Evaluation of p-amidinophenyl esters as potential antithrombotic agents.

Three p-amidinophenyl esters have been synthesized and characterized as irreversible inhibitors of the vitamin-K dependent proteinases; factors IXa, Xa and thrombin (Turner et al. [4]).+ In the present report we describe the in vitro and in vivo effects of these agents on standard coagulation tests in vitro and in blood from animals treated with the compounds. At a concentration of 500 microM, the three esters increased the activated partial thromboplastin time (PTT) of pooled human plasma 3 to 5-fold. The prothrombin time increased 1.4 to 3.7-fold under similar conditions. The p-amidinophenyl ester of cinnamic acid (CINN) showed the most pronounced effect on both assays. This ester also is the best inhibitor of human factors IXa and Xa, while the p-amidinophenyl ester of benzoic acid (BENZ) is a slightly better alpha-thrombin inhibitor (4). The effect of these esters on the thrombin clotting time correlated with in vitro kinetic measurements of alpha-thrombin inhibition rates. Both BENZ and CINN increased the assay endpoint more than 6-fold. The three esters also were studied using mouse plasma. A comparable effect on the PTT was noted. Intravenous administration of 300 microliter of 1 mM CINN as a single bolus in mice caused a 2.3-fold increase in the PTT which remained 1.2-fold normal 2 h later. The BENZ and alpha-methyl-cinnamic acid (MECINN) esters were somewhat less effective as predicted from their in vitro effect on the PTT. This investigation and previous studies indicate that these compounds demonstrate low toxicity at therapeutic levels. It is concluded that the p-amidinophenyl esters may be useful in antithrombotic therapy.

High-performance affinity chromatography of trypsins on Asahipak GS-gel coupled with p-aminobenzamidine.

An adsorbent for high-performance affinity chromatography of trypsins was prepared, based on a micro-particulate polyvinyl alcohol gel for high-performance liquid chromatography, Asahipak GS-gel. After the hydroxyl groups had been activated with 1,1'-carbonyldiimidazole, 6-aminohexanoic acid was coupled as a spacer, then p-aminobenzamidine, a specific ligand for trypsin-family enzymes, was immobilized on the spacer. Fluorometric detection of eluted protein and on-line assay of enzyme activity using a fluorogenic substrate, peptidylmethylcoumarylamide, made it possible to attain very high sensitivity. Microgram amounts of bovine trypsin and Streptomyces griseus trypsin could easily be analyzed in a short time (less than 1 h).