Targeting the GPIbα binding site of thrombin to simultaneously induce dual anticoagulant and antiplatelet effects.

Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis-Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin.

Novel low molecular weight lignins as potential anti-emphysema agents: In vitro triple inhibitory activity against elastase, oxidation and inflammation.

No molecule has been found to be effective against emphysema to date primarily because of its complex pathogenesis that involves elastolysis, oxidation and inflammation. We here describe novel unsulfated or sulfated low molecular weight lignins (LMWLs) chemo-enzymatically prepared from 4-hydroxycinnamic acids monomers, as the first potent triple-action inhibitors of neutrophil elastase, oxidation and inflammation. The inhibitory potencies of three different cinnamic acid-based LMWLs were determined in vitro using chromogenic substrate hydrolysis assays, radical scavenging and lung cellular oxidative biomarker reduced glutathione (rGSH) assays, and lung cellular inflammatory biomarker NFκB and IL-8 assays, respectively. Each LWML uniquely displayed triple-action inhibition, among which CDSO3, a sulfated caffeic acid-based LMWL, was most potent. The half-maximal anti-human neutrophil elastase (HNE) potency of CDSO3 was 0.43 µM. This high potency arose from lignin-like oligomerization, which was further potentiated by 6.6-fold due to sulfation. Mechanistically, this elastase inhibition was of mixed-type, time-dependent and more selective to positively charged elastases. The half-maximal anti-oxidative potency of CDSO3 was 3.52 µM, 4.8-fold potentiated from that of the monomer, caffeic acid (CA). In contrast, the half-maximal inhibitory potency to TNFα-induced inflammation was 5-10 µM, despite no activity with the monomer. More intriguingly, this anti-inflammatory activity was essentially identical with different stimuli, okadaic acid and hydrogen peroxide (H(2)O(2)), which implied that CDSO3 acts directly on inflammatory cascades within the cells. Overall, oligomerization and sulfation produced or significantly potentiated the activity, in comparison to the monomer. Thus, sulfated and unsulfated LMWLs are novel non-peptidic 2.8-4.1 kDa macromolecules that exhibit for the first time potent triple inhibitory activity against elastase, oxidation and inflammation, the three major pathogenic mechanisms known to cause emphysema.

Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin.

Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I(35), I(55) and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I(35) possessed good anticoagulation potential (APTT=4.2µM; PT=6.8µM), while I(55) and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4m/z, which most probably arise from variably functionalized ß-O4ß-ß-linked trimers and/or a ß-O4ß-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants.

Sulfated, low molecular weight lignins inhibit a select group of heparin-binding serine proteases.

Sulfated low molecular weight lignins (LMWLs), designed as oligomeric mimetics of low molecular weight heparins (LMWHs), have been found to bind in exosite II of thrombin. To assess whether sulfated LMWLs recognize other heparin-binding proteins, we studied their effect on serine proteases of the coagulation, inflammatory and digestive systems. Using chromogenic substrate hydrolysis assay, sulfated LMWLs were found to potently inhibit coagulation factor XIa and human leukocyte elastase, moderately inhibit cathepsin G and not inhibit coagulation factors VIIa, IXa, and XIIa, plasma kallikrein, activated protein C, trypsin, and chymotrypsin. Competition studies show that UFH competes with sulfated LMWLs for binding to factors Xa and XIa. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold.

Ascorbic acid attenuates lipopolysaccharide-induced acute lung injury.

