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Skin necrosis associated with acquired protein C deficiency in patients with renal failure and calciphylaxis
Skin Necrosis Associated with Acquired Protein C Deficiency in Patients with Renal Failure and Calciphylaxis RAVINDRAL. MEHTA,M.B.,B.S.,M.D.,GLYNISSCOTT,M.D.,JAMESA. FRANCIS,M.D., Rochester, New York
PURPOSE:To determine if the natural anticoagulant protein C plays a role in the pathogenesis of systemic calciphylaxis, a syndrome characterized by extensive vascular and soft tissue calcification and skin necrosis, which is similar to that seen in warfarin-induced skin necrosis. PATIENTS AND METHODS: The study population included five patients with end-stage renal disease and systemic calciphylaxis undergoing hemodialysis, 12 patients without evidence of calciphylaxis undergoing dialysis, eight patients with nephrotic syndrome, and eight normal healthy volunteers. Protein C antigen levels were measured by rocket immunoelectrophoresis, and functional activity was quantitated by a chromogenic assay and an anticozn8ay utilixing the venom of Agkistrodon RESULTS:Skhr biopsy specimens of involved areas in three patients showed thrombotic occlusion of venules identical to that seen in warfarin-induced skin necrosis. Protein C antigen levels were normal in all groups. However, protein C activity was significantly reduced as measured by chromogenic (p
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atients with end-stage renal disease have a high P incidence of vascular calcification [l-4] that has been related to several metabolic abnormalities including secondary hyperparathyroidism, hypertriglyceridemia, and hypomagnesemia [5]. Although clinical evidence of associated vascular insufficiency is uncommon, rare patients with end-stage renal disease develop a syndrome of progressive vascular and soft tissue calcification and ischemic skin necrosis, initially described by Selye, and termed calciphylaxis [6-81. The pathogenesis of the extensive metastatic calcification and the skin necrosis is not understood, although hyperparathyroidism, hyperphosphatemia, and therapy with vitamin D or steroids have been suggested as contributing factors [9]. We were impressed by the similarity between the clinical presentation of the skin necrosis seen in patients with calciphylaxis and that in warfarin-induced skin necrosis, a complication that develops occasionally during the initiation of oral anticoagulant therapy and that is characterized by extensive skin and soft tissue necrosis resulting from thrombotic occlusion of venules in the dermis and subcutaneous tissues [lo]. The apparently paradoxical development of thrombosis after administration of warfarin has been related to reduced plasma levels of protein C, a vitamin K-dependent natural anticoagulant [ll-131. During initiation of therapy, a disproportionate reduction in activity of the anticoagulant protein C system compared with procoagulant vitamin K-dependent factors may result in a hypercoagulable state [14,X]. Patients with heterozygous congenital protein C deficiency have an average 50% reduction in plasma protein C levels, and this results in an increased risk of venous thrombotic disease [16,17]. They may also have a greater tendency to develop warfarin-induced skin necrosis because of the lower baseline protein C levels [18,19]. In the current report, we describe five patients with end-stage renal disease, calciphylaxis, and skin necrosis. We report levels of protein C and compare the pathologic findings to those seen in warfarin-induced skin necrosis. PATIENTS AND METHODS Patients Samples from 12 patients with end-stage renal disease were obtained before hemodialysis (10 patients) or peritoneal dialysis (two patients), and samples were obtained in two patients after dialysis. Patients with nephrotic syndrome had proteinuria of at least 3.5 gl day. Healthy ambulatory adults served as normal control subjects. All patients with calciphylaxis had endstage renal disease requiring dialysis. In addition, they had radiographic evidence of extensive vascular and soft tissue calcification and skin necrosis. None of the
From the Departments of Medicine and Pathology, University of Rochester School of Medicine and Dentistry, Rochester, New York. This work was supported in part by Grant HC-30616 from the National Heart, Lung, and Blood Institute. National Institutes of Health, Bethesda, Maryland. Dr. Francis is the recipient of an Established Investigator Award from the Amencan Heart Association with funds provrded by the New York State Affiliate. Requests for reprints should be addressed to Ravindra L. Mehta, M.D., University of California, San Diego, Medical Center H-781-D. 225 Dickinson Street, San Drego. California 92103. Manuscript submitted September 8, 1989, and accepted in revised form January 3, 1990.
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TABLE I Clinical and Pathologic Features of Patients with Calciphylaxis
Patient Number
Age/Sex
Renal Disease/ Duration (months)
1
48/F
Hypertension, nephrosclerosrs/ 48
2
63/F
3
Duration of Dialysis before Skin Necrosis
Other Clinical Features
Involved Areas
Histopathology
25
Fingers, thighs, buttacks
Treated with sterords. quired skrn grafting.
Diabehc nephropathy, hypertensron/36
1
Frngers. toes, thighs, calf, breast
26/F
Drabettc nephropathy, hypertensron/36
5
Fingers
4
71/F
Diabetic nephropathy, hypertension/24
5
Thrgh
Insulin-dependent diabetes. Treated wrth steroids. Requrred bilateral amputations above knees and fingertip amputations. Insulin-dependent drabetes. Granulomatous hepatitis. Poor compliance with medical regimen. Insulin-dependent diabetes. Pseudoxanthoma elasticum.
