Activation of human factor IX (Christmas factor).

Abstract

A B S T R A C T Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXao, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt-11,000) from the internal region of the precursor molecule and the generation of Factor IXaO (mol wt -46,000). Factor IXa, is composed ofa light chain (mol wt -18,000) and a heavy chain (mol wt -28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXa, also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly -Gly -Arg -Asp -SerCys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXa,,: by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IX,,3 was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.

Posttranslational modifications of recombinant myotube-synthesized human factor IX

“…28,50 Sixty percent of the carbohydrate in factor IX is at N-linked sites located within the activation peptide. 55 We analyzed the glycosylation of the myotube-synthesized factor IX using enzymatic cleavage of N-linked glycans and oligosaccharide profiling. Band patterns after enzymatic removal of N-linked oligosaccharides or of sialic acid residues suggest a similar amount of carbohydrate in myotubesynthesized F.IX and plasma-derived F.IX, though the patterns are not identical.…”

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

“…FIX is a 56-kDa polypeptide comprised of C-terminal trypsinlike catalytic (heavy chain) domain and an N-terminal noncatalytic (light chain) region separated by an 11-kDa activation peptide (1,11). Two proteolytic cleavages are required to liberate the activation peptide from the remainder of the molecule to produce FIXa␤ (11,12).…”

“…Two proteolytic cleavages are required to liberate the activation peptide from the remainder of the molecule to produce FIXa␤ (11,12). Activation may occur by two distinct mechanisms mediated by the plasma serine proteases factor VIIa (EC 3.4.21.21) and factor XIa (EC 3.4.21.27) (2,3,13,14).…”

“…The importance of the FIX Gla domain to FIX activation by factor XIa is less certain because the reaction appears to involve a mechanism distinctly different from typical vitamin K-dependent protease-substrate interactions. Although calcium is required (1,21,22), phospholipid has little influence on the process (1,11,23). Indeed, factor XIa lacks a Gla domain, suggesting that it may interact poorly with phospholipid (3,24,25).…”

The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…The Ca 2ϩ -dependent activation of FIX by FXIa (8,9) requires the exposure of a substrate-binding site within the Apple 2 and/or Apple 3 domain of FXIa and the ␥-carboxyglutamic acid domain of FIX, as well as an extended, macromolecular substrate-binding exosite in the protease domain of FXIa (10 -14). The activation of FIX to FIXa␤ involves two cleavages by FXIa, one after Arg 145 and another after Arg 180 , which releases an 11-kDa activation peptide (8,9,15). FIXa␤ is also produced by the tissue factor-factor VIIa complex (16).…”