Thromboinflammation as bioactivity assessment of H2O2-alkali modified titanium surfaces.

The release of growth factors from platelets, mediated by the coagulation and the complement system, plays an important role in the bone formation around implants. This study aimed at exploring the thromboinflammatory response of H2O2-alkali soaked commercially pure titanium grade 2 discs exposed to whole human blood, as a way to assess the bioactivity of the discs. Commercially pure titanium grade 2 discs were modified by soaking in H2O2, NaOH and Ca(OH)2. The platelet aggregation, coagulation activation and complement activation was assessed by exposing the discs to fresh whole blood from human donors. The platelet aggregation was examined by a cell counter and the coagulation and complement activation were assessed by ELISA-measurements of the concentration of thrombin-antithrombin complex, C3a and terminal complement complex. The modified surface showed a statistically significant increased platelet aggregation, coagulation activation and complement activation compared to unexposed blood. The surface also showed a statistically significant increase of coagulation activation compared to PVC. The results of this study showed that the H2O2-alkali soaked surfaces induced a thromboinflammatory response that indicates that the surfaces are bioactive.

Analytical comparison of a US generic enoxaparin with the originator product: The focus on comparative assessment of antithrombin-binding components.

Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.

Nucleic acid aptamers for target validation and therapeutic applications.

In the simplest view, aptamers can be thought of as nucleic acid analogs to antibodies. They are able to bind specifically to proteins, and, in many cases, that binding leads to a modulation of protein activity. New aptamers are rapidly generated through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process and have a very high target affinity and specificity (picomoles to nanomoles). Furthermore, aptamers composed of modified nucleotides have a long in vivo half-life (hours to days), are nontoxic and nonimmunogenic, and are easily produced using standard nucleic acid synthesis methods. These properties make aptamers ideal for target validation and as a new class of therapeutics. As a target validation tool, aptamers provide important information that complements that provided by other methods. For example, siRNA is widely used to demonstrate that protein knock-out in a cellular assay can lead to a biological effect. Aptamers extend that information by showing that the dose-dependent modulation of protein activity can be used to derive a therapeutic benefit. That is, aptamers can be used to demonstrate that the protein is a good target for drug development. As a new class of therapeutics, aptamers bridge the gap between small molecules and biologics. Like biologics, biologically active aptamers are rapidly discovered, have no class-specific toxicity, and are adept at disrupting protein-protein interaction. Like small molecules, aptamers can be rationally engineered and optimized, are nonimmunogenic, and are produced by scalable chemical procedures at moderate cost. As such, aptamers are emerging as an important source of new therapeutic molecules.

Quality assessment of heparin coatings by their binding capacities of coagulation and complement enzymes.

In vitro testing of blood contacting materials before clinical application is generally advisable. Four heparin coatings from different manufacturers were tested for adsorbed proteins and soluble activation markers. The surface with the highest antithrombin, thrombin, high-molecular-weight-kininogen (HMWK) and the lowest fibrinogen binding capacity (Carmeda, Medtronic) showed significantly lower levels of granulocytes and platelet activation (beta-TG, PMN-elastase release). No statistically significant differences in soluble markers of the coagulation system could be detected (F1 + 2, TAT). Interestingly, complement activation (TCC) was significantly reduced within the group of the lowest adsorption of the complement factor C3. Our data demonstrate that there is a relation between the binding affinity of proteins (C1-inhibitor, C3-complement) and the consecutive changes in complement activation (TCC). Therefore, measuring adsorbed proteins on artificial surfaces is a suitable, sensitive and very reproducible method for assessing the thrombogenicity of biomaterials.

Antithrombin agents as anticoagulants and antithrombotics. Implications in drug development.

Synthetic and recombinant thrombin inhibitors have undergone several clinical evaluations for thrombotic and cardiovascular indications. While the initial trials were focused in coronary indications, more recently, these agents are also developed for the prophylaxis and therapeutic management of thromboembolic disorders. Hirudin, PEG-hirudin and argatroban are in advanced clinical development. Recombinant hirudin has been approved in Europe as a substitute anticoagulant for the management of HIT patients. Several additional clinical trials are currently carried out to demonstrate the usefulness of these agents in thrombotic and cardiovascular indications. Despite these developments such issues as dosage optimization, laboratory monitoring, neutralization and drug interactions require additional studies for the optimal development of these drugs.

Binding affinities for sulfonamide inhibitors with human thrombin using Monte Carlo simulations with a linear response method.

The binding of sulfonamide inhibitors to human thrombin is examined to evaluate the viability of calculating free energies of binding, deltaGb, utilizing Monte Carlo (MC) statistical mechanics with a linear response approach. Coulombic and van der Waals energy components determined from MC simulations of the bound and unbound inhibitors solvated in water plus a solvent-accessible surface area term, as an index for cavity formation, were correlated with the free energies of binding for the inhibitor MD-805 and six derivatives. The best correlations yield an average error of 0.8 kcal/mol for the seven binding affinities, which cover an observed range of 6.0 kcal/mol. The MC simulations also provided insights into the interactions occurring in the active site and the origins of variations in deltaGb. Equatorial placement of the carboxylate group at C2 in the piperidine ring of the inhibitors causes electrostatic destabilization with the side chain of Glu-H192, while axial disposition of the C4-methyl group reduces favorable hydrophobic interactions in the P-pocket of the enzyme.