5
22/F
Focal glomerulosclerosis/24
20
Calf
patients was receiving warfarin or had clinically significant liver disease, and none had a past medical history or family history of venous thromboembolic disease. Samples were obtained from all patients with calciphylaxis 5 to 23 months after parathyroidectomy. One patient (Patient 2) had active skin necrosis at the time both samples were obtained, whereas samples from the other patients were obtained during or after healing of lesions at intervals of 4 to 8 weeks. Protein C Assays After informed consent was obtained, venous blood was collected into sodium citrate (0.4% final concentration), placed immediately on melting ice, centrifuged within 1 hour at 2,500g for 20 minutes at 4°C and frozen at -70°C until assayed. Antigenic protein C was measured by rocket immunoelectrophoresis using a polyclonal antibody (American Diagnostica, New York, New York). Protein C functional activity was assayed using kits provided by American Bioproducts (Parsippany, New Jersey). For the chromogenic assay, protein C was activated by a specific activator extracted from the venom of Agkistrodon contortrir, and the enzyme formed was measured by its ability to cleave the chromogenic substrate, 2AcOH,H-D-Lys(Cbo)Pro-Arg-pNA. The anticoagulant assay used the same activator, but activated protein C was measured by its ability to degrade factors V and VIII, thereby prolonging the activated partial thromboplastin time of mixtures of patient and protein C-deficient plasma [20]. For samples tested repeatedly in the same assay, the standard deviations for chromogenic and clotting assays were 2.2% and 14.9% for normal subjects and 4.2% and 9.9%, respectively, for patients with calciphylaxis. The variance in assays done on different days was 6.1% by clotting and 16.5% by chromogenic assay using samples from patients with calciphylaxis. Specific activity was calculated as protein C activity by functional assay divided by protein C antigenic assay.
Re-
Renal transplantation twice. Poor compliance with medical regimen.
Extensrve cutaneous necrosrs with stromal calcification and calcification of small and medrum-srzed vessels. Extensfve cutaneous necrosis wrth vascular and stromal calcificatton. Numerous fibrin thrombi In the lumen of ventries in dermis and subcutis. Eprdermal necrosis with calcifrcation of vessels and stroma. Multiple fibrin thrombi present In venules of subcutrs. Epidermal necrosis with vascular calcification and swollen and irregularly clumped calcified elastic fibers, diagnostic of pseudoxanthoma elasticum. Multiple fibrin thrombr within venules in the subcutts. Fat necrosis with stromal and vascular calcification.
Histopathology Tissue specimens were fixed in formalin, embedded in paraffin, sectioned at 5 to 6 p, and stained with hematoxylin-eosin. Frazer-Lendrum stain for fibrin and elastin-von-Giesen stain for elastin were used in each case. Statistical Analysis Statistical significance dent’s t-test.
was calculated
using Stu-
RESULTS All five patients with calciphylaxis were women with end-stage renal disease requiring dialysis for between 1 and 25 months before the onset of skin necrosis (Table I). All patients were hypertensive and had radiologically evident vascular calcification and secondary hyperparathyroidism. Three patients had diabetes mellitus, and one each granulomatous hepatitis and pseudoxanthoma elasticurn. One patient had received two renal transplants, and two were poorly compliant with medical regimens. Skin lesions typically began with tingling and burning pain in the affected site. Red or violaceous discoloration with a papular or nodular eruption preceded development of ulceration and skin necrosis with eschar formation (Figure 1). Ulcerated lesions were slow to heal, and skin grafting was needed in one patient, whereas amputation was required in a second patient. Patient 1 had extensive skin grafting over a period of 1 year after parathyroidectomy, whereas Patient 2 required 11 months for her lesion to heal. The lesions in Patients 3,4, and 5 took more than 4 months to .heal. Therapy with prednisone in one patient or prednisone and methotrexate in another were without apparent effect. All patients underwent total parathyroidectomy with forearm implantation of parathyroid tissue. Histopathologic examination of sections from areas of skin necrosis from each patient showed extensive March
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pared with levels in normal subjects (p
COMMENTS The findings presented in this report demonstrate similarities between the clinical presentation, pathologic features, and pathogenesis of the skin lesions in calciphylaxis and warfarin-induced skin necrosis. Warfarin-induced skin necrosis is more common in female patients and typically begins with burning pain in an area with abundant subcutaneous fat such as thighs, buttocks, or breasts. The affected areas develop a violaceous or hemorrhagic discoloration, and the appearance of petechiae or bullae may precede the Figure
1. Skin necrosis
involving
calf of Patient
2
stromal and arterial wall calcification (Figure 2). In addition, skin biopsy specimens from Patient 4 showed characteristic changes of pseudoxanthoma elasticurn, which consisted of clumping and calcification of elastin fibers. Ulceration of the epidermis and dermis was seen in all cases (Figure 3), and in Patients 2,3, and 4, multiple thrombi were identified in smalldiameter vessels in the dermis and subcutaneous tissue (Figures 4 and 5), with no associated inflammatory cell infiltrate. Stains for elastin showed that the occluded vessels were venules, and stains for fibrin confirmed the presence of fibrin thrombi. The results of the protein C antigenic assay were normal or elevated in all samples except one from Patient 4 (Table II). However, there was reduced functional protein C measured by both chromogenic and anticoagulant assay, with a greater reduction by the latter. With both assays, there was wide variability between patients and in the same patient at different times, with a range of 11% to 120% by chromogenic assay and 17% to 70% by anticoagulant assay. Specific functional activity was also reduced, and this was also more prominent with the anticoagulant assay. Protein C levels and specific activities in patients with calciphylaxis were compared with levels and activities in normal subjects and patients with renal disease (Table III). In patients with calciphylaxis, the functional protein C level measured by chromogenic assay was significantly reduced compared with that in normal subjects (p
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Figure 2. Subcutis near an area of necrosis showing deposition of calcium in the wall of an artery (hematoxylin and eosin stain; bar 40~ before 30% reduction).
