Chemistry: molecular biology and microbiology – Kit
Reexamination Certificate
2002-04-04
2004-10-26
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Kit
C435S007400, C435S023000, C435S024000, C435S219000, C424S094630
Reexamination Certificate
active
06808927
ABSTRACT:
1. FIELD OF THE INVENTION
The invention relates to a purified form of stabilized thrombin-activatable fibrinolysis inhibitor (TAFI), a method of producing stabilized TAFI, methods for therapeutic, prophylactic and diagnostic use of stabilized TAFI compositions and inhibitors thereof and kits comprising stabilized TAFI useful in measuring TAFI and other carboxypepetidase activity using the stabilized TAFI compositions of the invention as a standard.
2. BACKGROUND OF THE INVENTION
A proper balance between the activities of coagulation and fibrinolytic cascades is needed both to protect an organism from excessive blood loss upon injury and to maintain blood flow within the vascular system. The two opposing coagulation and fibrinolytic cascades are recognized to comprise a series of zymogen to enzyme conversions which terminate in the two respective proteolytic enzymes, thrombin and plasmin. These enzymes catalyze the formation and removal of fibrin within the circulatory system. Imbalances are characterized by either bleeding or thrombotic tendencies which may result in heart attacks or strokes in the organism.
Thrombin activatable fibrinolytic inhibitor (TAFI) is a 60 KDa glycoprotein present in human plasma that modulates fibrinolyisis in vivo. TAFI present in plasma is a proenzyme form which is most efficiently activated by proteolytic cleavage at Arg-92 with a thrombin-thrombomodulin complex. The proenzyme form of TAFI may also be activated by proteolytic cleavage by other proteolytic enzymes including, but not limited to, thrombin or plasmin. Upon activation of the TAFI proenzyme by proteolytic cleavage with thrombin-thrombomodulin, an active enzyme of 35 KDa is formed with carboxypeptidase-like activity (TAFIa). This molecule has also been referred to in the literature as plasma procarboxypeptidase B (PCPB), or plasma carboxypeptidase U (PCPU).
Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginine and lysine residues of partially degraded fibrin, thereby inhibiting the stimulation of tissue plasminogen activator (t-PA) modulated plasminogen activation. The fibrinolytic system is activated primarily by t-PA which is provided by damaged cells in the blood vessel wall. t-PA converts circulating plasminogen to the active protease plasmin and can produce either slow enhancement of fibrinolysis or, when combined with fibrin, rapid enhancement of fibrinolysis. The effect of t-PA on fibrinolysis can be blocked by a class of inhibitors termed plasminogen activator inhibitors (PAIs), of which several have been identified.
Thrombomodulin is a component of the blood vessel wall which binds thrombin and changes its specificity from fibrinogen to protein C, resulting in a molecule possessing anticoagulant, rather than procoagulant, activity. The thrombin-thrombomodulin complex catalyzes cleavage of protein C to activated protein C, which results in down-regulation of the coagulation cascade by proteolytically inactivating the essential cofactors, Factor Va and VIIIa. In this manner, the body regulates coagulation cascade.
Studies such as that by Taylor et al.,
Thromb. Res.
37:639 (1985) have suggested that activated protein C is not only an anticoagulant, but also profibrinolytic, both in vivo and in vitro. Subsequently, it was determined that protein C only appears profibrinolytic because it prevents the thrombin-catalyzed activation of a previously unknown fibrinolysis inhibitor, whose precursor was isolated from plasma and designated as being TAFI.
TAFI was discovered independently in three different laboratories. In initially appeared as an unstable carboxypeptidase B-like molecule in human serum and was described by Hendriks et al.,
Biochim. Biophys. Acta
1034:86 (1990). A year later the cDNA for the molecule was cloned, its amino acid sequence was described, its activation by trypsin and its enzymatic properties toward synthetic carboxypeptidase B substrates was reported (see U.S. Pat. No. 5,206,161). In 1994, Wang et al., (
J. Biol. Chem.
269:15937 (1994)) isolated the activated molecule and named it carboxypeptidase U (“U” being designated for unstable). Subsequently, Nesheim et al. (
J Biol. Chem.
