn) to activated platelets and inhibition of this binding blocks platelet aggregation. Synthetic peptides modeled after the platelet binding sequence on fgn block the platelet glycoprotein IIb/IIIa receptor and effectively inhibit aggregation. SC-47643 (SC) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet IIb/IIIa receptor. SC inhibited fgn binding to activated platelets (IC50: 1.0 x 10(-5) M) and prevented platelet aggregation in response to a variety of platelet agonists in both washed human platelets and platelet rich plasma (IC50's ranging from 4 x 10(-6) to 1 x 10(-5) M, respectively). SC inhibited collagen induced thrombocytopenia in the rat (ED50 0.07 mg/kg and t1/2 36 min). In dogs ex vivo collagen induced platelet aggregation was inhibited 50% after a bolus injection of 1.7 mg/kg. After a steady state infusion (2 hr), the ED50 was 0.03 mg/kg/min, with no effects on blood pressure, heart rate or platelet count. These data demonstrate that SC, a peptide mimetic of the natural fgn binding sequence, is capable of blocking platelet-fgn interactions and platelet aggregation.

SC-49992--a potent and specific inhibitor of platelet aggregation.

Fibrinogen binding is required for platelet aggregation and subsequent thrombus formation. SC-49992 (SC), an RGDF mimetic, is a potent and specific inhibitor of the binding of fibrinogen to its receptor on activated platelets, glycoprotein IIb/IIIa (IC50 0.7 microM). SC was more potent (1-5 microM) than either RGDS, RGDF or the gamma chain dodecapeptide in blocking platelet aggregation to a variety of agonists in both dog and human platelet rich plasma. SC was more potent as an inhibitor of GP IIb/IIIa on platelets than it was against other integrin and non-integrin receptors, including the RGD-dependent vitronectin receptor and other non-RGD-dependent integrins such as CDII/CD18. SC had little effect on ristocetin induced agglutination. SC blocked ex vivo collagen induced aggregation in dogs and collagen induced thrombocytopenia in rats. These data suggest that elimination of the Arg-NH2 and the Arg-Gly amide bond of RGDF provided increased inhibitory potency and specificity. This structural modification may be of value in the development of other more potent RGDF mimetics for the inhibition of platelet aggregation.

Species variation in the effect of glycoprotein IIb/IIIa antagonists on inhibition of platelet aggregation.

Differences exist between platelets of different species in their reaction to pharmaceutical agents, such as inhibitors of platelet aggregation. Understanding these differences is critical in the interpretation of data from experimental animal models of thrombosis. Platelet aggregation, essential in the hemostatic process, requires that fibrinogen (fgn) bind to activated platelets. Analogs of Arginine-Glycine-Aspartic acid-Phenylalanine (RGDF), a peptide sequence of fgn, block fgn binding to its receptor known as glycoprotein (GP) IIb/IIIa on activated platelets and prevent aggregation. We studied the inhibition resulting from Arginine-Glycine-Aspartic acid-Serine (RGDS) and two analogs of RGDF, (SC-46749 and SC-47643) on aggregation of human, rat, guinea pig, dog, and rhesus monkey platelets in vitro using ADP as the agonist. The inhibitory potency of RGDS, SC-46749, ad SC-47643 was species dependent. The rank order of potency was rhesus monkey, dogs, and human followed by guinea pig and rat. In order to study the relative inactivity of the compounds in rat platelets compared to human, we diluted rat platelet-rich plasma (PRP) to yield platelet levels approximating that of humans. Platelet inhibition was not significantly changed in diluted rat PRP nor did changing concentration appear to affect activity in human PRP. Our data suggest that the platelet response of some species may better represent human response with regard to inhibition of GP IIb/IIIa by (RGDX) analogs.

Hemispheric involvement in shadowing vs. interpretation: a time-sharing study of simultaneous interpreters with matched bilingual and monolingual controls.

