Productive recognition of factor IX by factor XIa exosites requires disulfide linkage between heavy and light chains of factor XIa.
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.
Sol Sherry Thrombosis Research Center, University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Mariola M
MM
Marcinkiewicz
Dipali
D
Sinha
Peter N
PN
Walsh
D001667:Binding, Competitive / Q000502:physiology; D001777:Blood Coagulation / Q000502:physiology*; D020134:Catalytic Domain / Q000502:physiology; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation / Q000502:physiology; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015945:Factor XIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
D004220:Disulfides; D005164:Factor IX; D010447:Peptide Hydrolases; D015945:Factor XIa