Alterations of primary haemostasis in mixed connective tissue disease (MCTD)

THROMBOSIS RESEARCH 63; 281-286,199l 0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

ALTERATIONS OF PRIMARY HAEMOSTASIS IN MIXED CONNECTIVE TISSUE DISEASE (MCTD) M . Udvardy, E. Bodolayx, Gy. szegedi J. Hbrsfalvi, Z. Boda, and K. Rak

X

,

2nd and 3rdx Department of Medicine, University Medical School, 4012 Debrecen, Hungary (Received

13.12.1990;

accepted

in revised form 15.4.1991

by Editor O.N. Ulutin)

ABSTRACT Willebrand-factor antigen level and structure analysis, ristomycin-cofactor assay, beta-thromboglobulin and thromboxane metabolite estimations were performed in 22 patients with mixed connective tissue disease to evaluate the incidence and the possible role of haemostatic alterations in the complications occurring during the course of the disease. High levels of Willebrand-factor antigen and ristomycin-cofactor acitivity were detected in patients with throm.bocytopenia, previous thrombotic event, pulmonary vascular lesions and usually in the presence of circulating antiendothelial antibodies. Increased platelet activation could have been found in antibody positive cases and in patients with thrombocytopenia as well. The documented alterations of endothelial and platelet funtions may play important role in the vascular complications of mixed connective tissue disease.

INTRODUCTION-

Mixed connective tissue disease (MCTD) is characterized by the simultaneous appearance of overlapping clinical symptoms of systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis and polymyositis (1, 2, 6, 9). The most frequent symptom-complex consists of arthritis, Raynaud's phenomenon and swollen hands. Impaired pulmonary functions, myositis, dermatological signs, neurological involvement may illustrate the diverse clinical picture. The sera of MCTD patients contain autoantibodies to nuclear ribonucleoprotein (1, 9).

Key words: Mixed connective tissue disease, anti-endothelial antibodies, Willebrand factor, platelet activation 281

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One of us (E. Bodolay, 4) suggested previously that the sera of MCTD patients frequently contain antibodies which are cytotoxic to human umbilical vein endothelium in vitro at 370 C. The presence of antibodies was associated with higher frequency of disturbed cardiac, lung and cerebral functions, and therefore may be predictive of ongoing tissue damage. TWO cases of haemolytic-uraemic syndrome were also registered among the anti-endothelial antibody positive MCTD patients. In the present work some parameters of primary haemostasis, such as Willebrand-factor antigen (vWFAg) level and structure, ristomycin-cofactor activity (vWFRCof), beta-thromboglobulin (B-TG) and thromboxane-B2 (TXB-2) were investigated in MCTD patients to collect own experience upon their influence on the complications and clinical course of MCTD. Special attention was paid to the presence of anti-endothelial antibodies, owing to the frequent occurrence and postulated pathogenetic role of these type of antibodies in haemolytic-uraemic syndrome (7).

PATIENTS AND METHODS The samples of 22 MCTD patients were examined. The diagnosis of MCTD was made on the basis of arthritis/arthralgia, Raynaud's phenomenon, decreased lung ventillation capacity, myositis and skin involvement. The presence of four out of five clinical criteria and positivity for anti-nuclear ribonucleoprotein antibodies were necessary to satisfy the diagnosis of MCTD (6). Mean age was 34.9 years. 19 patients were female, three males. At the time of testing the mean follow-up time was 8,5 years. At the time of investigations the pathologic process was active in four patients and indicated a daily administration of 35 to 75 mg of prednisone. 18 patients were in the inactive stage (inactive period meant that there was no evidence of disease activity for at least one year, and no progressive organ damage ensued). Eight of 18 patients in the inactive stage were treated with 5-15 mg/day of prednisone and 10 patients were received non-steroid antiinflammatory agent (pyroxicam). Steroid and non-steroid treatments were interrupted 72 hours prior blood sampling. The vWFAg content of platelet-poor plasmas were assayed with immunoelectrophoretic method described by Zimmerman et al (12). Normal range of plasma vWFAg in our laboratory proved to be 62-128%. Two-dimension electrophoresis and SDS-agarose multimeric analysis were also performed (8). To assess vWFRCof-activity isolated washed platelets were used. 50~1 Ristomycin (Aggristin, Reanal, Hungary) was added to 400 ul washed platelet suspension altogether with 50,ul diluted patient plasma sample (Ristomycin final concentration: 1 mg/ml). Aggregation slopes were determined in a Chronolog dual channel aggregometer (3). The normal range achieved by this method was 70-130%. B-TG and TXB-2 contents of plasma samples were measured with commercially available radioimmunoassay kits (RCC IM 88 United Kingdom and MTA IRBO 17, Hungary, respectively) as it had been described previously (10). Our normal range for B-TG may be estimated as 20-60 ng/ml, whereas for TXB-2 30-70 pg-ml.

