Activity of a synthetic hexadecasaccharide (SanOrg123781A) in a pig model of arterial thrombosis.

The activity of SanOrg123781A, a new synthetic antithrombotic drug inhibiting both factor Xa and thrombin through antithrombin (AT), was compared to that of unfractionated heparin (UFH) and of the synthetic pentasaccharide (fondaparinux, SP) in an ex vivo arterial thrombosis model in the pig. Six groups of four pigs were administered intravenously with SanOrg123781A (1, 3, 10 and 30 nmol kg(-1)), UFH (30 nmol kg(-1)) or SP (30 nmol kg(-1)). In this arterial model in which platelet thrombus was formed on a thrombogenic surface under a constant high shear rate, UFH and SP had moderate antithrombotic effects while SanOrg123781A exhibited a strong, dose-dependent inhibitory activity on platelet adhesion and platelet thrombus formation. In contrast to UFH, SanOrg123781A did not modify the activated partial thromboplastin time (aPTT) even at 30 nmol kg(-1), but strongly inhibited thrombin generation. At the same dose, despite a lower antithrombotic activity than SanOrg123781A, UFH significantly affected all the coagulation parameters. Taken together, these results show that SanOrg123781A, due to its potent and selective antifactor Xa and antifactor IIa activities is a promising new antithrombotic agent even in arterial setting.

The effect of a novel glycoprotein IIb/IIIa antagonist, SR 121566A, on platelet aggregation and activation in rhesus monkeys.

SR 121566A represents a peptidomimetic glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitor 3-[N- 4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl ) -N-(1-carboxymethylpiperid-4-yl) amino] propionic acid, trihydrochloride. To investigate the intravenous and subcutaneous pharmacodynamics of this agent, a primate model ( Macaca mulatta) was used. The IC50 for adenosine diphosphate (ADP) (10 micromol/L)-induced platelet aggregation in this primate platelet system was found to be 45 +/- 6 nmol/L. Comparatively in the human platelet rich plasma system, SR 121566A demonstrated an IC50 of 39 +/- 4 nmol/L. Graded doses of SR 121566A in the range of 25-400 microg/kg were administered intravenously. Blood samples were drawn from individual groups of primates (n = 4-6) at varying periods of time up to 24 hours after administration of SR 121566A. The pharmacodynamic effects were measured by platelet aggregation using ADP (10 micromol/L) as an agonist. In addition, flow cytometric methods were used to measure thrombin receptor-activating peptide (TRAP) (6.25 micromol/L)-induced platelet activation. In the subcutaneous studies, 50, 100, 250, and 400 microg/kg of SR 121566A was administered with an identical blood-drawing schedule and analysis as with the intravenous studies. In the intravenous studies, all doses of SR 121566A produced > 80% inhibition of platelet aggregation 5 minutes after the administration of the drug. The duration of the inhibitory effect is proportional to the dose administered and the 50% recovery time ranged from 2 to 15 hours. By flow cytometry, TRAP-induced P-selectin expression was also blocked for a varying duration of time in a dose-dependent fashion. The subcutaneous studies showed > 90% inhibition of platelet aggregation, which was observed at 15 minutes after administration of both 50 and 100 microg/kg of the drug. The recovery time after the subcutaneously administered doses was found to be shorter than the intravenously administered doses. These studies demonstrate that SR 121566A is an effective platelet inhibitor with predictable pharmacokinetic and pharmacodynamic characteristics.

Laboratory monitoring of pentasaccharide in a dog model of hemodialysis.

Varying dosages of pentasaccharide (400-800 nmol/kg) were compared to a 250-U/kg single bolus dosage of unfractionated heparin (UFH) in a dog model of hemodialysis. Several laboratory assays were used to monitor the effects of pentasaccharide and UFH. The pentasaccharide did not produce any anticoagulant effects as measured by the activated partial thromboplastin time. However, in the anti-Xa chromogenic assay and the Heptest assays, there was a dose-dependent prolongation after pentasaccharide administration. In the group of dogs administered 800 nmol/kg of pentasaccharide, there was a 50% decrease in the thrombin antithrombin (TAT) complex level after 60 minutes on dialysis. In the UFH-treated dogs, wide variations in assays were observed. There was a marked elevation in the activated partial thromboplastin time and Heptest assays up to 6 hours after UFH administration. Both anti-Xa and anti-IIa activity was measured up to 4 hours. In the TAT assay, UFH was found to have a stronger effect in suppressing the formation of TAT in comparison to the pentasaccharide. These results suggest that pentasaccharide can be used as a replacement for UFH in a dog model of hemodialysis to keep the dialysis circuit patent. In addition, the anti-Xa-based assays such as the Heptest and the chromogenic anti-Xa assays can be used to monitor the effects of pentasaccharide in this model.

