Free Protein S Levels Are Elevated in Familial C4b-Binding Protein Deficiency

By Philip C. Comp, Judith Forristall, Clark D. West, and Robert G. Trapp

In plasma, 40% of the protein S is free and functions as a cofactor for the anticoagulant effects of activated protein C. The remaining 60% of protein S is complexed to C4b-binding protein and is functionally inactive. A family with hereditary C4b binding protein deficiency has been identified with C4b-binding protein levels in an affected father and daughter of 37 pg/mL and 23 pg/mL. respec- tively; these values are significantly below the normal range for this protein of 180 pg/mL f 4 4 pg/mL (mean & 2 SD). The total protein S (free + bound) is normal in these

ROTEIN S IS A COFACTOR for the anticoagulant P effects of activated protein C.’ In normal individuals approximately 40% of protein S is free in plasma and is functionally active. The remaining 60% is complexed to C4b-binding protein, an inhibitor of the complement system, and is functionally ina~ t ive .~ -~ Patients with decreased levels of protein S have an increased risk of thromboembolic disease.’.’

C4b-binding protein inhibits the classic complement cas- cade by blocking the formation and promoting the decay of the C3 convertase, C4bC2a. C4b-binding protein binds C4b and makes the C4b susceptible to cleavage by the protease factor I.* The C4b-binding protein-protein S complex also promotes the degradation of C4b by factor I.9

The physiologic significance of the C4b-binding protein- protein S complex is unknown. It has been proposed that the association of C4b-binding protein with protein S may facilitate the binding of C4b-binding protein to membrane surfaces, which display negatively charged phospholipid groups and thus serve to regulate activation of the classic complement pathway on or near the membrane surface.” Protein S bound to C4b-binding protein is resistant to leukocyte proteases.” This resistance has prompted the suggestion that the complex formation of protein S and C4b-binding protein may protect protein S from proteolytic degradation at sites of inflammation. C4b-binding protein is an acute phase p r~ te in~ . ’~ ; acute elevation of C4b-binding protein can result in a reduction in free protein S level^.'^ However, it is unknown how the concentration of C4b- binding protein-protein S complex affects the free protein S level, or the total protein S level, under steady state condi- tions. The discovery of a family with hereditary C4b-binding protein defi~iency’~ permits the evaluation of protein S status in the absence of the C4b-binding protein-protein S complex.

METHODS

C4bbinding protein and protein S were determined by radioimmu- noassay as previously described.’* Free protein S was determined by the polyethylene glycol precipitation previously de~cribed,’~ followed by radioimmunoassay to measure the free protein S. Qualitative estimation of the distribution of protein S between free and com- plexed forms was made by crossed immunoelectrophoresis as previ- ously described.I5

Case history. The proband is a 33-year-old white woman who presented with facial pain and swelling to the Southern Illinois University School of Medicine (Springfield), and details of the case history have been previously reported.14 In summary, the patient

individuals (23.2 pg/mL and 17.8 pg/mL, respectively: normal 19.1 pg/mL + 6.0 pg/mL). The free protein S levels are markedly increased at 22.5 pg/mL and 17.4 pg/mL, respectively (normal 5.9 pg/mL & 2.4 pg/mL). This experi- ment of nature shows that total protein S levels in plasma are not affected by the absence of C4b-binding protein and that chronic elevation of free protein S is not associated with increased hemorrhagic tendencies. 0 1990 by The American Society of Hematology.

experienced repeated episodes of ulcers on the soft pallet, cutaneous vasculitis with evidence of immunoglobulin M (IgM) and C3 deposition on the basement membrane of arterioles, and synovitis. Antinuclear antibodies, anti-DNA, and rheumatoid factor were negative. Complement components Clq, C2, C3, C4, C5, C6, C7, C8, C9, factor B, factor I, factor H, and C1-inhibitor were normal. Properdin was 1.6 mg/dL (normal range 1.7 to 3.5 mg/dL). The proband, her asymptomatic father, and asymptomatic sister were found to be deficient in C4b-binding protein (Fig 1). Family members have not experienced increased bleeding or bruising. The proband previously underwent cholecystectomy and two cervical laminectomy procedures without excessive blood loss and did not require transfusion with any of these procedures. The proband did not exhibit excessive bleeding from skin biopsy sites.