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Endothelial cell were derived from umbilical cord veins for anti-endothelial antibody estimations. The details were described previously (4); in brief: the cultured endothelial cells were incubated with 30 ,ul of patient's fresh-frozen serum, mixed with non-cytotoxic rabbit complement and incubated further at ambient room temperature for 50 minutes. At the end of the incubation period cell death was determined by trypan-blue exclusion test using a phase-contrast inverse microscope. The test was considered to be positive if more than 25% of the cells could take up trypan blue. 12 out of the 22 MCTD patients proved to be anti-endothelial antibody positive. Comparisons were made with "chi-square" test. RESULTS Summarizing the results of the 22 MCTD patients the following values have been obtained: the mean concentration of plasma vWFAq proved to be moderately elevated (133.69+ 56.55%). The relative motility of vWFAq,assessed by two-dimension electrophoresis showed only minor increase (1.14+ 17%) and also the multimeric analysis of vWFAg have simiiar or identical pattern to the healthy control group. The mean vWFRCof-activity was significantly high (200.36+159.20%). B-TG concentrations were also elevated in MCTD patients (mean: 147.91z67.31 ng/ml). Plasma TXB-2 levels were also high, 87.04243.12 pq/ml. The patients were divided into subgroups according to the detectability of anti-endothelial antibodies, to the presence or absence of thrombocytopenia (less than 100 G/l) and according to some of the important clinical features and complications (previous thrombosis, pulmonary vascular lesions, neurological signs, myositis, thyroid disorder, arthritis). The data of primary haemostasis parameters in the several groups are summarized in Table 1. Neurological complications were registered in eight of MCTD patients. Comparing their data with the other Pn.CTDpatients no differences could have been seen in vWFAg, vWRCof, B-TG or TXB-2 values between the two groups. Typical myositis, or thyroid disorder could have been seen only in few instances (3 and 2 respectively), and on the other hand dermatological signs, Raynaud's phenomenon were detected in almost all cases, SO the comparison on these bases would not have been releva.nt. DISCUSSION In the past few years it has become obvious that the prognosis of MCTD is not as good as was once thought (5, 9, 11). Lung biopsy materials revealed highly altered small arteries, endothelial proliferation. Consequently it is suspected that the damaged blood vessels may play an important role in disease mechanism. In the recent years anti-endothelial cytotoxic antibodies have been detected in the sera of patients with systemic lupus erythematodes, rheumatoid arthritis and progressive systemic

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Table 1. Primary haemostasis

vVWAg %(+SD) Antiendoth. antibody pos. (12) neg.

(10)

<0.02

P Platelets (100 G/l (7) >lOO

G/l

(15)

P Thrombosis ---detected (13) not detected (9) P Pulm. compl. detected (5) not detected (17) P

P

160.16 (70.67) 116.25 (35.83)

parameters

in MCTD

vWFRCof %(+SD)-

B-TG ng/ml(+SD)

TXB-2 pg/ml(+sD)

286.54 (175.26) 117.00 (69.74)

170.90 (45.06) 128.50 (56.29)

96.53 (44.87) 63.62 (32.13)

(0.05

)0.05

>0.05

148.16 (36.76) 121.93 (77.15)

359.15 (155.64) 155.56 (110.65)

205.42 (34.87) 120.53 (61.26)

122.71 (36.33) 62.61 (32.01)

(0.05

<0.02

>0.05

< 0.001

153.15 (66.90) 106.44 (26.42)

2.55.50 (169.87) 125.60 (64.77)

166.07 (63.38) 130.66 (57.32)

94.57 (44.94) 62.43 (31.00)

(0.05

( 0.02

172.80 (98.83) 126.27 (38.21)

343.00 (186.96) 173.88 (137.25)