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Figure 3. Section from involved area with full-thickness necrosis of the skin (hematoxylin and eosin stain: bar 160 p before 30% reduction).
development of deep necrotic lesions. An eschar forms over the defect, which may heal slowly, often with scarring. Skin grafting may be needed, and amputation of extremities or breast has been required for nonhealing lesions [lo]. All of the patients in this report with calciphylaxis were women with lesions involving thigh, buttock, or breasts in three patients. Burning pain was a frequent presentation. The clinical appearance of the lesion was typical of warfarin-induced skin necrosis (Figure l), as was the progression with slow healing that required skin grafting in one patient and amputation in another. The histologic appearance was also similar (Figures 2,3,4, and 5) with fibrin thrombi in otherwise normal venules without surrounding inflammation, indicating that local thrombosis plays an important role in the development of skin lesions in calciphylaxis as it does in warfarin skin necrosis. The protein C abnormalities that we found in renal patients without calciphylaxis are consistent with prior reports, Normal or elevated levels of protein C measured antigenically or functionally have been found in patients with nephrotic syndrome [21-241. In patients undergoing hemodialysis, Sorensen et al [25] have described decreased protein C activity with normal protein C antigen levels, and Vaziri et al [26] found elevated protein C antigen levels with reduced protein C activity in patients with spinal cord injury and endstage renal disease. We found an increase in protein C antigen in patients undergoing dialysis or with nephrotic syndrome, but functional levels within the normal range resulting in an overall decrease in specific activity (Table III), but the reduction in functional activity and specific activity was greater in patients on dialysis with calciphylaxis. The decreased levels of functional protein C in patients with calciphylaxis (Tables II and III) suggest that the reduced activity of this natural anticoagulant pathway may play a pathogenetic role in the development of the skin lesions. Protein C is activated at the surface of endothelial cells by a complex of thrombin and thrombomodulin [27]. With protein S serving as a co-factor [28], activated protein C cleaves and inactivates factors V, and VIII,, thereby inhibiting coagulation [29,30]. Activated protein C also enhances fibrino-
lysis by inactivating plasminogen activator inhibitor [31]. Congenital homozygous deficiency of protein C results in purpura fulminans with hemorrhagic necrosis of the skin and a frequently fatal outcome due to widespread vascular thrombosis [32-341. Heterozygous deficiency results in a reduction of plasma protein C levels to approximately 50% of normal and is associated with a high risk of venous thromboembolic disease in some families [16,17]. Since protein C is a vitamin K-dependent protein [ll-131, warfarin administration results in reduction of functional levels, and a disproportionate reduction in the anticoagulant activity of protein C compared with the procoagulant activities of factors II, VII, IX, and X may contribute to the development of warfarin-induced skin necrosis during initiation of therapy [14,15]. This may occur more frequently in patients with heterozygous congenital deficiency who have lower baseline levels of protein C [18,19]. Patients with calciphylaxis had reduced levels of functional protein C but normal levels of protein C measured antigenically (Tables II and III), reflecting circulation of dysfunctional or inactivated protein C. A possible explanation for the lower clotting than chromogenic protein C activity is that dysfunctional enzyme or enzyme inhibitor complexes may cleave the small peptide in the chromogenic assay while having greatly reduced activity with the natural protein substrate. Congenital protein C deficiency with the presence in blood of immunologically reactive but dysfunctional protein C has been reported [35], but the clinical presentation was different than in our patients with calciphylaxis. In a patient with a fatal thrombotic disorder, an acquired IgG inhibitor of protein C was described resulting in normal antigenic but low functional protein C levels [36]. However, incubation studies with normal plasma showed no evidence of such an inhibitor in our patients. Two physiologic inhibitors of activated protein C have been described [37,38] one of which is accelerated by heparin [38]. In patients with disseminated intravascular coagulation, complexes of activated protein C with these inhibitors have been identified in plasma [39,40], and these inactivated complexes may result in elevated antigenic compared March
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Figure 4. Section from involved skin showing thrombi (arrows) in dermal venules in the absence of an inflammatory response .(hematoxylin and eosin stain; bar 60 Jo before 30% reduction).
1
TABLE II Protein
Patient Number 1
2 3 4 5
Figure 5. Involved skin showing two venules containing (arrows) with an adjacent patent medium-sized artery and eosin stain; bar 10 p before 30% reduction).
