Localization of a Heparin Binding Site in the Catalytic Domain of Factor XIa

Abstract

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.

Mechanism of Catalysis of Inhibition of Factor IXa by Antithrombin in the Presence of Heparin or Pentasaccharide

“…Ca 2ϩ enhances the rate of f.Xa inhibition by antithrombin, presumably by inducing the exposure of a heparin-binding site on the protease, thereby allowing formation of a ternary heparin-antithrombin-f.Xa complex (27,28). This concept is supported by surface plasmon resonance studies demonstrating that the binding of heparin to f.Xa is Ca 2ϩ -dependent (29). In the same study, it also was determined that heparin binds to f.IXa with a K d of 17.8 nM, but only in the presence of Ca 2ϩ .…”

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

“…Increasing concentrations of DxS, DS, HS, CS-C, CS-A, and CS-A/C were spotted on the membrane and subsequently probed with FXIIa. Heparin was employed as a positive control (17). Although we observed association of FXIIa with heparin, HS, DS, DxS, and CS-A (Fig.…”

“…In general, the protein HS-binding platforms are organized into large, positively charged surfaces composed of several basic amino acids that do not display a common sequence pattern (24). Although several structural domains of FXII/FXIIa have been described to bind negatively charged surfaces, the precise location of the FXII site involved in the interaction with GAG chains has not yet been identified (1,17). Analysis of heparin binding to a number of other coagulation factors may provide mechanistic and structural insights as to how FXIIa associates with HS-type GAGs (25).…”

“…Accumulation of FXIIa on CHO-A745 was reduced by 45% and on CHO-D677 by 22% when compared with wild-type cells. Although CHO-D677 cells are HS-deficient, this cell line expresses increased amounts of CS at the cell surface (17). Because the reduction of FXIIa binding to CHO-D677 was less pronounced than to CHO-A745, we assumed that elevated CS levels compensate for the lack of HS and assist in the interaction of FXIIa with the cell surface.…”

A Catalytic Domain Exosite (Cys527−Cys542) in Factor XIa Mediates Binding to a Site on Activated Platelets

“…Based on this comparison, the 7 subdomains with the greatest dissimilarities (boxed sequences in Figure 5) were identified, and 8 different peptides (Table 1; Figure 5) were synthesized (the 8 th peptide being a scrambled peptide). An alternative approach to identifying FXIa residues that mediate binding to activated platelets comes from previous studies carried out in our laboratory (33 and/or a heparin binding consensus sequence (BXBBXBX, where B represents a basic residue and X is a hydrophilic residue); comprising the same sequence as Peptide 6). As shown in Figure 6A, 5 of the catalytic domain peptides (peptides 1, 2, 3, 5 and 6) were ineffective in displacing FXIa from the platelet surface.…”