Thrombin-like enzyme from Lachesis muta muta venom: isolation and topographical analysis of its active site structure by means of the binding of amidines and guanidines as competitive inhibitors.

A serine protease enzyme was purified from Lachesis muta muta venom, with 40% yield, by gel filtration on Sephadex G-100 and affinity chromatography on Sepharose-agmatin. Homogeneity of the enzyme preparation was demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the enzyme had a relative mol. wt of 45,000. The molar extinction coefficient at 280 nm was 62,127 (M x cm)-1. The enzyme hydrolysed Bz-Arg-Nan with Ks = 0.233 +/- 0.08 mM and kcat = 2.80 +/- 0.07 sec-1. All the amidines and guanidines tested for their inhibitory effect on thrombin-like enzyme behaved as competitive inhibitors of the enzyme with Ki values in the range 6.2 microM to 42.3 mM for amidines and 0.19 mM to 9.31 mM for guanidines. Dissociation constant values were analyzed in terms of the binding of the inhibitors with the subsite S1, the specificity pocket of the enzyme, Ki values were discussed in accordance with those for trypsin inhibition. beta-Naphthamidine was the strongest inhibitor, while guanidine was the weakest. The differences among the Ki values were interpreted in terms of the shape of the enzyme active site. For meta- and para-substituted benzamidinium ions a good correlation was found between log l/Ki and sigma Hammett values of the substituents. The substituent effects in the pi-electrons of the benzamidine ring were considered in the frame of Hückel molecular orbital theory. A model for the binding of p-benzamidine derivatives with the primary specificity S1 subsite of the enzyme active site was proposed.

Surgical closure of the patent ductus arteriosus with an intravascular prosthesis: clinical experience.

The authors developed a new prosthesis for patent ductus arteriosus (PDA) closure, using a delivery device inserted through the main pulmonary artery (MPA) avoiding ductal dissection and use of cardiopulmonary bypass. The prosthesis was inserted in 19 consecutive patients between 1985 and 1992. They have been followed for a mean of 4.8 years (minimum 30 days, maximum 7.5 years). There were 14 women (72%) and the average age was 11 years (16 months to 38 years). All patients presented with pulmonary hypertension (4 severe, 5 moderate, and 10 mild). Simultaneous surgical procedures for congenital heart disease were performed in two cases. One patient had a diffuse calcified PDA. The average diameter of the inserted prosthesis was 7.5 mm (3.5 to 12.5 mm). Neither hemorrhage nor prosthesis dislocation/embolization occurred during the implantation or in the postoperative period. In a newborn (30 days) with severe cardiomegaly and thin MPA, we decided to ligate the ductus. Chronic cor pulmonale contributed to death in one patient 3.7 years after operation. The remaining patients recovered well, without clinical evidence of residual shunt. Therefore, we recommend the use of this new prosthesis for PDA closure in cases of large ductus or ductus complicated with calcification, pulmonary hypertension, and when associated open heart surgery is required.