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Upon vascular damage platelet activation and blood coagulation are initiated. Interference at the initial level of the activation of the coagulation cascade can result in effective inhibition of thrombus formation. The in vivo antithrombotic properties of a series of bovine pancreatic trypsin inhibitor mutants (BPTI, aprotinin) 4C2, 7L22, 5L15, 5L15-PEG, 6L15 and 5L84, as described in the accompanying paper, with a combined inhibitory activity on factor Xa, factor VIIa-tissue factor complex, factor XIa and plasma kallikrein were compared to rTAP, r-hirudin, heparin and enoxaparin in a platelet rich thrombosis model in hamsters. Platelet dependent thrombus deposition was quantified by dedicated image analysis after transillumination of the femoral vein to which a standardised vascular trauma was applied. After increasing intravenous bolus injections all tested agents, except for aprotinin, induced a dose dependent decrease of thrombus formation and a concomitant prolongation of the aPTT. From the linear correlation between these two parameters it was found that 5 out of the 6 tested aprotinin analogues, rTAP and r-hirudin completely inhibited thrombus formation at a therapeutical (2- to 3-fold) aPTT prolongation while 4C2, heparin and enoxaparin only inhibited thrombus formation for 40 to 50 percent at a 2-fold aPTT prolongation. Based on the calculated IC50 values for thrombus formation rTAP was found to be the most active compound in this model. It is concluded that acceptable interference at the initial level of the blood coagulation, e.g. within a therapeutical aPTT prolongation, can significantly inhibit platelet deposition at a site of vascular injury.

Cyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-alpha-aspartyl- cyclic (1-->5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-propyl-L-arginyl-glycyl-L-alpha- aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1-->9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor. The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 micrograms/kg, ex vivo ADP-induced platelet aggregation with ID50 of 10 micrograms/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 +/- 9 to 1,100 +/- 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 micrograms/kg, ex vivo platelet aggregation with an ID50 of 50 micrograms/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 micrograms/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induced ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 +/- 0.4 to 12 +/- 2 min.(ABSTRACT TRUNCATED AT 250 WORDS)

The synergistic antithrombotic properties of G4120, a synthetic Arg-Gly-Asp (RGD) containing peptide which strongly inhibits platelet aggregation, and of Argatroban, a synthetic thrombin inhibitor, were examined in a reproducible quantitative hamster femoral vein platelet-rich mural thrombosis model. Bolus injections of G4120 and Argatroban inhibit thrombus formation in a dose-dependent way; 50% inhibition (ID50) is obtained with 11 micrograms/kg G4120 and with 2 mg/kg Argatroban. Combined bolus injections of 3 micrograms/kg G4120 with 0.5, 0.75 or 1 mg/kg Argatroban and of 1 mg/kg Argatroban with 1.5 or 3 micrograms/kg G4120 caused linear dose-dependent inhibition of thrombus formation, whereas 3 micrograms/kg G4120 or 1 mg/kg Argatroban alone had very little effect (less than 20% inhibition). ID50 was obtained with the combination of 3 micrograms/kg G4120 and 0.5 mg/kg Argatroban, corresponding to an equi-effective fractional combination of 0.62 with a 95% confidence interval of 0.50 to 0.74. Alternatively the ID50 was obtained with the combination of 1 mg/kg Argatroban and 1.3 micrograms/kg G4120, corresponding to an equi-effective fractional combination of 0.52 with a 95% confidence interval of 0.18 to 0.86. In both instances these results are indicative of a significant synergistic interaction. Bolus injection of 10 mg/kg aspirin, 100 U/kg heparin or the combination did not inhibit thrombus formation. The synergistic effect of the combination of platelet inhibiting RGD-peptides and synthetic thrombin inhibitors could be useful in the prevention of arterial occlusion with platelet-rich thrombus in patients with ischemic heart disease following thrombolytic therapy or angioplasty, although this combination is not expected to reverse platelet thrombus formation.