Protein C Deficiency in Liver Disease*

FREDERICK J. WALKER, Ph .D.

American Red Cross Blood Services, and Departments o f Laboratory Medicine and Medicine, University of Connecticut School o f Medicine, Farmington, CT 06032

ABSTRACT Protein C is a vitamin K-dependent zymogen of a serine protease that is found in blood plasma. The active form, activated protein C, can inhibit blood coagulation and stimulate fibrinolysis. Protein C is synthesized in the liver as a single chain protein. Its synthesis requires several post- translational modifications including carboxylation of glutamic acid resi dues, hydroxylation of aspartic acid residues, and glycosylation. Plasma protein C levels are sensitive to liver function. Protein C levels fall more rapidly than other vitamin K-dependent proteins when synthesis is altered by the administration of oral anticoagulants. In addition, low protein C levels are highly indicative of abnormal liver function. In one case, homo zygous protein C deficiency has been corrected by liver transplantation. In liver transplantation for end-stage liver failure, plasma protein C levels may be a good indicator of the success of the transplantation.

Introduction arin.42 A substantial amount of work has been carried out on the molecular basis Protein C is a vitamin K-dependent of the anticoagulant and profibrinolytic protein found in blood plasma.44 Protein activities of activated protein C, as well C can be converted to a serine protease as the clinical consequences of inherited that is able to inhibit blood coagulation34 and acquired defects in the protein. The and stimulate fibrinolysis.3 56 Protein C mechanism of the inhibition of blood was originally discovered as a vitamin K- coagulation by activated protein C has dependent protein that had no known been shown to be through the proteo function.44 Work by several groups led to lytic inactivation of factors Va34,54 and the discovery of its effects on coagulation VU.36,47,53 The activity of both of these and fibrinolysis, raising the possibility proteins is lost through the cleavage of that protein C may be an essential regu their heavy chains.20 lator of blood coagulation along with A number of studies have also indi antithrombin III, a plasma protease cated that activated protein C can poten inhibitor that can be potentiated by hep tiate the fibrinolytic activity of tissue plasminogen activator.910 Some have * This work was supported by a Grant from the suggested that this is through a mecha National Institutes of Health, (N.H.L.B.I. HL40328) nism by which plasminogen activator 106 0091-7370/90/0300-0106 $01.20 © Institute for Clinical Science, Inc. PROTEIN C DEFICIENCY IN LIVER DISEASE 1 0 7 inhibitor (PAI) is inactivated by activated its anticoagulant activity.14 In addition, protein C;7,9 but, it is still unclear as to protein C contains a modified aspartic the actual mechanism by which fibrino acid, beta-hydroxy aspartic acid, and is lysis is enhanced.21 Recently, a third also glycosylated. The beta-hydroxy- effect of activated protein C, inhibition aspartic acid appears to be important in of endotoxin induced septic shock in the interaction between protein S and baboons, has been observed. High doses protein C.39 The liver enzyme involved of activated protein C have been ob in the hydroxylation has not been de served to block the effects of lethal scribed. No definite function has been doses of endotoxin.46 The mechanism of described for the glycosylated regions of this effect is unclear. These observations activated protein C, but they may alter and a growing list of clinical studies, its rate of clearance in the hepatic sys indicate that protein C is an important tem. regulator of hemostasis and also plays a The primary structure of protein C role in a number of immunologic phe resembles that of other vitamin K- nomena. dependent proteins.22 The amino-termi- Like other vitamin K-dependent pro nal region contains the gammacarboxy- teins, protein C is synthesized only in glutamic acid residues. This is generally the liver.24 Hepatocytes have been ob considered to be the membrane binding served to synthesize protein C,18 secret domain of the protein. The second ing it as a single-chain protein. This sug domain in the sequence is the epidermal gests that there is a protease that can growth factor domain. This region has a cleave an asp-lys bond and convert the two-fold repeat characterized by a large single chain into the two-chain protein.18 number of disulfide bonds and probably In plasma, most of the circulating pro involved in the interaction with protein tein C is the two-chain variety, although S.39 The third domain is the protease some single chain has also been domain. This region contains significant observed.18 The gene for protein C has sequence homology with trypsin and been cloned and sequenced by two includes the active site serine that is groups.24,35 It was cloned from a human involved in proteolysis.23 liver mRNA library and identified either Protein C is converted to its active by the synthesis of protein C24 or by use