Gerald F. Smith

The heparin-thrombin complex in the mechanism of thrombin inactivation by heparin.

Heparin and thrombin, at very low concentrations, are shown to form a complex with a very high association constant. The heparin-thrombin complex contains an altered thrombin structure, as evidenced by a 70% decrease in the thrombin arnidase activity. In the presence of α 2 -antithrombin the heparin-thrombin complex can be completely inactivated. It is shown that heparin and α 2 -antithrombin do not interact under conditions in which the heparin-thrombin complex is formed and observed. Therefore, it is proposed that the first molecular event in the heparin-mediated thrombin inactivation process in blood is the rapid formation of the heparin-thrombin complex. The complete inactivation of the thrombin in this complex is achieved by further reaction with α 2 -antithrombin in blood, the latter reaction probably being the rate limiting step in the thrombin inactivation process.

D-Phe-Pro-p-Amidinobenzylamine: A potent and highly selective thrombin inhibitor

Abstract The design, synthesis, and enzyme inhibitory profile of D-Phe-Pro-p-Amidinobenzylamine are presented. This compound has inhibitory activity equivalent to D-Phe-Pro-Arg-H, two orders of magnitude more potent than D-Phe-Pro-Agmatine. The results indicate that binding energy provided by the covalent bond of a transition-state analog can be replaced with noncovalent interactions.

1,2-Disubstituted indole, azaindole and benzimidazole derivatives possessing amine moiety : A novel series of thrombin inhibitors

A novel series of 1,2-disubstituted indole, azaindole and benzimidazole derivatives possessing an amine moiety was identified as thrombin inhibitors. An indole with basic diamine moieties (12a) was the most potent thrombin inhibitor in the series with Kass= 197 x 10(6) L/mol.

Serine protease selectivity of the thrombin inhibitor D-Phe-Pro-Agmatine and its homologs

Abstract Analogs of D-Phe-Pro-Agmatine were assayed for inhibititory activity versus thrombin, trypsin, plasmin, n-tPA and urokinase. The X-ray structure of the thrombin/D-Phe-Pro-Agmatine co-crystal revealed that the agmatine and analogous arginals have very similar bound conformations.

Correlation of the in vivo anticoagulant, antithrombotic, and antimetastatic efficacy of warfarin in the rat

Fibrin formation has been hypothesized to be an element of the metastatic process in cancer, and pharmacological interference with such fibrin formation has been proposed as a means of antimetastatic therapy. We have tested this hypothesis through an in vivo study of warfarin in two independent rat disease models--a model of chemical-injury-induced arterial thrombosis, and a model of spontaneous metastasis. We found 0.50 mg/kg-day warfarin to be uniformly lethal after two weeks treatment. The chronic dose of 0.25 mg/kg-day was non-toxic and produced effective anticoagulation and marked antithrombotic and antimetastatic activity. The 0.125 mg/kg-day dose produced a reduction in factor IIc (50%) and factor VIIc (70%), and resulted in statistically significant antithrombotic and antimetastatic activity. The 0.0625 mg/kg-day dose failed to reduce the vitamin K-dependent clotting factors, and failed to produce any antithrombotic or antimetastatic effects. The substantial correlation (very similar dose-response effects) among the anticoagulant, antithrombotic and antimetastatic efficacies of warfarin in the rat suggests that anticoagulation provides the pharmacological mechanism underlying both the antithrombotic and the antimetastatic effects. The poor therapeutic index we observed in the rat may be the attribute which limits the efficacy of warfarin in the treatment of human cancer.

Chapter 11. Future Antithrombotic Therapy

Publisher Summary This chapter summarizes the recent advancement in the synthesis and characterization of select agents that hold the promise of being future antithrombotic therapeutics. Hemostasis is the natural physiological response of blood damage to the vasculature, ultimately leading to cessation of blood loss. Thrombosis that occurs in both arteries and veins is the virtual or total occlusion of a blood vessel by the elements of the hemostatic system. As the pathogenesis of arterial thrombosis clearly differs from that of venous thrombosis, the agents that inhibit platelet activity have the most benefit in the treatment of arterial thrombosis and, until recently, agents that inhibit the coagulation pathways have been used primarily for venous disease. From the widespread clinical study, aspirin has been generally accepted as a useful antithrombotic with a variety of arterial indications. Aspirin inhibits platelet aggregation by inhibiting generation of thromboxane A 2 (TXA 2 ) by activated platelets via acetylation of the enzyme cyclooxygenase. An alternative means of controlling TXA 2 -dependent aspects of platelet aggregation is by specific inhibition of thromboxane synthase. Ticlopidine is a thienopyridine derivative that has undergone extensive clinical testing and is a moderately active inhibitor of platelet aggregation. Factor Xa is an arginine directed endopeptidase responsible for the conversion of prothrombin to thrombin. Inhibition of this enzyme is a potential means to control aberrant coagulation and resulting thrombosis. The fibrinogen binding site on thrombin is also being explored as a potential site of action of therapeutic agents. Irreversible active-site inhibitors of thrombin, which target the serine in the catalytic site of thrombin, have also been developed. Based on the opportunities afforded by the advancement in molecular biology and by elucidation of the molecular basis of receptor/ligand and enzyme/substrate interactions, great strides have been made in the understanding of hemostasis and thrombosis.