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Antigenie 85 100 100 180 150 110 120 200 40 120 9”;
in Patients
with Calciphylaxis
Protein C (%) ChromoAntigenie coagulant 63 96 92 87 iI! 120 11 35 -. 8’: 90
70 17 i: 28 :“6 38 32 ;i 70
Specific Activity ChromoAntigenie coagulant 0.74 0.96 0.92 0.48 0.12 0.85 1 .oo 0.06 0.88 0.75 1.31 0.95
0.82 0.17 0.27 0.25 0.19 0.17 0.22 0.19 0.80 0.58 0.92 0.74
C or, alternatively, to reduced clearance of activated protein C-inhibitor complexes due to renal disease. Another potential explanation for reduced protein C levels is consumption due to intravascular thrombosis. However, this is an unlikely explanation in our patients, since reduced protein C levels persisted when skin lesions were healing at a time when there was no evidence of a thrombotic process. Wide variation was seen in functional protein C assays in patients with calciphylaxis (Table II) that could not be explained by variability in the assay systems. We would hypothesize that the thrombotic skin lesions develop at times of lowest protein C activity and that the variable protein C levels (Table II) and reduced concentrations present after healing of the skin lesions suggest additional factors contribute to their pathogenesis. Hyperparathyroidism is a feature in most patients with calciphylaxis, and a role in the development of skin necrosis is suggested by the healing of lesions after parathyroidectomy. However, some patients with calciphylaxis have had normal parathyroid hormone levels [7], and no prothrombotic effect of this hormone has been described. Since parathyroid hormone stimulates uptake of extracellular calcium, it may influence platelet aggregation, but recent studies
with functional protein C levels. Although the explanation for reduced functional protein C in calciphylaxis is unclear, the presence in plasma of complexes of activated protein C with inhibitor would explain the reduced protein C specific activity that we have found. This could result from either accelerated formation of such complexes due to increased activation of protein 256
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TABLE III
Comparison of Protein C Levels in Normal Subjects and Patients with Renal Disease with or without Calciphylaxis*
I
Group
Number
Antigenic
Protein C (%) Chromogenic
Anticoagulant
Chromogenic
Specific Activity Anticoagulant
I
Normal Dialysis Nephrotic syndrome Calclphylaxis
1; 8
119zk8 99f3 148f 15
97 94&2f 9 121 f 10
95 f 10 93fll 77f 19
0.81 f 0.03 0.96 0.05 0.84 f 0.07
0.79 f 0.10 0.96f0.11 0.55 f 0.14
0.75 f 0.14
0.47 f 0.13
* Mean f SEM.
do not support this [41]. The vascular calcification that is a hallmark of the syndrome may predispose to thrombosis by narrowing the vessel lumen and injuring the vascular wall. However, vascular calcification is common in uremia, occurring in up to 46% of patients undergoing dialysis [2], whereas calciphylaxis occurs rarely. It is likely that several factors interact in contributing to the pathogenesis of calciphylaxis. Our findings suggest that a reduced functional protein C level may be an important element in the pathogenesis, resulting in a hypercoagulable state and contributing to thrombosis in the presence of vascular damage.
ACKNOWLEDGMENT We thank Geraldine Quigley and M. Julianne Brown for technrcal assistance and L. Taylor-Donald and Carol Weed for help In the preparatron of the manuscript
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309: 340-344. 18. McGehee WG, Klotz TA. Epstein DJ, Rapaport SI: Coumann necrosrs assocrated wrth heredrtary protein C deficiency. Ann Intern Med 1984; 100: 59-60. 19. Samama M, Horellou MH, Soria J, Conard J, Nicolas G: Successful progressive anticoagulation in a severe protein C deficiency and previous skin necrosis at the Initiation of oral anticoagulant therapy. Thromb Haemost 1984; 51: 132-133. 20. Francrs RC, Patch MJ: A functional assay for protein C rn human plasma. Thromb Res 1983; 32: 605-613. 21. Pabinger-Fasching I, Lechner K, Niessner H. Schmidt P, Balzar E, Mannhalter Ch: High levels of plasma protein C rn nephrotic syndrome. Thromb Haemost 1985; 53: 5-7. 22. Cosio FG. Harker C, Batard MA, Brandt JT, Griffin JH: Plasma concentrations of the natural anticoagulants protein C and protern S rn patients with proteinuria. J Lab Clan Med 1985; 106: 218-222. 23. Mannuccio Mannucci P. Valsecchi C, Bottasso B, D’Angelo A, Casati S, Ponticelli C: Hugh levels of protern C activrty and antigen In the nephrotic syndrome. Thromb Haemost 1986; 55: 31-33. 24. Vazin ND, Alikhani S, Pate1 B, Nguyen Q. Barton CH, Gonzales EV: Increased levels of protein C activity, protein C concentratron. total and free protein S rn nephrotic syndrome. Nephron 1988; 49: 20-23. 25. Sorensen PJ, Hielsen AH, Knudsen F. Dyerberg J: Defective protein C in uraemia. Blood Purrfication 1987; 5: 29-32. 26. Vaziri ND, Pate1 B, Alikhanr A, et al: Protein C abnormalibes in spinal cord iniured oatients with end-stage renal dtsease. Arch Phys Med Rehabrl 1987: 68: 791-793. 27. Esmon NL, Owen WG, Esmon CT: Isolation of a membrane-bound cofactor for thrombrn-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864. 28. Walker FJ: kegulabon of activated protein C by protein S. the role of phospholrpid in factor V, Inactivation. J BIOI Chem 1981; 256: 11128-l 1131. 29. Marlar RA, Kleiss AJ, Gnffrn JH: Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 10671072. 30. Kisiel W, Canfield WH, Ericsson LH. Davie EW: Anticoagulant properties of bovine plasma protein C following activation by thrombin. Brochemistry 1977; 16: 58245831. 31. Sakata Y. Koskutoff DJ. Gladson CL, Hekman CM, Griffin JH: Mechanism of protein C-dependent clot lysis: role of plasmrnogen acbvator inhibitor. Blood 1986; 68: 1218-1223. 32. Branson HE, Katz R, Grifbn JH: Inherited protein C deficrency and Coumarinresponsive chronic relapsing purpura fulminans in a newborn infant. Lancet 1983; 2: 1165-1168. 