Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

Annals of the Rheumatic Diseases 1985, 44, 614-620

Raised serum hyaluronate levels in : an effect of growth factor induced activation of connective tissue cells?

ANNA ENGSTROM-LAURENT,12 NILS FELTELIUS,2 ROGER HALLGREN,2 AND AKE WASTESON3 From the 'Department ofMedical and Physiological Chemistry, the Biomedical Center, Uppsala University; the 2Department ofInternal , University Hospital, Uppsala, Sweden; and the 3Clinical Research Center, University ofLinkoping, Sweden

SUMMARY The circulating levels of hyaluronate were determined in 36 patients with scleroderma and in 36 control subjects matched for age and sex. The mean serum hyaluronate concentration in patients with progressive systemic sclerosis (n=25) was 131±67 (SD) ,g/l and significantly greater (p<0001) than that of the controls (mean level 49±21 (SD) pg/i). Hyaluronate levels in patients with localised scleroderma (n=4) were 141±47 (SEM) pg/l and in patients with scleroderma-associated overlap syndromes (n=7) 202±54 (SEM) [ig/l. The increase in serum hyaluronate probably reflected an enhanced synthesis or outflow of hyaluronate from the connective tissue, or both; it could not be explained by affection of the liver, which is thecopyright. catabolic site of hyaluronate. The hyaluronate values were not related to certain serological indicators of inflammatory activity or to the extent of the lesions or the severity of internal organ manifestations. A positive correlation was noted between circulating platelet counts and hyaluronate levels (p<0-001). Plasma ,3-thromboglobulin was measured in 15 of the patients with systemic sclerosis and found to correlate positively with platelet counts. Raised levels of 3-thromboglobulin were associated with the highest hyaluronate values. Platelet-derived growth

factor, which stimulates connective tissue cells and is stored in the a-granules of platelets togetherhttp://ard.bmj.com/ with ,-thromboglobulin, was shown to enhance hyaluronate synthesis in fibroblast cultures. The results suggest an involvement in scleroderma of connective tissue activating substances released from platelets. Key words: hyaluronic acid, systemic sclerosis, platelets, fibroblasts, platelet-derived growth factor. on September 30, 2021 by guest. Protected

Systemic scleroderma is an inflammatory connective in the skin of scleroderma patients,7 and Fleisch- tissue disease with unpredictable but often un- majer and Perlish found an increase of dermatan favourable prognosis. Immunological aberrationsl4 sulphate.' Increased urinary excretion of glycosamino- and affection of the microvascular system5 have glycans in scleroderma has also been reported, been detected, but the aetiology remains unknown. possibly reflecting the severity of the disease.9 A striking manifestation in scleroderma is the Cultured skin fibroblasts from patients with sclero- replacement of subcutaneous fat by abnormal con- derma were recently shown to synthesise considerably nective tissue and accumulation of .6 Uitto more hyaluronate than normal skin fibroblasts.10 et al. reported an excess glycosaminoglycan content Hyaluronate seems to enter the circulation via the lymph12 and is rapidly eliminated by the hepatic Accepted for publication 5 March 1985. pathway. 14 Although the resulting concentrations Correspondence to Dr Anna Engstrom-Laurent, Section of , Dept of , University Hospital, of circulating hyaluronate are normally quite low, S-751 85 Uppsala, Sweden. the development of a new sensitive assay for 614 Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

