LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa

Abstract

Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, or FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation by factor XIIa suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed by examinations of FIX-activation showing that FIX-activation by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXI-R5 and FXI-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity in assays utilizing a kallikrein-specific chromogenic substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 4% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen utilizing FXIa-R4 as the enzyme were similar to those achieved with kallikrein but not with FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. The residues of FXIa catalytic domain (R3704, Y5901, E98, Y143, I151, and K192 in chymotrypsin numbering) that are possibly involved in the interactions with its inhibitors have been identified based on the co-crystal structure of the FXIa catalytic domain with the KPI domain of the kunitz-type inhibitor, protease nexin 2, (PN2KPI). A single mutation comprising Y5901A in the R2 region of FXIa displayed resistance to the inhibition by PN2KPI indicating that Y5901 is involved in the interaction with PN2KPI. In conclusion, these studies of FXI/PK chimeric and mutant proteins implicate residues in the R1, R2 and R3 regions in the maintenance of FXIa structure; residues in the R5 and R6 regions involved in active site architecture; residues within the R4 region (99-loop) of FXIa in the determination of amidolytic substrate specificity; residues within the R6 region (autolysis loop) of FXIa in the interaction with the macromolecular substrate, FIX; and the residue Y5901 in the R2 region of FXIa is involved in the interaction of FXIa with PN2KPI. FXIa activates FIX by facilitating the sequential cleavages first at R145-A146 resulting in FIXα and then at R180-V181 producing FIXa. Calcium binding to the γ-carboxyl glutamate-rich (Gla) domain of FIX is required for the interaction of FIX with FXIa and presence of FIX binding exosites have been implicated within both heavy and light chains of FXIa. To explore the mechanism by which calcium affects the interactions between the two FIX binding sites within the heavy and light chains of FXIa, the time course of FIX- activation by FXIa or the light chain of FXIa (FXIa-LC) in the presence or absence of calcium ions was examined. When FIX was activated by FXIa in the presence of calcium ions, the first cleavage at R145-A146 occurred at early time points when FIXa formation was also observed suggesting that the two scissile bonds of FIX were cleaved almost simultaneously. FIX activation either by FXIa in the absence of calcium ions or by the catalytic domain of FXIa (i.e. in the absence of the heavy chain of FXIa) showed the accumulation of the inactive intermediate FIXα produced from the cleavage at R145- A146, indicating a slower cleavage rate at R180-V181. Thus, both calcium and the heavy chain of FXIa are essential for optimal FIX activation. To determine the roles of calcium and the two FIX-binding exosites within the heavy and light chains of FXIa in the cleavages of the two scissile bonds of FIX respectively, FXI-R145A (in which only the cleavage at R180-V181 available) and FIX-R180A (in which only the cleavage at R145-A146 available) were prepared. The cleavage rate at R145-A146 of FIX-R180A in the absence of calcium was slower than in the presence of calcium ions. The cleavage rate at R180-V181 of FIX-R145A without the prior cleavage at R145 by FXIa in the presence of calcium ions was much slower than that of wild-type FIX indicating that the efficient cleavage at R180-V181 is facilitated by the prior cleavage at R145-A146. The scissile bond R180-V181 of FIX-R145A was hardly cleaved by FXIa in the absence of calcium ions or the heavy chain of FXIa from which we conclude that both calcium and the heavy chain of FXIa are required for efficient cleavage at R180-V181 of FIX. Based on these results, we propose a model of the mechanism of FIX-activation by FXIa: When FIX is activated by FXIa in the presence of calcium ions, FIX binds to both heavy and light chains of FXIa to bring the two scissile bonds of FIX, R145-A146 and R180-V181, close enough to be cleaved almost simultaneously for FIXa generation. When FIX is activated by FXIa in the absence of calcium ions or by FXIa-LC lacking the heavy chain of FXIa, FIX binds only to the light chain of FXIa to facilitate cleavage of the first scissile bond (R145-A146) producing the inactive intermediate FIXα whereas the second cleavage at R180-V181 is slow resulting in the accumulation of FIXα.