33. Seligsohn V. Berger A, Abend M, et al: Homozygous protein C deficiency manifested by inactivated venous thrombosrs rn the newborn, N Engl J Med 1984; 310: 5599562. 34. Marcrniak E, Wilson HD, Marlar RA: Neonatal purpura fulmrnans: a genetic disorder related to the absence of protein C in blood. Blood 1985: 65: 15-20. 35. Matsuda M. Sugo T. Sakata Y. et al: A thrombotic state due to an abnormal protein C. N Engl J Med 1988; 319: 1265-1268. 36. Mitchell CA, Rowell JA, Hau L, Young JP, Salem HH: A fatal thrombotrc disorder associated with an acquired inhibitor of protein C. N Engl J Med 1987; 317: 16381642. 37. Suzuki K, Nishioka J. Hashimoto S: Protein C inhibitor. Purification from human plasma and characterization. J Biol Chem 1983; 258: 163-168. 38. Heeb MJ, Espana F. Griffin JH: lnhibrtion and complexation of activated protein C by two major Inhibitors in plasma. Blood 1989; 73: 446-454. 39. Marlar RA, Endres-Brooks J, Miller C: Serial studies of protein C and its plasma inhibitor in patients with disseminated intravascular coagulation. Blood 1985; 66: 59-63. 40. Heeb MJ, Mosher D, Griffin JH: Acbvation and complexation of protern C and cleavage and decrease in protein S in plasma of patients with intravascular coagulabon. Blood 1989; 73: 455-461. 41. Docci D, Turcr F. Delvecchio D. Gollrnr C. Baldratr L, Pistocci E: Lack of evtdence for the role of secondary hyperparathyroidism in the pathogenesis of uremtc thrombocytopathy. Nephron 1982; 30: 237-239.
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PURPOSE:To determine if the natural anticoagulant protein C plays a role in the pathogenesis of systemic calciphylaxis, a syndrome characterized by extensive vascular and soft tissue calcification and skin necrosis, which is similar to that seen in warfarin-induced skin necrosis. PATIENTS AND METHODS: The study population included five patients with end-stage renal disease and systemic calciphylaxis undergoing hemodialysis, 12 patients without evidence of calciphylaxis undergoing dialysis, eight patients with nephrotic syndrome, and eight normal healthy volunteers. Protein C antigen levels were measured by rocket immunoelectrophoresis, and functional activity was quantitated by a chromogenic assay and an anticozn8ay utilixing the venom of Agkistrodon RESULTS:Skhr biopsy specimens of involved areas in three patients showed thrombotic occlusion of venules identical to that seen in warfarin-induced skin necrosis. Protein C antigen levels were normal in all groups. However, protein C activity was significantly reduced as measured by chromogenic (p
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atients with end-stage renal disease have a high P incidence of vascular calcification [l-4] that has been related to several metabolic abnormalities including secondary hyperparathyroidism, hypertriglyceridemia, and hypomagnesemia [5]. Although clinical evidence of associated vascular insufficiency is uncommon, rare patients with end-stage renal disease develop a syndrome of progressive vascular and soft tissue calcification and ischemic skin necrosis, initially described by Selye, and termed calciphylaxis [6-81. The pathogenesis of the extensive metastatic calcification and the skin necrosis is not understood, although hyperparathyroidism, hyperphosphatemia, and therapy with vitamin D or steroids have been suggested as contributing factors [9]. We were impressed by the similarity between the clinical presentation of the skin necrosis seen in patients with calciphylaxis and that in warfarin-induced skin necrosis, a complication that develops occasionally during the initiation of oral anticoagulant therapy and that is characterized by extensive skin and soft tissue necrosis resulting from thrombotic occlusion of venules in the dermis and subcutaneous tissues [lo]. The apparently paradoxical development of thrombosis after administration of warfarin has been related to reduced plasma levels of protein C, a vitamin K-dependent natural anticoagulant [ll-131. During initiation of therapy, a disproportionate reduction in activity of the anticoagulant protein C system compared with procoagulant vitamin K-dependent factors may result in a hypercoagulable state [14,X]. Patients with heterozygous congenital protein C deficiency have an average 50% reduction in plasma protein C levels, and this results in an increased risk of venous thrombotic disease [16,17]. They may also have a greater tendency to develop warfarin-induced skin necrosis because of the lower baseline protein C levels [18,19]. In the current report, we describe five patients with end-stage renal disease, calciphylaxis, and skin necrosis. We report levels of protein C and compare the pathologic findings to those seen in warfarin-induced skin necrosis. PATIENTS AND METHODS Patients Samples from 12 patients with end-stage renal disease were obtained before hemodialysis (10 patients) or peritoneal dialysis (two patients), and samples were obtained in two patients after dialysis. Patients with nephrotic syndrome had proteinuria of at least 3.5 gl day. Healthy ambulatory adults served as normal control subjects. All patients with calciphylaxis had endstage renal disease requiring dialysis. In addition, they had radiographic evidence of extensive vascular and soft tissue calcification and skin necrosis. None of the
From the Departments of Medicine and Pathology, University of Rochester School of Medicine and Dentistry, Rochester, New York. This work was supported in part by Grant HC-30616 from the National Heart, Lung, and Blood Institute. National Institutes of Health, Bethesda, Maryland. Dr. Francis is the recipient of an Established Investigator Award from the Amencan Heart Association with funds provrded by the New York State Affiliate. Requests for reprints should be addressed to Ravindra L. Mehta, M.D., University of California, San Diego, Medical Center H-781-D. 225 Dickinson Street, San Drego. California 92103. Manuscript submitted September 8, 1989, and accepted in revised form January 3, 1990.
