Approaches to Selective Peptidic Inhibitors of Factor Xa

Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro‐ and anti‐coagulant roles and thus fXa is thought to represent an ideal target. Discrete k cat and K m values for cleavage of a library of fluorescence‐quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa‐specific peptidic inhibitors. We hypothesized that K m rather than k cat / K m values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence‐quenched substrates, standard α ‐peptides and peptides containing a β ‐homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence‐quenched substrates to be the best inhibitors, while the inhibitory effect of the β ‐homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the β ‐peptides than the corresponding α ‐peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a β ‐amino acid at the cleavage site.