Protein S Deficiency and Skin Necrosis Associated With Continuous Ambulatory Peritoneal Dialysis

Protein S Deficiency and Skin Necrosis Associated With Continuous Ambulatory Peritoneal Dialysis K. Shashi Kant, MD, Helen I. Glueck, MD, Macie C. Coots, MS, Vonnie A. Tonne, BS, MT (ASCP), Ronald Brubaker, MD, and Israel Penn, MD (Editor's Note: Editorial review was performed by Dr John Curtis in Birmingham)

• Skin necrosis associated with protein C deficiency has recently been reported to occur in hemodialysis patients. The clinical presentation and course of this syndrome appears indistinguishable from skin necrosis (purpura fulminans) seen in other settings with inherited or acquired deficiency of the naturally occurring anticoagulant proteins, protein C and S. Patients on maintenance hemodialysis may have low levels of these factors. However, patients on peritoneal dialysis have normal or elevated levels of these proteins despite documented peritoneal losses. We report two patients in whom the occurrence of protein S deficiency and subsequent skin necrosis can be related to demonstrated peritoneal dialysis-associated losses. We suggest that these losses may become critical under appropriate conditions and suggest caution in peritoneal dialysis patients requiring warfarin therapy. © 1992 by the National Kidney Foundation, Inc. INDEX WORDS: Protein S; peritoneal dialysis; skin necrosis.

C

ONGENITAL DEFICIENCIES of the naturally occurring anticoagulant proteins C and S are well known. 1-4 Decreased levels of these naturally occurring vitamin K-dependent inhibitors are associated with recurrent thromboembolic disorders. I -6 Coumarin therapy decreases both proteins C and S and may result in skin necrosis when administered to patients with already compromised levels of those factors. 7 Patients with congenital defects can likewise develop skin necrosis when given coumarin. 8,9 Skin necrosis has recently been reported in five patients with calciphylaxis on maintenance hemodialysis who were not receiving coumarin.1O Low levels of functional protein C were demonstrated in these patients. Our interest in the role of continuous ambulatory peritoneal dialysis (CAPD) was stimulated by observations on an earlier patient 11 with monoclonal IgA primary amyloidosis and a factor VIII:C inhibitor, which became manifest only after therapy was changed from peritoneal dialysis to hemodialysis. We showed that the inhibitor was removed by the peritoneal dialysate. CAPD is known to cause significant loss of a From the Departments ofMedicine-Nephrology, Pathology and Laboratory Medicine, and Surgery, College ofMedicine. University of Cincinnati. Cincinnati, OB. Address reprint requests to K. Shashi Kant, MD, Department of Medicine-Nephrology, College of Medicine, 231 Bethesda Ave. Cincinnati, OH 45267-0585. © 1992 by the National Kidney Foundation. Inc. 0272-6386/92/1903-0008$3.00;0 264

whole host of proteins in the dialysatey,13 A recent study measured plasma levels of protein C in patients undergoing hemodialysis and found functional protein C to be reduced. 10 We report two patients on CAPD with severe skin necrosis. The signal patient presented while on coumarin therapy. A depressed plasma level offree protein S was found. Moreover, these levels remained depressed for several months after warfarin was discontinued. The second patient, who had received no coumarin, likewise presented with skin necrosis and a low free protein S value. When the peritoneal dialysates of these two patients were concentrated and analyzed, both proteins C and S could be demonstrated. Subsequently, an additional five asymptomatic patients on CAPD were similarly studied. All of the latter group had normal plasma values with the exception of one who had a moderate depression of protein C. No evidence of systemic thrombosis or skin necrosis was found. Nevertheless, all seven of the peritoneal dialysates contained measurable quantities of both protein Sand C. These studies are the subject of the present communication. CASE REPORTS

Patient 1 Patient I, age 64, had a past history of rheumatic heart disease with aortic valve involvement, non-insulin-dependent diabetes for approximately 10 years, and hypertension for almost 40 years. When seen in 1987, she had symptoms relating to the valvular disease (left ventricular failure, syncope), and renal insufficiency with an elevated serum creatinine, 132.6

