1. Field of the Invention
This invention relates to proteins which inhibit the coagulation of the blood, processes for preparing these proteins, and their use.
2. Description of the Background Art
Anti-coagulant proteins, which are present in most mammals, can be divided into three groups based on their different mechanisms of activity.
One group of proteins form a complex with a coagulation factor and thereby render the coagulation factor inactive. Proteins in this category include antithrombin III (Thromb. Res., 5:,439-452 (1974)), alpha.sub.1 -protease inhibitor (Ann. Rev. Biochem., 52:655-709 (1983)), alpha.sub.2 -macroglobulin (Ann. Rev Biochem., 52: 655-709 (1983)), C.sub.1 -inhibitor (Biochemistry 20: 2738-2743 (1981)), and protease nexin (J. Biol. Chem., 258: 10439-10444 (1983)).
A second group of proteins act proteolytically on a coagulating factor and thereby inactivate it. The only protein of this kind that has been described is protein C (J. Biol. Chem., 251: 355-363 (1976)).
The third category to which anti-coagulant proteins can be grouped are those which screen and/or hydrolyze the negatively charged phospholipids so that the phospholipid-dependent reactions of the blood coagulation mechanism are inhibited. Thus far, only phospholipases isolated from various types of snake venom have been described as having this mode of action (Eur. J. Biochem., 112: 25-32 (1980)).
In recent years, the step-wise coagulation system has been investigated thoroughly. It is understood to be an intensifying multi-stage system of different interconnected proteolytic reactions in which an enzyme converts a zymogen into the active form (cf. Jackson, C. M. and Nemerson, Y., Ann. Rev. Biochem., 49: 765-811 (1980)). The speed of this reaction is decisively increased by the presence of phospholipids and other cofactors such as factor V.sub.a and factor VIII.sub.a. In vivo, the procoagulation reactions are regulated by a variety of inhibitory mechanisms which prevent an explosively thrombotic trauma after slight activation of the coagulation cascade.
The mechanisms by which the anti-coagulation proteins of these three groups act have been described (Rosenberg, R. D. and Rosenberg, J. S., J. Clin. Invest., 74: 1-6 (1984)).
In Group 1, serine-protease factor X.sub.a and thrombin are inactivated as a result of their binding to antithrombin III or to the antithrombin/heparin complex. Both the prothrombin activation and also the formation of fibrin can be inhibited in this way. In addition to antithrombin III, there are also various other plasmaprotease inhibitors such as alpha2-macroglobulin and antitrypsin, the activity of which is dependent on time.
In Group 2, the discovery of protein C led to another anti-coagulation mechanism. Once protein C is activated, it acts as an anti-coagulant by selective proteolysis of the protein cofactors V.sub.a and VIII.sub.a, by which prothrombinase and the enzyme which converts factor X are deactivated.
In Group 3, plasmin cleaves monomeric fibrin 1, a product of the effect of thrombin on fibrinogen, thereby preventing the formation of an insoluble fibrin (Nossel, H. L., Nature, 291: 165-167 (1981)).
Of the above-mentioned native proteins involved in the coagulation process, at present only antithrombin III is clinically used. However, the increase in the tendency to bleed when this protein is administered has proven to be a serious disadvantage.
All the agents previously used as anticoagulants, whether native to the body or synthetic, in some way render the coagulation factors ineffective and thereby lead to side effects which have a disadvantageous effect on the coagulation process.