Abstract
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 IC50 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 VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM 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.
Highlights
Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, Department of Chemistry and Biochemistry, Washington and Lee University, Lexington, VA 24450, USA; Abstract: The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study
A higher concentration of LSAS was needed to double the activated partial thromboplastin time (APTT) of human plasma deficient of factor XI (FXI) which was about 428.9 ± 45.6 μg/mL, an increase of about 1.5-fold
Under our conditions, unfractionated heparins (UFH) were found to affect both APTT as well as prothrombin time (PT) at concentrations of 0.68 μg/mL and 2.53 μg/mL, respectively, while 10.1 μg/mL was needed from UFH to double APTT in human plasma deficient of antithrombin confirming that antithrombin is the clinical target of UFH
Summary
Fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. The coagulation process is triggered by the intrinsic pathway or the extrinsic pathway (Figure 1A) and is highly regulated by serpins, serine protease inhibitors Examples of these endogenous serpins are antithrombin (AT; inhibitor of FXa and thrombin), tissue factor pathway inhibitor (TFPI; inhibitor of FXa), and activated protein C (APC; inhibitor of factors V and VIIIa), among others. Thrombotic diseases include venous thrombosis such as pulmonary embolism and deep vein thrombosis, or arterial thrombosis such as ischemic heart attacks and stroke [1] Their treatment/prevention entails the use of anticoagulants which includes heparins. Porcine heparinsare arethe theonly onlyanticoagulants anticoagulants approved in the for open surgeries and heparins approved in the US US for open heartheart surgeries and kid‐
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