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mmoljL (1.5 mg/dL), attributed to diabetic nephropathy. Serologic studies showed evidence for both hepatitis A and B. Progressive left ventricular failure led to a porcine aortic valve replacement in January 1988. She tolerated the procedure well and was discharged on furosemide, verapamil, c10nidine, and warfarin. While on outpatient therapy, prothrombin times (PTs) were maintained at approximately 1.5 times the control level of 13 seconds. Renal function deteriorated progressively over the next 9 months, and she was readmitted in October 1988. Serum creatinine was 344.7 mmoljL (3.9 mg/dL), the antinuclear antibody test was negative, and the complement profile was normal. Renal biopsy confirmed diabetic glomerulosclerosis and some suggestive features of acute interstitial nephritis (tubulorrhexis, foci of interstitial infiltration by polymorphs, lymphocytes, plasma cells, and eosinophils). Volume overload and azotemia necessitated initiating CAPO in December 1988. Over the ensuing 6 months, she did well except for one admission for fluid overload. In mid July 1989, she complained of pain in both lower legs, with findings of extensive superficial thrombophlebitis. A duplex venous scan ruled out deep venous thrombosis, and she was treated unsuccessfully with leg elevation, rest, nonsteroidal antiinflammatory agents, and warfarin. Surgical removal of the latter veins was performed on September 1, 1989. Heparin followed by warfarin was prescribed. However, 5 days postoperatively, she developed frank necrotic areas in the skin of the left medial thigh. Questions were raised about calciphylaxis. However, previous records showed reasonable phosphate control and parathyroid hormone (PTH) levels (midmolecule) that were only modestly elevated. Protein C and/or S deficiency was suspected because of the hemorrhagic necrotic lesions. Unequivocal evidence of protein S deficiency was obtained on September 22, 1989 (Table 1, patient I). Warfarin was discontinued on September 30, 1989. The necrotic areas in her upper thigh were treated with debridement and subsequent skin grafting. She was finally discharged on

November 2,1989. Medications included subcutaneous heparin (10,000 U every 12 hours), and CAPO. She returned approximately 2 weeks later while on heparin only. Two new areas of skin necrosis had developed in the upper thighs. Biopsies of involved skin were obtained, and she was administered a short course of prednisone. The peritoneal dialysate of December 5, 1989 was concentrated and studied, and CAPO was discontinued on December 7, 1989. Surgical treatment for the necrotic skin ulcers was again performed. She was changed to hemodialysis and nutritional support. However, the skin ulcers became superinfected, and she ultimately died of systemic fungal infection on January 23,1990.

Patient 2 Patient 2, age 48, with chronic glomerulonephritis, developed end-stage renal disease that required maintenance hemodialysis from March 1986 until April 1989, at which time she elected to change to CAPO. She did relatively well until March 1990, when she developed severe burning pain and violaceous skin lesions on the outer aspect of the left thigh. This episode was followed by several other such lesions. The underlying subcutaneous fat was indurated, very tender, and reminiscent of the lesions seen in patient 1. She referred herself to the dermatology clinic, where a punch biopsy showed mild septal panniculitis. When seen 2 weeks later in the nephrology clinic, the necrotic lesions were tender and required narcotic analgesics. Based on the earlier experience of patient 1, peritoneal dialysis was discontinued and samples of blood and dialysate were obtained. Within a few days the pain subsided and no new lesions developed. However, the subsequent development of infection in these lesions required hospitalization for debridement (May 1, 1990). During the next few days new lesions became evident on her calves. The PTH level was elevated at 29.6. Mean calcium phosphate product for the preceding 6 months was 72. A parathyroidectomy was performed on May 6, 1990. After several weeks of wound care with de-

Table 1. Coagulation Studies and Clinical Events in Two Patients With Skin Necrosis Protein C%

Protein S%

Antigen (normal,

Functional (normal,

Total (normal,

Free (normal,

C..Bp% (normal,

Date

80-120)

70-150)

70-150)

65-135)

77-135)

Clinical Events

Patient 1 9/22/89 9/29/89 11/22/89 12/7/89 1/12/90

55 69 80 109 93

NO 75 NO NO NO

65 73 141 133 64

12.5 51 29 41 72

NO NO NO 209 NO

Warfarin stopped PTH 3.6 On heparin CAPO stopped Plasma, RBC transfused

Patient 2 4/27/90 5/18/90 6/8/90 9/21/90

113 96 157 132

109 132 NO NO

226 212 166 93

41 45 50 69

356 NO NO NO

CAPO stopped On hemodialysis Parathyroidectomy Skin necrosis resolved

Abbreviation: NO, not determined.

