Thrombotic disorders afflict a large number of people. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US alone. Even more alarming is that thrombotic disorders are ~3-fold more likely in people with cancer. Anticoagulants, including heparins and coumarins, are the mainstay of treatment and prevention of thromboembolic disorders. Yet, the current anticoagulation therapy is beset with a significant number of adverse reactions including enhanced bleeding risk, immunological reaction, genetic variation in metabolism, food or drug interactions and liver toxicity. In addition, problems such as patient-to-patient response variability, narrow therapeutic index, inadequate duration of action, poor oral bioavailability, the need for frequent coagulation monitoring, and high cost to benefit ratio further complicate the treatment of thrombotic conditions. We reasoned that to reduce the problems associated with the current anticoagulation therapy, molecules radically different from all the current agents (heparins, warfarins, hirudins, and peptidomimetics) should be discovered. We have discovered that chemo-enzymatically synthesized lignins, represented by three sulfated dehydropolymer (DHP) molecules, named CDs, FDs and SDs, possess extremely interesting anticoagulant properties and a novel mechanism of action. 1) Sulfated DHPs (CDs, FDs and SDs) prolong prothrombin time at concentrations 2-6-fold below that of the clinically used LMWH enoxaparin, while in the activated partial thromboplastin time assay they required 2-6-fold higher concentration. 2) Whole blood clotting studies using thromboelastography and hemostasis analysis system reveal that our novel anticoagulants inhibit clotting with potency only 18-30-fold weaker than enoxaparin. 3) Mechanistically, the new molecules inhibit thrombin, factor Xa and factor XIa with IC50 values in the range of 10-240 nM. 4) In contrast, they inhibit factor IXa and factor VIIa with IC50 values 60-170-fold and >840-fold higher, respectively suggesting high selectivity for thrombin and factor Xa. 5) This potent inhibition arises primarily from direct inhibition of thrombin and factor Xa, although indirect inhibition mediated by antithrombin may also contribute. 6) Direct inhibition arises from an allosteric disruption of thrombin's catalytic apparatus (reduction in kCAT). 7) Competitive binding studies suggest that CDs interacts with exosite II of thrombin, a site not typically associated with inhibition. 8) A chemically synthesized CDs-based monomer inhibits thrombin and factor Xa with an IC50 of ~30

Public Health Relevance

Nearly 576000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US alone. Thrombotic disorders are even more prevalent in people with cancer. The proposed research on novel dual direct inhibitors of thrombin and factor Xa aims to improve current anticoagulation therapy, which is beset with significant number of adverse reactions and limitations.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL090586-04
Application #
8267015
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
2009-07-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$370,013
Indirect Cost
$122,513
Name
Virginia Commonwealth University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
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Al-Horani, Rami A; Gailani, David; Desai, Umesh R (2015) Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants. Thromb Res 136:379-87
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Liang, Aiye; Desai, Umesh R (2015) Studying glycosaminoglycan-protein interactions using capillary electrophoresis. Methods Mol Biol 1229:355-75
Sarkar, Aurijit; Desai, Umesh R (2015) A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins. PLoS One 10:e0141127
Wang, Congzhou; Jin, Yingzi; Desai, Umesh R et al. (2015) Investigation of the heparin-thrombin interaction by dynamic force spectroscopy. Biochim Biophys Acta 1850:1099-106
Al-Horani, Rami A; Karuturi, Rajesh; Verespy 3rd, Stephen et al. (2015) Synthesis of glycosaminoglycan mimetics through sulfation of polyphenols. Methods Mol Biol 1229:49-67

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