Glinical Investigator
Clin Investig (1992) 70:529-534
Molecular Medicine Review
© Springer-Verlag 1992
Acquired protein S deficiency B. Kemkes-Matthes Zentrum f~r Innere Medizin der Justus Liebig Universit/it Giel3en
Summary. Hereditary deficiencies of coagulation inhibitors like antithrombin III, protein C and protein S lead to an enhanced incidence of thromboembolic complications. Recently, acquired deficiencies of protein S were described in several disease states in which thromboembolic complications frequently occur. These acquired protein S deficiencies reach - in part - the extent realised by hereditary protein S deficiency. Thus, acquired protein S deficiencies seem to be one source of thromboembolic complications occurring in nephrotic syndrome, acute phase reactions, malignancy and pregnancy. In this presentation disease states accompanied by acquired protein S deficiency and the mechanisms leading to these alterations are discussed.
cascade and behaves like an acute phase reactant [61]. Recently, acquired deficiencies of protein S have been described in nephrotic syndrome, malignancy, oral contraceptive treatment and pregnancy. It is well-known that patients with nephrotic syndrome or malignant diseases as well as women taking oral contraceptives or during pregnancy have an increased risk of suffering from thromboembolic complications. Thus, acquired deficiencies of protein S are described in several disease states. Dependent upon the underlying disorders, distinct mechanisms leading to protein S deficiency are discussed.
Synthesis dysfunction
Key words: Acquired protein S deficiency Thrombosis
Hereditary deficiencies of coagulation inhibitors AT III, protein C and protein S [5, 6, 10, 11, 18, 38, 56] are known to be risk factors for thromboembolic diseases. Protein C and protein S are vitamin-K-dependent proteins. After activation [8, 19-21] by thrombin/thrombomodulin, protein C acts as a major inhibitor of blood coagulation: it degrades Factors Va and VIIIa [8, 58, 71] and additionally has profibrinolytic properties [9, 17, 28, 62, 66] (Fig. 1). In contrast to AT III and protein C, only about 40% of protein S, the so-called free protein S, acts as a coagulation inhibitor: free protein S has a cofactor function for activated protein C concerning the degradation of Factors Va and VIIIa [70]. The remaining 60% of protein S is bound to C4bbinding protein (C4b-BP) and does not have any function in the coagulation cascade [14, 34, 35]. C4b-BP is a regulatory protein of the complement Abbreviations: AT I I I = antithrombin III; C4b-BP = C4b-binding protein; DIC=disseminated intravascular coagulation; SLE = systemic lupus erythematosus
Liver disorders
Liver protein synthesis dysfunction occurs in severe liver diseases, leading to a diminution of all plasma proteins synthesized by the liver. In severe liver disorders, a diminution of the vitamin K-dependent protein S down to 67% of the normal level in patients with decompensated liver cirrhosis can be observed [15, 42-44].
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Protein Ca t
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Fig. 1. The protein C-protein S system. Protein C is activated by thrombin/thrombomodulin complex at the endothelial surface. Activated protein C inhibits factors Va and VIIIa; free protein S acts as cofactor. Protein S bound to C4b-binding protein (C4b-BP) has no cofactor function for activated protein C. Mutual inhibition exists between activated protein C (protein Ca) and factors PAI-I and PAI-3 (plasminogen activator inhibitors)
530 The decrease of protein S in liver diseases is not as strong as the diminution of other coagulation inhibitors like protein C and AT III [41, 65], because protein S is not only synthesized by the liver but also by endothelial cells [22] and megakaryocytes [59]. Severe liver disorders lead to a diminution of procoagulatory active coagulation factors as well as of coagulation inhibitors. Therefore, thromboembolic complications are extremely rare in liver diseases, while bleeding complications, which are, in addition to the diminution of coagulation factors, due to portal hypertension and thrombocytopenia, are relatively frequent problems. C4b-BP does not play an important role concerning alterations of the protein S levels in liver disorders [44] : patients suffering from inflammatory liver diseases tend to present elevated C4b-BP levels, and patients with very severe liver diseases like cirrhosis have slightly reduced C4b-BP levels, but no significant differences between these groups were found [44]. Takahashi et al. [65] found reduced levels of total and free protein S as well as of C4b-BP in patients suffering from liver disorders, but he did not give the results for the distinct groups. Treatment with coumarin derivatives
Coumarin treatment as well as vitamin K deficiency results in a diminution of prothrombin complex, proteins C and S. During stable ant±coagulation, no thrombotic tendency results because both procoagulatory active coagulation factors and coagulation inhibitors are diminished. In hereditary protein C deficiency, an increased risk exists of suffering from coumarin necrosis [4, 7, 45, 53]: during the first days of coumarin treatment, a very rapid decrease of protein C occurs because of its short half-life in comparison with the procoagulatory active coagulation factors. Coumarin necrosis is also known to occur in protein S deficiency [30].