Comparison of a synthetic antithrombin III-binding pentasaccharide and standard heparin as an adjunct to coronary thrombolysis.

The effects on alteplase-induced thrombolysis of the synthetic ATIII-binding pentasaccharide SR90107A/ORG 31540 (synthetic pentasaccharide, SP) and of standard heparin (SH) were compared in a copper coil model of coronary artery thrombosis in 6 groups of 10 dogs. After 1 h of occlusion, all animals received intravenously alteplase and aspirin, and were randomly assigned to a 2 h infusion of either saline, or one of two doses of SH (100 IU/kg bolus plus 50 IU/kg/h infusion, or 200 IU/kg bolus plus 100 IU/kg/h infusion), or one of three doses of SP (100 nmol/kg bolus plus 50 nmol/kg/h infusion, 200 nmol/kg bolus plus 100 nmol/kg/h infusion, or 400 nmol/kg bolus plus 200 nmol/kg/h infusion). Coronary angiography was performed every 10 min for 4 h. Appropriate doses of SP and SH enhanced alteplase-induced thrombolysis to a similar extent. In contrast, SP was devoid of any anti-IIa activity or aPTT prolongation.

Ultrastructural studies of platelet aggregates from human subjects receiving clopidogrel and from a patient with an inherited defect of an ADP-dependent pathway of platelet activation.

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti-ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

Protamine sulphate inhibits pentasaccharide (SR80027)-induced bleeding without affecting its antithrombotic and anti-factor Xa activity in the rat.

The neutralization of a potent anti-factor Xa pentasaccharide (SR 80027) and heparin-induced bleeding enhancement by protamine sulphate was studied in vivo. Bleeding time, as measured by transection of the tail of anaesthetized rats, increased after the administration of standard heparin and SR 80027. Doses of 0.6 and 2.5 mg/kg of heparin and SR 80027, respectively, were required to enhance blood loss to the same extent (6-fold increase). Protamine sulphate (10 mg/kg i.v.) reduced blood loss induced by both compounds but also neutralized the anti-factor Xa activity as well as the antithrombotic activity of standard heparin measured in a venous thrombosis model. However, protamine sulphate did not affect the anti-factor Xa activity or the antithrombotic activity of SR 80027. These data suggest that protamine sulphate may be an effective antidote for the bleeding side-effects of SR 80027 but they also indicate that the bleeding tendency associated with this type of compounds cannot be attributed to their anti-factor Xa activity.

Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long-term heparin treatment.

Vascular smooth muscle cells (SMC) do not represent a homogeneous population (Schwartz et al., 1990, Am. J. Pathol. 136: 1417-1428). Cellular clones resistant to the antiproliferative activity of heparin were isolated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435-443; San Antonio et al., 1993, Arterioscler. Thromb., 13:748-757) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341-342). We have shown in the present study that long-term treatment (growth medium supplemented with 200 micrograms/ml heparin, from the second to the tenth passage) of rat aortic SMC, without cell cloning, resulted in a significant loss of sensitivity to the growth inhibition by heparin and its derivatives. The heparin resistance was stable after growing cells for two passages in heparin-free medium, suggesting the selection of a particular phenotype. We tried to characterize these cells and to determine the causes of the resistance to the growth inhibition by heparin. Heparin-treated SMC (HT-SMC) were smaller than their control culture at the same passage, expressed less alpha-SM actin, and did not overgrow after reaching confluence. As in the heparin-resistant clones (San Antonio et al., 1993, Cell Regul., 1:435-443) expression of alpha-SM actin could be increased in HT-SMC by heparin addition before Western blotting. Heparin resistance was associated with a tenfold decrease in [3H]-heparin binding capacity (Bmax = 1.9 x 10(6) sites per cell) compared to control cultures (Bmax = 1.7 x 10(7) sites per cell), which was irreversible after growing the cells for two additional passages in heparin-free medium. We also investigated protein kinase C (PKC) in HT-SMC in terms of both enzymatic activity and protein expression (evaluated by [3H]-staurosporine and [3H]-phorbol-12,13-dibutyrate binding). We found that HT-SMC had only half the PKC activity and expression as control SMC. Therefore, long-term treatment of rat aortic SMC with heparin allowed the selection of a less differentiated subpopulation of cells, exhibiting low sensitivity to the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or expression.