Independence of Factor XIa Subunits in Factor IX Activation.
Authors: Smith Stephen B. , Sun Mao-fu , Bock Paul E. , Gailani David 
Published: October 2019

Abstract
Coagulation factor XI (FXI) is the zymogen of FXIa, a plasma protease composed of two identical polypeptide subunits linked by a disulfide bond. Each FXIa subunit contains a catalytic domain and a binding domain. The dimeric structure of FXIa is unique among coagulation proteases, but its importance to FXIa function is not known. During hemostasis, FXIa cleaves factor IX (FIX) to generate the protease FIXaβ. FIX is also activated by the factor VIIa/tissue factor (FVIIa/TF) complex. Both FXIa and FVIIa/TF activate FIX by proteolytic cleavage at Arg145-Ala146 and Arg180-Val181 to generate FIXaβ, and release an 11 kDa activation peptide. FVIIa/TF preferentially cleaves FIX at Arg145-Ala146, generating significant amounts of the catalytically inactive intermediate FIXα, followed by cleavage at Arg180-Val181 to generate FIXaβ. In contrast, only trace amounts of FIXα are generated when FIX is activated by FXIa. It has been postulated that the dimeric structure of FXIa accounts for the difference in mechanism compared to FVIIa/TF. The two catalytic domains of a single FXIa dimer could cleave the two activation cleavage sites of one FIX molecule without releasing an intermediate. The present study was designed to determine the importance of the dimeric structure of FXIa to interactions with, and activation of, FIX. We studied the cleavage of FIX (100 nM) by FXIa or FVIIa/TF (1 nM of protease active sites) using western immunoblotting. FIXα accumulates over time during activation by FVIIa/TF, but is barely detectable in reactions with FXIa. We constructed a monomeric variant of FXIa by replacing the dimerization (A4) domain with the homologous domain from the related, but monomeric protease plasma prekallikrein (PK). The resulting chimeric protease, FXIa-PKA4, activates FIX with a modest accumulation of FIXα, but the cleavage pattern is more similar to that seen with FXIa than FVIIa/TF. When FXI is activated to FXIa by factor XIIa, the process proceeds through an intermediate in which only one subunit has been activated (FXIa1/2). FXIa1/2 can be separated from FXI by chromatography on benzamidine-Sepharose, and has slightly greater than 50% of the activity of an equimolar concentration of FXIa towards a chromogenic substrate (S2366). At equal active site concentrations (1 nM), there was no apparent difference in cleavage pattern of FIX on western blots by FXIa1/2 compared to FXIa, with little generation of FIXα for either protease. To determine the affinity and stoichiometry of the FXIa-FIX interaction, we inhibited the active sites of FXIa with a fluorescent inhibitor, dansyl-EGR-chloromethyl-ketone, and measured changes in fluorescence on FIX binding, representing FXIa exosite-mediated interactions involved in substrate recognition. The results demonstrate that FIX binds to FXIa with KD of 62 ± 20 nM and a stoichiometry of 1.73 ± 0.14 FIX molecules per FXIa dimer, strongly suggesting that each FXIa subunit binds one FIX molecule. In the recently elucidated crystal structure of zymogen FXI, the catalytic domains are on opposite sides of the molecule and the active site clefts point away from each other. It seems unlikely that a single FIX molecule could be engaged by the two protease domains without a substantial change in conformation upon activation to FXIa. Cumulatively, the data indicate that each subunit of the FXIa homodimer binds to and activates one molecule of FIX.