A mechanism for exosite-mediated factor IX activation by factor XIa

Abstract

Factor XI (FXI) is the zymogen of a protease (FXIa) that contributes to blood coagulation by activating factor IX (FIX). This thesis presents several novel observations regarding the structure and function of FXI that will enhance our understanding of this molecule. First, a new mechanism is described for FIX activation by FXIa based on a rigorous kinetic analysis. FXIa cleaves FIX initially after Arg145 to form FIXá, which is released, and then quickly rebinds to FXIa, allowing cleavage after Arg180 to form the active protease FIXaâ. Catalytic efficiency for cleavage after Arg180 is significantly greater than after Arg145, limiting FIXá accumulation. Using site-directed mutagenesis and protein modeling, the structural basis for the interaction of FIX and FXIa was identified. The ù-loop of FIX appears to bind to an area on FXIa comprised of residues from the N- and C-termini of the A3 domain in the heavy chain. These residues are buried in zymogen FXI, and are exposed upon activation to permit FIX binding. FXI is a homodimeric zymogen that is converted to a protease with one (1/2-FXIa) or two (FXIa) active subunits by factor XIIa (FXIIa) or thrombin. The in vitro and in vivo studies presented in this thesis indicate that FXIIa and thrombin activate FXI by different mechanisms. The dimeric structure of FXI is essential for normal activation by FXIIa, but is not required when thrombin is the activating protease. Activation of FXI is accelerated by polyanions such as polyphosphate released from platelet dense granule. Indeed, FXI can undergo autoactivation in the presence of polyphosphate. We determined that polyphosphate enhances FXI activation through a template-type mechanism requiring FXI and an activating protease to binding in proximity to each other on the polyphosphate. Two anion binding sites on the A3 and catalytic domains of FXI are required for optimal enhancement. Work with mice indicates that the anion binding sites contribute to FXI-dependent thrombotic processes in vivo. The work in this thesis provides a foundation for future studies to understand the role of FXI in normal and pathologic coagulation, and for developing therapeutic agents to treat or prevent thrombotic disorders.