form, activated protein C, by the of synthetic oligonucleotides as probes.35 removal of a dodecapeptide from the Expression systems for protein C, as well amino-terminus of the heavy chain.34 as other vitamin K-dependent proteins, The cleavage of this peptide is catalyzed require mammalian cells which, unlike by thrombin.34 Thrombin, alone, is a prokaryotic cells, contain the enzymes poor activator of protein C and is inhib needed for the post-translational modifi ited by physiological levels of calcium cations required for the formation of an ion.15 However, when thrombin is active enzyme. bound to the surface of endothelial cells, There are at least three post-transla it becomes a potent activator of protein tional modifications made to protein C. C that requires the presence of calcium First is the carboxylation of 10 glutamic ions.12 This effect is due to the presence, acid residues near the amino terminus on the surface of endothelial cells, of by a vitamin K-dependent liver carboxyl thrombomodulin, an intrinsic membrane ase.14 The resulting residues, gamma- protein,12 which has a high affinity for carboxyglutamic acids, are essential for thrombin. When thrombin is in complex the calcium-dependent, lipid binding with thrombomodulin, its substrate properties of protein C, responsible for specificity changes such that protein C 108 WALKER becomes a better substrate than fibrino studies have relied on measurements of gen,11 platelets,13 or factor V.11 Hence, protein C using functional assays, all of the partitioning of thrombin onto the which require two steps. The first step is endothelial surface provides a mecha the conversion of protein C to its active nism by which the clotting process can form, activated protein C. Three be limited. By this mechanism, throm methods which have been widely used bin, the final protease formed in the are (1) activation by thrombin,26,40 (2) coagulation cascade, appears to be able activation by the thrombin-thrombomo- to limit its own production. dulin complex,4,43 and (3) activation by a The anticoagulant activity of activated protein C activator from the venom of protein C is dependent upon the pres the Southern Copperhead snake (Agkis- ence of a membrane surface where it can trodon contortrix contortrix).17,27 form a complex with a second vitamin In One problem that arises in the use of dependent protein, protein S.31 Protein either thrombin or the thrombin-throm- S is a cofactor for activated protein C. It bomodulin complex is that thrombin will is required for the maximal expression of react with other plasma proteins. This the anticoagulant activity of activated means that it is necessary to separate the protein C on the surface of platelets,30 protein C from some of these compo endothelial cells,45 or membrane vesi nents such as fibrinogen prior to activa cles.52 Protein S is also synthesized in tion and to inactivate the thrombin after the liver,18 but it is also found in endo activation so it will not interfere with the thelial cells19 and megakaryocytes.38 In detection of activated protein C. Two plasma, it is found free and in complex methods have been used for the separa with C4b-binding protein.6 Only free tion of protein C from other plasma com protein S is active as a cofactor for acti ponents. The first is barium adsorption vated protein C.5 The anticoagulant of plasma with barium citrate.26 Protein activity of activated protein C can be C absorbs to barium citrate which can be altered by acute phase reactants, of washed free of fibrinogen and other which C4b-binding protein is one, plasma proteins, and protein C then through changes in plasma levels of free eluted with ethylenediamine tetraacetic protein S. acid (EDTA). The second method is immunoabsorption using calcium depen Detection of Protein C in Plasma dent monoclonal antibodies.48 Protein C is absorbed in the absence of calcium Over the past few years, there has and then eluted from the antibody by been considerable evolution in the the addition of calcium. In the second methods used for the detection of pro step, following activation, thrombin is tein C in plasma. The earliest studies, neutralized with antithrombin III, the including some of the largest to date, plasma protease inhibitor which will have measured antigenic levels of protein block thrombin activity but not the activ C by radioimmunological assays16,31,32 or ity of activated protein C. Detection of enzyme linked immunosorbent assay activated protein C is accomplished by (ELISA) methodology.1,26 For example, using an amidolytic assay, in which one in the study of Militech, who deter of several synthetic substrates can be mined protein C levels in 4,723 individ converted to colored compounds by acti uals, a double antibody technique was vated protein C. The second method is used in which the first antibody captured to measure the anticoagulant activity. In protein C and the second antibody pro this assay, the inhibitory effect of acti vided the signal.26 The majority of recent vated protein C on prothrombin time PBOTEIN C DEFICIENCY IN LIVER DISEASE 1 0 9