Enhanced consumption/loss Enhanced consumption
During disseminated intravascular coagulation (DIC), consumption of coagulation factors and coagulation inhibitors AT III and protein C occurs. Alterations of protein S in DIC are still being discussed controversely. Takahashi et al. [65] found normal free and total protein S levels in 39 patients with DIC, while the C4b-BP levels were elevated up to 149.5% ±
33.8% of normal. These results are confirmed by the observations of Hesselvik et al. [33], who also found normal free and total protein S levels in 3 patients with DIC due to septic shock. The C4bBP levels in these patients were 118%+_40% of normal. In contrast, D'Angelo et al. [15] observed normal total (112% of normal) and reduced free protein S levels (80% of normal) in 20 patients with laboratory evidence of DIC secondary to different clinical conditions (obstetrical, posttraumatic, infective). C4b-BP was elevated up to 150% of normal in these patients. Low total protein S levels during DIC, except in patients suffering from malignancy, were observed by Heeb et al. [31]. Madden et al. [50] examined 2 patients suffering from purpura fulminans and DIC and concluded protein S to be consumed during DIC. The observations concerning the behaviour of protein S in DIC patients are not quite comparable, because free protein S, total protein S and C4b-BP were not measured by all authors, and different methods were used. Alterations of the protein S level in D]C seem to be dependent upon the disorder leading to DIC : Heeb et al. [31] observed reduced total protein S levels in patients suffering from infectious diseases and DIC, while patients suffering from malignancy and DIC presented with normal total protein S levels. Elevated C4b-BP levels and reduced protein S activity [15] in DIC patients led to the conclusion that increases of C4b-BP cause a shift from free to bound protein S, resulting in a thrombotic tendency. Loss
In the nephrotic syndrome, an enhanced risk of suffering from thromboembolic complications is recognised. Vigano-D'Angelo et al. [69] observed low free protein S levels ( 6 9 % ! 2 7 % of normal) in patients suffering from nephrotic syndrome despite having elevated levels of total protein S antigen (139%___42% of normal). He could demonstrate an urinary loss of free protein S and increases of C4b-BP up to 170% ± 52% of normal, leading to a shift from free to bound protein S, resulting in a pronounced decrease of free protein S. Thus, in the nephrotic syndrome, two causes for thrombosis risk due to lowered free protein S levels are f o u n d urinary loss of low molecular weight free protein S as well as increased high molecular weight C4b-BP-bound protein S. This leads to alterations discussed in the next paragraph on C4b-BP-induced alterations.
531 C4b-BP-induced alterations
Inflammatory diseases In patients suffering from systemic lupus erythematosus (SLE), significantly reduced free protein S levels were found [40, 57]. Keeling et al. [40] observed free protein S levels in SLE patients to be lower (0.21 U/ml) in patients presenting without than in patients with (0.25 U/ml) phospholipidspecific antibodies. These antibodies cause an additional down-regulation of the protein C-protein S system by inhibiting the catalytic activity of thrombomodulin [24, 68]. Moreb and Kitchens [57] observed 2 patients with SLE and unusual thrombosis. They found reduced concentrations of free protein S (2.0 and < 1.0 gg/ml; normal range 3.5 8.5 gg/ml) because of excessive binding to C4b-BP at the time the thrombosis occurred. Comparable to the results of Moreb and Kitchens [57], SLE patients examined by my own group [32] showed a tendency to reduced free and elevated bound protein S levels, but there was no significant difference versus the normal control group because of wide standard deviations. In patients suffering from inflammatory diseases like SLE, sepsis, pneumonia and fever, a highly positive correlation was found between bound protein S and C4b-BP [32], indicating that C4b-BP plays an important role concerning the distribution between the free and the bound protein S fraction. In contrast, Schved et al. [64] examined patients suffering from non-acute inflammatory syndromes including patients with suppuration, tuberculosis, cancer and rheumatoid arthritis. They found a tendency to increased levels of free protein S and C4b-BP, yet the extremely high standard deviations do not allow definite conclusions. Transient deficiencies of protein S, protein C and AT III were observed by Jorens et al. [39] in a patient suffering from colitis ulcerosa who experienced cerebral arterial thrombosis. Wyshock et al. [73] observed a patient with severe inflammatory bowel disease complicated by 10 episodes of deep venous thrombosis and nonfamilial deficiency of protein S.
We could demonstrate reduced free protein S levels in patients suffering from metastasising carcinoma [46-48]. The decrease of free protein S was dependent upon the severity of the disease [48]: patients without metastases showed normal (48%_+11% of normal total protein S), patients with local metastases slightly (39%_+ 8% of normal total protein S) and patients with disseminated malignant disease significantly decreased free protein S levels (32%_+ 10% of normal total protein S ; normal range 44% _+11% of normal total protein S). Patients with malignancy and thromboembolic complications showed the lowest free protein S levels: 23%_+ 3% of normal total protein S. In metastatic disease, the diminutions of free protein S reached protein S levels known in hereditary protein S-deficient patients. In malignant diseases, the distribution between free and bound protein S seems to be regulated by C4b-BP [49]: in patients suffering from pulmonary carcinoma, a highly positive correlation was found between bound protein S levels and C4b-BP. Total protein S was slightly elevated in cancer patients examined by my group, but there was no significant difference to the healthy control group [46-48]. These observations confirm the results of Heeb et al. [31]. Troy et al. [67] found normal total and significantly decreased levels of free protein S in patients suffering from acute myelocytic leukemia compared with those in remission, while patients with acute lymphocytic leukaemia presented with normal free protein S levels. Low free and total protein S levels were observed in 2 patients with essential thrombocythemia and thrombosis [13], but no information was given concerning C4b-BP levels. To my knowledge, no further publications exist concerning protein S levels in cancer patients except for one [74] on polycythemia vera patients, who are known to suffer frequently from arterial or venous thrombotic complications; low free protein S levels were observed in 5 cases [74]. In contrast to our results in carcinoma patients, C4b-BP levels in these patients were within the normal range. Oestrogen-dependent alterations
Malignancy
Pregnancy
More than 100 years ago, Trousseau observed patients suffering from malignancy to have an increased risk of suffering from thrombotic complications [60]. The source of the so-called Trousseau's phenomenon is not completely clarified.
In pregnancy, an enhanced risk of suffering from thromboembolic complications exists [37]. This thrombotic tendency is related in part to immobilization during the last weeks of pregnancy and/or to (partial) compression of the vena cava inferior.