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TABLE I Clinical and Pathologic Features of Patients with Calciphylaxis
Patient Number
Age/Sex
Renal Disease/ Duration (months)
1
48/F
Hypertension, nephrosclerosrs/ 48
2
63/F
3
Duration of Dialysis before Skin Necrosis
Other Clinical Features
Involved Areas
Histopathology
25
Fingers, thighs, buttacks
Treated with sterords. quired skrn grafting.
Diabehc nephropathy, hypertensron/36
1
Frngers. toes, thighs, calf, breast
26/F
Drabettc nephropathy, hypertensron/36
5
Fingers
4
71/F
Diabetic nephropathy, hypertension/24
5
Thrgh
Insulin-dependent diabetes. Treated wrth steroids. Requrred bilateral amputations above knees and fingertip amputations. Insulin-dependent drabetes. Granulomatous hepatitis. Poor compliance with medical regimen. Insulin-dependent diabetes. Pseudoxanthoma elasticum.
5
22/F
Focal glomerulosclerosis/24
20
Calf
patients was receiving warfarin or had clinically significant liver disease, and none had a past medical history or family history of venous thromboembolic disease. Samples were obtained from all patients with calciphylaxis 5 to 23 months after parathyroidectomy. One patient (Patient 2) had active skin necrosis at the time both samples were obtained, whereas samples from the other patients were obtained during or after healing of lesions at intervals of 4 to 8 weeks. Protein C Assays After informed consent was obtained, venous blood was collected into sodium citrate (0.4% final concentration), placed immediately on melting ice, centrifuged within 1 hour at 2,500g for 20 minutes at 4°C and frozen at -70°C until assayed. Antigenic protein C was measured by rocket immunoelectrophoresis using a polyclonal antibody (American Diagnostica, New York, New York). Protein C functional activity was assayed using kits provided by American Bioproducts (Parsippany, New Jersey). For the chromogenic assay, protein C was activated by a specific activator extracted from the venom of Agkistrodon contortrir, and the enzyme formed was measured by its ability to cleave the chromogenic substrate, 2AcOH,H-D-Lys(Cbo)Pro-Arg-pNA. The anticoagulant assay used the same activator, but activated protein C was measured by its ability to degrade factors V and VIII, thereby prolonging the activated partial thromboplastin time of mixtures of patient and protein C-deficient plasma [20]. For samples tested repeatedly in the same assay, the standard deviations for chromogenic and clotting assays were 2.2% and 14.9% for normal subjects and 4.2% and 9.9%, respectively, for patients with calciphylaxis. The variance in assays done on different days was 6.1% by clotting and 16.5% by chromogenic assay using samples from patients with calciphylaxis. Specific activity was calculated as protein C activity by functional assay divided by protein C antigenic assay.
Re-
Renal transplantation twice. Poor compliance with medical regimen.
Extensrve cutaneous necrosrs with stromal calcification and calcification of small and medrum-srzed vessels. Extensfve cutaneous necrosis wrth vascular and stromal calcificatton. Numerous fibrin thrombi In the lumen of ventries in dermis and subcutis. Eprdermal necrosis with calcifrcation of vessels and stroma. Multiple fibrin thrombi present In venules of subcutrs. Epidermal necrosis with vascular calcification and swollen and irregularly clumped calcified elastic fibers, diagnostic of pseudoxanthoma elasticum. Multiple fibrin thrombr within venules in the subcutts. Fat necrosis with stromal and vascular calcification.
