Site‐specific functional roles of the Factor X activation peptide in the intrinsic tenase‐mediated Factor X activation

Abstract

The conversion of zymogen Factor X (FX) to an active protease involves the removal of a 52‐residue long activation peptide (AP). Through site‐directed mutagenesis, we investigate the role of the AP and demonstrate that the high abundance of proline residues is important for efficient proteolysis of FX. Moreover, we identify an essential interaction site for Factor IXa (FIXa) between residues 22 and 30 (AP numbering) and find that the residues between 31 and 41 may provide an important interaction site for the intrinsic tenase complex, composed of Factor IXa (FIXa) and Factor VIIIa (FVIIIa). Finally, we suggest that the carbohydrate chain at Asn‐39 restricts the activator specificity, as elimination of this glycosylation site increases the activation rate for activation by FIXa and FXa.

Comparative sequence analysis of vitamin K‐dependent coagulation factors

“…Before secretion into circulation, the AP linkers of FX and PC are processed intracellularly at a proprotein convertase cleavage site, producing two‐chain zymogens whose light and heavy chains remain connected through a disulfide bond (Figure S1 ). 43 , 44 , 45 , 46 , 47 An AP is then released when the zymogens are activated through cleavage at position 15. 43 , 44 , 45 , 46 , 47 In the case of PC, a proprotein convertase cleavage site with consensus sequence K‐K‐R‐3X‐K‐ R is only found in the AP linker of placental mammals, with bats being an exception because of a cysteine substitution that replaces the scissile arginine (Figure 4A,B ).…”

“…43 , 44 , 45 , 46 , 47 An AP is then released when the zymogens are activated through cleavage at position 15. 43 , 44 , 45 , 46 , 47 In the case of PC, a proprotein convertase cleavage site with consensus sequence K‐K‐R‐3X‐K‐ R is only found in the AP linker of placental mammals, with bats being an exception because of a cysteine substitution that replaces the scissile arginine (Figure 4A,B ). All other vertebrates, including monotremes and marsupials, lack the K‐K‐R‐3X‐K‐ R consensus sequence in the AP linker (Figure 4A,B ).…”

“…All VK-dependent zymogens contain an activation peptide (AP) linker between the EGF2 and protease domains (Figure S1). 8,9,12,[43][44][45] The N-terminal border of the AP linker is defined by the perfectly conserved cysteine that covalently cross-links the EGF2 and serine protease domains, whereas the C-terminal border is delineated by the zymogen activation site (i.e., residue 15). Among vertebrates, the AP linkers of FIX and FX are significantly longer than those of FVII and PC, with average lengths that can be nearly four times greater (Figure 3A).…”

O-glycosylation and its role in therapeutic proteins

“…The N-glycan at N49 on the activation peptide seems to function to prevent the unregulated action of free (extrinsic) FVIIa and FIXa to activate FX but has no effect on the intrinsic co-factor dependent proteolytic reaction. A very recent paper from a group at Novo Nordisk in Denmark [30] reinvestigated some of these findings through a detailed site-directed mutagenesis study. They ignored the N-glycan at N49 and instead looked at the N-glycan at N39, which was not considered in the previous paper.…”