Localization of a heparin binding site in the catalytic domain of factor XIa

Inhibition of factor XIa by protease nexin II (K-i approximate to 450 pM) is potentiated by heparin (KI x 30 pM), The inhibition of the isolated catalytic domain of factor XIa demonstrates a similar potentiation by heparin (Ki decreasing from 436 +/- 62 to 88 +/- 10 pM) and also binds to heparin on surface plasmon resonance (K-d 11.2 +/- 3.2 nM vs Kd 8.63 +/- 1.06 nM for factor XIa). The factor XIa catalytic domain contains a cysteine-constrained or-helix-containing loop: (527)CQKRYRGHKITHKMIC(542), identified as a heparin-binding region in other coagulation proteins. Heparin-binding studies of coagulation proteases allowed a grouping of these proteins into three categories: group A (binding within a cysteine-constrained loop or a C-terminal heparin-binding region), factors XIa, IXa, Xa, and thrombin; group B (binding by a different mechanism), factor XIIa and activated protein C; and group C (no binding), factor VIIa and kallikrein. Synthesized peptides representative of the factor XIa catalytic domain loop were used as competitors in factor XIa binding and inhibition studies. A native sequence peptide binds to heparin with a K-d 86 +/- 15 nM and competes with factor XIa in binding to heparin, K-i = 241 +/- 37 nM. A peptide with alanine substitutions at H-534, K-535, H-538, and K-539 binds and competes with factor XIa for heparin-binding in a manner nearly identical to that of the native peptide, whereas a scrambled peptide is similar to 10-fold less effective, and alanine substitutions at residues K-529, R-530, and R-532 result in loss of virtually all activity. We conclude that residues K-529, R-530, and R-532 comprise a high-affinity heparin-binding site in the factor XIa catalytic domain.