Antithrombin agents: the new class of anticoagulant and antithrombotic drugs.

Antithrombin drugs represent a wide group of natural agents, recombinant agents equivalent to some of the naturally occurring proteins, and synthetic agents. This group of drugs is characterized by marked structural and functional heterogeneity. Several of these drugs are currently in various phases of development. Argatroban represents the first clinically approved antithrombin agent, which was made available in Japan several years ago. Two recombinant hirudin preparations, Revasc (Novartis) and Refludan (Aventis), are available for postsurgical DVT prophylaxis and alternate anticoagulant use in patients with heparin-induced thrombocytopenia. A synthetic antithrombin agent based on the combined structures of hirudin and antithrombin peptides, hirulog (Bivalirudin), is undergoing clinical trials in cardiovascular indications. Additional studies on the hirudins are being carried out to test their efficacy as surgical and interventional anticoagulants as replacements for heparin. However, the need for a proper antagonist is one of the limiting factors for the optimal development of hirudin in this indication. Several of the synthetic thrombin inhibitors are also being developed for oral use for the prophylaxis of DVT in surgical patients. Since the therapeutic index of thrombin inhibitors is narrower than that of heparin, this route may not be an optimal approach for the development of these agents. Despite several unresolved developmental issues, the thrombin inhibitors provide a useful alternative to heparin anticoagulation and may prove to be useful in validated clinical use.

In vitro effects of a novel hemoglobin-based oxygen carrier on routine chemistry, therapeutic drug, coagulation, hematology, and blood bank assays.

Red blood cell (RBC) replacement solutions are being developed as alternatives to allogeneic RBC use in blood transfusions in the treatment of massive trauma, to achieve hemodynamic stability during elective surgery, and to increase oxygen-carrying capacity in anemia. Hemoglobin-based oxygen carrier (HBOC)-201 (Biopure Corp.) is a purified, sterile, isosmotic glutaraldehyde-polymerized bovine hemoglobin. Because this product is acellular, blood components containing this substance appear hemolyzed. This study reports on the interferences produced by the presence of HBOC-201 in a variety of clinical assays. This product was added in vitro at concentrations up to 60 g/L (6.0 g/dL) to normal human serum, plasma, or whole blood before testing for serum chemistries, coagulation profiles, and hematology and blood bank assays. In addition, a set of normal human sera containing HBOC-201 was supplemented with various therapeutic drugs and assayed for these agents. The results of these studies demonstrate that the presence of HBOC-201 in blood components does not result in significant analytical interference that would be of concern with many clinical assays at HBOC-201 concentrations encountered during routine clinical use of this RBC replacement solution in patients.

Tissue factor antigen levels in various biological fluids.

Tissue factor (TF), a transmembrane surface protein, is known to initiate thrombogenesis through plasmatic and cellular activation processes. Besides complexing with factor VII, eventually leading to fibrin generation via the extrinsic pathway, TF can also activate factor IX, resulting in the intrinsic activation of coagulation. Other functions of TF are currently unknown, although various cells are believed to have TF receptors. Many of the post-surgical and post-interventional thrombotic events are due to the release of TF. Increased levels of TF are associated with several pathologic conditions such as cancer, sepsis and inflammation. Cellular necrosis also results in an increase of TF as the cells in the traumatized area lyse and release endogenous cell surface-bound TF. An ELISA method (American Diagnostica, Greenwich, CT) has been developed to assay TF antigen levels in various biological fluids. This ELISA employs a murine monoclonal antibody raised against native human TF for antigen capture. In this study, cerebrospinal fluid, peritoneal fluid, pleural effusion and urine from patients were assayed for their TF content using this ELISA method. Normal individual serum and plasma were also assayed as controls against which the levels of TF in the patients' body fluids could be compared. The amount of TF antigen in normal human plasma and serum was 165 +/- 139 pg/ml and 165 +/- 110 pg/ml, respectively. Concentrations of TF antigen in other fluids were: cerebrospinal fluid 868 +/- 721 pg/ml, peritoneal fluid 124 +/- 247 pg/ml, pleural effusion 385 +/- 569 pg/ml, synovial fluid 97 +/- 23 pg/ml, seminal plasma 11,485 +/- 875 pg/ml and urine 86 +/- 57 pg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the thrombogenic effects of recombinant tissue factor. In vivo versus ex vivo findings.