Raised serum hyaluronate levels in scleroderma 615 hyaluronate'5 has enabled its determination not acid (Sigma); (2) 0-5 R1 of hyaluronate-substituted only in plasma or serum but in various biological gel'4 suspended in 400 p11 of 0-55 M sodium chloride fluids and tissue samples.'5'8 In the present work and 0-05 M sodium borate, pH 7-0; (3) 100 RlI of 125I1 this method was applied to the determination of labelled cartilage protein in 4 M guanidinium circulating hyaluronate levels in scleroderma, and to chloride,'4 about 20 000 cpm. After the incubation a study of whether raised levels correlated with the the gel was collected by centrifugation and washed severity or progress of the disease. twice. Standard curves for hyaluronate were obtained by the use of sodium hyaluronate (Healon) dis- Patients and methods solved in saline containing bovine serum albumin (Sigma). PATIENT DATA The acute phase plasma proteins, haptoglobin and Thirty-six inpatients with sclerodermatous skin orosomucoid, were measured by nephelometry at changes were included in the study. They had a the Department of and the serum mean age of 52 years and a mean disease duration of levels of IgG, IgA, and IgM by immunodiffusion at 7*5 years. Twenty-five patients qualified for the the Department of Clinical , University diagnosis of progressive systemic sclerosis (PSS).19 Hospital, Uppsala. White cells were measured Four patients suffered from morphea, i.e., a purely by means of a Coulter counter and platelets by dermatological affection with no systemic compo- means of a Thrombocounter (Coulter Electronics nent. Seven patients possessed clinical hallmarks Ltd, Great Britain). f-Thromboglobulin was assayed common to PSS but showed high antibody titres by a commercial kit (Amersham International, against extractable nuclear antigen and met the Amersham, UK). The normal mean plasma clinical criteria for mixed connective tissue disease 3-thromboglobulin value in our laboratory is 24 ,ug/l, (MCTD) established by Sharp.20 At the time of two SD ranges <10-50 ig/l. Blood samples were blood collection 11 of the patients with PSS were also subjected to the following analyses: alanine 100-500 alone or in aminotransferase, aspartate aminotransferase, bili- treated with cyclofenil mg/day, copyright. combination with prednisolone (n=5) 2-5-10 mg/day. rubin, and creatinine by means of a multichannel Two patients received penicillamine 500 mg/day, analyser (Greiner Electronics, Switzerland) with and another two received prednisolone 5-7-5 mg/day original reagents. as the only drug. Ten patients were without drug treatment, as were all patients with morphea. One CLINICAL FUNCTIONAL TESTS patient with MCTD was treated with prednisolone Pulmonary function tests were performed at the 7 5 mg/day. Department of Clinical Physiology, University Hospital, Uppsala and included determinations of ANALYTICAL METHODS total lung capacity, residual volume, vital capacity, http://ard.bmj.com/ Hyaluronate was determined according to the prin- and forced expiratory volume. Oesophageal func- ciples previously outlined15 and recently adapted for tion was assessed by radiography of the oesophagus measurements of serum samples. 18 In short, protein in the supine position and by cineradiography. with specific affinity for hyaluronate was prepared Renal function was evaluated by creatinine and from cartilage by affinity chromatography and [51Cr]EDTA clearance measurements. labelled with 1251. The radioactive protein was

incubated with varying amounts of free hyaluronic IN-VITRO EXPERIMENTS on September 30, 2021 by guest. Protected acid and a fixed amount bound to a Sepharose gel. Human normal foreskin fibroblasts (AG 1523, The protein was allowed to partition between free obtained from the Human Genetic Mutant Cell and bound polysaccharide. The amount of radio- Repository, Institute for Medical Research, activity pelleted with the gel is a function of the Camden, New Jersey) were plated in Eagle's mini- amount of free hyaluronate in the sytem. 500 ,ul of mal essential medium at a split ratio of 1:10 in Nunc the test sample was incubated with the following 35 mm dishes (A/S Nunc, Roskilde, Denmark). reagents for 20 h at 4°C during slow rotation: (1) 400 After four days of incubation the medium was pl of 1-5 M sodium chloride, 0-025 M phosphate removed, and the cultures were incubated for two buffer, pH 7-0, containing 33 mM ethylenediamine- days in serum-free MCDB 105 medium21 22; at this tetra-acetic acid (EDTA) (Merck, Darmstadt, FRG), stage the cultures had not yet reached confluency. 3 mg/l soybean trypsin inhibitor (Worthington, Pure platelet-derived growth factor (PDGF1 available Freehold, NJ), 1 mM iodoacetic acid (Sigma, St in our laboratory23) or epidermal growth factor Louis, MO), 3 mM phenylmethylsulphonyl fluoride (EGF; Collaborative Research Incorporated, (Sigma), 17 mM benzamidine (Sigma), 3 mg/l Waltham, Massachusetts) was then added at 10 Rig/l, pepstatin A (Sigma), and 0.33 M e-amino-n-caproic and the incubation was prolonged for up to 30 h. Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

