Determination of the P′1, P′2 and P′3 subsite-specificity of factor Xa

Factor Xa is a central protease in the coagulation cascade and the target for many anticoagulant compounds currently under development. The preferences of the enzyme for substrates incorporating residues N-terminal to the cleavage site (P-1, P-2, etc.) have been elucidated, but little is known of its preferences for residues C-terminal to the cleavage site (P-1', P-2' etc.). The preferences of bovine factor Xa for substrate residues in the P-1', P-2' and P-3' positions were mapped using fluorescence-quenched substrates. Bovine factor Xa, often used as a model for factor Xa, was most selective for the P-2' position, less selective at the P-1', position and almost completely non-selective at the P-3' position. It appears that while the prime side subsites of factor Xa impose some selectivity towards substrates, the influence of these sites on factor Xa cleavage specificity is relatively low in comparison to related enzymes such as thrombin. (C) 2002 Elsevier Science Ltd. All rights reserved.