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bridement and antibiotic therapy, she recovered fully and was discharged on May 24, 1990.

METHODS The studies on the plasma and dialysate of these patients were approved by the institutional review board at the University of Cincinnati.

Pathology Tissue debridement including skin, subcutaneous tissue, and superficial veins was formalin-fixed, paraffin-embedded. Sections (4 Jl) were stained with hematoxylin-eosin, Verhoeff, Lendrum, and Mallory's trichrome, and examined by light microscopy.

Coagulation Routine studies included the PT and the partial thromboplastin time (PTT)'4 using Thromboscreen thromboplastin and Thromboscreen-Kontact, respectively (Pacific Hemostasis, Ventura, CA).

Antithrombin III Tests for antithrombin III used a Coatest kit manufactured by KabiVitrum Diagnostica (Helen Laboratories, Beaumont, TX). The method measures the heparin cofactor activity of antithrombin after the addition of both heparin and thrombin in excess. The antithrombin-heparin complex is formed and the residual thrombin then splits off the paranitroaniline from the chromogenic substrate S2238. Absorbancy is measured at 425 nm. The change in absorbance per minute is linear in the 5% to 125% range of normal plasma.

Parathyroid Hormone PTH levels were measured by a midmolecule PTH assay using an antibody obtained from Incstar, Minneapolis, MN (normal value, 0.3 to 1.08 mgjmL).

Total Protein S Total protein S is measured by EID in which the protein S migrated in an electric field in an agarose gel containing a specific antibody to protein S (Diagnostica Stago, Ansieressur-Seine, France). At the conclusion of the electrophoresis, the height of each rocket stained with Coomassie brilliant blue is directly proportional to the total protein S in the sample. Normal pooled plasma similarly treated serves as a control. Included in each run are normal and abnormal plasma controls supplied with the kit.

Free Protein S Free protein S measurement quantifies the protein S not bound to the C4b binding protein (C.bBP). The 300 JlL of citrate plasma is added to 25 JlL of 25% polyethylene glycol. This precipitates the C4bBP, leaving the free protein S in the supernate. Following this procedure, the free protein S is measured using the same kit employed for measuring the total protein S.

C4b Binding Protein C4~P measurement was performed using a rocket technique and an Assera-Plate C4bBP(Diagnostica Stago).

Concentration of Peritoneal Dialysate Before testing, the peritoneal dialysates were concentrated in an Amicon 8200 concentrator (Amicon Corp, Lexington, MA) with a Diaflo PM 10 ultrafiltration membrane. The average volume of fluid used for peritoneal dialysis was 9,400 mL/24 h. Samples of approximately 160 mL were concentrated to a final volume of 2 to 4 mL. The values for functional and antigenic protein C, free and total protein S and C4~P, were expressed as percent of normal plasma. The values for normal plasma used in the final calculations were protein C, 4 JlgjmL's; protein S, total 25 JlgjmL'6; protein S free, 10 Jlgj mL 16 ; and C4~P, 200 JlgjmL. 16

Protein C Antigen

Calculation for Protein C, S, and Binding Protein in the Peritoneal Dialysate

Protein C antigen was measured using a kit supplied by Helena Laboratories, and electroimmunodiffusion (EID) with agarose gels containing antisera specific for protein C. Following electrodiffusion, the rocket-shaped precipitin pattern, stained with Coomassie brilliant blue, is proportional to the protein C concentration. Dilutions of a normal pool are used to construct a standard curve for quantitation of protein C antigen in the patient's plasma or peritoneal dialysate. Normal and abnormal control plasma samples furnished by the manufacturer are likewise included.

To estimate the total loss of each in the peritoneal dialysate, the value expressed in percent of normal (from the standard plasma curve) was multiplied by the number of micrograms of each factor in I mL of normal plasma divided by the concentration factor of the peritoneal dialysate and finally multiplied by the average total volume of peritoneal dialysate, ie, 9,400 mL/24 h. Hence, the values for the peritoneal dialysate are expressed in Jlg lost/24 h: [Jlg lost/24 h = (% in normal plasma X Jlg offactor in I mL normal plasma) X 9,400 mL]/ concentration factor.

Functional Protein C Functional protein C was measured using the chromogenic substrate CBS 65.25 and an activator, a highly purified extract of the venom, Agkistrodon contortrix Stachrom Protein C (Diagnostica Stago, Ansieres-sur-Seine, France). Protein C is activated by the venom and the quantity thus formed is measured by its amidolytic activity on the chromogenic substrate CBS 65.25, which releases paranitroaniline.

