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Thromboembolism, Coumarin Necrosis, and Protein C
Laboratory Medicine Thromboembolism, Coumarin Necrosis, and Protein C Familial bleeding disorders such as hemophilia are well known both to physicians and to the public. Further more, bleeding disorders due to deficiencies of clotting factors or platelets are fairly well understood. A much more difficult problem has been that of recurrent throm bosis, particularly recurrent familial venous thromboem bolism. Elucidation of the role of protein C, however, seems to be a "breakthrough" in this area. Normal hemostatic integrity involves interaction among blood vessels, blood platelets, and plasma fac tors. Early in this century, Morawitz postulated that clot ting occurs when thrombin converts fibrinogen to fibrin, a concept that has been.greatly amplified by the identifi cation of numerous plasma clotting factors and the de velopment of a "cascade" hypothesis of coagulation. Proper regulation of coagulation necessitates a finely tuned balance of both promoters and inhibitors of coagu lation. Until recently, coagulation promoters had re ceived considerably more attention than had inhibitors, but this emphasis may now be changing.
activated protein C is increased by a factor of 20,000. 6 Protein C activation is effected, in the presence of Ca + + and thrombin, by an endothelial cell receptor called thrombomodulin. Protein C also seems to stimulate fibrinolysis. 7 When purified bovine-activated protein C is injected intravenously in dogs, a prompt and specific fibrinolytic state is induced, together with a decrease in whole blood clot lysis time but without systemic activa tion of plasminogen. Activated protein C may enhance fibrinolysis by changing the balance between plasmin ogen activator and its fast-acting inhibitor and thus mak ing more endothelial plasminogen activator available. 8
In 1960, Mammen and associates1 identified a potent anticoagulant activity in a mixture of the known vitamin K-dependent factors that had been treated with thrombin. They named the inhibitor that caused this anticoagulant activity autoprothrombin Ila. More than a decade later, γ-carboxyglutamic acid, an unusual amino acid, was found in all the vitamin K-dependent clotting factors. Concurrently, additional vitamin K-dependent proteins were discovered besides the classic coagulation factors II, VII, IX, and X. 2 One of these γ-carboxyglutamic acid proteins was purified from bovine plasma and named protein C.3 The inhibitor originally described as autopro thrombin Ila seems to be identical to activated protein C. The rate of activation of protein C is very slow when blood clots in vitro (only traces of protein C are activated during clotting in a test tube). Consequently, a physiolog ic significance for protein C was thought unlikely. Investi gators discovered, however, that activated protein C is a potent anticoagulant that selectively inactivates the ac tive cofactors Va and Villa and inhibits platelet coagulant activity by inactivation of platelet factor Va. 4 " 6 When protein C and thrombin are perfused through an isolated heart, the rate of conversion of protein C to Mayo Clin Proc 60:673-674, 1985
673
Activated protein C is, in turn, neutralized by yet another specific plasma protease inhibitor. The function of activated protein C is optimized by the presence of a cofactor, protein S, which is also a vitamin K-dependent protein. Protein S enhances the binding of activated protein C to phospholipid membranes and accelerates the inactivation of factor Va by activated protein C. 9 Protein S exists both free in blood and reversibly bound to a component of the complement system, C4b-binding protein. Bound protein S complex shows little functional anticoagulant activity, and patients whose protein S is primarily bound to complement components might be anticipated to have recurrent thrombosis. 10 Thus, both protein C and protein S are important regulators of coagu lation, and persons who lack these substances should have an increased tendency for recurrence of thromboembolic disease. Clinical studies are confirming this prediction. Members of one family who had less than 50% of the normal blood concentration of protein C antigen suffered recurrent venous thromboembolism. 1 1 Both sexes were affected. The disorder seems to be inherited as an autosomal-dominant trait. Not all family members with de creased levels of protein C have recurrent thrombosis. Some persons with plasma protein C values less than 10% of normal have survived, at least thus far, into their 20s. Severe protein C deficiency, however, has been associated with massive venous thrombosis shortly after birth. 1 2 In addition, some protein C-deficient patients have functionally abnormal protein C but normal amounts of protein C antigen. Coumarin-associated skin necrosis is a very serious, rare, and poorly understood disorder. Necrosis of skin, especially of the extremities and the breasts, usually occurs within the first few days after initiation of therapy.
