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Thrombotic disorders primarily arise due to dysfunction of the highly regulated coagulation cascade and may also be precipitated by cancer, pregnancy, and infection. Deep vein thrombosis and pulmonary embolism, two major thrombotic disorders, are reported in ~3 million patients in the US annually. Heparin, a polyanionic polysaccharide introduced some eight decades ago, and low-molecular-weight heparins (LMWHs), introduced in the mid-1990s, have become major anticoagulant drugs for use in the thrombotic disorders with an estimated combined annual sale of more than $ 3 billion. Despite its success, heparin therapy suffers from a number of side effects, primarily bleeding complications and patient-to-patient response variability. In addition, heparin cannot nullify clot-bound thrombin and factor Xa. Consensus is growing that LMWHs also suffer from similar problems, although the frequency of incidences may be lower. A sequence-specific heparin pentasaccharide, fondaparinux, was introduced in 2001 and initial studies suggest that it has much reduced patient response variability and lowered number of bleeding episodes.
Heparin (and LMWH) therapy is primarily based on its ability to accelerate the inhibition of three critical coagulation enzymes (thrombin, factor Xa and factor IXa) by antithrombin, a plasma serine proteinase inhibitor (serpin). At the molecular level, two distinct, but simultaneous, mechanisms are involved. In the first mechanism, heparin pentasaccharide binding induces an allosteric rearrangement in antithrombin (‘conformational activation’) to greatly enhance its ability to recognize and inhibit factor Xa, while in the second, the polysaccharide chain of heparin provides a template to bridge antithrombin and its target enzyme (thrombin and factor IXa). Each of these interactions, heparin-inhibitor and heparin-enzyme, depends on heparin’s polyanionic nature. Yet, it is the highly anionic character of heparin (and LMWH) that introduces non-specific binding to plasma proteins, platelets, and endothelial cells resulting in the majority of adverse effects. With the smaller, and more specific, heparin pentasaccharide these non-specific interactions are much reduced. However, its synthesis is challenging, laborious and not cost-effective. Thus, new anticoagulants with synthetic advantage that reduce these adverse effects and possibly add advantages, such as oral activity and/or inhibition of clot-bound enzymes, are highly desirable.
Thrombotic disorders afflict a large number of people. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the common thrombotic conditions, are diagnosed every year in the US. Thrombotic disorders are also 3-fold more likely in people with cancer. Anticoagulants are the mainstay of treatment and prevention of thromboembolic disorders. The most widely used anticoagulants include the heparins (unfractionated heparin and low molecular weight heparin) and the coumarins (warfarin). Newer agents introduced, or likely to be introduced, include the hirudins (lepirudin, desirudin and bivalirudin), the pentasaccharides (fondaparinux and idraparinux), and the peptidomimetic small molecules (argatroban, dabigatran, ximelagatran, razaxaban, and others). Yet, current therapy is beset with a 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.
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, CDs, FDs and SDs 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) Chemically synthesized CDs-based monomer and dimer, b-5MSC and b-5DSC, respectively, potently inhibit thrombin / factor Xa. 9) Studies using A549 lung and HepG2 liver cell lines show no induction of toxicity by CDs, FDs, and b-5MSC at concentrations as high as 50 mg/L. In combination, these results demonstrate a phenomenon of potent anticoagulant activity by DHPs and their synthetic derivatives. The novel molecular mechanism of action and the radically different structural scaffold of these anticoagulants suggest a strong possibility for discovering novel anticoagulants.
Heparan sulfate (HS) is widely expressed proteoglycan in animal and human tissues and has diverse roles in development, differentiation and homeostasis. HS is a linear polysaccharide composed of alternating glucosamine and uronic acid residues. The glucosamines is N-acetylated while the uronic acid can be either glucuronic acid or iduronic acid. The polymeric chain is modified heterogeneously through de-acetylation and O- or N-sulfation. Introduction of sulfate groups introduces significant protein binding capacity in the polysaccharide. Whereas the overall sulfate density of HS is much less than that of related glycosaminoglycan heparin, there might be regions in the HS polymer with high sulfate densities. These sulfate groups, in combination with specific structure and sequence of saccharide residues, provide specific binding site for a variety of proteins, including cell adhesion molecules, growth factors, chemokines, and factors regulating angiogenesis.
A number of viruses use sites on HS as receptors for binding to cells. Viral entry may require interactions with other cell surface receptors as well. Herpes simplex virus types 1 and 2 (HSV-1, HSV-2), which are human herpes viruses, bind to cells through interactions of envelope glycoproteins gB and/or gC with cell surface HS. Following this binding, a third viral glycoprotein, gD, interacts with one of the multiple specific receptors facilitating viral entry through fusion of the viral envelop and cell membrane.
It is presumed that different HS sequences are required to interact with glycoproteins gB, gC and gD. Mimics of these sequences are likely to interact with the virus in solution thereby tying it up and disrupting the virus's interaction with cell surface HS. This competitive inhibition is hypothesized to prevent entry of the virus in cells. We design non-sugar sulfated molecules to mimic certain domains of heparan sulfate. These HS mimics function as inhibitors of HSV infection.
Low molecular weight heparins (LMWHs) are obtained through chemical or enzymatic depolymerization of natural glycosaminoglycan heparin. Chemical or enzymatic treatment reduces the average molecular weight from ~15,000 to about 5,000. A three-fold reduction in MR enhances the bioavailability of the species and reduces patient-to-patient response variability. However, chemical and enzymatic methods introduced non-native structures in the LMWH preparations. In addition, these methods may destroy active sequences in parent heparin.
The US market of LMWHs reached ~$2 billion in the year 2000. Numerous different manufacturers have several different products in the clinic. Many more are expected to arrive in the near future. Coupled to these LMWHs from other countries are expected to become available. No technique(s) exist for analysis of these LMWH preparations. Rapid analytical techniques are needed to assess the biochemical, biophysical and structural profiles of the preparation.
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©2003 VCU School of Pharmacy
Revised: March 10, 2003
Questions or Comments : Dr. Umesh
R. Desai