RESULTS

Skin lesions began with tingling and burning. Red and/or violaceous patches appeared before the development of the lesions, which were exquisitely tender, somewhat elevated, and purpuric. The lesions shown in Fig 1 (patient 2) are typical of those observed in both patients.

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PROTEINS AND SKIN NECROSIS IN CAPO

Fig 1. Purpuric cutaneous lesions (patient 2) typical of those found in both patients. (Composite of four different lesions.)

Histologic findings were similar for both patients. On every occasion, there was extensive fat necrosis with variable degrees of inflammation. Small caliber vessels in the subcutaneous adipose tissue frequently showed prominent calcification of the vascular wall and, in multiple sites, contained luminal fibrin thrombi (patient 1, Fig 2). Extensive colonization of ulcerated skin by Aspergillus sp was noted on one occasion. Proteins C and S, and C4bBP were assayed using the electroimmunologic methods. Figure 3 shows the quantitation of protein S in the peritoneal dialysate and is representative of all of the gels. Table 1 .summarizes the therapy, clinical events, and measurements of plasma proteins C and S, and C4bBP in patients 1 and 2. Of note was the diminished free protein S plasma levels in both patients associated with relatively high levels of the C4~P. Of particular interest was the normalization of free protein S when patient 2 was changed from peritoneal dialysis to hemodialysis. The plasma values for free protein S of patients 1 and 2 were significantly decreased (Table 2). In contrast, patients 3 to 7, without skin necrosis who served as controls, had values for proteins C and S that fell within the normal range (Table 2), with the exception of patient 3 in whom functional protein C was marginal (Table 2). Anti-

thrombin III was within the normal range in all seven patients. Following concentration of the peritoneal dialysates (40- to 80-fold), it was possible to measure proteins C and S, and C4bBP, expressed as percent of normal plasma. The values appeared insignificant until the total quantities of each lost over a 24-hour period were calculated (Table 3). The depression of free protein S was noted between September 22, 1989 and January 12,1990 (patient 1, Table 2). The later normalization of protein S can be attributed to the transfusion of 2 U of fresh frozen plasma and 7 U of red blood cells on January 10 and 11, 1990, respectively. Normalization of these factors in patient 2 occurred only after changing to hemodialysis. DISCUSSION

The protein C anticoagulant pathway involves the generation of functional protein C at the vascular endothelium. Thrombin, acting via thrombomodulin in the presence of protein S as a cofactor causes the vascular endothelium to generate functional protein C from protein C antigen. Functional protein C acts as an anticoagulant via proteolytic inactivation of factors Va and VIlla, as well as by inactivating plasminogen activator inhibitor, a inhibitor of fibrinolysis. Defects in the protein C anticoagulant pathway may result from inherited deficiency of the protein C. 7-9 In-

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KANT ET AL

Fig 2. Extensive subcutaneous fat necrosis (patient 1). Small thrombosed vessel (arrow) is shown enlarged in inset. (Hematoxylin & eosin; original magnification X15. Inset, Mallory's trichrome; original magnification X150.)

dividuals with partial deficits of this protein are predisposed to skin necrosis during the early phases of coumarin therapy when there is a disproportionately rapid decrease in the vitamin Kdependent anticoagulant proteins. Other mechanisms for the development of skin necrosis include protein S deficiencyl? and endothelial injury produced by other factors such as gramnegative sepsis. Heparin may also result in skin necrosis.1 8,19 The usual association of thrombocytopenia suggests a role for clotting mediated small vessel injury and hence a deficient protein

C generation in heparin-associated skin necrosis. 18,20 The pathology of skin necrosis occurring in association with the above syndromes is identical-dermal venular thrombosis with overlying skin and fat necrosis due to hemorrhogic infarction. This implies a common pathogenesis-deficient levels of functional protein C leading to venular thrombosis. Until recently, protein C deficits associated with warfarin therapy were recognized as the leading cause of skin necrosis. However, other factors such as liver disease, dietary inadequacy

