Heparin affinity was determined for a series of alanine substitutions in the factor IXa protease domain via a soluble binding assay that utilizes a fluoresceinated super-sulfated LMWH (Fl-ssLMWH) (5
). The binding assay revealed that the mutations K126A, K132A, and R233A dramatically (10–20 fold) reduced factor IXa heparin affinity, while the mutations N129A and R165A moderately (5-fold) reduced affinity (). These results suggest that the core heparin-binding site is located in the groove between R233 in the C-terminus α-helix and residues K126 and K132 in the c126–132 α-helix, with additional contributions from N129 and the R165 side chain (c164–170 α-helix). The significant contribution of the R233 side chain to heparin affinity is in agreement with previous reports analyzing the interaction of factor IXa with immobilized heparin (3
). In contrast to studies employing immobilized heparin, our solution binding assays demonstrated that the side chains of K132 and K126 dominated the interaction relative to R165, and that R170 did not significantly contribute to the binding of ssLMWH (3
). Modeling the interaction of a heparin pentasaccharide with this protease exosite using Autodock 4 revealed a preferred binding mode consistent with the binding and inhibition data (). Hydrogen bonding between the sulfate and carboxylate oxygens of heparin and the amino groups of K126, N129, K132, R165, and R233 was predicted. The model also predicted that residue K230, located in the bottom of the ligand-binding pocket, participates in heparin binding. This prediction agrees with the previous report that K230 contributed to gla-domainless factor IXa binding to immobilized heparin (36
). This pocket likely represents the minimal size of the heparin binding site. LMWH, with an average molecular weight of 5000, is much longer than the pentasaccharide and may also bind to adjacent residues. The binding of heparin oligosaccharide in this manner appears well-positioned to block important cofactor interactions with the c164–170 α-helix, including the critical interaction between the factor VIIIa A2 domain and the side chain of R165 (12
Similar to other heparin oligosaccharides, ssLMWH demonstrated the ability to directly inhibit factor X activation by the factor IXa-PL complex activated with ethylene glycol. The ssLMWH did not significantly affect the rate of p-nitroanilide cleavage from the substrate, CH3
-D-CHG-Gly-Arg-pNA, consistent with a lack of oligosaccharide effect on the S3–S1 subsites. In contrast, factor X activation by the factor IXa-PL complex was inhibited by ssLMWH with an apparent affinity (KI
) approximately seven-fold greater than the wild type protease-heparin interaction in solution. Addition of PL vesicles +/− ethylene glycol increased protease-heparin affinity two to four–fold (), partially explaining the increased affinity within the enzyme complex. In principle, factor X may also contribute to the apparent increased affinity via direct oligosaccharide binding or indirectly via effects on protease conformation. Since the affinity of the factor Xa-ssLMWH interaction appears almost five-fold less than the factor IXa-ssLMWH interaction (5
), a conformational effect on the protease appears more likely. Alanine substitutions within the protease exosite resulted in increased resistance to inhibition of factor X activation by the LMWH derivatives (), but their specific contributions to the inhibition were significantly different from the results of protease-ssLMWH binding in solution. In particular, K132 and N129 demonstrated a significantly reduced contribution to the ssLMWH interaction within the factor IXa-PL complex, while R233 continued to make a dominant contribution. The effect of alanine substitutions for R233 and K126 was relatively greater in the enzyme complex compared to protease-LMWH affinity in solution, while the effect of R165 was relatively consistent. The enhanced overall affinity for ssLMWH and reduced contribution of the c129-132 α-helix to the interaction within the factor IXa-PL complex suggests that the heparin-binding exosite undergoes a significant conformational change upon incorporation into the membrane-bound enzyme complex.
