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

BACKGROUND: Factor (F) IX/IXa inactivation by plasmin has been studied; however, whether plasmin converts FIXa to a fibrinolytic enhancer is not known. OBJECTIVE: Investigate plasmin proteolysis site(s) in FIXa that inactivates and transforms it into a fibrinolytic enhancer. METHODS: NH2 -terminal sequencing, mass spec analysis and functional assays. RESULTS: Plasmin in the presence of Ca(2+) /phospholipid (PL) rapidly cleaved FIXabeta at Lys316 downward arrowGly317 to yield FIXagamma followed by a slow cleavage at Lys413 downward arrowLeu414 to yield FIXadelta. FIXagamma/FIXadelta migrated indistinguishably from FIXabeta in nondenaturing gel system indicating that C-terminal residues 317-415/317-413 of heavy chain remain noncovalently associated with FIXagamma/FIXadelta. However, as compared to FIXabeta, FIXagamma or FIXagamma/FIXadelta (25-75 mixture, 8-hour/24-hour incubation analysis by Mass Spec) 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 Ca(2+) /PL, and ~650-fold in a system containing Ca(2+) /PL and FVIIIa. Further, FIXagamma or FIXagamma/FIXadelta bound FVIIIa with ~60-fold reduced affinity as compared to FIXabeta. Additionally, in ligand blots, plasminogen or Diisopropylfluorophosphate-inhibited plasmin (DIP-plasmin) bound FIXagamma and FIXadelta but not FIXabeta. This interaction was prevented by epsilon-aminocaproic acid or carboxypeptidase B treatment suggesting that plasminogen/DIP-plasmin binds to FIXagamma/FIXadelta through newly generated C-terminal Lys316 and Lys413. Importantly, FIXagamma/FIXadelta mixture but not FIXagamma enhanced tissue plasminogen activator (tPA)-mediated plasminogen activation in a concentration dependent manner. Similarly, FIXagamma/FIXadelta mixture but not FIXagamma enhanced tPA-induced clot lysis in FIX-depleted plasma. CONCLUSION: Plasmin cleavage at Lys316 downward arrowGly317 abrogates FIXabeta coagulant activity, whereas additional cleavage at Lys413 downward arrowLeu414 converts it into a fibrinolytic enhancer.