270:14477 (1995)) showed that the protein was both activated by thrombin and inhibits fibrinolysis, and designated the molecule TAFI. The co-identity of PCPB, PCPU, and TAFI has been established by their independent chromatographic behavior on plasminogen Sepharose® and the amino acid sequences present at the activation cleavage site.
The mechanism of TAFI inhibition of fibrinolysis can be schematically described as depicted in FIG.
1
.
Diagnostic assays for TAFIa may be useful in the prognosis or diagnosis of certain hemorrhagic or thrombotic diseases or disorders. Accordingly, there is a need in the art for effective carboxypeptidase activity assays.
As TAFIa is believed to play a central regulatory role in the fibrinolytic cascade, the manipulation of TAFIa levels or activity in biological fluids may have important therapeutic applications with respect to hemorrhagic disorders including, but not limited to, vascular and heart pathologies, and stroke. Inhibitors of TAFIa would enhance fibrinolysis and have an anti-coagulant effect (see U.S. Pat. No. 5,993,815). Inhibitors of TAFIa could also be effective at treating or preventing the inflammation associated with arthritis as vascular endothelial growth factor (VEGF) is a potential substrate of TAFIa. VEGF has been linked with arthritis (Farva, R. A.,
J. Exp. Med.
180:341-6 (1994)).
Conversely, due to its ability to inhibit fibrinolysis, TAFIa itself may be useful as a therapeutic protein capable of acting as a procoagulant. In hemophilia A, a disease characterized by excessive bleeding, the addition of a protein mixture containing TAFIa to hemophilia A plasma has been reported to increase plasma clot lysis times (Mosnier, L. O. et al.,
Thromb. Haemost.
86:1035-9 (2001)). Additionally, thrombolytic therapy with tissue plasminogen activator (t-PA) for acute ischemic stroke remains complicated by risks of hemorrhagic transformation (Sumii T. and Lo E. H.,
Stroke
33:831-6 (2002)).
Unfortunately, the use of TAFIa as a therapeutic protein has not been feasible due to the inherent instability of the enzyme which has a half-life of less than 10 minutes at 37° C. Consequently, TAFIa is also extremely difficult to purify due to its highly unstable nature. Thus, there remains a need in the art for a method of producing a more thermally stable form of TAFIa, including purified forms of stabilized TAFIa, which could have widespread diagnostic use as well as therapeutic use in the treatment, prevention or management of hemorrhagic diseases or disorders.
Citation or identification of any reference in this section is not to be construed as such reference being prior art to the present application.
3. SUMMARY OF THE INVENTION
The present invention is based in part on the surprising discovery that TAFIa can be prepared in a stabilized form by activation of TAFI by proteolytic cleavage in a substantially calcium-free environment. In brief, the present invention is related to a method of preparing a stabilized form of TAFIa, methods for diagnostic and/or therapeutic use of stabilized TAFIa, methods of therapeutic use of TAFIa inhibitors, and kits comprising stabilized TAFIa useful in measuring carboxypepetidase activity, particularly TAFI activity.
In one embodiment, the present invention encompasses a method of producing a stabilized form of TAFIa, or a pharmaceutical composition thereof. In particular, the method of producing stabilized TAFIa comprises protease catalyzed activation of TAFI to TAFIa in a substantially calcium-free environment. In one embodiment, the proteolytic cleavage is catalyzed by thrombin. In another embodiment, the proteolytic cleavage is catalyzed by plasmin. In another embodiment, the proteolytic cleavage is catalyzed by a thrombin-thrombomodulin complex.
In one embodiment, the TAFIa is prepared from wild-type TAFI (e.g., purified from a native or recombinant source). The wild-type TAFI can be
An Seong Soo A.
Greenfield Robert S.
American Diagnostica Inc.
Hanley S
Jones Day
Witz Jean C.
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