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Fig 3. Total protein 5 in peritoneal dialysate (patient 1). Left to right Wells 1 to 4, standard, normal pooled plasma, 100-12.5%; Wells 5 to 10, plasmas from unrelated patients; wells 11 to 14, patient 1's peritoneal dialysate: undiluted 1-2, 1-4, 1-8, respectively; well 15, universal normal control, undiluted; well 16, universal abnormal control.

with respect to vitamin K biliary obstruction, malabsorption, and the administration of broad spectrum antibiotics may all play a permissive role. 21 We can also add peritoneal dialysis-associated losses of anticoagulant proteins to the above list. Finally, functional protein C defects may be related to an acquired inhibitor of this protein. 2i Free protein S levels have been variably described to be 10w23 or elevated24,25 in patients with the nephrotic syndrome. Patients on CAPD have been found to have higher than normal levels of protein C and S.26,27

Protein S exists in the plasma in both bound and free (active) forms. The bound form exists in a complex with C4bBP. It is conceivable that elevated levels of C4bBP would cause decreased levels of free protein S even when total protein S levels are in the normal range. Elevated levels ofC4~P have been reported in peritoneal dialysis patients28 and confirmed in our report. Although the control patients also had elevated C4bBP levels, they were probably able to maintain normal protein S (free) levels by increasing synthesis. Our signal patient is interesting for several reasons: she took warfarin for a year before starting

Table 2. Plasma Values for Protein C, Antigen and Functional; Protein 5, Total and Free; and C4b BP in CAPO Patients Protein C Ag % (normal, 70-150)

Functional Protein C % (normal. 65- 135)

Total Protein S % (normal,70-15O)

Free Protein S % (normal. 65-135)

C•.,BP% (normal, 77-135)

With skin necrosis Patient 1 (12f7189) Patient 2 (4/27/90)

209 113

ONS 109

133 226

41 41

209 375

Without skin necrosis -controls Patient 3 Patient 4 Patient 5 Patient 6 Patient 7

80 123 142 158 76

53 137 140 140 143

81 133 129 208 217

89 103 119 134 134

146 194 223 221 231

Abbreviation: ONS, quantity not sufficient.

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KANT ET AL Table 3. Protein C Antigen, Functional Protein C, Total Protein S, and Free Protein S Lost in 24 Hours in Peritoneal Dialysate (l'g/24 h) Protein C

Ag

Functional Protein C

Total Protein S

Free Protein S

C.bBP

With skin necrosis Patient 1 (12/5/89) Patient 2 (4/23/90)

1,072 207

QNS 66

1,557 764

1,172 916

QNS 660

Without skin necrosis -controls Patient 3 Patient 4 Patient 5 Patient 6 Patient 7

761 216 1,382 151 120

263 178 857 385 86

5,522 1,007 4,645 1,538 420

2,350 705 3,192 1,094 502

0 1,880 33,665 2,735 0

CAPD with no problems; a low plasma level of free protein S was associated with both skin necrosis and documented loss in the peritoneal dialysate. Despite discontinuing warfarin, she developed new lesions while on heparin. Studies on the plasma and the peritoneal dialysates of the five asymptomatic CAPD patients showed that plasma levels of protein C and S were not depressed despite demonstrable peritoneal losses, implying that these patients were able to compensate for losses by increasing synthesis. This is supported by the studies of Vaziri et a1 26 and Lai et al.27 The term calciphylaxis has been used to describe a syndrome of vascular calcification in association with elevated calcium phosphate products as a result of hyperparathyroidism. 29 The syndrome consists of painful violaceous mottling (livedo reticularis) followed by a painful gangrene of fingers, toes, or skin. Vascular calcification as a result of hyperparathyroidism and attendant thrombosis is felt to be a cause of this, and emergent parathyroidectomy has been advocated. 29 However, several factors point to likely involvement of the protein CIS pathways in the genesis of this lesion. (1) Vascular calcification occurs in 67% of dialysis patients,30 whereas skin/extremity necrosis of this variety is rare. (2) Patient 1 in this report (who had only modest elevations of the calcium-phosphate product and PTH), and the patients described by Mehta et allO developed this syndrome despite previous parathyroidectomy, factors that point away from hyperparathyroidism and vascular calcification as the sole factors leading to this syndrome.

