Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2002-06-07
2004-03-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S320100, C435S325000, C435S252330, C435S023000, C435S214000, C536S023200, C424S214100
Reexamination Certificate
active
06706512
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to variant prothrombins and thrombins capable of activating protein C and having substantially reduced fibrinogen cleavage activity. More specifically, the invention relates to antithrombotic variant prothrombins and thrombins that have substantially reduced procoagulant activity, and to methods of reducing thrombus formation by administering the antithrombotic variant prothrombins or thrombins to an animal or human.
BACKGROUND
Thrombosis is caused by fibrin and platelet deposits that occlude blood vessels in various organs. The role of thrombosis in morbidity and mortality has been documented in many diseases, including, among others, deep vein thrombosis, pulmonary thrombo-embolism, myocardial infarction, ischemic stroke, anthrax and meningococcal sepsis, and heparin-induced thrombocytopenia. Macrovascular thrombosis can be prevented or successfully treated with anticoagulants, antiplatelet agents, and/or profibrinolytic agents. The antithrombotic therapy for microvascular thrombosis, however, presents a greater medical challenge. Pharmacological use of activated protein C (APC), a naturally circulating anticoagulant enzyme (Gruber et al. Blood 79: 2340-2348 (1992)) has been shown to reduce the mortality of severe sepsis (Bernard et al. New Eng. J. Med. 344: 699-709 (2001)). Clinical use of APC is now medically justifiable. However, manufacturing of injectable dosage forms of natural or recombinant APC for therapeutic use is expensive, especially in view of the large doses, such as, for example, administering 0.024 mg/kg/hour of recombinant human APC for the several days required for effective treatment.
The activation of naturally occurring physiologic systems leading to the production of endogenous therapeutic proteins can be as efficacious and more economical than administering the directly acting agent itself For example, relatively inexpensive plasminogen activators, such as streptokinase, are valuable in the systemic treatment of thrombosis, while the directly acting enzyme, plasmin, is suitable for topical therapy only (Marder et al. Thromb. Haemost. 86: 739-745 (2001)). An affordable alternative modality is needed to make APC-therapy accessible to a broader patient population, including those who suffer from septic disseminated intravascular coagulation due to sepsis.
Low dose wild-type human thrombin (WT) is a relatively safe antithrombotic agent in baboons (Hanson et al. J. Clin. Invest. 92: 2003-2012 (1993)) that is capable of binding to thrombomodulin and generating endogenous APC. However, the low-grade fibrin formation and platelet activation that accompany the infusion of WT have potentially adverse side-effects. WT not complexed with thrombomodulin can cause potentially fatal disseminated intravascular coagulation (Gresele et al. J. Clin. Invest. 101: 667-676 (1998)). Thrombomodulin deficiency or poor microcirculation in a patient pose additional safety risks. The use of guanidinobenzoyl thrombin for protein C activation has addressed several of these problems of WT because acyl-thrombin yields active thrombin by delayed deacylation after binding to endothelial thrombomodulin (McBane et al. Thromb. Haemost. 74: 879-885 (1995)). Acyl-thrombin is effective with a wider safety margin than WT in a pig model of thrombosis. The acylation approach, however, reduces, but does not eliminate, the potentially disastrous consequences of an inadvertent overdose, when simultaneous deacylation of unbound acyl-thrombin would suddenly clot all circulating blood.
Activation of the circulating protein C pool by a suitable snake venom activator has also been shown to produce antithrombotic effects in an arterio-venous (AV) shunt model (Kogan et al. Thromb. Res. 70: 385-393 (1993)). There are several potential advantages to snake enzymes, such as high specificity, long half-life, and stability. Immunogenecity, however, does present a problem if used repeatedly. Also, these enzymes are not readily available. In search for newer, specific, and safe protein C activators, a number of thrombin mutations have been reported to compromise cleavage of fibrinogen more than the activation of protein C (Cantwell et al. J. Biol. Chem. 275:39827-39830 (2000); Wu et al. Proc. Natl. Acad. Sci. U.S.A. 88:6775-6779 (1991); Gibbs et al. Nature 378: 413-416 (1995); Arosie et al. Biochemistry 39: 8095-8101 (2000)).
What is still needed, however, is an antithrombotic thrombin with a substantially reduced procoagulant activity and a compromised platelet activation activity, but having an effective capability to activate protein C.
What is also needed, therefore, is a variant thrombin that is practically devoid of activity towards fibrinogen and the platelet receptor PAR-1, but retains a significant capability to activate protein C in the presence of thrombomodulin.
What is still further needed are methods of administering to a patient variant thrombins capable of activating protein C but not of inducing thrombus formation.
What are also needed are methods to determine the antithrombotic potential and the status of activated protein C in the blood of an animal or human that do not necessitate the use of expensive protein C activators or the use of a protein C activator capable of inducing thrombus formation.
SUMMARY OF THE INVENTION
Briefly described, the present invention relates to novel antithrombotic variants of thrombin capable of proteolytically activating protein C, but which are substantially free of fibrinogen cleavage activity. The present invention further relates to variant prothrombins that may be cleaved to yield active thrombin variants. The present invention also relates to methods of inhibiting thrombus formation in an animal or human subject by delivering an antithrombotic variant thrombin of the present invention to the blood of the subject. The present invention relates also to methods that use the novel variant thrombins for determining the level of protein C activation in a blood sample or the thrombogenic potential of a patient.
The present invention provides variant prothrombins and thrombins that have substantially reduced fibrinogen cleavage activity and retain protein C activation activity. Nucleic acid encoding the variant prothrombins or thrombins of the present invention may be inserted into an expression vector and expressed in eukaryotic host cells. The secreted, glycosylated polypeptides may then be cleaved to the active variant thrombins and purified. The variant prothrombins and thrombins of the present invention are useful for administering to a patient as antithrombotic agents lacking a potent thrombus formation capability.
One aspect of the present invention provides variant prothrombins and thrombins that have a tryptophan-alanine substitution at the W215 position of the wild-type thrombin. The single mutant variant thrombin W215A has a substantially reduced fibrinogen cleavage activity and, therefore, a significantly reduced capability of thrombus formation in vivo, or procoagulant activity if contacted with blood in vitro. The variant thrombin W215A retains much of the activity of protein C activation seen with wild-type thrombin, and has an increased relative specificity with respect to PAR-1 cleavage.
Another aspect of the present invention provides a double mutant variant prothrombin and thrombin, WE, that have alanine substitutions at the W215 and E217 positions. The variant thrombin WE of the present invention has a substantially reduced fibrinogen cleavage activity, and significantly reduced PAR-1 cleavage activity (and hence reduced platelet activation activity). The WE variant retains a significant level of protein C activation activity. This variant thrombin, WE (W215A/E217A), is particularly useful for activating protein C, without either significantly cleaving fibrinogen or activating the PAR-1 under in vitro or in vivo conditions. Variant thrombin WE of the present invention has a significantly reduced capability to induce thrombus formation when delivered to the blood of animal or human
De Cera Enrico
Gruber Andras
Hanson Stephen R.
Emory University
Prouty Rebecca E.
Swope Sheridan L.
Womble Carlyle Sandridge & Rice PLLC
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