Robert J. Broersma

The effect of thrombin inhibition in a rat arterial thrombosis model

The effect of heparin and the synthetic irreversible antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl) was studied on FeCl3-induced thrombotic occlusion of rat carotid arteries. Thrombocytopenia prevented occlusion in five of 7 rats for the 60 min observation period after FeCl3 injury demonstrating platelet dependence in this model of thrombosis. Intravenous injection of heparin (250 units/kg) followed by continuous infusion (250 units/kg/hr) failed to prevent occlusion in four of 6 rats whereas intravenous FPRCH2Cl infusion prevented occlusion at a dose of 200 nmol/kg/min during infusion in 6/6 rats. These findings indicate that thrombin plays a principle role in the platelet-dependent process of arterial thrombosis in FeCl3-damaged rat carotid arteries. Neutralization of the thrombogenic stimulus in this model by the thrombin inhibitor FPRCH2Cl suggests selective thrombin inhibition may be useful in the treatment of arterial thrombosis.

Pharmacological profile of 8-amino octanoic acid substituted atrial natriuretic factor analogs

A series of six analogs of rat atrial natriuretic factor have been prepared by the solid-phase method. The modified analogs contain 8-amino octanoic acid (a simple alkyl spacer) in place of selected tripeptides. Binding affinity to cultured aortic smooth muscle cell membranes suggests that the sequence Arg11-Gly16 is important for binding. Vasorelaxant activity on serotonin contracted rabbit aortic rings indicates that the Phe8-Gly16 sequence must be present for vasorelaxation. In anesthetized rats, the natriuretic and diuretic effects of an IV bolus dose correlate with in vitro vasodilatory activity. The alkyl spacer approach provides a facile method to quickly determine key regions of a large peptide involved in molecular recognition.

Design and synthesis of metabolically stable atrial natriuretic factor analogs: Amino- and carboxy-terminal stabilization

Two analogs of rat atrial natriuretic factor, rANF7–28‐NH2 and [Mpr7, Ala2O,D‐Arg27]rANF7–27‐NH2 were prepared by the solid‐phase method. These peptides had 2‐fold and 7‐fold less affinity, respectively, than rANF1–28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5‐fold less potent than rANF1–28 in relaxing serotonin‐contracted rabbit aortic rings. rANF7–28‐NH2 was rapidly degraded by rat kidney homogenates but [Mpr7Ala2O,D‐Arg27]rANF7–27‐NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.

Short-term studies of MDL 19,660-induced canine thrombocytopenia.

After 2 days of dosing, platelet counts progressively declined in dogs treated orally with 30 mg/kg/day of the antidepressant compound MDL 19,660 for 8 days. Accompanying the decrease in platelet counts was an increase in both large and vacuolated degenerating platelets. Upon cessation of dosing, the platelet counts returned to levels equal to or exceeding predosing levels within 4–7 days. Co-administration with aspirin, a known antiaggregating agent, had no protective effect on the drug-induced thrombocytopenia. In vitro testing of normal canine platelets in the presence of MDL 19,660 further revealed that spontaneous aggregation did not occur and that ADP-induced aggregation was inhibited. Drug-related platelet loss was also not prevented by the co-administration of prednisone, a steroid with immunosuppressive effects and inhibitory properties against reticuloendothelial cell phagocytosis of platelets. The results of the present investigation indicate that MDL 19,660 may produce in the dog a reversible thrombocytopenia in the form of vacuolar degeneration and subsequent destruction of the platelet by means other than aggregation or steroid-responsive mechanisms.