The genesis of skin necrosis in patient 2 is probably due to different causes at different times. A permissive/precipitative role of peritoneal dialysis is suggested by more than 3 years of uneventful hemodialysis before the initiation of CAPD. That CAPD and associated protein S depletion was responsible is supported by an initial partial response to cessation of CAPD and to the slow restitution of protein S levels to normal on maintenance hemodialysis. The latter was accompanied by slow healing of the skin ulcers. The initial response to cessation of CAPD was only partial-superinfection required debridement. In the postoperative period, multiple new lesions occurred that required prolonged wound care. Coincident with the wound care, she received a parathyroidectomy. It is conceivable that hyperparathyroidism and an elevated calcium phosphate product, as well as other causes of endothelial injury (surgery, anesthesia, sepsis), all led to further small vessel thrombosis and skin necrosis. It is clearly difficult to elucidate a direct role for all these causes, but the time course suggests a causative/permissive role for hyperparathyroidism. In summary, we believe the genesis of skin necrosis in dialysis patients is probably multifactorial. Decreased anticoagulant protein levels may be caused by dialysate losses or low-grade clotting in the dialyser. Repetitive complement activation and/or exposure to endotoxin in small levels (in hemodialysis patients) may lead to endothelial injury and defective generation offunctional protein C. Vitamin K deficiency from various causes, other infections (including viral

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processes), and elevated calcium phosphate/hyperparathyroidism may all play a contributing role at various times. Liver disease, as in patient 1, is possibly another such contributing factor. Finally, what distinguishes patients who develop frank disease from asymptomatic CAPD patients warrants further study. Our studies suggest that in patients undergoing peritoneal dialysis, it is important to be aware

that these biological inhibitors are continuously . lost in the dialysate. Vigilant monitoring of CAPD patients receiving coumarin products would seem to be in order. ACKNOWLEDGMENTS We gratefully acknowledge the technical staff of the Coagulation Laboratory who performed all of the tests, Naomi Hayes and Mary Carol Menkhaus who prepared the manuscript, and Jay Card who did the photography.

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17. Grimaudo V, Gueissaz F, Hauert T, et al: Necrosis of skin induced by coumarin in a patient deficient in protein S. Br Med J 298:233-234, 1989 18. White PW, Sadd JR, Nensel RE: Thrombotic complications of heparin therapy including six cases of heparin-induced skin necrosis. Ann Surg 190:595-608, 1978 19. Ronglioletti F, Pisan S, Ciaccio AP, et al: Skin necrosis due to intravenous heparin. Dermatologia 178:47-50, 1989 20. Levine LE, Bernstein JE, Soltani K, et aI: Heparininduced cutaneous necrosis unrelated to injection sites. Arch Dermatol 119:400-403, 1983 21. Comp PC, Elrod JP, Karzenski S: Warfarin-induced skin necrosis. Semin Thromb Hemost 16:293-298, 1990 22. Suzuki K, Nishioka J, Hashimoto S: Protein C inhibitor-purification from human plasma and characterization. J Bioi Chern 258:163-168, 1983 23. Vigano-D'AngeIo SV, D'Angelo A, Kaufman CE Jr, et al: Protein S deficiency occurs in nephrotic syndrome. Ann Intern Med 107:42-47, 1987 24. Vaziri ND, Alikhani S, Patel B, et al: Increased levels of protein C activity, protein C concentration, total and free protein S in nephrotic syndrome. Nephron 49:20-23, 1988 25. Cosio FG, Harker C, Batard MA, et aI: Plasma concentrations of the natural anticoagulants protein C and protein S in patients with proteinuria. J Lab Clin Med 106:218-222, 1985 26. Vaziri ND, Shah GM, Winer RL, et al: Coagulation cascade fibrinolythic system, antithrombin III, protein C and S in patients maintained on continuous ambulatory peritoneal dialysis. Thromb Res 53:731-780, 1989 27. Lai KN, Yin JA, Yuen PMP, et al: Protein C, Protein S, and antithrombin III levels in patients on continuous ambulatory peritoneal dialysis and hemodialysis. Nephron 56: 271-276, 1990 28. Garcia-Maldonado M, Smith M, Klarich M: C 4bBP is elevated in patients in continuous ambulatory peritoneal dialysis. Clin Res 38:140A, 1990 (abstr) 29. Gipstein RM, Coburn JW, Adams DA, et al: Calciphylaxis in man: A syndrome of tissue necrosis with vascular calcification in II patients with chronic renal failure. Arch Intern Med 136:1273-1280, 1976 30. Ibels LS, Alfrey AC, Huffer WE, et aI: Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med 66:790-796, 1979