A NEW FACTOR THAT MAY CONTROL COLLAGEN RESORPTION

333 TABLE I-MEAN AND RANGE OF WOUND

DEPTH, AND

AGE, SEX, WOUND LENGTH,

WOUND SITE BY TREATMENT GROUP

wound (table I). Table II shows the distribution of minor and major haematomas in the three treatment groups. Haematomas developed in only three out of fifteen of the thrombin-irrigated wounds, whereas haematomas developed in twelve out of fifteen diluent-irrigated wounds and twelve out of fifteen non-irrigated wounds

(A2=10.68; P<0.01). DISCUSSION

Since the treatment groups were well matched for sex, age, wound length, wound depth, and wound site, the differences in the haematoma-rates in these groups must have been due to the topical application of thrombin. The present study is being extended to determine whether the routine use of topical thrombin could facilitate better management of all surgical wounds, not just those in patients who have received low-dose heparin. Requests TABLE

II-FREQUENCY

for

reprints should be addressed to G.

OF WOUND HAMATOMA

S. S.

REFERENCES 1. International Multicentre Trial. Lancet, 1975, ii, 45. 2. Sherry, S. New Engl. J. Med. 1975, 293, 300. 3. Tidrick, R. T., Seegers, W. H., Warner, E. D. Surgery, 1943,

14, 191.

Hypothesis A NEW FACTOR THAT MAY CONTROL

haemostatic agent in selected surgical conditions has been reviewed by Tidrick et al.,3 but to the best of our knowledge its prophylactic use in the management of wound hxmatoma in heparinised patients undergoing routine abdominal surgery has not previously been

COLLAGEN RESORPTION

JOHN J. REYNOLDS ANTHONY SELLERS

GILLIAN MURPHY ELIZABETH CARTWRIGHT

Cell Physiology Department,

Strangeways Research Laboratory, Cambridge CB1 4RN

reported. METHODS

patients (all over the age of 40 years and of either sex) undergoing elective abdominal surgery were included in the trial. In all these patients, any pre-existing bleeding tendency was initially excluded on clinical and haematological grounds. Each patient received deep subcutaneous injections of heparin (5000 i.u.) an hour before surgery, and twice a day thereafter for five days. Consecutive patients were allocated into three treatment groups in the following manner: the first patient received tOpical thrombin (Parke, Davis & Co; 5000 units in 5 ml); the second patient received the diluent of topical thrombin (5 ml of saline containing benzethonium chloride preservative); and the third patient did not receive topical irrigation. This patient-allocation cycle was repeated until 45 patients had been studied. In each case, the deeper layers of the wound were 45 consecutive

standardised manner with continuous monofilaThe skin was closed with interrupted silk sutures, no subcutaneous stitch being used. Before skin closure the length and depth of the wound were measured and recorded. The wound area was then flooded uniformly with 5 ml of the topical solution by means of a needle and a syringe. Wounds were examined for haematomas by an independent surgeon on the 3rd, 5th, 7th, and 10th postoperative days. A localised collection of frank blood resulting in wound breakdown or requiring evacuation was categorised as a major hæmatoma. Any amount of discoloration (caused by altered blood) of skin contiguous with the wound area was classified as minor hxmatoma.

sutured in ment

a

nylon.

RESULTS

Patients in the three treatment groups were well matched for age, sex, and the dimensions and site of the

Summary

Specific collagenases responsible for the enzymic step leading to degracollagen fibrils of connective tissues have initial

dation of the been found in both latent and active forms. The most important factor controlling the local activity of collagenase extracellularly may be an inhibitor that is synthesised by connective tissues, and it is proposed that latent enzymes are all enzyme-inhibitor complexes. INTRODUCTION

SINCE 1962, when a specific collagenase was first described in tadpole tails, many mammalian collagenases have been reported.2-4 Collagenases are capable of degrading native collagen (the major structural protein of connective tissue) under physiological conditions to produce characteristic fragments, usually by cleavage at one point in the helical region of collagen chains. Collagenases have mostly been isolated from the media of either tissue fragments or cells in culture. The enzyme can only be extracted from either tissues or cells in small amounts, if at all. The sole exception is the polymorphonuclear leucocyte which contains a collagenase in specific granules,s but this enzyme is different from the collagenase of connective tissues. 2-4 Although there is strong evidence that collagenase is involved in the first stage of collagen degradation, the biological mechanisms controlling the activity of this enzyme are not clear, but it seems likely that collagenolytic activity is regulated by multiple pathways. Colla-

