Plasmin-mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer.

Amy E Schmidt, Kanagasabai Vadivel, Julian Whitelegge, Satya Paul Bajaj

Journal of thrombosis and haemostasis : JTH 2020 May

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Factor (F) IX/IXa inactivation by plasmin has been studied; however, whether plasmin converts FIXa to a fibrinolytic enhancer is not known. Investigate plasmin proteolysis site(s) in FIXa that inactivates and transforms it into a fibrinolytic enhancer. NH2 -terminal sequencing, mass spectrometry analysis, and functional assays. Plasmin in the presence of Ca2+ /phospholipid (PL) rapidly cleaved FIXaβ at Lys316↓Gly317 to yield FIXaγ followed by a slow cleavage at Lys413↓Leu414 to yield FIXaδ. FIXaγ/FIXaδ migrated indistinguishably from FIXaβ in nondenaturing gel system indicating that C-terminal residues 317-415/317-413 of heavy chain remain noncovalently associated with FIXaγ/FIXaδ. However, as compared with FIXaβ, FIXaγ or FIXaγ/FIXaδ (25-75 mixture, 8-hour/24-hour incubation analysis by mass spectrometry) was impaired ~ 10-fold in hydrolyzing synthetic substrate CBS 31.39 (CH3-SO2-D-Leu-Gly-Arg-pNA), ~ 30-fold (~ 5-fold higher Km , ~ 6-fold lower kcat ) in activating FX in a system containing Ca2+ /PL, and ~ 650-fold in a system containing Ca2+ /PL and FVIIIa. Further, FIXaγ or FIXaγ/FIXaδ bound FVIIIa with ~ 60-fold reduced affinity compared with FIXaβ. Additionally, in ligand blots, plasminogen or diisopropylfluorophosphate-inhibited plasmin (DIP-plasmin) bound FIXaγ and FIXaδ but not FIXaβ. This interaction was prevented by ε-aminocaproic acid or carboxypeptidase B treatment suggesting that plasminogen/DIP-plasmin binds to FIXaγ/FIXaδ through newly generated C-terminal Lys316 and Lys413. Importantly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tissue plasminogen activator (tPA)-mediated plasminogen activation in a concentration dependent manner. Similarly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tPA-induced clot lysis in FIX-depleted plasma. Plasmin cleavage at Lys316↓Gly317 abrogates FIXaβ coagulant activity, whereas additional cleavage at Lys413↓Leu414 converts it into a fibrinolytic enhancer. © 2020 International Society on Thrombosis and Haemostasis.

Citation

Amy E Schmidt, Kanagasabai Vadivel, Julian Whitelegge, Satya Paul Bajaj. Plasmin-mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer. Journal of thrombosis and haemostasis : JTH. 2020 May;18(5):1171-1182