A time-sharing study used shadowing and interpretation/paraphrasing tasks to evaluate lateralization in professional interpreters individually matched to bilingual and monolingual controls. A two-step multivariate general linear model procedure was used to determine lateralized effects and extent of disruption produced by the tasks. Results revealed the monolingual group to be left lateralized for both tasks, but the two bilingual groups were lateralized in the LH only for shadowing. The monolingual group was significantly different from the bilingual groups in the pattern of hand asymmetry for interpretation/paraphrasing. The findings replicate outcomes of prior repeated measures analysis of variance procedures on percentage of change scores. However, new information is added by the more refined analysis. The findings are also discussed in terms of previous laterality studies using similar tasks and subject samples.

Evaluation of a stable 5-F prostacyclin analogue as an antithrombotic agent in haemodialysis.

Prostacyclin has been used as a substitute for heparin in renal dialysis because of its potent antiplatelet activity and its short half-life. However, it is difficult to use because of its instability in neutral buffer and its hypotensive activity at antithrombotic doses. SC-39902, a 5-F prostacyclin analogue, has an ED50 of 17.7 micrograms/kg i.v. against ADP-induced thrombocytopenia in rats. The ED25 for hypotensive activity is 7.0 micrograms/kg i.v. The therapeutic index (blood pressure ED25/antiplatelet ED50) of 0.40 is 3.6 times better than that of PGI2 (0.11). SC-39902 supports clot-free dialysis in dogs at 4.0 micrograms/kg/min (ED100 for dialysis) with a 26% change in blood pressure at the 100% effective dose as compared with 0.052 microgram/kg/min required for PGI2 with a 32% change in blood pressure. The authors conclude that SC-39902 will provide the desirable antiplatelet/antithrombotic activities of PGI2 without instability and buffer incompatability problems. However, at the ED100 for dialysis, SC-39902 does have haemodynamic activity and, thus, a therapeutic index which is greater than four times that of PGI2 in rats is required for a PGI2 analogue to have less hypotensive activity during dialysis.

Biotinidase deficiency: early neurological presentation.

Three patients with biotinidase deficiency are described. Two presented at eight weeks of age with anticonvulsant-resistant fits, developmental delay and hypotonia. Treatment has been effective. The third developed ataxia and alopecia at 14 months and died suddenly at 19 months of age. In all three cases the diagnosis was not considered quickly enough. Biotinidase deficiency is a treatable cause of severe neurological problems.

The protective dose of the potent GPIIb/IIIa antagonist SC-54701A is reduced when used in combination with aspirin and heparin in a canine model of coronary artery thrombosis.

BACKGROUND: Fibrinogen receptor antagonists block the fibrinogen-platelet interaction at the GPIIb/IIIa receptors and inhibit thrombus formation. SC-54701 is the active metabolite of SC-54684A, an orally fibrinogen receptor antagonist. We compared the efficacy of SC-54701A (SCa, hydrochloride salt) with that of aspirin (ASA) or heparin and with combination therapy in a canine model of continuous current injury. METHODS AND RESULTS: Sixty-six dogs were used (6 per treatment). SCa (15-minute loading dose followed by [//] infusion [microgram/kg per minute]: (0.87//0.39=1 X SCa; 0.52//0.23=0.6 X SCa; and 0.425//0.20= 0.5 X SCa), ASA (2.8 mg/kg), heparin (200 U/kg plus 1000 U/h), or saline (0.1 mL/kg) was administered intravenously. Experimental time was 180 minutes of current. Time to occlusion was increased (P < .05) by SCa (T=incidence of thrombosis) (1 X SCa, >180 minutes [T=0]; 0.6 X SCa, 158 +/- 15 minutes [T=2]; 0.5 X SCa, 130 +/- 22 minutes [T=4]), heparin (114 +/- 16 minutes [T=5]), and ASA plus heparin (130 +/- 11 minutes [T=5]) relative to saline (58 +/- 7 minutes [T=6]). Time to occlusion for the SCa treatments was increased compared with ASA (64 +/- 7 minutes [T=6]). When 0.5 X SCa was administered with ASA plus heparin, time to occlusion was >180 minutes [T=0]. SCa provided complete protection at > or = 90% inhibition of ex vivo collagen-induced platelet aggregation. Cyclic flow variations were minimal with SCa or any treatment involving 0.5 X SCa and ASA. CONCLUSIONS: SCa has dose-dependent antithrombotic efficacy and inhibits ex vivo platelet aggregation. ASA, heparin, or saline was ineffective in this model. SCa (0.5X) plus ASA and heparin maximized the antithrombotic effect of this lower dose of SCa.