334 genases exist in both latent and active forms in the media from a wide variety of tissues in culture. Explanations for this latency have broadly been of two kinds: the latent collagenase is either in a proenzyme (zymogen) form that needs proteinases for activation,6-10 or is an enzyme-inhibitor complex, formed by active collagenase combining with inhibitors.11-16 The serum proteins &agr;2-macroglobulin12,17 and &bgr;1,-anticollagenase,15 are welldefined inhibitors of collagenase, and inhibitors from extracts of a rabbit tumour, 14 from extracts of bovine and human cartilage and aorta,18 and from the media of human skin fibroblast" and chick skin16 cultures have also been described. However, the data about these. inhibitors have cast little light on the importance of collagenase latency. Factors involved in

LATENT COLLAGENASES

Latent

collagenases

EVIDENCE FOR LATENT

are

activated

by trypsin

COLLAGENASES, ACTIVATION

or

collagen resorption.

other

BY A.P.M.A.

(4-AMINOPHENYLMERCURIC ACETATE), AND SYNTHESIS OF COLLAGENASE INHIBITOR

proteinases,6-10,13,19-23 but we have demonstrated that a thiol-binding reagent, 4-aminophenylmercuric acetate (A.P.M.A.), activates latent collagenases just as effectively. 24-21 Since activation of latent collagenases by A.P.M.A. decreased their molecular weights, as did trypsin activation, we concluded that enzymic activation is not evidence that latent collagenases are in a proenzyme form.26 We suggest that reagents such as A.P.M.A., thioand iodide16 activate collagenase by causing dissociation of enzyme-inhibitor complexes, whereas proteinases act by preferential degradation of inhibitor. The accompanying table lists the tissues and cells which in culture have been shown to produce latent collagenase (i.e., enzyme unmasked by proteolytic treat-

cyanate,12

ment). We found that rabbit bones in culture synthesised a potent inhibitor of mammalian collagenases during the first few days24,26,27 when no collagenase was detectable. Later in the culture period the medium contained latent collagenase, while the inhibitory activity declined in a manner, suggesting that inhibitor combines with enzyme to give the latent complex. Much later in the culture period active enzyme was detected, when no free inhibitor was present. Partially purified bone inhibitor combined with active collagenase to give an inactive latent enzyme, with properties similar to those of the naturally occurring latent enzyme.26-28 The molecular weight of the inhibitor is about 30 000, as is that of rabbit collagenase, yet the molecular weight of either the naturallyoccurring latent enzyme from uterus and bone cultures, or that from recombination of active enzyme with inhibitor, is approximately 40 000.26-28 It is not known whether this discrepancy can be accounted for by conformation changes or whether the inhibitor, as isolated, is a polymeric form of a lower molecular weight inhibitor." As shown in the table (last column) we have found potent inhibitors (molecular weight fraction about 30 000) in the media of a wide variety of tissues in culture.

reciprocal

R.A.=rheumatoid arthritis. p.A.=proliferative arthritis. ..=No information available. This data comes from recent reviews,’-’ from publish’ed,24-28 and unpublished work of our own and from other workers.6,8,9,13,16,19,20-23,29,30 Data on latent enzymes that have so far been activated by A.P.M.A. are largely our own published24-28 and unpublished work with the exceptions of collagenases from rabbit cornea (R. Leary and M. Berman, personal communication), human synovium (J. Tyler and T. Cawston, personal communication), and human synovial cells (E. D. Harris, personal communication).