532
No alterations of coagulation factors or coagulation inhibitors such as AT III and protein C were found to be the cause for thromboembolic complications during pregnancy [16, 26, 29, 52, 63, 72]. However, decreasing levels of total and free protein S during pregnancy were observed recently [12, 16, 23, 26, 51, 72]: at delivery total and free protein S are diminished down to 50% of the normal range and reach normal values about 1 week later. Thus, during the last trimester of pregnancy, the decrease of protein S is comparable to the diminution in hereditary protein S-deficient patients. This is one explanation for the enhanced risk of suffering from thromboembolic complications during pregnancy. The cause for the decrease of total and free protein S during pregnancy is not yet clarified; Malta et al. [51] observed increasing levels of C4bBP up to 143% at delivery, which seem to downregulate free protein S values, while Comp et al. [12] did not find any alterations of C4b-BP during pregnancy. Yet it has to be noticed that C4b-BPinduced alterations of protein S cause an increase of bound and a decrease of the free protein S fraction, whereas the typical alteration of protein S during pregnancy is a diminution of the total, bound as well as free protein S fractions. Therefore, increased C4b-BP levels during pregnancy do not seem to be the main cause for decreasing values of free protein S. Furthermore, in pregnancy an increase of plasma oestrogen levels occurs [27], which might cause a decrease of protein S levels, similar to that discussed under oral contraceptive treatment.
Oral contraceptives and postmenopausal oestrogen substitution Women using oral contraceptives, too, have an increased risk of developing thromboembolic complications [3, 25, 54]. Boerger et al. [2] and Malta et al. [51] observed decreased total and free protein S levels in women taking oral contraceptives, while Huisfeld et al. [36] only found a reduction of total ( 8 4 . 5 % + 1 9 % ; normal range 111.7%+_21.8%) but not of free protein S (94.3% _+18.0% ; normal range 104.6% -t- 15.2%). Melissari and Kakkar [55] examined women using oral contraceptives in comparison with normal and postmenopausal women on a hormone replacement therapy with oestradiol valerate. They concluded that there is a relationship between steroid dosage and duration of treatment and the decrease of free protein S. These results are confirmed by my own observations (unpublished data) that in oral contraceptive users free
protein S levels were reduced depending upon the daily oestrogen dose. Concerning C4b-BP, no significant alterations were found in women taking oral contraceptive treatment in comparison with normal controls [2, 55]. Thus, the regulation of free protein S levels in oral oestrogen treatment and during pregnancy seems to be dependent upon plasma oestrogen levels. Conclusion
In this presentation distinct mechanisms leading to acquired protein S deficiency with or without thromboembolic tendency were discussed. No thromboembolic risk situations result in acquired protein S deficiency due to protein synthesis dysfunction, such as severe liver diseases and coumarin anticoagulation. In contrast, patients suffering from nephrotic syndrome have a high risk of experiencing thromboembolic complications. In these patients, a urinary loss of free protein S was observed. This, together with increased C4b-BP levels, results in a pronounced decrease of free protein S. Increase of C4b-binding protein in acute phase reactions induce a decrease of free protein S, possibly leading to thromboembolic events. C4b-binding protein-dependent alterations of protein S are typical in acute inflammatory diseases and in malignancy. Furthermore, plasma protein S levels seem to be oestrogen-dependent: in women on oestrogen medication such as oral contraceptives and during pregnancy, decreased free and total protein S levels were found. Thus, the regulation of protein S levels depends upon several different mechanisms. References 1. Bezeaud A, Venisse L, Conard J, Horellou MH, Salomon JL, Lebaleur A, Samama M, Guillin MC (1987) D6ficit h6r6ditaire en prot+ine S e t thromboses veineuses r6cidivantes. Presse Med 16 : 1895-1897 2. Boerger LM, Morris PC, Thurnau GR, Esmon CT, Comp PC (1987) Oral contraceptives and gender affect protein S status. Blood 69:692-694 3. B6ttiger LE, Boman G, Eklund G, Westerholm B (1980) Oral contraceptives and thromboembolic disease: effects of lowering oestrogen content. Lancet h 1099-1101 4. Broekmans AW, Bertina RM, Loeliger EA, Hofmann V (1983) Protein C and the development of skin necrosis during anticoagulant therapy. Thromb Haemost 49 : 244 5. Broekmans AW, Bertina RM, Reinalda-Poot J, Engesser L, Muller HP, Leeuw JA, Michiels JJ, Brommer EJP, Brier E (1985) Hereditary protein S deficiency and venous throm-