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 alpha IIb beta 3) 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 alpha-amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I-fibrinogen binding to activated platelets (IC50 = 10-30 microM) and inhibited ADP induced platelet aggregation (IC50 = 10-30 microM). 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.

Extended inhibition of platelet aggregation with the orally active platelet inhibitor SC-54684A.

BACKGROUND: Platelet aggregation is important in thrombotic events, and platelets play a major role in the etiology of several cardiovascular diseases. Platelet aggregation requires the binding of fibrinogen (fgn) to activated platelets. Synthetic peptides modeled after the RGD binding sequence on the fgn alpha-chain block the platelet glycoprotein (GP) IIb/IIIa receptor for fgn and effectively inhibit aggregation. SC-54684A (SCp, orally active prodrug of the active moiety SC-54701, SCa) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet GPIIb/IIIa receptor. SCa blocks the binding of fgn to the platelet and therefore prevents platelet aggregation in response to all agonists. METHODS AND RESULTS: SCp was administered orally at 1.25, 2.5, 5.0, and 7.5 mg/kg in a single-dose, dose-ranging study. Blood samples were taken periodically for 24 hours, and platelet-rich plasma was prepared and tested for inhibition of ex vivo collagen-induced platelet aggregation. The plasma concentration of active moiety was determined by bioassay. The time, inhibition, and concentration data were used to predict two doses that would result in minimum daily inhibition levels of 30% and 70% when administered twice daily (0.6 and 2.4 mg/kg, respectively). SCp was administered orally to conscious dogs twice daily for 14 days (after dose adjustment). Blood samples were obtained at daily peak and trough plasma levels (predicted from dose-ranging study). Inhibition of ex vivo collagen-induced platelet aggregation and concentration of active moiety in the plasma were determined. Average inhibition of platelet aggregation and plasma concentration of active moiety amounted to approximately 21% and 14 ng/mL at 1.5 mg/kg BID and 75% and 24 ng/mL at 2.4 ng/kg BID at daily minimum plasma levels (trough) in steady state. Platelet counts in the 2.4-mg/kg group declined from 3.2 x 10(5)/microL to 2.5 x 10(5)/microL in the first 9 days of dosing, with no further decline despite continued administration of compound. No changes were observed in the animals receiving 1.5 mg/kg. CONCLUSIONS: The results of the dose-ranging study show that oral administration of SCp results in dose-dependent inhibition of platelet aggregation. As shown in the 14-day administration, this dose-dependent inhibition can be maintained for an extended period while exhibiting no adverse effects. SCp is a leading candidate for development and is currently in clinical trials.

Characterization of Binding of an RGD Mimetic, [(3)H]-SC-52012, to Platelet GPIIb/IIIa.

In order to compare binding of small peptide mimetics on activated vs resting platelets and with fibrinogen (fgn) on activated platelets, the binding of [(3)H]-SC-52012, a low molecular weight (483) mimetic of the RGDF sequence present in fgn, was evaluated. This compound is a potent inhibitor of fgn binding to activated platelets, IC, 9.0 ± 0.6 nM (mean ± SEM), and inhibits ADP induced human platelet aggregation (IC, 44 ± 5 nM). The dissociation constant (Kd) of [(3)H]-SC-52012 was 21.6 ± 4.7 nM (n = 13) in ADP-induced human washed platelets while the Kd for resting platelets was 156 ± 8.3 nM (n = 3). The maximum number of binding sites on ADP-activated and resting platelets were 60846 ± 7158 and 59464 ± 5898 molecules/platelet, respectively. By comparison, results with [(125)I]-fgn binding to activated platelets gave values of 363 ± 73 nM and 58046 ± 6386 molecules/platelet (n = 8) for the Kd and receptor number, respectively. These data suggest that the small molecule binds regardless of activation state of the platelet with only a change in affinity. [(3)H]-SC-52012 could be displaced by unlabelled SC-52012 with an IC(50) of 135 ± 20 nM.