( 0.02

> 0.10

210.40 (32.21) 129.05 (63.51) (0.05

values were assessed upon "chi-square" test

so.20

104.51 (42.50) 74.27 (41.41) Bo.05

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sclerosis, and in half of MCTD cases (2, 4). On the other hand the presence of similar antibodies have been documented in haemolytic-uraemic syndrome, a well-known disease of widespread endothelial damage and increased platelet activation (7), so it seemed to be justified to analyze the clinical presentation, anti-endothelial antibodies and some primary haemostasis parameters in the MCTD patients. In the present investigation some signs of endothelial damage, indicated by plasmatic vWFAg and vWFRCof-activity, altogether with moderately enhanced in vivo platelet activation, documented with elevated B-TG and TXB-2 concentrations could have been well registered in almost all FZCTD patient. The presence of anti-endothelial antibodies was associated with elevated vWFAg levels and vWFRCof-activity, without alterations in the platelet function tests examined in this setting. A similar pattern of primary haemostatic alterations were detected in those MCTD patients who previously sufferedfrcm thrombotic disorders. This suggests, that the endothelial damage and its further consequences may be regarded as thrombosis promoting factors in MCTD. In thrombocytopenic patients vWFAg and vWFRCofactivity altoqether with B-TG and TXB-2 concentrations proved to be increased. The association was specially marked with TXB-2, so thrombocytopenia in VCTD may sometimes be the consequence of increased in vivo platelet activation mediated by thromboxaneA2. The association of pulmonary complications (a typical lifethreatening complication of KCTD with endothelial proliferation) with high levels of vWFAg and vWFRCof-activity was also very remarkable. Neurological complications were independent of the presence or absence of primary haemostatic alterations. The analysis of the possible correlation of endothelial and platelet function parameters with dermatological, joint, thyroid symptoms or Raynaud's phenomenon was not relevant in the investigated group. No doubt, plasmatic vWFAg may be regarded as one of the "acute phase reactants", and inflammatory processes are able to cause elevation of vWFAg levels directly, so our results do not reflect necessarily endothelial cell damage. The higher plasmatic vWFAg concentrations in our >lCTD cases were not accompanied by vWFAg structure alterations, and this may provide further evidence, that increased vWFAg levels were merely the consequence of some of the inflammatory processes of MCTD. On the other hand the enhanced vWFRCof-activity indicates that this elevation is coupled with functional changes, too. This, and the lack of clinical activity (only four exceptions with moderate alterations only) during the time of investigations however raise the possibility that vWFAg and activity changes may be regarded rather as a consequence and indicator of endothelial damage in MCTD. Endothelial dysfunction or damage, increased platelet activation proved to be quite common characteristic of MCTD cases. The presence of anti-endothelial antibodies possibly enhances endothelial impairment and may predispose to thrombotic complications. In some instances consumptive type thrombocytopenia might have been suspected in YCTD. An uniform pattern of grossly elevated vWFAg and vWFRCof-activity and increased platelet activation indicated by high B-TG concentration could have been documented in pulmonary complications of MCTD. The alterations of primary haemostatic functions may play some

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role in several and sometimes serious complications of MCTD, so further investigation of endothelial and platelet functions seems to be desirable.

REFERENCES -1. ALARCON-SEGOVIA, D., VILLAREL, M. Classification and diagnostic criteria for mixed connective tissue disease. In: Mixed connective tissue and antinuclear antibodies (Eds. Kasukawa, R., Sharp, G.C.) Elsevier Science Publishers B.V. Amsterdam, p. 33, 1987. 2. BENNET, R.M., CORNELL, D.J. Mixed connective tissue disease. A clinicopathologic study of 20 cases.Seminars in Arthritis and Rheum. 10, 25-40, 1980. 3. BODA, Z., SOLUM, N.O., SZTARICSKAI, F., RAK, K. Study of platelet agglutination induced by the antibiotics of the Vancomycin group: Ristocetin, Ristomycin, Actinoidin, Vancomycin.Thromb. Haemostas. 42, 1164-1168, 1979. 4. BODOLAY, E., BOJAN, F., SZEGEDI, GY., STENSZKY, V., FARID, N.R. Cytotoxic endothelial cell antibodies in mixed connective tissue disease. Immunol. Letters 20, 163-168, 1989. 5. EULDERINK, F., CATS, A. Fatal primary pulmonary hypertension in mixed connective tissue disease. Z. Rheumatol. 40, 25-32, 1981. 6. KASUKAWA, R. Preliminary diagnostic criteria for classification of mixed connective tissue disease. In: Mixed connective tissue disease and antinuclear antibodies (Eds. Kasukawa R, Sharp, G.C.) Elsevier Science Publishers B.V., Amsterdam, p. 41, 1987. 7. LEUNG, D.Y. Lytic anti-endothelial cell antibodies in haemolytic uraemic syndrome. Lancet 2, 183-188, 1988. 8. RUGGERI, Z.M., ZIMMERMAN, T.S. The complex multimeric composition of Factor VIII/van Willebrand factor. Blood 57, 1140-1143, 1981. 9. SHARP, G.C. Therapy and prognosis of MCTD, In: Mixed connective tissue disease and antinuclear antibodies. (Eds. Kasukaz R. Sharp, G.C.) Elsevier Science Publishers B.V. Amsterdam, p. 315, 1987. 10. UDVARDY, M., TOROK, I, RAK, K. Plasma thromboxane and prostacyclin metabolite ratio in atherosclerosis and diabetes mellitus. Thromb. Res. 47, 479-484, 1987. 11. LJEDA, N. Mixed connective tissue disease with fatal pulmonary hypertension and a review of literature. Virch. Arch. (Pathol.-Anat.) 404, 335-431. 1984. 12. ZIMMERMAN, T.S., HOYER, L.W. DICKSON, L., EDINGTON, T.S. Determination of the von Willebrand's disease antigen in plasma by quantitative immunoelectrophoresis. J. Lab. Clin. Med. 86, 152-159, 1975. --