An exposure of blood to tissue factor (TF) activates the coagulation system by the extrinsic pathway and may cause clot formation. Recombinant TF (r-TF) has been produced and subsequently reconstituted into phospholipid vesicles. The aim of these studies was to elucidate the in vitro procoagulant effects and the in vivo thrombogenicity of r-TF using a rabbit jugular vein stasis thrombosis model. The in vitro studies exhibited a clear concentration-dependent decrease in the clotting time when rabbit brain thromboplastin was replaced by r-TF in the prothrombin time assay. The in vivo studies revealed a dose-dependent thrombogenicity between 1.6 ng/kg and 50 ng/kg. Electron microscope scanning of the surface of representative clots revealed fibrin-rich structures of heterogeneous density. In comparison, thrombi obtained when FEIBA was utilized as the thrombogenic agent were more homogeneous. The injection of r-TF caused a slight transient drop in blood pressure with little or no effects on the pulse rate, complete blood count (CBC) profile, clotting and amidolytic assays when compared to sham control animals. In contrast, the whole blood clotting parameters (activated clotting time and thrombelastograph) were prolonged dose-dependently after r-TF injection. The antithrombotic activity of heparin was assessed in this model and compared to the antithrombotic activity when FEIBA is used as the thrombogenic agent. The apparent ED50 of heparin was found to be 4 times higher in the r-TF system. In control studies, no thrombogenic effects were observed by the phospholipid vesicles alone nor by r-TF not embedded in phospholipid vesicles. These data demonstrate that lipidated r-TF is a potent thrombogenic challenge that activates the hemostatic system by the extrinsic pathway.

Comparative studies on the anticoagulant and protease generation inhibitory actions of newly developed site-directed thrombin inhibitory drugs. Efegatran, argatroban, hirulog, and hirudin.

Site-directed thrombin inhibitors are being currently assessed clinically for their antithrombotic efficacy. Although these agents are claimed to be specific and direct thrombin inhibitors, their mechanism of inhibition varies. The objective of these studies was to compare four such agents in in vitro systems and to assess their relative anticoagulant efficacy. The four agents utilized in these studies were argatroban, Efegatran, hirulog, and hirudin. While hirulog and hirudin are specific irreversible inhibitors of thrombin, argatroban and Efegatran are reversible. All four agents were found to have a concentration-dependent anticoagulant effect when supplemented into normal human plasma, as assessed in the global clotting tests (PT, APTT, and Heptest). The most potent anticoagulant on a molar basis was hirudin in all three tests. The other three agents had similar anticoagulant actions. All four agents were also capable of inhibiting the generation of thrombin and factor Xa as determined by an amidolytic method after intrinsic or extrinsic activation of fibrinogen-deficient human plasma. Except for hirulog, all agents inhibited the extrinsic generation of thrombin, with hirudin being the most potent agent. The intrinsic generation of thrombin was blocked by the reversible thrombin inhibitors (argatroban and Efegatran) but not by the irreversible inhibitors (hirulog and hirudin). While all agents were capable of inhibiting the intrinsic generation of factor Xa at very low concentrations, only Efegatran was capable of blocking the extrinsic generation of the same factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular weight and biochemical profile of a chemically modified heparin derivative, Suleparoide.

Recently, a new chemically modified derivative of heparin (Suleparoide, Syntex Laboratories Buenos Aires, Argentina) was introduced for the prophylaxis of thrombosis and treatment of vascular disorders. This agent is claimed to contain a depolymerized, chemically modified, heparin derivative with similar biologic actions as heparan sulfate. To study the pharmacologic profile of this agent, we have defined its molecular weight distribution profile, utilizing a computerized gel permeation chromatographic system equipped with ultraviolet and refractive index detectors. Suleparoide exhibited a normal molecular distribution profile with no contaminants. It exhibited a weight average of 9.3 K DA and an apparent peak MW of 8.0 K DA. Approximately 50% of the molecular components were < 5.0 K DA and 40% > 5.0 K DA. The results from these studies on the mechanisms show that Suleparoide has anticoagulant activity primarily mediated through Heparin Cofactor-II (HC-II) and because of its novel mechanism of action, further investigations on the biochemical profile of Suleparoide are carried out. Global clotting tests such as Activated Partial Thromboplastin Time (APTT), Heptest and Thrombin Time (TT) revealed a concentration dependent effect in all assays. Plasma samples supplemented with Suleparoide exhibited no significant anti-Xa and anti-IIa activities. However, in the HC-II mediated inhibitory assay for IIa, Suleparoide exhibited significant activity. In contrast, the Antithrombin-III (AT-III) mediated inhibition of IIa was much weaker.