616 Engstrom-Laurent, Feltelius, Hallgren, Wasteson

The medium was harvested, and the remaining cell when patients with morphea and MCTD were layer was digested with 1-0 ml of trypsin (50 mg/l in included in the statistical analysis (r=0-55, p<0-001) phosphate-buffered saline). The medium and trypsin (Fig. 1). No relationship was seen between serum fractions were assayed for hyaluronic acid in the hyaluronate levels and polymorphonuclear or presence of protease inhibitors as described pre- monocyte counts. ViOUSIy.t4 17 When it became apparent that a relationship Statistical analyses were performed according to existed between platelet counts and serum hyaluron- Student's t test. ate levels we performed concomitant measure- ments of plasma ,B-thromboglobulin, serum hyalur- Results onate, and platelet counts in 15 of our patients with PSS. Their mean 3-thromboglobulin level was 108 SERUM HYALURONATE AND OTHER ig/l (range 32-299 ,ug/l). Eight of the patients had LABORATORY FINDINGS ,B-thromboglobulin levels exceeding 50 ,ug/l, i.e., the Table 1 lists the serum hyaluronate levels in patients upper normal range. Their mean serum hyaluronate with PSS, morphea, and MCTD. In PSS patients the value was 173 1g/l, whereas the mean hyaluronate average hyaluronate concentration was about three value was 109 ig/l in those PSS patients who had times higher than in age and sex matched healthy normal 3-thromboglobulin levels. There was a controls (p<0-001). Raised serum hyaluronate levels significant relationship between the 0-thrombo- of approximately the same magnitude were also globulin values and the platelet counts (r=0.51, observed in patients with morphea. The greatest p

Table 2 Laboratory findings' in patients with PSS, morphea, or MCTD Diagnosis Haptoglobin Orosomucoid IgG IgA IgM (gil) (gll) (gll) (gli) (gl) PSS 1-6±0-2 0-9+0-1 13-5±0-6 2-5±0-3 1-2+0-2 Morphea 2-3±0 4 0-9±0 2 11-2±0 8 2-4±0 9 1 0±0 2 MCTD 2-6±0-8 1-0±0-2 20-0+1 8 2-2±0-5 0-8±0-2 Reference ranges 0-3-2-0 0-4-1-2 8-2-19-9 0-7-3-2 0-3-2-2 Mean serum values ± SEM. Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

Raised serum hyaluronate levels in scleroderma 617

- 400-

0

C1 x:" 300- )I,

. . Fig. 1 Relationship between 200- 0 serum levels ofhyaluronate and 0. blood platelet counts, in patients 0 0 with PSS (0), MCTD (0), and .0 morphea (O). * a

* 0. 100- 0 0

0 00 0 0 a

100 200 300 400 500 600 Platelets 10719 copyright.

IN-VITRO EXPERIMENTS net synthesis of hyaluronic acid in the presence of The clinical evidence for increased platelet activity the growth factors, rather than to a shift in in scleroderma and the fact that platelets contain the distribution of hyaluronic acid between different growth factors PDGF and EGF acting on mesen- compartments of the culture. Determination of chymal cells induced us to test whether these factors hyaluronate after shorter periods of stimulation influenced the synthesis of hyaluronate. showed an increase in hyaluronic acid production at

Incubation of human foreskin fibroblasts with 20 h but not at 8 h (Fig. 2). http://ard.bmj.com/ PDGF or EGF in a completely synthetic medium caused an increase in hyaluronate accumulation Discussion both in the medium (3-7-5-2-fold stimulation) and in the cell layer (trypsin fraction; 1-6-1*7-fold stimula- The present findings of raised serum levels of tion) (Table 3). Since there was an increase in the hyaluronate in progressive systemic scleroderma are total amount of hyaluronate in the cultures, the in accordance with previous ideas of disturbed observed effect was ascribed to an increase in the glycosaminoglycan metabolism in this disease.7-10 However, the significance of such a postulated on September 30, 2021 by guest. Protected disturbance, at least when estimated by serum hyaluronate measurements, seems evident for Table 3 Determination of hyaluronic acid in fibroblast less cultures incubated in the presence or absence of PDGF and the disease process. Thus we were unable to link EGF for 30 hours serum hyaluronate values to the severity of internal organ manifestations or to the intensity of the Condition Compartment ng hyaluronatel Fold inflammatory process as reflected by serum con- dish stimulation centrations of acute phase plasma proteins or If it is that raised Medium 123-4 1 immunoglobulins. assumed NoNoadditionadition { Cell layer 129-1 1 hyaluronate levels in the circulation of scleroderma Medium 657-0 5-2 patients could originate from certain affected organs, PDGF.PDGF 10 s~lAgg/l i Cell layer 221 6 1-7 then one should particularly consider the sclero- Medium 459-0 3.7 EGF,10 g/l dermatous skin as a possible source, since raised EGF. 10 Cell 206 7 1-6 pg/lI{ layer serum hyaluronate levels were observed also in Mean of duplicatc detcrminations on duplicate dishes. patients with the morphea type of scleroderma who Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