RESULTS

Crossed immunoelectrophoresis of the proband’s plasma showed marked elevation of free protein S (Fig 2). The free protein S of the patient and her father exhibited a migration pattern identical with that observed for the free protein S in normal plasma. The small peak located between the free protein S and the site of sample application was observed in the patient’s plasma in a location anticipated for the C4b- binding protein-C4bprotein S complex.

The levels of free and total protein S and C4b-binding protein for the proband and her father are shown in Table 1.

From the Department of Medicine, University of Oklahoma Health Sciences Center, and the Oklahoma Medical Research Foundation, Oklahoma City, OK: the Southern Illinois University School of Medicine, Springfield: and Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH.

Submitted May 12, 1989: accepted August 27,1990. Supported by Grant Nos. HL30443 (to P.C.C.). and AM28315

and AM26279 (R.G.T.) from the National Institutes of Health. Presented in abstract form at the 61st Scientific Sessions of the

American Heart Association, Washington DC, 1988. Circulation, Part II78:319, 1988.

Address reprint requests to Philip C. Comp. MD. PhD, Depart- ment of Medicine, University of Oklahoma Health Sciences Center, 825 NE 13th St. Oklahoma City. OK 73104.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

0 1990 by The American Society of Hematology. 0OO6-4971/90/7612-0012$3.00/0

Mood, Vol76, No 12 (December 15). 1990: pp 2527-2529 2527

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COMP ET AL

Table 1. Total Protein S. Free Protein S. and C4b Binding Protein Levels

I Normal 180 f U pg/ml Mean f 2SD

Fig 1. C4b binding protein levels of the deficient family. The proband‘s mother was not available for testing.

The total protein S levels were normal. As anticipated from the crossed immunoelectrophoresis pattern, the free protein S levels were markedly increased in both father and daughter at 22.5 pg/mL and 17.4 pg/mL, respectively. C4b-binding protein levels were markedly decreased in the proband and her father at 23 pg/mL and 37 pg/mL (normal range 158 to 202 pg/mL). One sister had normal levels of C4b binding protein (142 pg/mL) and a second sister had low levels of C4b binding protein (38 pg/mL). The proband’s protein S functional activity was 220% determined as previously described.13 The proband’s activated partial thromboplastin time (aPlT) was within normal limits, the factor VI11 activity was 92% (normal range 62% to 154%), and the factor V activity was 104% (normal range 56% to 128%). Protein C antigen and protein C functional (chromogenic)

Normal Proband Father (mean f 2 SD)

Total S (pg/mL) 17.0 23.2 19.1 ~t: 6.0 Free S (pg/mL) 17.4 22.5 5.9 * 2.4 C4bBP (pg/mL) 23 37 180 f 44

activity were 90% and 8496, respectively, performed as previously described.16

DISCUSSION

Total protein S levels are normal in this C4b-binding protein deficient family. At least 95% of the protein S appears present as the free protein. The absence of C4b- binding protein and the C4b-binding protein-protein S com- plex has no appreciable effect on the total plasma protein S levels. The protein S status of the family here described suggests that the regulation of plasma protein S is based on the total protein S level. Because C4b-binding protein displays the characteristics of an acute phase protein,” inflammation could increase the C4b-binding protein level and shift the protein S to the bound and inactive form. This shift would result in a decrease in protein S activity in the plasma and the downregulation of the protein C system. A decrease in free protein S have been observed during acute inflammati~n.’~ The response of both total and free plasma protein S levels to prolonged inflammatory challenges will require further investigation.