533 bo-embolism. A study in three Dutch families. Thromb Haemost 37:273 277 6. Chafa O, Fischer AM, Meriane F, Chellali F, Rahal S, Sternberg C, Benabadji M (1989) A new case of "type II" inherited protein S deficiency. Br J Haematol 73 : 501 505 7. Cole MS, Minifee PK, Wolma FJ (1988) Coumarin necrosis a review of the literature. Surgery 103:271-277 8. Comp PC (1984) Animal studies on protein C physiology. Semin Thromb Haemost 10:149-153 9. Comp PC, Esmon CT (1981) Generation of fibrinolytic activity by infusion of activated protein C into dogs. J Clin Invest 68:1221-1228 10. Comp PC, Nixon RR, Cooper MR, Esmon CT (1984) Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 74:2082 2088 11. Comp PC, Doray D, Patton D, Esmon CT (1986) An abnormal plasma distribution of protein S occurs in functional protein S deficiency. Blood 67: 504-508 12. Comp PC, Thurnau GR, Welsh J, Esmon CT (1986) Functional and immunologic protein S levels are decreased during pregnancy. Blood 68:881-885 13. Conlan MG, Haire WD (1989) Low protein S in essential thrombocythemia with thrombosis. Am J Hematol 32:88 93 14. Dahlb/ick B (1984) Interaction between vitamin K-dependent protein S and the complement protein, C4b-binding protein. Semin Thromb Hemost 10:139-148 15. D'Angelo A, Vigano-D'Angelo S, Esmon CT, Comp PC (1988) Acquired deficiencies of protein S. J Clin Invest 81 : 1445-1454 16. De Boer K, Care JW ten, Sturk A, Borm JJJ, Treffers PE (1989) Enhanced thrombin generation in normal and hypertensive pregnancy. Am J Obstet Gynecol 160:95-100 17. De Fouw NJ, Hinsbergh VWM van, Jong YF de, Haverkate F, Bertina RM (1987) The interaction of activated protein C and thrombin with the plasminogen activator inhibitor released from human endothelial cells. Thromb Haemost 57:176-182 18. Engesser L, Broekmans AW, Brier E, Brommer EJP, Bertina RM (1987) Hereditary protein S deficiency: ctinical manifestations. Ann Int Med 106:677-682 19. Esmon CT (1983) Protein C: biochemistry, physiology, and clinical implications. Blood 62:1155-1158 20. Esmon CT, Esmon NL (1984) Protein C activation. Semin Thromb Haemost 10:122-130 21. Esmon NL, Owen WG, Esmon CT (1982) Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 257:859-864 22. Fair DS, Marlar RA, Levin EG (1986) Human endothelial cells synthesize protein S. Blood 67:1168-1171 23. Fern/mdez JA, Estell6s A, Gilabert J, Espana F, Aznar J (1989) Functional and immunologic protein S in normal pregnant women and in full-term newborns. Thromb Haemost 61:474-478 24. Freyssinet J-M, Cazenave J-P (1987) Lupus-like anticoagulants, modulation of the protein C pathway and thrombosis. Thromb Haemost 58:679 681 25. Gerstmann BB, Piper JM, Tomita DK, Ferguson WJ, Stadel BV, Lundin FE (1991) Oral contraceptive estrogen dose and the risk of deep venous thromboembolic disease. Am J Epidemiol 133:32-37 26. Gilabert J, Fernandez JA, Espana F, Aznar J, Estelles A (1988) Physiological coagulation inhibitors (protein S, protein C and antithrombin III) in severe preeclamptic states and in users of oral contraceptives. Thromb Res 49:319-329 27. Gips H (1986) Endokrinologie der Schwangerschaft. In: Kfinzel W, Wulf KH (eds) Klinik der Frauenheilkunde und
Geburtshilfe, vol 4. Die normale Schwangerschaft. Urban & Schwarzenberg, Munich, pp 351-371 28. Gladson CL, Schleef RR, Binder BR, Loskutoff D J, Griffin JH (1989) A comparison between activated protein C and des-l-41-1ight chain-activated protein C in reactions with type 1 plasminogen activator inhibitor. Blood 74:173 181 29. Gonzalez R, Alberca I, Vicente V (1985) Protein C levels in late pregnancy, postpartum and in women on oral contraceptives. Thromb Res 39:632640 30. Grimaudo V, Gueissaz F, Hauert J, Sarraj A, Kruithof EKO, Bachmann F (1989) Necrosis of skin induced by coumarin in a patient deficient in protein S. Br Med J 298:233 234 31. Heeb MJ, Mosher D, Griffin JH (1989) Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascutar coagulation. Blood 73:455-461 32. Heidinger K, Kemkes-Matthes B, Matthes KJ (1991) Ver/inderungen antikoagulatorisch wirksamer Plasmaproteine bei entz/indlichen Erkrankungen. Klin Wochenschr 69 [Suppl XXIII] : 130 33. Hesselvik JF, Malm J, Dahlb/ick B, Blombfick M (1991) Protein C, protein S and C4b-binding protein in severe infection and septic shock. Thromb Haemost 65:126-129 34. Hillarp A, Dahlb~ick B (1987) The protein S-binding site localized to the central core of C4b-binding protein. J Biol Chem 262:11300-11307 35. Hillarp A, Hessing M, Dahlb/ick B (1989) Protein S binding in relation to the subunit composition of human C4b-binding protein. FEBS Lett 259:53-56 36. Huisveld IA, Hospers JEH, Meijers JCM, Starkenburg AE, Erich WBM, Bouma BN (1987) Oral contraceptives reduce total protein S, but not free protein S. Thromb Res 45:109114 37. Jefl¥ies WS, Bochner F (1991) Thromboembolism and its management in pregnancy. Med J Aust 155:253558 38. Jespersen J, Gram J, Bertina RM (1989) The risk of thrombosis in hereditary protein S deficiency in a Scandinavian family. Fibrinolysis 3:37-40 39. Jorens PG, Hermanns CR, Haber I, Kockx MM, Vermylen J, Parizel GA (1990) Acquired protein C and S deficiency, inflammatory bowel disease and cerebral arterial thrombosis. Blut 61:307-310 40. Keeling DM, Campbell SJ, Mackie IJ, Machin SJ, Isenberg DA (1990) Total and free protein S in systemic lupus erythematosus. Thromb Res 60:23%240 41. Kemkes-Matthes B, Matthes KJ (1985) Zusammenhfinge zwischen Gerinnungsfaktoren und anderen Plasmaproteinen bei chronischen Lebererkrankungen. Med Welt 36:1223-1227 42. Kemkes-Matthes B, Matthes KJ (1988) Protein S bei Lebererkrankungen. Klin Wochenschr 66 [Suppl XIII] : 64 43. Kemkes-Matthes B, Matthes KJ (1989) Ver/inderungen antikoagulatorisch wirksamer Plasmaproteine bei Lebererkrankungen. Zeitschr Gastroenterot XXVII : 37 44. Kemkes-Matthes B, Matthes KJ (1990) Beziehungen zwischen Protein S und C4b-Bindungsprotein bei akuten und chronischen Lebererkrankungen. Med Welt 41:923-926 45. Kemkes-Matthes B, Heinrich D, Dapper F, Lasch HG (1988) Kumarinnekrose bei Protein C-Mangel. Med Welt 39 : 72-75 46. Kemkes-Matthes B, Matthes KJ, Pralle H (1988) Protein S bei metastasierten Carcinomen und Lymphomen. Klin Wochenschr 66 [Suppl XIII] : 269 47. Kemkes-Matthes B, Matthes KJ, Pralle H (1989) Protein S in pulmonary and intestinal cancer patients. Thromb Haemost 62: 273
