Abstract
In the intrinsic pathway of blood coagulation factor XIa (FXIa) activates factor IX (FIX) by cleaving the zymogen at Arg(145)-Ala(146) and Arg(180)-Val(181) bonds releasing an 11-kDa activation peptide. FXIa and its isolated light chain (FXIa-LC) cleave S-2366 at comparable rates, but FXIa-LC is a very poor activator of FIX, possibly because FIX undergoes allosteric modification on binding to an exosite on the heavy chain of FXIa (FXIa-HC) required for optimal cleavage rates of the two scissile bonds of FIX. However preincubation of FIX with a saturating concentration of isolated FXIa-HC did not result in any potentiation in the rate of FIX cleavage by FXIa-LC. Furthermore, if FIX binding via the heavy chain exosite of FXIa determines the affinity of the enzyme-substrate interaction, then the isolated FXIa-HC should inhibit the rate of FIX activation by depleting the substrate. However, whereas FXIa/S557A inhibited FIX activation of by FXIa, FXIa-HC did not. Therefore, we examined FIX binding to FXIa/S557A, FXIa-HC, FXIa-LC, FXIa/C362S/C482S, and FXIa/S557A/C362S/C482S. The heavy and light chains are disulfide-linked in FXIa/S557A but not in FXIa/C362S/C482S and FXIa/S557A/C362S/C482S. In an ELISA assay only FXI/S557A ligated FIX with high affinity. Partial reduction of FXIa/S557A to produce heavy and light chains resulted in decreased FIX binding, and this function was regained upon reformation of the disulfide linkage between the heavy and the light chains. We therefore conclude that substrate recognition by the FXIa exosite(s) requires disulfide-linked heavy and light chains.
Highlights
The heavy chain of factor XIa (FXIa) (FXIa-HC) is essential for efficient factor IX (FIX) activation, the light chain (FXIa-LC) contains the active site
Factor IX Binding to FXIai, Isolated FXIa-HC, FXIa-LCi, and Zymogen FXI—Because FXIa-HC was ineffective in potentiating FIX activation by FXIa-LC or inhibiting the rate of FIXa generation by full-length FXIa, we examined binding of the substrate, FIX to FXIa and to the isolated heavy and light chains of FXIa in an ELISA assay
Comparison of Binding to FIX of FXIa/S557A and FXIa/S557A/C362S/C482S—To rule out the possibility that the isolation procedure may have been responsible for the loss of the capacity of FIX to recognize FXIa-HC and FXIa-LC, we investigated the binding to FIX of FXIa/S557A and FXIa/
Summary
The heavy chain of FXIa (FXIa-HC) is essential for efficient FIX activation, the light chain (FXIa-LC) contains the active site. Each subunit consists of one N-terminal heavy chain joined to the C-terminal light chain (or catalytic domain) by a disulfide bond formed between Cys362 in the heavy chain and Cys482 in the light chain It was originally established in our laboratory [14, 15] and later confirmed by other investigators (16 –18) that binding of FIX to an exosite on the heavy chain of FXIa is essential for optimal activation of FIX. If the affinity of the enzymesubstrate interaction is determined solely by the binding of FIX to the heavy chain of FXIa, FIX activation by FXIa should be inhibited in the presence of excess of FXIa-HC because of substrate depletion These two hypotheses were examined in experiments, the results of which are reported in this manuscript
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