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 seque...
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Published in | Journal of thrombosis and haemostasis Vol. 18; no. 5; pp. 1171 - 1182 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
England
Wiley Subscription Services, Inc
01.05.2020
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Subjects | |
Online Access | Get full text |
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Summary: | 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 spectrometry analysis, and functional assays.
Results
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.
Conclusion
Plasmin cleavage at Lys316↓Gly317 abrogates FIXaβ coagulant activity, whereas additional cleavage at Lys413↓Leu414 converts it into a fibrinolytic enhancer. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally and are co-first authors Present Address: Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine, Rochester, NY 14642, USA |
ISSN: | 1538-7933 1538-7836 1538-7836 |
DOI: | 10.1111/jth.14773 |