Thrombolytic activity of a novel plasminogen activator, LY210825, compared with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis.

LY210825, a recombinant tissue-type plasminogen activator (rt-PA), which contains the kringle-2 and serine protease functional domains of native tissue-type plasminogen activator, was previously produced by site-directed mutagenesis in a Syrian hamster cell line. We studied the thrombolytic potential of this molecule in a canine thrombosis model. Male hounds (16-22 kg) were anesthetized; a 2.0-cm segment of the left circumflex coronary artery (LCX) was isolated proximal to the first main branch, and the dogs were instrumented with an electromagnetic flow probe to measure coronary blood flow. An occlusive thrombus was formed after injury of the intimal surface of the LCX with an electrical current applied by a needle-tipped anode placed distal to the electromagnetic flow probe. After 1 hour of occlusion, either LY210825 or rt-PA was administered intravenously according to the following protocols: 1) a 1-hour infusion of either 0.25 mg/kg LY210825 or 0.4 mg/kg rt-PA, 2) single injections of 0.15-0.6 mg/kg LY210825, and 3) a single injection of 0.45 mg/kg LY210825 and a 3-hour infusion of 1.0 or 1.7 mg/kg rt-PA. Plasma half-lives of LY210825 and rt-PA were 58 +/- 7 and 3.3 +/- 0.3 minutes, respectively. LY210825 produced more rapid reperfusion of the LCX than did rt-PA. In the third study, 90% of the rt-PA-treated vessels reoccluded within 1 hour after cessation of drug, whereas only 25% of the LY210825-treated vessels reoccluded during a 4-hour washout period. There were significant, but relatively small, reductions produced by both plasminogen activators on plasma fibrinogen and plasminogen (25-35% decreases). Because of its longer plasma half-life, LY210825 could be administered intravenously as a single injection. In a canine model of coronary artery thrombosis, LY210825 was a more effective thrombolytic agent than was rt-PA.

Inhibitory Effect of Warfarin on the Metastasis of the PAIII Prostatic Adenocarcinoma in the Rat

The PAIII rodent metastatic prostatic adenocarcinoma model was employed to evaluate the effects of dietary warfarin, a prototypic antagonist of thrombin generation on the lymphatic and pulmonary metastases of the tumor from the tail site of subcutaneous transplantation in male Lobund Wistar (LW) rats. In addition, the anticoagulant effects of warfarin were determined in the same animals. Warfarin, administered in the diet at concentrations equivalent to 0.063, 0.125 or 0.250 mg./kg. b.w. for 30 days had no effect on final body weight, gluteal or iliac lymph node weights. Significant (p less than 0.05) dose-dependent extensions of whole blood prothrombin (WBPT), activated partial thromboplastin (WBAPTT) and clotting times (WBCT) over control values were observed with warfarin treatment. Preliminary studies demonstrated that the 0.500 mg./kg. dose produced 50 per cent mortality at +14 days. Warfarin produced significant (p less than 0.05) dose-dependent decreases in the number of PAIII pulmonary metastases as indicated by reductions in dry lung weights and lung colony numbers when compared to untreated tumor-bearing controls. While the therapeutic index of warfarin is a limiting factor in clinical use as an antimetastatic agent, these results suggest that compounds capable of altering hemostatic mechanisms may be potential inhibitors of tumor metastasis. The PAIII prostatic adenocarcinoma model may be a useful system to quantitatively evaluate potential antimetastatic and cytotoxic agents.

The effects of 1-methyl-5-thiotetrazole in a rat liver vitamin K-dependent carboxylase assay

1-Methyl-5-thiotetrazole (NMTT), a metabolite of moxalactam (MoxamR), was studied for its potential inhibition of vitamin K-dependent carboxylation. The assay system utilized a detergent solubilized rat liver microsomal preparation. Vitamin K1H2 was artificially produced in situ by the NADH-dependent reduction of exogenous phylloquinone and the resultant carboxylation monitored by 14CO2 incorporation into a soluble peptide substrate. Warfarin, used as a reference inhibitor, gave results expected from the literature - 50% inhibition at a pharmacologically excessive level of 1.0 mM. Carboxylation was unaffected by 1.0 mM NMTT and was marginally (0-14%) diminished by 5.0 mM NMTT. Carboxylation was 25% diminished at 10.0 mM NMTT, a concentration far above that achieved in human testing of moxalactam. When NMTT was pre-incubated with the liver microsomal carboxylase enzyme preparation, 10.0 mM NMTT again caused merely a 25% diminution of carboxylation in the assay. These results do not support a role for NMTT as an inhibition of Vitamin K-dependent carboxylation which would produce pharmacological side effects during moxalactam therapy. During these studies it was found that dramatic consumption of NADH occurs in the presence of liver microsomal preparations (independent of vitamin K and of NMTT) and that NMTT effects on these processes may explain the small carboxylation diminution observed at 10.0 nM NMTT in the carboxylase assay.

A four component coupling strategy for the synthesis of d-phenylglycinamide-derived non-covalent factor Xa inhibitors

A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.