SYNTHETIC SUBSTRATES FOR HUMAN FACTOR-VIIA AND FACTOR-VIIA-TISSUE FACTOR

A series of 100 tripeptide fluorogenic substrates has been synthesized . These substrates contain Arg in the P1 position, various amino acids in the P2 and P3 positions, and different 6-amino-1-naphthalenesulfon amides (ANSN) as the detecting group (P'). The 38 compounds possessing the highest initial rates of factor VIIa hydrolysis were evaluated fo r substrate kinetic parameters in the presence and absence of tissue f actor (TF) and by factor Xa. Most of these substrates had a higher k(c at)/K(M) (k(eff)) value for the factor VIIa-TF complex than for factor Xa. Substitution of different amino acids in the P2 position showed t hat substrates with bulkier amino acids such as Leu, Pro, and Val have higher values for K(M) and k(cat) than those with smaller amino acids (Gly or Ser). The highest second-order rate constants were found for substrates with Val or Pro in the P2 position. A decrease or increase in volume of the P2 substituent (Gly, Ser, or Leu) resulted in a decre ase in this constant. Substrates with the highest k(eff) values have P he in the P3 position. As the hydrophobicity and volume of the amino a cid in the P3 position decreased, the k(eff) was reduced. The efficien cy of substrates for hydrolysis by factor VIIa was enhanced by an incr ease of hydrophobicity in the P' structure. TF enhanced the amidolytic activity of the ''family' of 38 substrates with ANSN in the P' positi on on an average of 58-fold.