| It has been demonstrated that activated FXI (FXIa) binds to high-affinity (KD ∼1.7 nM) saturable receptors (n =250 sites/platelet) on the activated platelet surface that have been shown to be distinct from the receptors for FXI (n = 1500 sites/platelet; KD ∼10 nM) (Sinha, Seaman et al. 1984; Baglia, Jameson et al. 1995; Baird and Walsh 2002). In order to investigate the differences between FXI and FXIa binding to activated platelets, a number of recombinant proteins were examined for their ability to compete with FXIa for binding to the activated platelet surface. A chimeric FXIa with the A3 domain of prekallikrein (FXIa/PKA3, Ki ∼2.7 nM) competes with 125I-FXIa for binding sites on activated platelets suggesting that the platelet binding site on FXIa is not located in the A3 domain of FXIa. The recombinant heavy chain was not able to inhibit 125I-FXIa binding to the activated platelet surface, whereas the recombinant catalytic domain (Ile370-Val670) did inhibit (Ki ∼3.5 nM) demonstrating that the FXIa platelet binding site is in the catalytic domain of FXIa. A conformationally constrained cyclic peptide (Cys527-Cys542) containing a high-affinity (KD ∼86 nM) heparin-binding site within the catalytic domain of FXIa displaced 125I-FXIa from the surface of activated platelets (Ki ∼5.8 nM), whereas a scrambled peptide of identical composition was without effect suggesting that the binding site in FXIa that interacts with the platelet surface exists not in the A3 domain of FXIa but resides in the catalytic domain near the heparin binding site of FXIa.;The present study also examines the interaction of FXIa with Factor IX (FIX), the natural macromolecular substrate of FXIa, the small synthetic substrate, S-2366, and a physiologically relevant FXIa inhibitor, Protease Nexin 2 (PN2). A number of FXIa catalytic domain (FXIac) residues (Glu98, Tyr143, Ile151, Lys192, Arg 3704, and Tyr5901, chymotrypsin numbering) were mutated in order to investigate these molecular interactions. All mutants bound S-2366 normally (i.e., with comparable Km values) when compared to plasma FXIa; however Glu98Asp,Tyr143Ala, Arg 3704Ala, and Lys192Arg all were >14-fold defective in their ability to cleave S-2366. Tyr143Ala, Glu98 Val and Lys192Gln displayed a decrease in their ability to hydrolyze FIX of ∼10-fold. It has been previously demonstrated that the catalytic domain of FXIa and the Kunitz protease inhibitory domain (PN2KPI) of PN2 contain all of the energy required for the inhibition of FXIa by PN2 (Badellino and Walsh 2000). In this dissertation, we show that the Lys 192Ala mutant had approximately a 3-fold decrease in inhibition constant (Ki) compared to plasma FXIa, while the Lys192Glu and Lys192Arg mutants and the Tyr5901Ala and Tyr5901Val mutants lost their capacity to be inhibited by PN2KPI. We conclude that the most important residues for the catalysis of both S-2366 and FIX are Tyr143, Glu98, and Lys192 and Lys192 and Tyr5901 are important for inhibition by PN2KPI. |