OBJECTIVE: Sepsis-induced lung injury is a persisting clinical problem with no direct therapy. Recent work suggests that intravenously infused ascorbic acid improves the circulatory dysfunction of sepsis. We used a model of endotoxin-induced acute lung injury to determine whether parenteral ascorbic acid modulates the dysregulated proinflammatory, procoagulant state that leads to lung injury. DESIGN: C57BL/6 mice were exposed to lethal lipopolysaccharide doses (10 µg/g of body weight) to induce acute lung injury. SETTING: Laboratory investigation. SUBJECTS: Wild-type C57BL/6 mice. INTERVENTIONS: Ascorbic acid or its oxidized form (dehydroascorbic acid) was administered intraperitoneally at 200 mg/kg 30 mins after the lethal lipopolysaccharide dose. MEASUREMENTS AND MAIN RESULTS: We quantified survival, lung capillary leak, proinflammatory chemokine expression, and lung microvascular thrombosis. Lipopolysaccharide induced 100% lethality in mice within 28 hrs of exposure and in lung we observed intense neutrophil sequestration, loss of capillary barrier function, exuberant pulmonary inflammation, and extensive microthrombus formation. A time-delayed infusion protocol of both ascorbic acid and dehydroascorbic acid significantly prolonged survival. Both ascorbic acid and dehydroascorbic acid preserved lung architecture and barrier function while attenuating proinflammatory chemokine expression and microvascular thrombosis. Ascorbic acid and dehydroascorbic acid attenuated nuclear factor kappa B activation and normalized coagulation parameters. CONCLUSIONS: Ascorbic acid administered in an interventional manner following lipopolysaccharide infusion attenuates proinflammatory, procoagulant states that induce lung vascular injury in an animal model of sepsis.

Nonsulfated, cinnamic acid-based lignins are potent antagonists of HSV-1 entry into cells.

In an effort to discover macromolecular mimetics of heparan sulfate (HS), we previously designed sulfated lignins (Raghuraman et al. Biomacromolecules 2007, 8, 1759-1763). To probe the relevance of sulfate groups of HS in viral entry, lignins completely devoid of sulfate moieties, and yet possessing an electrostatic surface equivalent to that of HS, were designed. Two carboxylated lignins based on a 4-hydroxy cinnamic acid scaffold were synthesized using enzymatic oxidative coupling in high yields, fractionated according to their sizes, and tested in cellular assays of herpes simplex virus-1 (HSV-1) infection. The two carboxylated lignins were found to not only inhibit HSV-1 entry into mammalian cells (IC(50) = 8-56 nM), but were more potent than sulfated lignins. In addition, shorter carboxylated lignins were found to be as active as the longer chains, suggesting that structural features, in addition to carboxylate groups, may be important. It can be expected that carboxylated lignins also antagonize the entry of other enveloped viruses, for example, HIV-1, Kaposi's sarcoma-associated herpes virus, and hepatitis C virus, that utilize HS to gain entry into cells. The results present major opportunities for developing lignin-based antiviral formulations for topical use.

Study of physico-chemical properties of novel highly sulfated, aromatic, mimetics of heparin and heparan sulfate.

Heparin (H) and heparan sulfate (HS) play major roles in a number of biological processes. Yet, H/HS-based pharmaceutical agents are also associated with multiple adverse effects. This has led to the concept of designing noncarbohydrate, aromatic mimetics that modulate H/HS function. In this work, we study a library of synthetic, aromatic H/HS mimetics for their capillary electrophoretic profiles, the acid and base stability, and aqueous-organic partitioning property. The nonsugar H/HS mimetics exhibit electrophoretic properties similar to sulfated oligosaccharides suggesting that the mimetics can be rapidly and quantitatively analyzed. Stability studies show that the mimetics are essentially stable under neutral and basic conditions in a manner similar to the heparins, but are considerably unstable under acidic conditions in contrast to heparins. The measurement of partition coefficients show major differences within the sulfated mimetics as well as between the measured and calculated log P values. Understanding these physico-chemical properties is expected to have significant implications in the pharmaceutical development of this growing class of molecules.

Characterization of the plasma and blood anticoagulant potential of structurally and mechanistically novel oligomers of 4-hydroxycinnamic acids.

Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties. Structurally and mechanistically, sulfated DHPs are radically different from all the anticoagulants studied to date. To assess whether their unique mechanism and structure is worth exploiting for further rational design of homogeneous DHP-based molecules, we investigated their anticoagulant potential in human plasma and blood using a range of clotting assays. Sulfated DHPs prolong plasma clotting times, prothrombin and activated partial thromboplastin times at concentrations comparable to the clinically used low-molecular-weight heparin, enoxaparin. Fibrin formation studies on human plasma show that there is a structural dependence of anticoagulant action. Human whole blood studies using thromboelastography and hemostasis analysis system indicate that they are 17-140-fold less potent than enoxaparin. These results demonstrate that sulfated DHPs possess good in-vitro and ex-vivo activity, which will likely be improved through a rational design.

Structural characterization of a serendipitously discovered bioactive macromolecule, lignin sulfate.

The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed beta-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.