Histopathology Tissue specimens were fixed in formalin, embedded in paraffin, sectioned at 5 to 6 p, and stained with hematoxylin-eosin. Frazer-Lendrum stain for fibrin and elastin-von-Giesen stain for elastin were used in each case. Statistical Analysis Statistical significance dent’s t-test.
was calculated
using Stu-
RESULTS All five patients with calciphylaxis were women with end-stage renal disease requiring dialysis for between 1 and 25 months before the onset of skin necrosis (Table I). All patients were hypertensive and had radiologically evident vascular calcification and secondary hyperparathyroidism. Three patients had diabetes mellitus, and one each granulomatous hepatitis and pseudoxanthoma elasticurn. One patient had received two renal transplants, and two were poorly compliant with medical regimens. Skin lesions typically began with tingling and burning pain in the affected site. Red or violaceous discoloration with a papular or nodular eruption preceded development of ulceration and skin necrosis with eschar formation (Figure 1). Ulcerated lesions were slow to heal, and skin grafting was needed in one patient, whereas amputation was required in a second patient. Patient 1 had extensive skin grafting over a period of 1 year after parathyroidectomy, whereas Patient 2 required 11 months for her lesion to heal. The lesions in Patients 3,4, and 5 took more than 4 months to .heal. Therapy with prednisone in one patient or prednisone and methotrexate in another were without apparent effect. All patients underwent total parathyroidectomy with forearm implantation of parathyroid tissue. Histopathologic examination of sections from areas of skin necrosis from each patient showed extensive March
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pared with levels in normal subjects (p
COMMENTS The findings presented in this report demonstrate similarities between the clinical presentation, pathologic features, and pathogenesis of the skin lesions in calciphylaxis and warfarin-induced skin necrosis. Warfarin-induced skin necrosis is more common in female patients and typically begins with burning pain in an area with abundant subcutaneous fat such as thighs, buttocks, or breasts. The affected areas develop a violaceous or hemorrhagic discoloration, and the appearance of petechiae or bullae may precede the Figure
1. Skin necrosis
involving
calf of Patient
2
stromal and arterial wall calcification (Figure 2). In addition, skin biopsy specimens from Patient 4 showed characteristic changes of pseudoxanthoma elasticurn, which consisted of clumping and calcification of elastin fibers. Ulceration of the epidermis and dermis was seen in all cases (Figure 3), and in Patients 2,3, and 4, multiple thrombi were identified in smalldiameter vessels in the dermis and subcutaneous tissue (Figures 4 and 5), with no associated inflammatory cell infiltrate. Stains for elastin showed that the occluded vessels were venules, and stains for fibrin confirmed the presence of fibrin thrombi. The results of the protein C antigenic assay were normal or elevated in all samples except one from Patient 4 (Table II). However, there was reduced functional protein C measured by both chromogenic and anticoagulant assay, with a greater reduction by the latter. With both assays, there was wide variability between patients and in the same patient at different times, with a range of 11% to 120% by chromogenic assay and 17% to 70% by anticoagulant assay. Specific functional activity was also reduced, and this was also more prominent with the anticoagulant assay. Protein C levels and specific activities in patients with calciphylaxis were compared with levels and activities in normal subjects and patients with renal disease (Table III). In patients with calciphylaxis, the functional protein C level measured by chromogenic assay was significantly reduced compared with that in normal subjects (p
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Figure 2. Subcutis near an area of necrosis showing deposition of calcium in the wall of an artery (hematoxylin and eosin stain; bar 40~ before 30% reduction).
PROTEIN
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Figure 3. Section from involved area with full-thickness necrosis of the skin (hematoxylin and eosin stain: bar 160 p before 30% reduction).
development of deep necrotic lesions. An eschar forms over the defect, which may heal slowly, often with scarring. Skin grafting may be needed, and amputation of extremities or breast has been required for nonhealing lesions [lo]. All of the patients in this report with calciphylaxis were women with lesions involving thigh, buttock, or breasts in three patients. Burning pain was a frequent presentation. The clinical appearance of the lesion was typical of warfarin-induced skin necrosis (Figure l), as was the progression with slow healing that required skin grafting in one patient and amputation in another. The histologic appearance was also similar (Figures 2,3,4, and 5) with fibrin thrombi in otherwise normal venules without surrounding inflammation, indicating that local thrombosis plays an important role in the development of skin lesions in calciphylaxis as it does in warfarin skin necrosis. The protein C abnormalities that we found in renal patients without calciphylaxis are consistent with prior reports, Normal or elevated levels of protein C measured antigenically or functionally have been found in patients with nephrotic syndrome [21-241. In patients undergoing hemodialysis, Sorensen et al [25] have described decreased protein C activity with normal protein C antigen levels, and Vaziri et al [26] found elevated protein C antigen levels with reduced protein C activity in patients with spinal cord injury and endstage renal disease. We found an increase in protein C antigen in patients undergoing dialysis or with nephrotic syndrome, but functional levels within the normal range resulting in an overall decrease in specific activity (Table III), but the reduction in functional activity and specific activity was greater in patients on dialysis with calciphylaxis. The decreased levels of functional protein C in patients with calciphylaxis (Tables II and III) suggest that the reduced activity of this natural anticoagulant pathway may play a pathogenetic role in the development of the skin lesions. Protein C is activated at the surface of endothelial cells by a complex of thrombin and thrombomodulin [27]. With protein S serving as a co-factor [28], activated protein C cleaves and inactivates factors V, and VIII,, thereby inhibiting coagulation [29,30]. Activated protein C also enhances fibrino-
