Localization of a heparin binding site in the catalytic domain of factor XIa.
Inhibition of factor XIa by protease nexin II (K(i) approximately 450 pM) is potentiated by heparin (K(I) approximately 30 pM). The inhibition of the isolated catalytic domain of factor XIa demonstrates a similar potentiation by heparin (K(i) 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 K(d) 8.63 +/- 1.06 nM for factor XIa). The factor XIa catalytic domain contains a cysteine-constrained alpha-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 (534)H, (535)K, (538)H, and (539)K 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 approximately 10-fold less effective, and alanine substitutions at residues (529)K, (530)R, and (532)R result in loss of virtually all activity. We conclude that residues (529)K, (530)R, and (532)R comprise a high-affinity heparin-binding site in the factor XIa catalytic domain.
Badellino
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Department of Physiology, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, Pennsylvania 19140, USA.
Walsh
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D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D004789:Enzyme Activation; D015949:Factor IXa / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011486:Protein C / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D011486:Protein C; D001710:Biotin; D006493:Heparin; D007610:Kallikreins; D015942:Factor VIIa; D015949:Factor IXa; D015945:Factor XIa; D013917:Thrombin; D015951:Factor Xa