HYPOTHESIS

We propose that all connective tissues

synthesise colinhibitors to control the local activity of the enzyme. We believe that the relative concentrations of active enzyme and inhibitor determine the sites and extent to which collagen resorption takes place (see accompanying figure). Because collagen usually has a lagenase

335

long half-life, and because it is difficult to extract much collagenase from tissues, it seems likely that collagenase is synthesised only under the control of specific stimulp-4 such as hormones, prostaglandins, or lymphokines.3O.31 Around stimulated cells, collagenase could be in excess of inhibitor, so that active resorption of collagen fibrils would take place there, but not elsewhere. Thus far there is only circumstantial evidence for the existence of the inhibitor in vivo, and the factors that control its synthesis are unknown. In our model (see figure), we suggest that the collagenase inhibitor is ubiquitous and normally provides a fail-safe mechanism to prevent resorption. The extent to which there may be tissue and species’ differences between inhibitors is uncertain, but there is already sufficient evidence to suggest that they are very closely related, if not identical. Latent human collagenase binds to collagen almost as well as does active collagenase,32 so it is possible that latent enzyme could under some circumstances become activated by proteinases. This might happen in an inflammatory condition with,an invasion by polymorphonuclear leucocytes, and if tissue cells were exposed to noxious stimuli. In such cases either activation of latent enzyme bound to collagen or a local decrease in inhibitor concentration could be related to the secretion of a

proteolytic activating enzyme.23,32 IMPLICATIONS FOR FUTURE RESEARCH

proposals are correct then previous investiga tions attempting to correlate collagenase synthesis and secretion with collagen resorption will have to be re-examined. In rheumatoid arthritis the loss of collagen can be equated with the permanent irreversible damage to articular cartilage,33 but the cellular origin of synovial collagenase is not yet certain. We suggest that the proliferating pannus synthesises either an excess of collagenase over inhibitor, or secretes enzymes that could either destroy inhibitor or activate latent enzyme. It seems quite possible that destruction of the inhibitor (or perhaps blockage of its synthesis) is the crucial event leading to articular damage, and we are currently testing these hypotheses. An imbalance between leakage of enzymes and provision of inhibitors by lining cells has recently been suggested as the primary lesion in osteoarthritis.34 The role of collagenase inhibitors in tumour invasion is also being investigated. Extracts of cartilage and aorta" inhibited tumour neovascularisation.35 The relation between the substances extracted from cartilage 18,35 and the inhibitor that we have isolated from bone26,27 is as yet unknown, but it does seem likely that collagenase inhibitors play a key role in preventing tumour cells from invading connective tissues. If

our

This work was supported by funds from the Medical Research Council, the Nuffield Foundation and the Smith, Kline and French Foundation. E. C. is a junior Beit fellow. Requests for reprints should be addressed to J. J. R. REFERENCES 1. Gross, J., Lapiere, C. M. Proc. natn. Acad. Sci. U.S.A. 1962, 48, 1014. 2. Harris, E. D., Krane, S. M. New Engl. J. Med. 1974, 291, 557, 605, 652. 3. Gross, J. in Biochemistry of Collagen (edited by G. N. Ramachandran and A. H. Reddi); p. 275. New York, 1976. 4. Harris, E. D., Cartwright, E. C. in Proteinases of Mammalian Cells and Tissues (edited by A. J. Barrett); p. 249. Amsterdam, 1977. 5. Murphy, G., Reynolds, J. J., Bretz, U., Baggiolini, M. Biochem. J. 1977, 162, 195.

Reviews of Books Female

Urinary Incontinence

STUART L. STANTON F.R.C.S., St George’s don : Lloyd-Luke. 1977. Pp. 118. £4.

Hospital, London.

Lon-

THIs book is most timely. The past decade has witnessed an step forward in our knowledge and understanding of the mechanisms involved in incontinence in women and consequently of the principles of management. The new science that has evolved is that of urodynamics. The work has been greatly fostered by the International Continence Society-formed seven years ago. The approach, rightly emphasised by Mr Innes Williams in his foreword, is interdisciplinary and involves neurologists, paediatricians, geriatricians, radiologists, . physicists, and engineers as well as gynaecologists and urologists. The book is divided simply into four chapters. The first reviews modern thinking on the functional anatomy and physiology of the bladder and urethra. The second chapter discusses clinical aspects of incontinence. The disparity that is often found between the history and the urodynamic data is emphasised. The third chapter, perhaps the most important, describes the modern investigational approach. The whole range of the urodynamic armamentarium is recorded in some detail. A frequent criticism voiced at postgraduate meetings is that the sophisticated and highly expensive equipment in use at specialist centres has little relevance for the ordinary "man on the spot". Simple cystometry with a central-venous-pressure set can, however, be done in all hospitals and might help to delineate that group of patients who, although presenting with stress incontinence, in reality suffer from detrusor instability. The last chapter discusses management. Although all the