674
LABORATORY MEDICINE
Paradoxically, many patients with coumarin necrosis have received the drug previously or receive it subse quently without experiencing problems. Examination of the veins in the necrotic areas shows fibrin thrombi and sometimes cellular infiltration of small veins. A toxic vasculitis has been proposed as the cause. Protein C deficiency has been disclosed in several patients with coumarin-associated skin necrosis. 13 The detection of protein C deficiency in patients with skin necrosis seems more than coincidental, and deficiency of this substance may prove to be a major predisposing factor. During the initial administration of coumarin drugs, protein C levels decline rapidly, in parallel with coagula tion factor VII. Although this reduction poses little threat to most patients, if the level of protein C is already low, coumarin therapy may result in venous thrombosis. Cur rently, routine testing of every patient for protein C defi ciency before anticoagulant therapy is impractical. At the onset of anticoagulant therapy, however, large doses of coumarin drugs should be avoided. If protein C deficien cy is suspected in a patient who is already taking couma rin drugs, the deficiency can be confirmed during main tenance anticoagulant therapy by relating the protein C level to the levels of other vitamin K-dependent factors (for example, factor X). Plasma concentrations of protein C also decrease in patients with liver disease and in those with decompensated intravascular clotting. 1 4 Assays for protein C may be either immunologic or functional and include Laurell "rocket" electroimmunoassay and radioimmunoassay. 1 1 , 1 5 Functional assays currently in use involve partial purification on insoluble salts (barium citrate or aluminum hydroxide) and subse quent activation with thrombin or thrombin-thrombo modulin complex or activation of protein C in plasma by using thrombin-thrombomodulin complex. The activat ed protein C is then adsorbed on anti-protein C immunoglobulin and quantitated by using synthetic substrates. These assay systems have been useful in detecting pa tients with functional deficiency of protein C and throm bosis. They test for distinct and different functions of the molecule, and neither assay precisely measures the anti coagulant activity of activated protein C. 1 6 Other causes of familial thrombosis include low plas ma levels of antithrombin III and molecular abnormalities offibrinogenand plasminogen. 17 With knowledge of the role of the endothelium and some of the regulatory proteins of the clotting mechanism, investigators can now evaluate recurrent venous thromboembolism in terms of the fundamental molecular abnormalities.
Mayo Clin Proc, October 1985, Vol 60
Francis J. Kazmier, M.D. Division of Cardiovascular Diseases and Internal Medicine
Address reprint requests to Dr. F.J. Kazmier, Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, Rochester, MN 55905. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
13. 14. 15. 16. 17.
Mammen EF, Thomas WR, Seegers WH: Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II or autoprothrombin ll-A). Thromb Diath Haemorrh 5:218-249, 1960 StenfloJ, Fernlund P, Egan W, Roepstorff P: Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sei USA 71:2730-2733, 1974 Stenflo J: A new vitamin K-dependent protein: purification from bovine plasma and preliminary characterization. J Biol Chem 251:355-363, 1976 Walker FJ, Sexton PW, Esmon CT: The inhibition of blood coagu lation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 571:333-342, 1979 Comp PC, Esmon CT: Activated protein C inhibits platelet prothrombin-converting activity. Blood 54:1272-1281, 1979 Esmon CT, Owen WG: Identification of an endothelial cell cofac tor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sei USA 78:2249-2252, 1981 Comp PC, Esmon CT: Generation of fibrinolytic activity by in fusion of activated protein C into dogs. J Clin Invest 68:1221-1228, 1981 Van Hinsbergh VWM, Bertina RM, van Wijngaarden A, van Tilburg NH, Emeis JJ, Haverkate F: Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-con ditioned medium. Blood 65:444-451, 1985 Walker FJ: Regulation of activated protein C by protein S: the role of phospholipid in factor Va inactivation. J Biol Chem 256:11128-11131, 1981 Comp PC, Nixon RR, Cooper MR, Esmon CT: Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 74:2082-2088, 1984 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C: Deficiency of protein C in congenital thrombotic disease. J Clin Invest 68:1370-1373, 1981 Seligsohn U, Berger A, Abend M, Rubin