Protective effect of oral xemilofiban in arterial thrombosis in dogs: increased activity in combination with aspirin.

BACKGROUND: Inhibition of platelet aggregation by preventing the binding of fibrinogen to glycoprotein (GP) IIb/IIIa on activated platelets results in antithrombotic activity. We report on the antithrombotic effect of xemilofiban (SC-54684A), an oral GP IIb/IIIa antagonist, administered alone or with aspirin (ASA) in an acute thrombosis model. METHODS AND RESULTS: Conscious dogs were treated with xemilofiban (1.25, 2.5, 5.0, or 6 mg/kg, n=6); low-dose (LD, 81 mg) ASA, n=7; high-dose (HD, 162 mg) ASA, n=6; xemilofibran 1.25 mg/kg plus LD ASA, n=6; xemilofibran 1.25 mg/kg plus HD ASA, n=6; or placebo, n=7. Dogs were anesthetized 60 minutes later, and the effects of the treatments were evaluated after electrolytic injury (250 microA for 180 minutes) in the left circumflex coronary artery. Bleeding time (BT) was assessed in a separate study. Incidence of thrombosis was reduced (P<0.05) by xemilofiban > or =2.5 mg/kg, HD ASA, or xemilofiban 1.25 mg/kg plus HD ASA compared with placebo. Xemilofiban > or =2.5 mg/kg or xemilofiban 1.25 mg/kg plus HD ASA significantly increased time to occlusion, inhibited ex vivo platelet aggregation to collagen >90%, and prevented or decreased (P<0.05) cyclic flow variations (CFVs) compared with placebo. BT was increased (P<0.05) with xemilofiban > or =2.5 mg/kg but not with xemilofiban 1.25 mg/kg plus HD ASA. CONCLUSIONS: Xemilofiban > or =2.5 mg/kg, HD ASA, or xemilofiban 1.25 mg/kg plus HD ASA significantly reduced the incidence of thrombosis. These doses of xemilofiban or xemilofiban 1.25 mg/kg plus HD ASA increased time to occlusion, inhibited ex vivo platelet aggregation by >90%, and prevented or reduced CFVs. Xemilofiban > or =2.5 mg/kg but not xemilofiban 1.25 mg/kg plus HD ASA significantly increased BT.

SC-49992, a mimetic of the peptide arginine-glycine-aspartic acid-phenylalanine that blocks platelet aggregation, enhances recombinant tissue plasminogen activator-induced thrombolysis and prevents reocclusion in a canine model of coronary artery thrombosis.

8-Guanidino-octanoyl-aspartic acid-phenylalanine (SC-49992), a mimetic of the tetrapeptide arginine-glycine-aspartic acid-phenylalanine, is a potent inhibitor of platelet aggregation. In this study, the authors examined the effects of SC-49992 on the time to lysis of thrombi and the time to reocclusion in the canine coronary artery, which had been treated with tissue plasminogen activator. A lysis/reocclusion model was used that was originally designed so that the reoccluding thrombus was platelet rich. SC-49992 decreased the time to lysis in response to recombinant tissue plasminogen activator in a dose-dependent manner. The reduction of the lytic time was significant at the highest dose of 0.08 mg kg-1 min-1 (from 22.8 +/- 8.2 to 7.4 +/- 1.4 min, P < .05). The reocclusion time was prolonged at all doses of SC-49992 (from a control level of 3.4 +/- 0.6 min to more than 50 min at all doses of SC-49992, P < .05). At the 0.06- and 0.08-mg kg-1 min-1 doses, all but one animal in each group did not have a reocclusion during the time of the experiment. In those animals that did have reocclusions in the presence of SC-4992, ex vivo platelet aggregation was inhibited 100% at the time of reocclusion. The bleeding times were prolonged in these animals at the levels of inhibition in which the compound was the most effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiplatelet and antithrombotic effects of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) inhibition by arginine-glycine-aspartic acid-serine (RGDS) and arginine-glycine-aspartic acid (RGD) (O-me)Y (SC-46749).