534 48. Kemkes-Matthes B, Pralle H, Matthes KJ (1989) Protein S - Zusammenh~inge zwischen Thromboseneigung und Metastasierung bei Karzinompatienten. Klin Wochenschr 67 [Suppl XVI] : 165 49. Kemkes-Matthes B, Plusczyk T, Lasch HG (1992) Coagulation inhibitors in pulmonary cancer patients. Thromb Res (manuscript submitted) 50. Madden RM, Gill JC, Marlar RA (1990) Protein C and protein S levels in two patients with acquired purpura fulminans. Br J Haematol 75:112-117 51. Malta J, Laurel1 M, Dahlbfick B (1988) Changes in the plasma levels of vitamin K-dependent proteins C and S and of C4b-binding protein during pregnancy and oral contraception. Br J Haematol 68: 437~443 52. Manucci PM, Vigano S (1982) Deficiencies of protein C, an inhibitor of blood coagulation. Lancet II:463-466 53. McGehee WG, Klotz TA, Epstein D J, Rapaport SI (1984) Coumarin necrosis associated with hereditary protein C deficiency. Ann Int Med 100:59-60 54. Melis GB, Fruzzetti F, Ricci C, Carmassi F, Fioretti P (1988) Oral contraceptives and venous thromboembolic disease: the effect of the oestrogen dose. Maturitas [Suppl] 1:131-139 55. Melissari E, Kakkar VV (1988) The effects of oestrogen administration on the plasma free protein S and C4b-binding protein. Thromb Res 49 : 489-495 56. Michiels JJ, Stibbe J, Bertina R, Broekmans A (1987) Effectiveness of long oral anticoagulation treatment in preventing venous thrombosis in hereditary protein S deficiency. Br Med J 295 : 641-643 57. Moreb J, Kitchens CS (1989) Acquired functional protein S deficiency, cerebral venous thrombosis, and coumarin skin necrosis in association with antiphospholipid syndrome: report of two cases. Am J Med 87:202210 58. Odegaard B, Mann K (1987) Proteolysis of factor Va by factor Xa and activated protein C. J Biol Chem 262:1123311238 59. Ogura M, Tanabe N, Nishioka J, Suzuki K, Saito H (1987) Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01). Blood 70:301306 60. Sack GH, Levin J, Bell W (1977) Trousseau's syndrome and other manifestations of chronic disseminated coagulopathy in patients with neoplasms: clinical, pathophysiologic, and therapeutic features. Medicine 56:1 37 61. Saeki T, Hirose S, Nukatsuda M, Kusunoki Y, Nagasawa S (1989) Evidence that C4b-binding protein is an acute phase protein. Biochem Biophys Res Commun 164:1446 1451 62. Sakata Y, LoskutoffDJ, Gladson CL, Hekman CM, Griffin JH (1986) Mechanism of protein C-dependent clot lysis: role of plasminogen activator inhibitor. Blood 68:12181223
63. Saleh AA, Bottoms SF, Welch RA, Ali AM, Mariona FG, Mammen EF (1987) Preeclampsia, delivery, and the hemostatic system. Am J Obstet Gynecol 157:331-336 64. Schved JF, Gris JC, Sarlat C, Amiral J, Brun S, Brunschwig C, Petris I, Lassonery M (1990) Study of quantitative modifications of the plasma protein S system components in nonacute inflammatory syndromes. Nouv Rev Hematol 32:271-276 65. Takahashi H, Takewaki W, Wada K, Shibata A (1989) Plasma protein S in disseminated intravascular coagulation, liver disease, collagen disease, diabetes mellitus, and under oral anti-coagulant therapy. Clin Chim Acta 182:195-208 66. Taylor FB Jr, Lockhart MS (1985) A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by release from mononuclear leucocytes-platelet suspensions stimulated by phorbol diester. Thromb Res 37:155-164 67. Troy K, Essex D, Rand J, Lema M, Cuttner J (1991) Protein C and S levels in acute leukemia. Am J Hemato137:159-162 68. Tsakiris DA, Settas L, Makris PE, Marbet GA (1990) Lupus anticoagulant-antiphospholipid antibodies and thrombophilia. Relation to protein C-protein S-thrombomodulin. J Rheumatol 17:785-789 69. Vigano-D'Angelo S, D'Angelo A, Kaufman CE, Sholer C, Esmon CT, Comp PC (1987) Protein S deficiency occurs in the nephrotic syndrome. Ann Int Med 107:42-47 70. Walker FJ (1984) Protein S and the regulation of activated protein C. Semin Thromb Hemost 10:131-138 71. Walker FJ, Chavin SI, Fay PJ (1987) Inactivation of factor VIII by activated protein C and protein S. Arch Biochem Biophys 252:322-328 72. Warwick R, Hutton RA, Goff L, Letsky E, Heard M (1989) Changes in protein C and free protein S during pregnancy and following hysterectomy. J R Soc Med 82:591-594 73. Wyshock E, Caldwell M, Crowley JP (1988) Deep venous thrombosis, inflammatory bowel disease, and protein S deficiency. Am J Clin Pathol 90:633-635 74. Zoppi M, Furlan M, Brun del Re G, Wuillemin W, L/immle B (1990) Decreased free protein S levels in polycythemia vera. Thromb Haemost 64:177-178
Received: September 17, 1991 Returned for revision: December 18, 1991 Accepted:March 12, 1992
Dr. Bettina Kemkes-Matthes Zentrum ffir Innere Medizin Klinikstrasse 36 W-6300 Giel3en
Clin Investig (1992) 70:529-534
Molecular Medicine Review
© Springer-Verlag 1992
