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Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides
Abstract:
The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. 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 five residues long spanning S4 to S1' and has favorable interactions over seven 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 or 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 four 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 either 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. However 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.