在钙/磷脂存在的情况下纤溶酶介导的人凝血因子IXa的蛋白水解：促凝血因子IXa转化为纤溶增强剂。
Plasmin-mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer.
作者信息
Schmidt Amy E, Vadivel Kanagasabai, Whitelegge Julian, Bajaj Satya Paul
机构信息
Department of Orthopaedic Surgery, David Geffen School of Medicine, University of California, Los Angeles, California.
The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, California.
出版信息
J Thromb Haemost. 2020 May;18(5):1171-1182. doi: 10.1111/jth.14773. Epub 2020 Mar 30.
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
NH -terminal sequencing, mass spectrometry analysis, and functional assays.
RESULTS
Plasmin in the presence of Ca /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 K , ~ 6-fold lower k ) in activating FX in a system containing Ca /PL, and ~ 650-fold in a system containing Ca /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.
背景
纤溶酶对因子（F）IX/IXa的灭活作用已得到研究；然而，纤溶酶是否将FIXa转化为纤溶增强剂尚不清楚。
目的
研究纤溶酶在FIXa中的蛋白水解位点，该位点可使其失活并转化为纤溶增强剂。
方法
N端测序、质谱分析和功能测定。
结果
在Ca²⁺/磷脂（PL）存在的情况下，纤溶酶迅速在Lys316↓Gly317处切割FIXaβ，产生FIXaγ，随后在Lys413↓Leu414处缓慢切割，产生FIXaδ。在非变性凝胶系统中，FIXaγ/FIXaδ与FIXaβ的迁移无法区分，表明重链的C端残基317 - 415/317 - 413与FIXaγ/FIXaδ保持非共价结合。然而，与FIXaβ相比，FIXaγ或FIXaγ/FIXaδ（25 - 75混合物，质谱分析8小时/24小时孵育分析）在水解合成底物CBS 31.39（CH3 - SO2 - D - Leu - Gly - Arg - pNA）方面受损约10倍，在含有Ca²⁺/PL的系统中激活FX方面受损约30倍（Kₘ约高5倍，kₐₜ约低6倍），在含有Ca²⁺/PL和FVIIIa的系统中受损约650倍。此外，与FIXaβ相比，FIXaγ或FIXaγ/FIXaδ与FVIIIa的结合亲和力降低了约60倍。另外，在配体印迹中，纤溶酶原或二异丙基氟磷酸抑制的纤溶酶（DIP - 纤溶酶）与FIXaγ和FIXaδ结合，但不与FIXaβ结合。ε - 氨基己酸或羧肽酶B处理可阻止这种相互作用，表明纤溶酶原/DIP - 纤溶酶通过新生成的C端Lys316和Lys413与FIXaγ/FIXaδ结合。重要的是，FIXaγ/FIXaδ混合物而非FIXaγ以浓度依赖的方式增强组织纤溶酶原激活剂（tPA）介导的纤溶酶原激活。同样，FIXaγ/FIXaδ混合物而非FIXaγ增强了tPA诱导的FIX缺乏血浆中的凝块溶解。
结论
在Lys316↓Gly317处的纤溶酶切割消除了FIXaβ的凝血活性，而在Lys413↓Leu414处的额外切割将其转化为纤溶增强剂。
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