(PT) or activated partial thromboplastin tases,57 with depressed protein C levels. time (APTT) is determined.25 More Protein C levels have also been observed recently it has been observed that the to be low in patients with disseminated venom of the Southern Copperhead intravascular coagulation (DIC).28,41 snake is able to activate protein C. This Though several investigators have enzyme appears to be specific for protein ascribed these low levels to be due to C and can be used to activate protein C consumption of protein C, it is more in plasma. Subsequently, activated pro likely indicative of underlying liver dis tein C can be detected in plasma by ease.41 In addition, low protein C levels using one of the previously mentioned associated with acute leukemia, are due detection methods. This simplification to liver dysfunction rather than DIC. In has resulted in a more widespread use of one study, patients with DIC had no the functional assay for protein C. lower levels of protein C than those without DIC.41 Because low protein C N o r m a l V a l u e s levels are highly correlated with impaired liver function, it is recom Protein C levels in normal individuals mended that the observation of low pro have been measured in a large number tein C levels should be followed up with of studies. The distribution of concentra liver function studies. tions in normals has been found to be log normal with a mean of 4.0 |uug per ml.37 P r o t e in C a n d A nticoagulant T h e r a p y A slight age dependence has been ob served. Individuals under 30 years of age Since protein C is a vitamin K-depen- have a mean concentration of 3.76 (xg dent protein, its synthesis is sensitive to per ml while those over 60 have a mean the oral anticoagulant, coumarin. Cou- concentration of 4.45 (Jig per ml. Two marin acts to prevent synthesis in the standard deviations of the mean encom liver of protein C as well as other vitamin passes 70 to 140 percent. Heterozygote K-dependent proteins by the inhibition deficiency is estimated at a frequency of of the liver vitamin K-dependent, glu between one in 200 to one in 300, which tamic acid carboxylase. Protein C levels is in line with the observed rate of tend to fall more rapidly than do the homozygosity (approximately one in levels of other procoagulant factors leav 160,000 to 360,000). However, upon ing a short period where the patient may testing, one in 60 will have levels below be hypercoagulable.8,50,55 The half-life of 65 percent. Only levels below 55 percent protein C has been found to be approxi are actually predictive of heterozygosity. mately 15 hours, while the half-life of The risk factors for individuals with factor X is 24 hours and prothrombin is levels between 55 percent and 65 per 42 hours.59 In some patients with protein cent are unclear.37 C deficiency administration of oral anti coagulants is associated with skin necro P r o t e in C a n d L iv e r D y s f u n c t io n sis.

Patients with poor liver function have P r o t e in C a n d L iv e r T r a n sp l a n t s significantly depressed plasma levels of protein C.33’40’49’57 A number of studies Since plasma protein C levels are very have examined protein C levels in pa sensitive to liver function, it was of inter tients with cirrhotic livers,57 acute viral est to follow protein C levels in individ hepatitis, alcoholic hepatitis,49 or with uals undergoing liver transplantation. In disseminated tumors with liver metas- children, a persistent deficiency of pro 110 WALKER tein C was observed with enhanced risk min K-dependent protein S and complement of portal vessel thrombosis between four component C4b-binding protein. Proc. Natl. Acad. Sci. 78:2512-2516, 1981. and ten days following transplantation. 7. D ’A n g e l o , A ., L o ckh a rt, M. S., D ’An g e l o , Plasma concentrations of protein C did S. V, and Taylor, F. B.: Protein S is a cofactor not reach normal levels until two weeks for activated protein C neutralization of an inhib itor of plasminogen activation released from following transplantation. Adult liver platelets. Blood 69:231-237, 1987. recipients did not have protein C levels 8. D a n g e l o , A ., V ig a n o -D ’An g e l o , S., E sm o n , fall out of the normal range and did not C ., and C o m p , P.: Acquired deficiencies of pro tein C-protein S activity during oral anticoagu appear to have as great a risk for throm lation, in liver disease and in disseminated in- bosis as did children.29 One liver trans travascular coagulation. J. Clin. Invest. 81: plant has been performed on a 20- 1445-1454, 1988. 9. D e F ouw, N. J., de Jong, Y. F., Haverkate, month-old child with homozygous F., and B ertin a, R. M .: Activated protein C protein C deficiency.2 This was the first increases fibrin clot lysis by neutralization of known transplant for the purpose of cor plasminogen activator inhibitor— no evidence for a cofactor role of protein S. Thromb. Hae- recting a specific defect rather than pre most. 60:328-333, 1988. venting death owing to end-stage liver 10. D e F o u w , N. J ., H a v e r k a t e , F ., B e r t in a , disease. In this individual, protein C R. M ., Ko o p m a n , J., v o n W ijn g a a r d e n , A., and van H in s b e r g h , V. W. M .: The cofactor levels were raised to normal levels. role of protein S in the acceleration of whole Protein C appears to be an important blood clot lysis by activated protein C in vitro. regulator of blood coagulation which can Blood 67:1189-1195, 1986. 11. E s m o n , C. T., E s m o n , N. L ., and H a r r is, act either by the inhibition of clot forma K. 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