Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-04-06
2004-09-21
Wax, Robert A. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S384000, C530S350000, C435S013000, C424S094640
Reexamination Certificate
active
06794493
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to factors involved in the coagulation pathway, and more particularly to serpins modified to selectively modulate the coagulation pathway.
BACKGROUND OF THE INVENTION
The regulation of blood clotting is an important process in animals with a developed cardiovascular system. Clotting is achieved through either an intrinsic or extrinsic pathway that involves a cascade of protein activations resulting in the conversion of soluble fibrinogen to insoluble fibrin.
The intrinsic pathway includes the following steps: (1) factor XII is activated; (2) activated factor XII activates factor XI; (3) activated factor XI activates factor IX; (4) activated factor IX, with interaction from activated factor VIII, activates factor X; (5) activated factor X converts prothrombin to thrombin in the presence of activated factor V; (6) thrombin cleaves fibrinogen to fibrin; (7) fibrin polymerizes to form fibrin strands.
The extrinsic pathway includes the following steps: (1) trauma to the vessel wall causes binding of factor VII in plasma to tissue factor present in non-vascular tissue cells; (2) factor VII is activated; (3) factor VII-tissue factor complex activates Factor X. The remaining steps are the same as steps 5-7 of the intrinsic pathway.
A fine line must be maintained between the activation and inactivation of coagulation (Gaffney et al., 1999). If clotting proteins are constantly inactivated, then blood loses the ability to clot which could lead to life-threatening bleeding events.
Hemophilia A is an inherited factor VIII deficiency that results in extensive bleeding after trauma and may involve spontaneous bleeding into joints and muscles. In normal individuals, factor VIII circulates in the plasma bound to von Willebrand factor. Patients with Hemophilia A exhibit reduced levels of factor VIII, which results in a reduction in blood clotting. Patients with a level of factor VIII less than 1% of normal have severe bleeding episodes throughout life. A level of about 5% of normal results in few, if any, spontaneous bleeding episodes, but severe bleeding can occur in these patients, for example, following surgery, if not properly managed. Patients with factor VIII levels of about 10 to 30% of normal have very mild hemophilia, but may still experience excessive bleeding, for example, following surgery.
Treatment of patients with hemophilia A generally involves administering factor VIII. The factor VIII may be obtained from human donors, or from animals, for example, porcine factor VIII. Recombinantly produced factor VIII may also be used. Severe hemophiliacs require frequent infusions of factor VIII to restore the blood's normal clotting ability. However, supplies of human factor VIII are often inadequate, and the time and expense involved in its isolation and purification from blood are considerable, especially in light of the risk of transmitting viruses such as AIDS and hepatitis.
About 15% of hemophilia A patients develop antibodies to factor VIII. These antibodies inhibit the anticoagulant activity of therapeutically administered factor VIII. Immune tolerance may be achieved through continuous exposure to factor VIII. This requires large and continuous infusions of factor VIII, which are costly, and, in the case of human-derived factor VIII, pose the risk of viral infection. Providing a means for extending the bioavailability of factor VIII would reduce the amount of factor VIII needed to treat hemophilia A patients.
In the opposite situation, if clotting factors are not degraded or inhibited once clotting begins, the blood clot can quickly spread within vessels and capillaries blocking blood flow to vital organs. Some of the leading causes of death in this country are the result of clotting diseases like stroke and myocardial infarction. Regulation of blood clotting will greatly influence therapeutic control over clotting and clotting diseases.
The steps involved in forming a clot include a series of zymogen activations (Roberts and Lozier, 1992). Zymogens are precursors of enzymes that are activated once they are proteolytically cleaved. The cleavage of the zymogen alters the protein structure exposing an active site and allowing enzymatic activity to occur. The coagulation process is interesting because many of the zymogens, when cleaved, become active serine proteases which have a specificity to cleave the next zymogen in the reaction. The activation of successive serine proteases provides a rapid response to a relatively small signal. Several steps in the process can be regulated to activate or inhibit clot formation.
Within the coagulation cascade, many of the serine proteases can also cleave inhibitors of the clotting process. The inhibitors are members of the family of proteins known as serpins (
ser
ine
p
rotease
in
hibitor
s
) (Potempa et al., 1994). Often, when a serpin is cleaved by a serine protease, the two proteins remain covalently bound, which inhibits the protease by preventing it from reacting with any other molecules (Mammen, 1998). One of the best examples of this is the interaction between the serine protease thrombin and the serpin antithrombin (AT).
Thrombin is the final protease generated in the clotting cascade, and is activated by the cleavage of prothrombin by Factor Xa (FIG.
1
A). Thrombin can then cleave fibrinogen into fibrin monomers. Polymerization of fibrin monomers forms the fibrin part of the clot. Though activating fibrin is the main function of thrombin, it also has other functions. Thrombin can activate a positive feedback pathway by proteolytically activating Factors V and VIII that assist in the activation of prothrombin into thrombin. Thrombin also proteolytically cleaves proteins that can serve as inhibitors of its action.
Antithrombin is the most important anticoagulant in the blood (FIG.
1
B). It has a high specificity for thrombin, but also weakly inhibits several other serine proteases. Antithrombin inhibits thrombin by inserting its reactive site loop into the active site of thrombin. The interaction forms a stable complex between the two molecules (Mammen, 1998).
The protease activity of thrombin cleaves the reactive site loop of antithrombin between an arginine and serine which are labeled P1 and P1′ for the site of thrombin cleavage. The cleavage results in a covalent bond between antithrombin and thrombin and prevents thrombin from carrying out any further proteolytic reactions (Olson et al., 1995).
The binding between AT and thrombin occurs very slowly (
k
2
=9×10
3
M
−1
s
−1
) (Olson and Shore, 1982). In its native form, the reactive site loop of AT is not easily accessible to thrombin (Jin et al., 1997). Heparin, a polysaccharide with a strong negative charge, helps to speed this reaction (Olson and Shore, 1982). A specific pentasaccharide in heparin binds to the positively charged D-helix of AT (Jin et al., 1997; Olson et al., 1992). This causes AT to go through a structural change allowing the reactive site loop on AT to be more accessible to thrombin (Ersdal-Badju et al., 1997; Huntington and Gettins, 1998; Meagher et al., 1996). Heparin also binds to thrombin and acts as a bridge to draw the two molecules together (Danielsson et al., 1986). When heparin is bound to AT, the rate of reaction with thrombin is increased significantly (500- to 1000-fold) (Olson and Shore, 1982).
Although thrombin usually functions as a procoagulant, it can also activate the protein C pathway, an anticoagulant pathway. Thrombin has the ability to bind to an endothelial cell receptor called thrombomodulin (TM). When thrombin is bound to TM, it goes through a conformational change that results in a change in substrate specificity for protein C instead of fibrinogen (
FIG. 1A
) (Ye et al., 1991). Protein C interacts with thrombin through Ca
2+
bridges (Rezaie and Esmon, 1992). The protein C is cleaved by the thrombin bound to TM, generating activated protein C (APC). APC proteolytically degrades two cofactors of clotting, Factor Va and Factor VIIIa, preventing the
Cooper Scott
Rezaie Alireza
Walston Timothy
Godfrey & Kahn S.C.
Mondesi Robert B.
Wax Robert A.
WiSYS Technology Foundation, Inc.
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