618 Engstrom-Laurent, Feltelius, Hdllgren, Wasteson

600- 0)n 0 0 a) 4- Cco 0 400- I3 a Fig. 2 The amount of I hyaluronate in the medium during incubation offoreskin fibroblasts for various times with PDGF IO [Lgll (0), with EGFJO gll (5), 200- and in the absence ofadditives (0). 0 8 0 80 0 0

4 8 12 16 20 24 28 Time of incubation h lacked other organ manifestations. However, no effect on hyaluronate synthesis of platelet-derived a relationship existed in PSS or morphea between growth factor (PDGF), defined growth factor fromcopyright. serum hyaluronate and the extent of the sclerosis platelets.27 Since according to a recent report in the skin. Experimental studies indicate that some epidermal growth factor EGF may also be a normal circulating hyaluronate could arise from the joint platelet constituent,28 the latter was also included in and more specifically the synovial fluid, which is the study. An in-vitro system was selected (cultured very rich in this polysaccharide.24 Increased synovial human skin fibroblasts) that may reflect the activities production of hyaluronate is likely in inflammatory of the same category of cells as that affected in arthritides owing to the greatly increased volumes of scleroderma, i.e., cells of the connective tissue type. synovial fluid found in these conditions. We have Both growth factors, in pure form and in the therefore attributed to the inflamed synovial tissue a absence of non-defined additives, markedly enhanced http://ard.bmj.com/ critical role for the increased blood concentrations the production of hyaluronate. Similar findings have recently detected in rheumatoid arthritis.25 An previously been made with the so-called connective enhanced outflow from the joint cavity might be one tissue activating peptides.29 30 PDGF is normally mechanism behind the raised serum hyaluronate stored in the platelet a-granules27 and would have to values found in our scleroderma patients with be released from this site in order to reach putative overlap syndrome, since all suffered from joint target cells. There is as yet no completely reliable complaints and various degrees of arthritis. How- technique for measuring the concentrations of PDGF on September 30, 2021 by guest. Protected ever, it seems less likely that such a mechanism in plasma, and therefore we have not attempted to could provide a general explanation for the raised determine this component in the plasma of our serum levels in scleroderma, since only three patients scleroderma patients. Recently, however, raised with systemic sclerosis had apparent joint involve- levels of circulating f-thromboglobulin, another ment. polypeptide of the platelet a-granules,31 were re- The mechanisms regulating the in-vivo synthesis ported in this disease, indicating in-vivo activation of hyaluronate are unknown, but studies in vitro of platelets and increased release of platelet granule indicate that certain inflammatory mediators like constituents.32 Under conditions of selective release prostaglandin E and interferon may stimulate fibro- PDGF is accompanied by j6-thromboglobulin,33 blasts to an enhanced hyaluronate production.26 suggesting that increased circulating levels of PDGF Such mediators might also be operative in inflam- also may be found in scleroderma. Analysis of matory conditions like scleroderma. The observa- ,-thromboglobulin in 15 of our patients confirmed tion that the blood platelet counts in our scleroderma that there are raised levels. The values were also patients were positively correlated to their serum positively correlated with the platelet counts. It is hyaluronate values prompted us to examine the tempting to suggest that both the proliferation of Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

