1 Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and prothrombolytic activity in animals. We describe the antithrombotic eects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate. 2 The activity of MA33H1 against rat PAI-1 was con®rmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in rat venous (thromboplastin+stasis in the abdominal vena cava) and arterial (electric current applied to a carotid artery) thrombosis models. The eects on tailtransection bleeding time were studied. 3 MA33H1 at 100 ng ml 71 inhibited both human (44.1%) and rat PAI-1 (49.7%). This eect was concentration-dependent. Its eect on human PAI-1 was not signi®cantly inhibited by 1 mg ml 71 ®brin or a &7 fold molar excess of vitronectin (1 nM). Inhibition of rat PAI-1 was unchanged by ®brin, but vitronectin reduced inhibition from 0.5 nM. 4 In the venous thrombosis model, MA33H1 signi®cantly reduced thrombus weights by 38 and 58.6% at 50 and 100 mg kg 71 min 71 i.v. respectively. This eect was inhibited by tranexamic acid. In the arterial model, MA33H1 signi®cantly increased the delay to occlusive thrombus formation by 58 and 142% at 50 and 100 mg kg 71 min 71 i.v., and did not aect bleeding time at 300 mg kg 71 min 71 i.v. 5 Thus, a monoclonal antibody which transforms PAI-1 to a t-PA substrate prevents thrombus formation in the rat with no eect on bleeding time at a higher dose.

ABSTRACT

SanOrg123781A is a synthetic hexadecasaccharide that displays antithrombin-dependent inhibition of factor Xa and thrombin and potent antithrombotic effects. The antithrombotic activity of SanOrg123781A has been studied in a new mouse model of arterial thrombosis, where thrombus formation was induced by the application of an electrical current to the adventitial surface of a carotid artery. In this model, antiplatelet agents such as the ADP-receptor antagonist clopidogrel (30 mg/kg, p.o. 2 h before stimulation) and the GpIIb/IIIa antagonist SR121566A [3-{N-[4-{4-[amino(imino)methyl]phenyl}-1,3-thiazol-2-yl]-N-[1-(carboxymethyl)piperidin-4-yl]amino}propionic acid, trihydrochloride] (0.3 mg/ kg, i.v. 5 min before stimulation) strongly prolonged the time to occlusion (TTO) (761 and 473% increases, respectively), whereas aspirin was devoid of antithrombotic activity. Standard heparin (2 mg/kg, i.v.), the low molecular weight heparin enoxaparin (20 mg/kg, i.v.), and the synthetic, antithrombin-dependent inhibitor of factor Xa fondaparinux (10 mg/kg, i.v.) were also active in this model (742, 707, and 602% TTO increases, respectively). Interestingly, SanOrg123781A was active at much lower doses than the other oligosaccharides (554% increase in TTO at 0.3 mg

SummaryThe antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-dihydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 μM 5-HT + threshold concentrations of ADP (0.5-1 M) or collagen (0.3 g/ml) with mean IC50 values of 49 ± 13 and 48 ± 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p. o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p. o. SL65.0472 (30 μg/kg i. v.) virtually abolished coronary cyclic flow variations (7.2 ± 1.0/h to 0.6 ± 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 ± 0.8 ml/ min to 31.8 ± 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p. o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

SummaryWe studied the use of the Ecarin Clotting Time (ECT) as a predictive assay of the antithrombotic effects of argatroban in a new tissue factor-dependent model of venous thrombosis and a model of arterial thrombosis in the rat. Heparin was used as a reference anticoagulant.Infusions of argatroban dose-dependently increased the ECT across the range of doses required for antithrombotic activity in models of venous and arterial thrombosis (1.25-40 μg/kg/min). The TT was only useful as a marker in the case of venous thrombosis, since, in the arterial thrombosis model, the clotting times were >200 s in the majority of animals receiving antithrombotic doses. The aPTT is not sufficiently sensitive to be predictive of an antithrombotic effect in the venous model, and shows only modest increases in the arterial thrombosis model. Heparin did not significantly increase the ECT at antithrombotic doses in the venous thrombosis model, and only increased the ECT by 53% at 40 μg/kg/min in the arterial model, despite a marked antithrombotic effect. Both the TT and aPTT were dose-dependently increased by heparin at doses active in the venous model, whereas both parameters were >200 s at doses active in the arterial thrombosis model.Thus, the ECT provides a predictive marker for the antithrombotic activity of argatroban in both venous and arterial thrombosis, at least in the rat.