Exogenous activated protein C and protein S may prove to be useful as systemic anticoagulants’* and in the treatment of septic shock.” The use of any anticoagulant is accompanied by potential bleeding complications. There is presently no information available in humans concerning the risks of bleeding following protein C or protein S administration. The

Fig 2. Crossed immunoelectrophoresis for protein S. The crossed immunoelectrophoresis patterns of normal plasma (left). the proband (middle). and her father (right) are shown. In the normal plasma. the free protein S peak migrates more rapidly toward the positiie electrode (+) than the protein S. which is in complex with C4b binding protein. The protein S of the proband and father is predominantly in the free form.

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PROTEIN S IN C46-BINDING PROTEIN DEFICIENCY 2529

family reported here with high free protein S levels has no history of bleeding complications. This lack of complication history suggests that chronic elevation of plasma free protein S levels to three to four times normal is relatively safe in terms of risk of hemorrhage.

REFERENCES

1. Walker FJ: Regulation of activated protein C by a new protein. A possible function for bovine protein S. J Biol Chem 255:5521, 1980

2. Dahlback B: Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S. Biochem J 209:847,1983

3. Comp PC, Nixon RR, Cooper MR, Esmon C T Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 742082,1984

4. Dahlback B: Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 261:12022,1986

5. Comp PC, Esmon CT: Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med 311:1525,1984

6. Schwarz HP, Fischer M, Hopmeier P, Batard MA, Griffin JH: Plasma protein S deficiency in familial thrombotic disease. Blood 641297,1984

7. Broekmans AW, Bertina RM, Reinalda-Poot J, Engesser L, Muller HP, Leeuw JA, Michiels JJ, Brommer EJ, Briet E: Heredi- tary protein S deficiency and venous thrombo-embolism. A study in three Dutch families. Thromb Haemost 53:273, 1985

8. Fujita T, Gigli I, Nussenzweig V: Human C4-binding protein. 11. Role in proteolysis of C4b by C3b-inactivator. J Exp Med 148:1044,1978

9. Dahlback B, Hildebrand B: Degradation of human comple-

ment component C4b in the presence of the C4b-binding protein- protein S complex. Biochem J 209:857, 1983

10. Dahlback B: Interaction between vitamin K-dependent pro- tein S and the complement protein, C4b-binding protein. A link between coagulation and the complement system. Semin Thromb Hemost 10139,1984

11. Nukatsuka M, Nagasawa S: Characterization of the interac- tion between human protein S and C4bbinding protein (C4bp). J Biochem (Tokyo) 102:599,1987

12. Boerger LM, Morris PC, Thurnau GR, Esmon CT, Comp PC: Oral contraceptives and gender affect protein S status. Blood 69:692,1987

13. D’Angelo A, Vigano-D’Angelo S, Esmon CT, Comp PC: Acquired deficiencies of protein S: Protein S activity during oral anticoagulation, in liver disease and in disseminated intravascular coagulation. J Clin Invest 81:1445, 1988

14. Trapp RG, Fletcher C, Forristall J, West C D C4 binding protein deficiency in a patient with atypical Behcet’s disease. J Rheumatol 14:135,1987

15. Comp PC, Doray D, Patton D, Esmon C T An abnormal plasma distribution of protein S occurs in functional protein S deficiency. Blood 67:504, 1986

16. Vigano-DAngelo S, Comp PC, Esmon CT, DAngelo A: Relationship between protein C antigen and anticoagulant activity during oral anticoagulation and in selected disease states. J Clin Invest 77:416, 1986

17. Kurosawa S, Galvin JB, Esmon NL, Esmon CT: Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem 262:2206,1987

18. Comp PC: Clinical implications of the protein C/protein S system. Ann NY Acad Sci 509:149,1987

19. Taylor FB Jr, Chang A, Esmon CT, DAngelo A, Vigano- D’Angelo S, Blick KE: Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 79:918, 1987

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PC Comp, J Forristall, CD West and RG Trapp deficiencyFree protein S levels are elevated in familial C4b-binding protein 

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