Arginine-glycine-aspartic acid (RGD) is the minimal sequence in fibrinogen that leads to recognition and binding to the glycoprotein IIb/IIIa platelet receptor during aggregation. Analogs of tetrapeptides containing the RGD sequence have been previously shown to block fibrinogen binding to activated platelets in vitro. SC-46749 is an analog of arginine-glycine-aspartic acid-phenylalanine in which the phenylalanine is replaced by O-methyltyrosine. In this study the biological activities of SC-46749 were examined and its actions compared with the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS), one of the natural sequences on the fibrinogen alpha chain that binds to platelets. In vitro, SC-46749 was more potent than RGDS in inhibiting fibrinogen binding (IC50: SC-46749, 27 microM; RGDS, 47 microM), in preventing ADP-induced aggregation in human platelet-rich plasma (IC50: SC-46749, 32 microM; RGDS, 95 microM) and in inhibiting thrombin-induced aggregation in washed human platelets (IC50: SC-46749, 23 microM; RGDS, 64 microM). In rats, SC-46749 prevented collagen-induced thrombocytopenia with an ED50 of 0.87 mg/kg whereas RGDS did not inhibit the response by 50% at doses up to 10 mg/kg. SC-46749 inhibited thrombus formation in an electrically damaged rat carotid artery in a dose-dependent fashion whereas the effects of RGDS were biphasic. RGDS appeared to delay thrombus formation at lower doses but had no effect at higher doses. When infused in dogs for 15 min, SC-46749 prevented ex vivo collagen-induced aggregation at 4 mg/kg/min. These data demonstrate that SC-46749 is a potent inhibitor of platelet aggregation and platelet-dependent thrombus formation.

SC-54684A: an orally active inhibitor of platelet aggregation.

BACKGROUND: Intravenous therapy has been shown to be beneficial in the prevention of acute platelet-associated thrombotic events. However, orally active agents would be advantageous for chronic therapy. Fibrinogen receptor antagonists block the fibrinogen/platelet interaction and thus inhibit a step required for thrombus formation. To date, no orally active fibrinogen binding inhibitors have been characterized. SC-54684A, now in clinical trial, is the orally active prodrug of a potent and specific fibrinogen binding antagonist. METHODS AND RESULTS: We measured inhibition of 125I-fibrinogen binding to activated platelets and inhibition of aggregation in platelet-rich plasma to selected agonists and showed IC50s of 1.0 x 10(-8) and 3 to 7 x 10(-8) mol/L, respectively. Specificity of the active moiety was determined by studying its effect on the binding of (1) neutrophils to interleukin (IL)-1 beta-stimulated endothelial cells, (2) endothelial cells to fibronectin, and (3) vitronectin to isolated vitronectin and fibrinogen receptors. No effect was observed on the binding neutrophils to IL-stimulated endothelial cells or endothelial cell binding to fibronectin. There was a fivefold separation between binding to isolated receptors of vitronectin and fibrinogen. Collagen-induced aggregation was inhibited by 80%, and bleeding time was increased approximately 2.5-fold when the active moiety was infused to steady state at 0.2 micrograms/kg per minute in dogs. When the ester prodrug was given orally and the active moiety was given intravenously, the oral systemic activity was approximately 20%. Pharmacokinetic analysis after intravenous infusion of the prodrug or active moiety showed that the prodrug was rapidly converted to the active moiety; the active moiety had a t1/2 of 6.5 hours. When the prodrug was administered both orally and intravenously, the systemic availability of the active moiety was 62%. CONCLUSIONS: SC-54684A, an orally active antiplatelet drug now in clinical trial, is shown to be a potent, specific fibrinogen binding inhibitor that blocks platelet aggregation to a wide variety of known stimuli and has good bioavailability in animals.