Acquired protein S deficiency B. Kemkes-Matthes Zentrum f~r Innere Medizin der Justus Liebig Universit/it Giel3en
Summary. Hereditary deficiencies of coagulation inhibitors like antithrombin III, protein C and protein S lead to an enhanced incidence of thromboembolic complications. Recently, acquired deficiencies of protein S were described in several disease states in which thromboembolic complications frequently occur. These acquired protein S deficiencies reach - in part - the extent realised by hereditary protein S deficiency. Thus, acquired protein S deficiencies seem to be one source of thromboembolic complications occurring in nephrotic syndrome, acute phase reactions, malignancy and pregnancy. In this presentation disease states accompanied by acquired protein S deficiency and the mechanisms leading to these alterations are discussed.
cascade and behaves like an acute phase reactant [61]. Recently, acquired deficiencies of protein S have been described in nephrotic syndrome, malignancy, oral contraceptive treatment and pregnancy. It is well-known that patients with nephrotic syndrome or malignant diseases as well as women taking oral contraceptives or during pregnancy have an increased risk of suffering from thromboembolic complications. Thus, acquired deficiencies of protein S are described in several disease states. Dependent upon the underlying disorders, distinct mechanisms leading to protein S deficiency are discussed.
Synthesis dysfunction
Key words: Acquired protein S deficiency Thrombosis
Hereditary deficiencies of coagulation inhibitors AT III, protein C and protein S [5, 6, 10, 11, 18, 38, 56] are known to be risk factors for thromboembolic diseases. Protein C and protein S are vitamin-K-dependent proteins. After activation [8, 19-21] by thrombin/thrombomodulin, protein C acts as a major inhibitor of blood coagulation: it degrades Factors Va and VIIIa [8, 58, 71] and additionally has profibrinolytic properties [9, 17, 28, 62, 66] (Fig. 1). In contrast to AT III and protein C, only about 40% of protein S, the so-called free protein S, acts as a coagulation inhibitor: free protein S has a cofactor function for activated protein C concerning the degradation of Factors Va and VIIIa [70]. The remaining 60% of protein S is bound to C4bbinding protein (C4b-BP) and does not have any function in the coagulation cascade [14, 34, 35]. C4b-BP is a regulatory protein of the complement Abbreviations: AT I I I = antithrombin III; C4b-BP = C4b-binding protein; DIC=disseminated intravascular coagulation; SLE = systemic lupus erythematosus
Liver disorders
Liver protein synthesis dysfunction occurs in severe liver diseases, leading to a diminution of all plasma proteins synthesized by the liver. In severe liver disorders, a diminution of the vitamin K-dependent protein S down to 67% of the normal level in patients with decompensated liver cirrhosis can be observed [15, 42-44].
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~
Endothelial surface
Fig. 1. The protein C-protein S system. Protein C is activated by thrombin/thrombomodulin complex at the endothelial surface. Activated protein C inhibits factors Va and VIIIa; free protein S acts as cofactor. Protein S bound to C4b-binding protein (C4b-BP) has no cofactor function for activated protein C. Mutual inhibition exists between activated protein C (protein Ca) and factors PAI-I and PAI-3 (plasminogen activator inhibitors)
530 The decrease of protein S in liver diseases is not as strong as the diminution of other coagulation inhibitors like protein C and AT III [41, 65], because protein S is not only synthesized by the liver but also by endothelial cells [22] and megakaryocytes [59]. Severe liver disorders lead to a diminution of procoagulatory active coagulation factors as well as of coagulation inhibitors. Therefore, thromboembolic complications are extremely rare in liver diseases, while bleeding complications, which are, in addition to the diminution of coagulation factors, due to portal hypertension and thrombocytopenia, are relatively frequent problems. C4b-BP does not play an important role concerning alterations of the protein S levels in liver disorders [44] : patients suffering from inflammatory liver diseases tend to present elevated C4b-BP levels, and patients with very severe liver diseases like cirrhosis have slightly reduced C4b-BP levels, but no significant differences between these groups were found [44]. Takahashi et al. [65] found reduced levels of total and free protein S as well as of C4b-BP in patients suffering from liver disorders, but he did not give the results for the distinct groups. Treatment with coumarin derivatives
Coumarin treatment as well as vitamin K deficiency results in a diminution of prothrombin complex, proteins C and S. During stable ant±coagulation, no thrombotic tendency results because both procoagulatory active coagulation factors and coagulation inhibitors are diminished. In hereditary protein C deficiency, an increased risk exists of suffering from coumarin necrosis [4, 7, 45, 53]: during the first days of coumarin treatment, a very rapid decrease of protein C occurs because of its short half-life in comparison with the procoagulatory active coagulation factors. Coumarin necrosis is also known to occur in protein S deficiency [30].
