Christopher T. Dunwiddie

Purification and characterization of recombinant antistasin: A leech-derived inhibitor of coagulation factor Xa

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.

Pathways of coagulation activation in situ in rheumatoid synovial tissue.

Immunohistochemical techniques were applied to rheumatoid synovium in order to detect components of coagulation and fibrinolysis pathways within these tissues. These techniques revealed an intact coagulation pathway and plasminogen activator inhibitor-2 associated with macrophage-like cells that were present throughout these tissues, especially in subsurface areas. Cell-associated thrombin generation appeared to account for conversion of abundant fibrinogen to fibrin. Occasional macrophage-like cells also stained for urokinase but tissue-type plasminogen activator and plasminogen activator inhibitor-1 were restricted to vascular endothelium. Intense synovial fibrin deposition (with the limited evidence for associated fibrinolysis) may contribute to local inflammation and explain certain clinical features of rheumatoid arthritis. These findings suggest novel treatment hypotheses for this disease.

Selective factor Xa inhibition by recombinant antistasin prevents vascular graft thrombosis in baboons.

A baboon model of high-shear, platelet-dependent vascular graft thrombosis was used to assess the antithrombotic effect of recombinant antistasin (rATS), a 119-amino acid protein with selective, subnanomolar inhibitory potency against coagulation factor Xa. In this model, a Dacron vascular graft segment of a femoral arteriovenous (AV) shunt provided the thrombogenic stimulus. Antithrombotic efficacy of rATS was assessed by continuous monitoring of 111In-labeled platelet and 125I-labeled fibrin(ogen) deposition onto the graft surface and blood flow through the vascular shunt. Systemic intravenous administration of rATS (2 or 4 micrograms/kg.min-1) dose dependently decreased both platelet and fibrin(ogen) deposition onto the graft. Vascular graft thrombus formation was completely inhibited at a systemic dose of rATS of 4 micrograms/kg.min-1. None of the AV shunts in animals receiving rATS at either dose occluded, and blood flow was maintained at 81 +/- 4% (2 micrograms/kg.min-1 rATS) or 96 +/- 3% (4 micrograms/kg.min-1 rATS) of basal flow. Systemic fibrinopeptide A elevations in response to exposure to the Dacron graft segment were completely suppressed by both doses of rATS. The ex vivo activated partial thromboplastin times were extended to greater than 150 seconds during infusion of both doses of rATS; however, even at fully antithrombotic doses, template bleeding times were not significantly increased. Thus, in this baboon model, rATS is a potent antithrombotic agent that inhibits both platelet and fibrin(ogen) deposition onto a Dacron vascular graft segment. Furthermore, these results demonstrate that selective inhibition of coagulation factor Xa by rATS can completely prevent vascular graft thrombus formation without significantly compromising primary hemostasis as measured by template bleeding time.

[18] Purification and characterization of inhibitors of blood coagulation factor Xa from hematophagous organisms

Publisher Summary This chapter describes the purification and characterization of inhibitors of blood coagulation factor Xa from Hematophagous organisms. Hematophagous organisms, such as leeches and ticks possess in their salivas an extensive repertoire of biochemical factors that act in concert to maintain blood in a liquid state, both during the ingestion of a blood meal and during its prolonged storage in the gut. The therapeutic potential of leeches or leech-derived substances is recognized because of antiquity. The most extensively studied leech-derived substance, hirudin, the extremely potent anticoagulant found in the saliva of the leech Hirudo medicinalis , is a selective, stoichiometric inhibitor of thrombin. Activation of coagulation through either the intrinsic or extrinsic pathway results in the formation of factor Xa (fXa), which catalyzes the conversion of prothrombin to thrombin. Salivary gland extracts from the Mexican leech Haementeria officinalis are shown to contain a potent antimetastatic and anticoagulant activity. The protein responsible for both of these activities has been purified and named antistasin (ATS).

The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.