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.