LMWH derivatives demonstrated significantly lower apparent affinity for factor IXa within the intrinsic tenase versus the factor IXa-PL complex. Factor IXa wild type demonstrated KI
values nearly 4-fold higher for ssLMWH in the intrinsic tenase compared to the factor IXa-PL complex. With the notable exception of R170A (see below), this relationship was consistent for the remainder of factor IXa mutants which displayed KI
values 2–7 fold higher for ssLMWH in the intrinsic tenase relative to the factor IXa-PL complex (–). These results are consistent with the conclusion that LMWH derivatives and cofactor compete for binding to the factor IXa protease domain (2
). Factor IXa R170, similar to wild type protease with regard to affinity for ssLMWH in solution and inhibitor potency in the factor IXa-PL complex, demonstrated moderate resistance to inhibition by ssLMWH in the intrinsic tenase complex. The relative increase in KI
values between the factor IXa-PL and intrinsic tenase complexes for factor IXa R170A was significantly larger than the other mutant proteases - over 24-fold for ssLMWH. Thus, the effect of the R170A mutation on apparent heparin affinity was cofactor specific. This result is consistent with the four-fold increase in factor IXa-cofactor affinity reported for this protease, enhancing the ability of cofactor to compete with LMWH for binding to the protease domain (3
). In summary, LMWH derivatives interact with the factor IXa-PL and intrinsic tenase complexes in a similar fashion, and the presence of cofactor reduces factor IXa-LMWH affinity based on competition for overlapping binding sites on the protease domain.
In principle, inhibition of the intrinsic tenase complex by heparin oligosaccharide may result from disruption of the factor IXa-cofactor interaction, destabilization of the cofactor within the enzyme complex (loss of the A2 domain), or allosteric modulation of factor IXa proteolytic activity. To address the contribution of cofactor destabilization, the ability of LMWH derivatives to inhibit intrinsic tenase activity was examined in the presence of a B-domainless factor VIIIa containing mutations that stabilize the A2 domain within the activated cofactor (20
). The stabilized cofactor had only a minor effect on inhibitor potency relative to wild type protein, suggesting that cofactor destabilization does not contribute significantly to this inhibition mechanism. Similarly, an allosteric effect of heparin oligosaccharide on factor X catalysis may contribute to the inhibition (based on results with factor IXa-PL complex), however, the relative contribution of this mechanism is likely to be dwarfed by the effect of disrupting the cofactor-protease domain interaction.
These results provide a detailed view of the molecular target for antithrombin-independent inhibition of the intrinsic tenase complex by heparin oligosaccharides. A recent crystal structure of the complex between the human factor IXa protease-EGF2 fragment and pentasaccharide-activated antithrombin III contains an additional pentasaccharide-factor IXa interaction that is located on the periphery of the binding site identified by our results, utilizing some but not all of the implicated residues (39
). Intermolecular contacts were observed for K126, R165, and R233 as predicted, but not N129 or more importantly, K132, which had profound effects on heparin affinity when mutated to alanine. On the contrary, crystal contacts were observed for N178, which did not affect heparin binding or inhibition when mutated to alanine. While our results suggest that this interaction does not represent the favored binding mode of a pentasaccharide in solution, the complete extent of the heparin binding site is not addressed in our studies and potentially includes the region identified in the crystal structure. Additionally, a degree of flexibility in the protease-heparin interaction is suggested by the differences observed between the binding of free factor IXa in solution versus the factor IXa-PL complex. These results suggest that the heparin-binding exosite undergoes a conformational change within the enzyme complex.
The factor IXa-factor VIIIa complex represents an extensive, high affinity interaction that involves nearly the full-length of the protease () (17
). Interaction between the factor IXa EGF domains and the factor VIIIa light chain (A3-C1-C2 domains) contributes substantially to the overall affinity, but completely lacks cofactor activity (16
). In contrast, the interaction between the protease domain and the isolated factor VIIIa A2 domain is relatively low affinity, but required for cofactor activity (17
). Analysis of alanine substitutions within the protease domain heparin-binding exosite suggests an extensive cofactor interactive site that includes contributions from K126, N129, K132, R165, and R233 (3
). Our current results demonstrate substantial overlap between the heparin and cofactor binding sites on the protease domain, which includes residues critical to the A2 domain interaction () (12
). Mutagenesis of this protease exosite modulates cofactor affinity, with dramatic effects on recombinant factor IX phenotype in the setting of tissue-factor stimulated thrombin generation in human plasma, and venous thrombosis following saphenous vein injury in the hemophilia B mouse (18
). Allosteric communication between this protease exosite and the catalytic machinery of factor IXa is suggested by the role of the R165 side chain in cofactor-induced protease activation, and the ability of LMWH to both inhibit factor X activation by the factor IXa-PL complex, and modify accessibility of the factor IXa active site to bovine pancreatic trypsin inhibitor (9
). Thus, the critical but unstable interaction between this protease domain exosite and the cofactor A2 domain represents a highly vulnerable protein-protein interaction that may be therapeutically exploited.
Comparison of the factor VIIIa and heparin binding sites on factor IXa