L, Attias D, Zivelin A, Rapaport SI: Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med 310:559-562, 1984 McGehee WG, Klotz TA, Epstein DJ, Rapaport SI: Coumarin necrosis associated with hereditary protein C deficiency. Ann Intern Med 101:59-60, 1984 Griffin JH, Mosher DF, Zimmerman TS, Kleiss AJ: Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood 60:261-264, 1982 Bertina RM, Broekmans AW, van der Linden IK, Mertens K: Protein C deficiency in a Dutch family with thrombotic disease. Thromb Haemost 48:1-5, 1982 Esmon CT: Protein C. Prog Hemost Thromb 7:25-54, 1984 Aoki N, Moroi M, Sakata Y, Yoshida N, Matsuda M: Abnormal plasminogen: a hereditary molecular abnormality found in a patient with recurrent thrombosis. J Clin Invest 61:1186-1195, 1978
activated protein C is increased by a factor of 20,000. 6 Protein C activation is effected, in the presence of Ca + + and thrombin, by an endothelial cell receptor called thrombomodulin. Protein C also seems to stimulate fibrinolysis. 7 When purified bovine-activated protein C is injected intravenously in dogs, a prompt and specific fibrinolytic state is induced, together with a decrease in whole blood clot lysis time but without systemic activa tion of plasminogen. Activated protein C may enhance fibrinolysis by changing the balance between plasmin ogen activator and its fast-acting inhibitor and thus mak ing more endothelial plasminogen activator available. 8
In 1960, Mammen and associates1 identified a potent anticoagulant activity in a mixture of the known vitamin K-dependent factors that had been treated with thrombin. They named the inhibitor that caused this anticoagulant activity autoprothrombin Ila. More than a decade later, γ-carboxyglutamic acid, an unusual amino acid, was found in all the vitamin K-dependent clotting factors. Concurrently, additional vitamin K-dependent proteins were discovered besides the classic coagulation factors II, VII, IX, and X. 2 One of these γ-carboxyglutamic acid proteins was purified from bovine plasma and named protein C.3 The inhibitor originally described as autopro thrombin Ila seems to be identical to activated protein C. The rate of activation of protein C is very slow when blood clots in vitro (only traces of protein C are activated during clotting in a test tube). Consequently, a physiolog ic significance for protein C was thought unlikely. Investi gators discovered, however, that activated protein C is a potent anticoagulant that selectively inactivates the ac tive cofactors Va and Villa and inhibits platelet coagulant activity by inactivation of platelet factor Va. 4 " 6 When protein C and thrombin are perfused through an isolated heart, the rate of conversion of protein C to Mayo Clin Proc 60:673-674, 1985
673
Activated protein C is, in turn, neutralized by yet another specific plasma protease inhibitor. The function of activated protein C is optimized by the presence of a cofactor, protein S, which is also a vitamin K-dependent protein. Protein S enhances the binding of activated protein C to phospholipid membranes and accelerates the inactivation of factor Va by activated protein C. 9 Protein S exists both free in blood and reversibly bound to a component of the complement system, C4b-binding protein. Bound protein S complex shows little functional anticoagulant activity, and patients whose protein S is primarily bound to complement components might be anticipated to have recurrent thrombosis. 10 Thus, both protein C and protein S are important regulators of coagu lation, and persons who lack these substances should have an increased tendency for recurrence of thromboembolic disease. Clinical studies are confirming this prediction. Members of one family who had less than 50% of the normal blood concentration of protein C antigen suffered recurrent venous thromboembolism. 1 1 Both sexes were affected. The disorder seems to be inherited as an autosomal-dominant trait. Not all family members with de creased levels of protein C have recurrent thrombosis. Some persons with plasma protein C values less than 10% of normal have survived, at least thus far, into their 20s. Severe protein C deficiency, however, has been associated with massive venous thrombosis shortly after birth. 1 2 In addition, some protein C-deficient patients have functionally abnormal protein C but normal amounts of protein C antigen. Coumarin-associated skin necrosis is a very serious, rare, and poorly understood disorder. Necrosis of skin, especially of the extremities and the breasts, usually occurs within the first few days after initiation of therapy.