lysis by inactivating plasminogen activator inhibitor [31]. Congenital homozygous deficiency of protein C results in purpura fulminans with hemorrhagic necrosis of the skin and a frequently fatal outcome due to widespread vascular thrombosis [32-341. Heterozygous deficiency results in a reduction of plasma protein C levels to approximately 50% of normal and is associated with a high risk of venous thromboembolic disease in some families [16,17]. Since protein C is a vitamin K-dependent protein [ll-131, warfarin administration results in reduction of functional levels, and a disproportionate reduction in the anticoagulant activity of protein C compared with the procoagulant activities of factors II, VII, IX, and X may contribute to the development of warfarin-induced skin necrosis during initiation of therapy [14,15]. This may occur more frequently in patients with heterozygous congenital deficiency who have lower baseline levels of protein C [18,19]. Patients with calciphylaxis had reduced levels of functional protein C but normal levels of protein C measured antigenically (Tables II and III), reflecting circulation of dysfunctional or inactivated protein C. A possible explanation for the lower clotting than chromogenic protein C activity is that dysfunctional enzyme or enzyme inhibitor complexes may cleave the small peptide in the chromogenic assay while having greatly reduced activity with the natural protein substrate. Congenital protein C deficiency with the presence in blood of immunologically reactive but dysfunctional protein C has been reported [35], but the clinical presentation was different than in our patients with calciphylaxis. In a patient with a fatal thrombotic disorder, an acquired IgG inhibitor of protein C was described resulting in normal antigenic but low functional protein C levels [36]. However, incubation studies with normal plasma showed no evidence of such an inhibitor in our patients. Two physiologic inhibitors of activated protein C have been described [37,38] one of which is accelerated by heparin [38]. In patients with disseminated intravascular coagulation, complexes of activated protein C with these inhibitors have been identified in plasma [39,40], and these inactivated complexes may result in elevated antigenic compared March
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Figure 4. Section from involved skin showing thrombi (arrows) in dermal venules in the absence of an inflammatory response .(hematoxylin and eosin stain; bar 60 Jo before 30% reduction).
1
TABLE II Protein
Patient Number 1
2 3 4 5
Figure 5. Involved skin showing two venules containing (arrows) with an adjacent patent medium-sized artery and eosin stain; bar 10 p before 30% reduction).
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fibrin thrombi (hematoxylin
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Antigenie 85 100 100 180 150 110 120 200 40 120 9”;
in Patients
with Calciphylaxis
Protein C (%) ChromoAntigenie coagulant 63 96 92 87 iI! 120 11 35 -. 8’: 90
70 17 i: 28 :“6 38 32 ;i 70
Specific Activity ChromoAntigenie coagulant 0.74 0.96 0.92 0.48 0.12 0.85 1 .oo 0.06 0.88 0.75 1.31 0.95
0.82 0.17 0.27 0.25 0.19 0.17 0.22 0.19 0.80 0.58 0.92 0.74
C or, alternatively, to reduced clearance of activated protein C-inhibitor complexes due to renal disease. Another potential explanation for reduced protein C levels is consumption due to intravascular thrombosis. However, this is an unlikely explanation in our patients, since reduced protein C levels persisted when skin lesions were healing at a time when there was no evidence of a thrombotic process. Wide variation was seen in functional protein C assays in patients with calciphylaxis (Table II) that could not be explained by variability in the assay systems. We would hypothesize that the thrombotic skin lesions develop at times of lowest protein C activity and that the variable protein C levels (Table II) and reduced concentrations present after healing of the skin lesions suggest additional factors contribute to their pathogenesis. Hyperparathyroidism is a feature in most patients with calciphylaxis, and a role in the development of skin necrosis is suggested by the healing of lesions after parathyroidectomy. However, some patients with calciphylaxis have had normal parathyroid hormone levels [7], and no prothrombotic effect of this hormone has been described. Since parathyroid hormone stimulates uptake of extracellular calcium, it may influence platelet aggregation, but recent studies
with functional protein C levels. Although the explanation for reduced functional protein C in calciphylaxis is unclear, the presence in plasma of complexes of activated protein C with inhibitor would explain the reduced protein C specific activity that we have found. This could result from either accelerated formation of such complexes due to increased activation of protein 256
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TABLE III
Comparison of Protein C Levels in Normal Subjects and Patients with Renal Disease with or without Calciphylaxis*
I
Group
Number
Antigenic
Protein C (%) Chromogenic
Anticoagulant
Chromogenic
Specific Activity Anticoagulant
I
Normal Dialysis Nephrotic syndrome Calclphylaxis
1; 8
119zk8 99f3 148f 15
97 94&2f 9 121 f 10
95 f 10 93fll 77f 19
0.81 f 0.03 0.96 0.05 0.84 f 0.07
0.79 f 0.10 0.96f0.11 0.55 f 0.14
0.75 f 0.14
0.47 f 0.13
* Mean f SEM.
do not support this [41]. The vascular calcification that is a hallmark of the syndrome may predispose to thrombosis by narrowing the vessel lumen and injuring the vascular wall. However, vascular calcification is common in uremia, occurring in up to 46% of patients undergoing dialysis [2], whereas calciphylaxis occurs rarely. It is likely that several factors interact in contributing to the pathogenesis of calciphylaxis. Our findings suggest that a reduced functional protein C level may be an important element in the pathogenesis, resulting in a hypercoagulable state and contributing to thrombosis in the presence of vascular damage.
ACKNOWLEDGMENT We thank Geraldine Quigley and M. Julianne Brown for technrcal assistance and L. Taylor-Donald and Carol Weed for help In the preparatron of the manuscript
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