Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by deactivating Factors V.sub.a and VIII.sub.a in the coagulation cascade. Human protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. This precursor molecule undergoes multiple post-translational modifications including 1) cleavage of a 42 amino acid signal sequence; 2) proteolytic removal from the one chain zymogen of the lysine residue at position 155 and the arginine residue at position 156 to make the 2-chain form of the molecule, (i.e., a light chain of 155 amino acid residues attached through a disulfide bridge to the serine protease-containing heavy chain of 262 amino acid residues); 3) vitamin K-dependent carboxylation of nine glutamic acid residues clustered in the first 42 amino acids of the light chain, resulting in 9 gamma-carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). The heavy chain contains the well established serine protease triad of Asp 257, His 211 and Ser 360. Finally, the circulating 2-chain zymogen is activated in vivo by thrombin at a phospholipid surface in the presence of calcium ion. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing enzymatic activity.
In conjunction with other proteins, protein C functions as perhaps the most important down-regulator of blood coagulation. In other words the protein C enzyme system represents a major physiological mechanism of anticoagulation.
The coagulation system is best viewed as a chain reaction involving the sequential activation of zymogens into active serine proteases eventually producing the enzyme, thrombin, which through limited proteolysis converts plasma fibrinogen into the insoluble gel, fibrin. Two key events in the coagulation cascade are the conversion of clotting factor X to Xa by clotting factor IXa and the conversion of prothrombin into thrombin by clotting factor Xa. Both of these reactions occur on cell surfaces, most notably the platelet surface. Both of these reactions require cofactors. The major cofactors, factors V and VIII, in the system circulate as relatively inactive precursors, but when the first few molecules of thrombin are formed, thrombin loops back and activates the cofactors through limited proteolysis. The activated cofactors, Va and VIIIa, accelerate both the conversion of prothrombin into thrombin and also the conversion of factor X to factor Xa by approximately five orders of magnitude. Activated protein C overwhelmingly prefers two plasma protein substrates which it hydrolyzes and irreversibly destroys. These plasma protein substrates are the activated forms of the clotting cofactors, Va and VIIIa. Activated protein C only minimally degrades the inactive precursors, clotting factors V and VIII. Activated protein C in dogs has been shown to sharply increase circulating levels of the major physiological fibrinolytic enzyme, tissue plasminogen activator (tPA). Activated protein C has been shown in vitro to enhance the lysis of fibrin in human whole blood. Therefore, activated protein C represents an important adjunct to in vivo fibrinolysis in man.
Today, there are few effective treatments available for vascular occlusions, including thrombotic stroke. Treatment with tPA, if administered within three hours from the onset of the stroke, has been recently approved by the FDA. Treatment of strokes with either heparin or oral anticoagulants, although occasionally beneficial, carries a high risk for bleeding into the infarcted brain area.
The use of recombinant aPC (r-aPC) in the treatment of thrombotic occlusion or thromboembolism in a baboon model has been presented by Griffin, et al. in U.S. Pat. No. 5,084,274 and European Patent Specification EP 0 318 201 B1. Griffin claimed dose levels in the range of 0.07 mg/kg/hr to 1.1 mg/kg/hr for the treatment of thrombotic occlusion. However, applicants have found that these dose levels are in a range above the toxicological level of r-aPC. For example, pre-clinical toxicology studies in non-human primates indicate the safety of r-aPC for a 96 hour infusion is limited at a top dose of around 0.05 mg/kg/hr. Therefore, the lowest dose level taught by Griffin, et al., i.e. 0.07 mg/kg/hr, is at a level greater than the toxic dose established by applicants for humans. Thus, even the lowest dose level taught by Griffin would carry a high risk for bleeding into the infarcted brain area, thereby aggravating the neurological deficit accompanying the stroke. Accordingly, even in view of the teaching of Griffin, et al., there remains a need to identify an effective therapy of arterial thrombus formation in humans with aPC.
Contrary to the teachings of prior investigators, applicants have discovered that only low dose therapy with r-aPC is useful in the treatment of thrombotic stroke. The administration of aPC is also beneficial in preventing the local extension of the microvascular and macrovascular occluding arterial thrombus, thereby reducing the neurological deficit resulting from the stroke.