674
LABORATORY MEDICINE
Paradoxically, many patients with coumarin necrosis have received the drug previously or receive it subse quently without experiencing problems. Examination of the veins in the necrotic areas shows fibrin thrombi and sometimes cellular infiltration of small veins. A toxic vasculitis has been proposed as the cause. Protein C deficiency has been disclosed in several patients with coumarin-associated skin necrosis. 13 The detection of protein C deficiency in patients with skin necrosis seems more than coincidental, and deficiency of this substance may prove to be a major predisposing factor. During the initial administration of coumarin drugs, protein C levels decline rapidly, in parallel with coagula tion factor VII. Although this reduction poses little threat to most patients, if the level of protein C is already low, coumarin therapy may result in venous thrombosis. Cur rently, routine testing of every patient for protein C defi ciency before anticoagulant therapy is impractical. At the onset of anticoagulant therapy, however, large doses of coumarin drugs should be avoided. If protein C deficien cy is suspected in a patient who is already taking couma rin drugs, the deficiency can be confirmed during main tenance anticoagulant therapy by relating the protein C level to the levels of other vitamin K-dependent factors (for example, factor X). Plasma concentrations of protein C also decrease in patients with liver disease and in those with decompensated intravascular clotting. 1 4 Assays for protein C may be either immunologic or functional and include Laurell "rocket" electroimmunoassay and radioimmunoassay. 1 1 , 1 5 Functional assays currently in use involve partial purification on insoluble salts (barium citrate or aluminum hydroxide) and subse quent activation with thrombin or thrombin-thrombo modulin complex or activation of protein C in plasma by using thrombin-thrombomodulin complex. The activat ed protein C is then adsorbed on anti-protein C immunoglobulin and quantitated by using synthetic substrates. These assay systems have been useful in detecting pa tients with functional deficiency of protein C and throm bosis. They test for distinct and different functions of the molecule, and neither assay precisely measures the anti coagulant activity of activated protein C. 1 6 Other causes of familial thrombosis include low plas ma levels of antithrombin III and molecular abnormalities offibrinogenand plasminogen. 17 With knowledge of the role of the endothelium and some of the regulatory proteins of the clotting mechanism, investigators can now evaluate recurrent venous thromboembolism in terms of the fundamental molecular abnormalities.
Mayo Clin Proc, October 1985, Vol 60
Francis J. Kazmier, M.D. Division of Cardiovascular Diseases and Internal Medicine
Address reprint requests to Dr. F.J. Kazmier, Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, Rochester, MN 55905. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
13. 14. 15. 16. 17.
Mammen EF, Thomas WR, Seegers WH: Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II or autoprothrombin ll-A). Thromb Diath Haemorrh 5:218-249, 1960 StenfloJ, Fernlund P, Egan W, Roepstorff P: Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sei USA 71:2730-2733, 1974 Stenflo J: A new vitamin K-dependent protein: purification from bovine plasma and preliminary characterization. J Biol Chem 251:355-363, 1976 Walker FJ, Sexton PW, Esmon CT: The inhibition of blood coagu lation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 571:333-342, 1979 Comp PC, Esmon CT: Activated protein C inhibits platelet prothrombin-converting activity. Blood 54:1272-1281, 1979 Esmon CT, Owen WG: Identification of an endothelial cell cofac tor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sei USA 78:2249-2252, 1981 Comp PC, Esmon CT: Generation of fibrinolytic activity by in fusion of activated protein C into dogs. J Clin Invest 68:1221-1228, 1981 Van Hinsbergh VWM, Bertina RM, van Wijngaarden A, van Tilburg NH, Emeis JJ, Haverkate F: Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-con ditioned medium. Blood 65:444-451, 1985 Walker FJ: Regulation of activated protein C by protein S: the role of phospholipid in factor Va inactivation. J Biol Chem 256:11128-11131, 1981 Comp PC, Nixon RR, Cooper MR, Esmon CT: Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 74:2082-2088, 1984 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C: Deficiency of protein C in congenital thrombotic disease. J Clin Invest 68:1370-1373, 1981 Seligsohn U, Berger A, Abend M, Rubin L, Attias D, Zivelin A, Rapaport SI: Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med 310:559-562, 1984 McGehee WG, Klotz TA, Epstein DJ, Rapaport SI: Coumarin necrosis associated with hereditary protein C deficiency. Ann Intern Med 101:59-60, 1984 Griffin JH, Mosher DF, Zimmerman TS, Kleiss AJ: Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood 60:261-264, 1982 Bertina RM, Broekmans AW, van der Linden IK, Mertens K: Protein C deficiency in a Dutch family with thrombotic disease. Thromb Haemost 48:1-5, 1982 Esmon CT: Protein C. Prog Hemost Thromb 7:25-54, 1984 Aoki N, Moroi M, Sakata Y, Yoshida N, Matsuda M: Abnormal plasminogen: a hereditary molecular abnormality found in a patient with recurrent thrombosis. J Clin Invest 61:1186-1195, 1978