Reactivity of bovine blood coagulation factor IXa.beta., factor Xa.beta., and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

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Abstract
The published activation site sequences of bovine factors IX and X were utilized to synthesize a number of peptides specifically designed as substrates for bovine factors XIa and IXa.beta., respectively. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/Km value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least 5 residues long spanning S4 to S1'' and has favorable interactions over 7 subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa.beta., Xa.beta. or thrombin. Factor IXa.beta. failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved 4 hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3''. Factor Xa.beta. poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa.beta. substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of factors IXa.beta. or Xa.beta. toward synthetic substrates. Both factor IXa.beta. and Xa.beta. cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. The rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa.beta. and Xa.beta.