A cross-reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect

Summary. Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXIP161L. kcat for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is � 3-fold lower for activated FXIP161L (FXIaP161L) than for wild-type FXIa (FXIaWT), consistent with an abnormal protease active site. Inhibition of FXIaP161L by diisopropyl fluorophosphate is 2.4-fold slower than for FXIaWT, suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIaWT and FXIaP161L. Rates of carbamylation of Ile16 were also similar for FXIaWT and FXIaP161L, indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.