Kar Srabani, Bankston Page, Afosah Daniel K, Al-Horani Rami A
Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA 70125, USA.
Department of Chemistry and Biochemistry, Washington and Lee University, Lexington, VA 24450, USA.
Pharmaceuticals (Basel). 2021 Aug 31;14(9):886. doi: 10.3390/ph14090886.
The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC value of ~8 μg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis-Menten kinetics, LSAS decreased the V of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its K indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa-antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure-activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.
本研究对木质素磺酸钠（LSAS）这一非糖类肝素模拟物的抗凝活性进行了研究。LSAS是一种相对安全的工业副产品，具有与肝素相似的聚阴离子特性。采用人体血浆凝血试验、纤维蛋白聚合试验和酶抑制试验来研究LSAS的抗凝活性。在正常人血浆中，LSAS在约308μg/mL时可使活化部分凝血活酶时间（APTT）选择性加倍。同样，在抗凝血酶缺乏的血浆中，LSAS在约275μg/mL时使APTT加倍。然而，使用缺乏因子XI的血浆时，LSAS在429μg/mL的较高浓度下使APTT加倍。LSAS在1000μg/mL时不影响FXIIIa介导的纤维蛋白聚合。酶试验表明，LSAS抑制因子XIa（FXIa），IC值约为8μg/mL。在测试的最高浓度下，LSAS不抑制凝血酶、因子IXa、因子Xa、因子XIIIa、胰凝乳蛋白酶或胰蛋白酶，并且对因子XIIa和纤溶酶具有显著的选择性。在米氏动力学中，LSAS降低了FXIa对三肽发色底物的水解反应速度（V），而未显著改变其米氏常数（K），表明其为变构抑制机制。该抑制剂还破坏了FXIa-抗凝血酶复合物的生成，抑制了因子XIIa介导的和凝血酶介导的酶原因子XI向FXIa的激活，并与肝素竞争与FXIa的结合。硫酸鱼精蛋白似乎可逆转其作用。构效关系研究表明，与LSAS的乙酰化和去磺化衍生物相比，LSAS具有有利的选择性和变构行为。LSAS是一种磺化肝素模拟物，在人体血浆中表现出显著的抗凝活性。总体而言，LSAS似乎是一种有效的、选择性的FXIa变构抑制剂，在人体血浆中具有显著的抗凝活性。总之，本研究将LSAS作为一种有前景的抗凝剂进一步开发的先导物进行了介绍。