enormous

6. Harper, E., Block, K. J., Gross, J. Biochemistry, 1971, 10, 3035. 7. Kruze, D., Wojtecka, E. Biochim. biophys. Acta, 1972, 285, 436. 8. Vaes, G. Biochem. J. 1972, 126, 275. 9. Hook, R. M., Hook, C. W., Brown, S. I. Invest. Opthal. 1973, 12, 771. 10. Oronsky, A. L., Perper, R. J., Schroder, H. C. Nature, 1973, 246, 417. 11. Bauer, E. A., Eisen, A. Z., Jeffrey, J. J. J. invest. Derm. 1972, 59, 50. 12. Nagai, Y. Molec. cell. Biochem. 1973, 1, 137. 13. Bauer, E. A., Stricklin, G. P., Jeffrey, J. J., Eisen, A. Z. Biochem. Biophys. Res. Comm. 1975, 64, 232. 14. McCroskery, P. A., Richards, J. F., Harris, E. D. Biochem. J. 1975, 152, 131. 15. Woolley, D. E., Roberts, D. R., Evanson, J. M. Nature, 1976,261,325. 16. Shinkai, H., Kawamoto, T., Hori, H., Nagai, Y. J. Biochem. 1977, 81, 261. 17. Werb, Z., Burleigh, M. C., Barrett, A. J., Starkey, P. M. Biochem. J. 1974,

139, 359. Kuettner, K. E., Hiti, J., Eisenstein, R., Harper, E. Biochem. Biophys. Res. Commun. 1976, 72, 40. 19. Harris, E. D., Reynolds, J. J., Werb, Z. Nature, 1975, 257, 243. 20. Birkedal-Hansen, H., Cobb, C. M., Taylor, R. E., Fullmer, H. M. Biochim. biophys. Acta, 1976, 429, 229. 21. Dayer, J. M., Krane, S. M., Russell, R. G. G., Robinson, D. R. Proc. natn.

18.

Acad. Sci. 22.

U.S.A. 1976, 73, 945.

Horwitz, A. L., Crystal, R. G. Biochem. Biophys. Res. Commun. 1976, 69,

296. 23. Woessner, J. F. Biochem. J. 1977, 161, 535. 24. Sellers, A., Cartwright, E. C., Murphy, G., Reynolds, J. J. Biochem. Soc. Trans. 1977, 5, 227. 25. Cartwright, E., Murphy, G., Sellers, A., Reynolds, J. J. ibid. p. 229. 26. Sellers, A., Cartwright, E., Murphy, G., Reynolds, J. J. Biochem. J. 1977, 163, 303. 27. Sellers, A., Reynolds, J. J. ibid. (in the press). 28. Murphy, G., Cartwright, E. C., Sellers, A., Reynolds, J. J. Biochim. biophys.

Acta, 1977, 483, 493. Birkedal-Hansen, H., Cobb, C. M., Taylor, R. E., Fullmer, H. M. Scand. J. dent. Res. 1975, 83, 302. 30. Wahl, L. M., Wahl, S. M., Mergenhagen, S. E., Martin, G. R. Science, 1975, 187, 261. 31. Dayer, J. M., Russell, R. G. G., Krane, S. M. Science, 1977, 195, 181. 32. Werb, Z., Mainardi, C. L., Vater, C. A., Harris, E. D. New Engl. J. Med. 1977. 296, 1017. 33. Harris, E. D. Arthritis Rheum. 1976, 19, 68. 34. Glynn, L. E. Lancet, 1977, i, 574. 35. Langer, R., Brem, H., Falterman, K., Klein, M., Folkman, J. Science, 1976,

29.

193, 70.