Enhanced consumption/loss Enhanced consumption
During disseminated intravascular coagulation (DIC), consumption of coagulation factors and coagulation inhibitors AT III and protein C occurs. Alterations of protein S in DIC are still being discussed controversely. Takahashi et al. [65] found normal free and total protein S levels in 39 patients with DIC, while the C4b-BP levels were elevated up to 149.5% ±
33.8% of normal. These results are confirmed by the observations of Hesselvik et al. [33], who also found normal free and total protein S levels in 3 patients with DIC due to septic shock. The C4bBP levels in these patients were 118%+_40% of normal. In contrast, D'Angelo et al. [15] observed normal total (112% of normal) and reduced free protein S levels (80% of normal) in 20 patients with laboratory evidence of DIC secondary to different clinical conditions (obstetrical, posttraumatic, infective). C4b-BP was elevated up to 150% of normal in these patients. Low total protein S levels during DIC, except in patients suffering from malignancy, were observed by Heeb et al. [31]. Madden et al. [50] examined 2 patients suffering from purpura fulminans and DIC and concluded protein S to be consumed during DIC. The observations concerning the behaviour of protein S in DIC patients are not quite comparable, because free protein S, total protein S and C4b-BP were not measured by all authors, and different methods were used. Alterations of the protein S level in D]C seem to be dependent upon the disorder leading to DIC : Heeb et al. [31] observed reduced total protein S levels in patients suffering from infectious diseases and DIC, while patients suffering from malignancy and DIC presented with normal total protein S levels. Elevated C4b-BP levels and reduced protein S activity [15] in DIC patients led to the conclusion that increases of C4b-BP cause a shift from free to bound protein S, resulting in a thrombotic tendency. Loss
In the nephrotic syndrome, an enhanced risk of suffering from thromboembolic complications is recognised. Vigano-D'Angelo et al. [69] observed low free protein S levels ( 6 9 % ! 2 7 % of normal) in patients suffering from nephrotic syndrome despite having elevated levels of total protein S antigen (139%___42% of normal). He could demonstrate an urinary loss of free protein S and increases of C4b-BP up to 170% ± 52% of normal, leading to a shift from free to bound protein S, resulting in a pronounced decrease of free protein S. Thus, in the nephrotic syndrome, two causes for thrombosis risk due to lowered free protein S levels are f o u n d urinary loss of low molecular weight free protein S as well as increased high molecular weight C4b-BP-bound protein S. This leads to alterations discussed in the next paragraph on C4b-BP-induced alterations.
531 C4b-BP-induced alterations
Inflammatory diseases In patients suffering from systemic lupus erythematosus (SLE), significantly reduced free protein S levels were found [40, 57]. Keeling et al. [40] observed free protein S levels in SLE patients to be lower (0.21 U/ml) in patients presenting without than in patients with (0.25 U/ml) phospholipidspecific antibodies. These antibodies cause an additional down-regulation of the protein C-protein S system by inhibiting the catalytic activity of thrombomodulin [24, 68]. Moreb and Kitchens [57] observed 2 patients with SLE and unusual thrombosis. They found reduced concentrations of free protein S (2.0 and < 1.0 gg/ml; normal range 3.5 8.5 gg/ml) because of excessive binding to C4b-BP at the time the thrombosis occurred. Comparable to the results of Moreb and Kitchens [57], SLE patients examined by my own group [32] showed a tendency to reduced free and elevated bound protein S levels, but there was no significant difference versus the normal control group because of wide standard deviations. In patients suffering from inflammatory diseases like SLE, sepsis, pneumonia and fever, a highly positive correlation was found between bound protein S and C4b-BP [32], indicating that C4b-BP plays an important role concerning the distribution between the free and the bound protein S fraction. In contrast, Schved et al. [64] examined patients suffering from non-acute inflammatory syndromes including patients with suppuration, tuberculosis, cancer and rheumatoid arthritis. They found a tendency to increased levels of free protein S and C4b-BP, yet the extremely high standard deviations do not allow definite conclusions. Transient deficiencies of protein S, protein C and AT III were observed by Jorens et al. [39] in a patient suffering from colitis ulcerosa who experienced cerebral arterial thrombosis. Wyshock et al. [73] observed a patient with severe inflammatory bowel disease complicated by 10 episodes of deep venous thrombosis and nonfamilial deficiency of protein S.
We could demonstrate reduced free protein S levels in patients suffering from metastasising carcinoma [46-48]. The decrease of free protein S was dependent upon the severity of the disease [48]: patients without metastases showed normal (48%_+11% of normal total protein S), patients with local metastases slightly (39%_+ 8% of normal total protein S) and patients with disseminated malignant disease significantly decreased free protein S levels (32%_+ 10% of normal total protein S ; normal range 44% _+11% of normal total protein S). Patients with malignancy and thromboembolic complications showed the lowest free protein S levels: 23%_+ 3% of normal total protein S. In metastatic disease, the diminutions of free protein S reached protein S levels known in hereditary protein S-deficient patients. In malignant diseases, the distribution between free and bound protein S seems to be regulated by C4b-BP [49]: in patients suffering from pulmonary carcinoma, a highly positive correlation was found between bound protein S levels and C4b-BP. Total protein S was slightly elevated in cancer patients examined by my group, but there was no significant difference to the healthy control group [46-48]. These observations confirm the results of Heeb et al. [31]. Troy et al. [67] found normal total and significantly decreased levels of free protein S in patients suffering from acute myelocytic leukemia compared with those in remission, while patients with acute lymphocytic leukaemia presented with normal free protein S levels. Low free and total protein S levels were observed in 2 patients with essential thrombocythemia and thrombosis [13], but no information was given concerning C4b-BP levels. To my knowledge, no further publications exist concerning protein S levels in cancer patients except for one [74] on polycythemia vera patients, who are known to suffer frequently from arterial or venous thrombotic complications; low free protein S levels were observed in 5 cases [74]. In contrast to our results in carcinoma patients, C4b-BP levels in these patients were within the normal range. Oestrogen-dependent alterations
Malignancy
Pregnancy
More than 100 years ago, Trousseau observed patients suffering from malignancy to have an increased risk of suffering from thrombotic complications [60]. The source of the so-called Trousseau's phenomenon is not completely clarified.