Raised serum hyaluronate levels in scleroderma 619 connective tissue and the increased level of serum 10 Cabral A, Castor C W. Connective tissue activation. XXVII. hyaluronate in scleroderma are due to a The behavior of skin fibroblasts from patients with scleroderma. pathological Arthritis Rheum 1983; 26: 1362-9. release of PDGF, a growth factor known specifically 11 Bashey R I. Millan A, Jiminez S A. Increased biosynthesis of to activate mesenchymal cells.27 glycosaminoglycans by scleroderma fibroblasts in culture. Other factors which might influence the circulating Arthritis Rheum 1984; 27: 1040-5. hyaluronate levels in scleroderma include 12 Laurent U B G, Laurent T C. On the origin of hyaluronate in impaired blood. Biochem Int 1981; 2: 195-9. hepatic elimination of polysaccharide and abnormal 13 Fraser J R E, Laurent T C, Pertoft H, Baxter E. Plasma metabolism due to pharmacological therapy. It has clearance, tissue distribution and metabolism of hyaluronic acid been demonstrated in vivo that the uptake and injected intravenously in the rabbit. Biochem J 1981; 200: degradation of hyaluronate occur in the liver 415-24. 14 Fraser J R E. Laurent T C, Engstrom-Laurent A, Laurent sinusoids'3 34; experiments in vitro have shown that U B G. Elimination of hyaluronic acid from the blood stream this occurs in the endothelial cell fraction.3536 in the human. Clin Exp Pharmacol Phvsiol 1984; 11: 17-25. Observations of very high serum values of hyaluronate 15 Laurent U B G, Tengblad A. Determination of hyaluronate in in cirrhotic patients further central biological samples by a specific radioassay technique. Anal emphasise the Biochem 1980; 109: 386-94. role of the liver in hyaluronate metabolism.37 Our 16 Laurent U B G. Hyaluronate in aqueous humour. Exp Eye Res patients with scleroderma had no laboratory signs of 1981; 33: 147-55. liver disease, but this does not rule out the theoretical 17 Dahl L, Hopwood J J, Laurent U B G. Lilja K, Tengblad A. of an the endothelial The concentration of hyaluronate in amniotic fluid. Biochem possibility affection of liver Med 1983; 30: 280-3. cells in this disease. A number of our patients with 18 Engstrom-Laurent A. Laurent U B G, Lilja K, Laurent T C. PSS were treated with low dosages of coiticosteroids. Concentration of sodium hyaluronate in serum. Scand J Clin Such treatment but at higher dosages reduces Lab Invest (in press). increased serum at 19 Masi A T. Rodnan G P. Preliminary criteria for the classifica- pathologically hyaluronate levels, tion of systemic sclerosis (scleroderma). Arthritis Rheum 1980; least in patients with active rheumatoid arthritis.25 23: 581-90. In conclusion this study has shown that there are 20 Sharp G C, Irwin W S, Tan E M, Gould R G, Holman H R. Mixed connective tissue disease; an apparently distinct rheuma- raised levels of hyaluronate in serum from patients copyright. with scleroderma and suggests that this may be due tic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 1972; 52: to abnormal stimulation by connective tissue activat- 148-59. ing substances from platelets. 21 McKeehan W L, McKeehan K A, Hammond S L. Ham R G. Improved medium for clonal growth of human diploid fibro- Supported by grants from the Swedish Medical Research Council, blasts at low concentrations of serum proteins. In Vitro 1977; Gustaf V 80 Year Fund, the Swedish Association against Rheuma- 13:399-416. tism, the Medical Faculty of Uppsala. and Signe and Reinhold 22 McKeehan W L, Ham R G. Assay and partial purification of Sunds, Foundation. factors from serum that control multiplication of human diploid fibroblasts. Biochem Biophys Res Commun 1978; 80: 1013-21.

23 Johnsson A, Heldin C H, Westermark B, Wasteson A. Platelet http://ard.bmj.com/ derived growth factor: identification of constituent polypeptide References chains. Biochem Biophys Res Commun 1982; 104: 66-8. 1 Chaves F C, Rodrigo F G, Franco M L, Esteves J. Systemic 24 Antonas K N, Fraser J R E, Muirden K D. Distribution of sclerosis associated with auto-immune haemolytic anaemia. BrJ biologically labelled radioactive hyaluronic acid injected into Dermatol 1970; 82: 298-302. joints. Ann Rheum Dis 1973; 32: 103-11. 2 Murray-Lyon I M, Thompson R P H, Ansell I D, Williams R. 25 Engstrom-Laurent A, Hallgren R. Circulating hyaluronate in Scleroderma and primary biliary cirrhosis. Br Med J 1970; iii: rheumatoid arthritis: relationship to inflammatory activity and 258-9. the effect of therapy. Ann Rheum Dis 1985; 44:

3 Winkelmanni R K. Carapeto F J, Jordon R E. Direct immuno- 83-8. on September 30, 2021 by guest. Protected fluorescence in the diagnosis of scleroderma syndromes. Br J 26 Yaron M, Yaron I, Wiletzki C, Zor U. Interrelationship Dermatol 1977; 96: 231-8. between stimulation of prostaglandin E and hyaluronate produc- 4 Heinzerling R H, Weyer R, Dziuba D S, Belviso H M, tion by poly (I), poly (C) and interferon in synovial fibroblast Gurnham T K. Elevated levels of antibodies to polyuric acid culture. Arthritis Rheum 1978; 21: 694-8. detected and quantitated in systemic scleroderma patients by 27 Westermark B, Heldin C-H, Ek B, et al. Biochemistry and solid phase radioimmunoassay. J Invest Dermatol 1980; 75: biology of platelet-derived growth factor. In: Guroff G, ed. 224-7. Growth and maturation factors. New York: Wiley, 1983: 5 Norton W L, Nardo J M. Vascular disease in progressive 73-1 14. systemic sclerosis. Ann Intern Med 1970; 73: 317-24. 28 Oka Y, Orth D N. Human plasma epidermal growth factor/ 6 Fleischmajer R, Damiano V, Nedwich A. Scleroderma and the fl-urogastrone is associated with blood platelets. J Clin Invest subcutaneous tissue. Science 1971; 171: 1019-21. 1983; 72: 249-59. 7 Uitto J, Ohlenschlager K, Lorenzen I B. Solubility of skin 29 Castor C W, Ritchie J C, Williams C H Jr, et al. Connective collagen in normal human subjects and in patients with tissue activation. XIV. Composition and action of a human generalized scleroderma. Clin Chim Act 1971; 31: 13-8. platelet autocoid mediator. Arthritis Rheum 1979; 22: 260-72. 8 Fleischmajer R, Perlish J. Glycosaminoglycans in scleroderma 30 Sisson J C, Castor C W, Klavons J A. Connective tissue and scleredema. J Invest Dermatol 1972; 58: 129-32. activation. XVIII. Stimulation of hyaluronic acid synthetase 9 Hardy K H, Rosevear J W, Sams W M, Winkelmann R K. activity. J Lab Clin Med 1980; 96: 189-97. Scleroderma and urinary excretion of acidic glycosamino- 31 Moore S, Pepper D S, Cash J D. The isolation and characterisa- glycans. Mayo Clin Proc 1971; 46: 119-27. tion of a platelet specific ,B-globulin (fr.thromboglobulin) and Ann Rheum Dis: first published as 10.1136/ard.44.9.614 on 1 September 1985. Downloaded from

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the detection of antiurokinase and antiplasmin from thrombin- circulating hyaluronic acid. A whole body autoradiographic aggregated washed human platelets. Biochim Biophys Acta study. Cell Tissue Res 1983; 233: 285-93. 1975; 379: 360-9. 35 Eriksson S, Fraser J R E, Laurent T C, Pertoft H, Smedsr0d B. 32 Kahalek M B, Osborn I, Leroy E C. Elevated levels of Endothelial cells are a site of uptake and degradation of circulating platelet aggregates and beta-thromboglobulin in hyaluronic acid in the liver. Exp Cell Res 1983; 144: 223-8. scleroderma. Ann Intern Med 1982; 96: 610-3. 36 Smedsr0d B, Pertoft H, Eriksson S, Fraser J R E, Laurent T C. 33 Kaplan K L, Broekman M J, Chernoff A, Lesznik G R, In vitro studies on the uptake and degradation of sodium Drillings M. Platelet alpha-granule proteins: studies on release hyaluronate in rat liver endothelial cells. Biochem J (in press). and subcellular localization. Blood 1979; 53: 604-9. 37 Engstrom-Laurent A, Nyberg A, Loof L. Increased serum 34 Fraser J R E, Appelgren L-E, Laurent T C. Tissue uptake of hyaluronate in liver diseases. (in press). copyright. http://ard.bmj.com/ on September 30, 2021 by guest. Protected