In pregnancy, an enhanced risk of suffering from thromboembolic complications exists [37]. This thrombotic tendency is related in part to immobilization during the last weeks of pregnancy and/or to (partial) compression of the vena cava inferior.
532
No alterations of coagulation factors or coagulation inhibitors such as AT III and protein C were found to be the cause for thromboembolic complications during pregnancy [16, 26, 29, 52, 63, 72]. However, decreasing levels of total and free protein S during pregnancy were observed recently [12, 16, 23, 26, 51, 72]: at delivery total and free protein S are diminished down to 50% of the normal range and reach normal values about 1 week later. Thus, during the last trimester of pregnancy, the decrease of protein S is comparable to the diminution in hereditary protein S-deficient patients. This is one explanation for the enhanced risk of suffering from thromboembolic complications during pregnancy. The cause for the decrease of total and free protein S during pregnancy is not yet clarified; Malta et al. [51] observed increasing levels of C4bBP up to 143% at delivery, which seem to downregulate free protein S values, while Comp et al. [12] did not find any alterations of C4b-BP during pregnancy. Yet it has to be noticed that C4b-BPinduced alterations of protein S cause an increase of bound and a decrease of the free protein S fraction, whereas the typical alteration of protein S during pregnancy is a diminution of the total, bound as well as free protein S fractions. Therefore, increased C4b-BP levels during pregnancy do not seem to be the main cause for decreasing values of free protein S. Furthermore, in pregnancy an increase of plasma oestrogen levels occurs [27], which might cause a decrease of protein S levels, similar to that discussed under oral contraceptive treatment.
Oral contraceptives and postmenopausal oestrogen substitution Women using oral contraceptives, too, have an increased risk of developing thromboembolic complications [3, 25, 54]. Boerger et al. [2] and Malta et al. [51] observed decreased total and free protein S levels in women taking oral contraceptives, while Huisfeld et al. [36] only found a reduction of total ( 8 4 . 5 % + 1 9 % ; normal range 111.7%+_21.8%) but not of free protein S (94.3% _+18.0% ; normal range 104.6% -t- 15.2%). Melissari and Kakkar [55] examined women using oral contraceptives in comparison with normal and postmenopausal women on a hormone replacement therapy with oestradiol valerate. They concluded that there is a relationship between steroid dosage and duration of treatment and the decrease of free protein S. These results are confirmed by my own observations (unpublished data) that in oral contraceptive users free
protein S levels were reduced depending upon the daily oestrogen dose. Concerning C4b-BP, no significant alterations were found in women taking oral contraceptive treatment in comparison with normal controls [2, 55]. Thus, the regulation of free protein S levels in oral oestrogen treatment and during pregnancy seems to be dependent upon plasma oestrogen levels. Conclusion
In this presentation distinct mechanisms leading to acquired protein S deficiency with or without thromboembolic tendency were discussed. No thromboembolic risk situations result in acquired protein S deficiency due to protein synthesis dysfunction, such as severe liver diseases and coumarin anticoagulation. In contrast, patients suffering from nephrotic syndrome have a high risk of experiencing thromboembolic complications. In these patients, a urinary loss of free protein S was observed. This, together with increased C4b-BP levels, results in a pronounced decrease of free protein S. Increase of C4b-binding protein in acute phase reactions induce a decrease of free protein S, possibly leading to thromboembolic events. C4b-binding protein-dependent alterations of protein S are typical in acute inflammatory diseases and in malignancy. Furthermore, plasma protein S levels seem to be oestrogen-dependent: in women on oestrogen medication such as oral contraceptives and during pregnancy, decreased free and total protein S levels were found. Thus, the regulation of protein S levels depends upon several different mechanisms. References 1. Bezeaud A, Venisse L, Conard J, Horellou MH, Salomon JL, Lebaleur A, Samama M, Guillin MC (1987) D6ficit h6r6ditaire en prot+ine S e t thromboses veineuses r6cidivantes. Presse Med 16 : 1895-1897 2. Boerger LM, Morris PC, Thurnau GR, Esmon CT, Comp PC (1987) Oral contraceptives and gender affect protein S status. Blood 69:692-694 3. B6ttiger LE, Boman G, Eklund G, Westerholm B (1980) Oral contraceptives and thromboembolic disease: effects of lowering oestrogen content. Lancet h 1099-1101 4. Broekmans AW, Bertina RM, Loeliger EA, Hofmann V (1983) Protein C and the development of skin necrosis during anticoagulant therapy. Thromb Haemost 49 : 244 5. Broekmans AW, Bertina RM, Reinalda-Poot J, Engesser L, Muller HP, Leeuw JA, Michiels JJ, Brommer EJP, Brier E (1985) Hereditary protein S deficiency and venous throm-
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Received: September 17, 1991 Returned for revision: December 18, 1991 Accepted:March 12, 1992
Dr. Bettina Kemkes-Matthes Zentrum ffir Innere Medizin Klinikstrasse 36 W-6300 Giel3en
