Mural thrombosis and atherogenesis in coronary arteries and aorta: An investigation using antifibrin and antiplatelet sera

Atherosclerosis, 19 (1974) 543-553 543 �9 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

M U R A L THROMBOSIS A N D A T H E R O G E N E S I S I N C O R O N A R Y

A R T E R I E S A N D A O R T A

AN INVESTIGATION USING ANTIFIBR1N AND ANTIPLATELET SERA

JANE HUDSON AND W. T. E. McCAUGHEY* School of Pathology, Trinity College, Dublin (Ireland)

(Revised, received November 9th, 1973)

SUMMARY

Using immunofluorescent methods fibrinogen/fibrin deposition was found to be a common occurrence in atherosclerotic lesions of the coronary arteries and aorta, and also in slight degrees of intimal thickening of the coronary arteries. With the coarser forms of deposit which were frequently present in macroscopically obvious intimal thickenings, evidence of platelet residues was often observed in corresponding areas. There were no obvious differences between subjects with occlusive coronary thrombi and those without as regards these findings. Fibrinogen/fibrin, and platelet residues, were found to be invariably piesent in organised occlusive coronary thrombi and some of the patterns of fibrin deposition were similar to those seen in many atheromatous lesions. The findings in this study support the view that repeated episodes of thrombosis contribute to the development of the atherosclerotic lesion. The reactions of the media of coronary arteries and aorta indicate that fibrinogen is frequently present in

the interstitial tissue.

Key words: Fibrin - lmmunofluorescence -Pathogenesis o f atherosclerosis- Platelets

INTRODUCTION

Using antifibrinogen or antifibrin serum a number of workers have shown that

various forms of reacting material aie often present in atheromatous lesions of the aorta 1-4. I t has also been found that fine deposits of this material frequently occur in

The authors wish to thank the Irish Heart Foundation for a grant in support of this work. * Present address: Department of Pathology, The University of Western Ontario, London 72,

Ont. (Canada).

544 J. HUDSON, W. T. E. MCCAUGHEY

fatty streaks 5,6 and in macroscopically normal intima 1. The question as to whether these findings are related to infiltration and trapping of plasma fibrinogen from the lumen or to deposition of thrombus remains unresolved. The coarser and especially the laminated form of reacting material has been held to be particularly suggestive of a thrombotic origin1,4; supporting this view is the finding that close on half of such banded deposits react with antiplatelet serum 4. However, in other work involving the use of antiplatelet serum, platelet antigen was not found to accompany the material reacting with antifibrinogen serum in uncomplicated atheromatous plaques 7. The banded form of fibrinogen/fibrin deposit has been observed to occur more frequently in the aortic plaques of subjects with coronary artery occlusion than in those with no significant stenosis 8.

The present report concerns an investigation of the frequency and character of fibrinogen/fibrin and platelet deposition in the intima of coronary arteries of subjects with coronary thrombosis and in matched controls.

MATERIALS AND METHODS

The material was obtained from 40 adult male subjects, 20 of whom had recent or old coronary thrombosis. The 20 cases without thrombosis were matched with cases of coronary thrombosis in respect of age (within 5 years), duration of storage of material, and the batch of antiserum used for staining. In collecting the material many cases with and without coronary thrombosis had to be rejected because of calcification in the coronary arteries. In each case two blocks were taken from areas of coronary artery showing no appreciable narrowing of the lumen. In the coronary thrombosis cases and wherever possible in the others a block was obtained from areas showing respectively slight, moderate (25-50 ~) , and severe narrowing. Samples of thrombosed segments of artery from these and other cases were also taken. Two blocks of aorta were obtained in most cases, at least one of which was from a fully developed non- ulcerated plaque. All blocks were snap-frozen at --70~ in a mixture of dry ice and isopentane and stored in air-tight containers at --20~

Preparation of antisera Anti-human fibrinogen/fibrin serum was produced in New Zealand white rab-

bits. Initially the method was that described by Crawford and Woolf 9, which uses both fibrinogen and fibrin as antigen. As the antisera prepared in this way were found to be weak when tested by the Ouchterlony technique, the rabbits were given 4 booster injections of human fibrinogen (Lister Institute, Elstree, Herts. 25 mg protein/ml) emulsified in Freund's complete adjuvans (1 ml:l ml) intramuscularly on alternate days. The rabbits were bled by cardiac puncture 7-10 days later, and the isolated serum was mixed with normal human serum. Following this adsorption the antiserum was found to be specific for fibrinogen when tested in double diffusion and immuno- electrophoretic systems. Platelet-rich plasma from Group O Rh-ve donors, freed as far as possible of red cells and lymphocytes, was used for the preparation of anti-

MURAL THROMBOSIS AND ATHEROGENESIS 545

human platelet serum. Platelet deposits were washed at least 6 times with saline and finally resuspended in saline to give a protein content of 1.5-2.0 mg/ml. 0.5 ml of this suspension, emulsified with an equal quantity of Freund's complete adjuvans, was injected intramuscularly into New Zealand rabbits on alternate days for 12 days and the rabbits bled by cardiac puncture 7-10 days later. The antiserum was inactivated by heating at 56~ for 30 min, and then absorbed repeatedly with washed human ery- throcytes of mixed ABO groups until no further agglutination occurred. The platelet agglutination titre was determined using the technique described by Dausset et al. ~o, but substituting platelet-rich plasma for washed platelets as antigen. All the antisera produced gave marked agglutination at dilutions of 1 in 256 and at least some agglutination at 1 in 512. Goat anti-rabbit serum was obtained commercially (Antibodies Inc.). Gamma globulin fractions of the 3 antisera described, and of non-immune rabbit serum, were separated by column chromatography on DEAE cellulose.

Conjugation The gamma globulin fraction of the goat anti-rabbit serum was conjugated with

fluorescein isothiocyanate absorbed on Celite (Calbiochem), at a rate of 1 mg dye to 100 mg protein. The unconjugated dye molecules were removed in a Sephadex (G-25- 80) column equilibrated with phosphate buffered saline (0.01 M, pH 7.1). To abolish non-specific staining all antisera were absorbed twice (20 min, 37 ~ with a well- washed homogenate of human renal cortex immediately before use.

Preparation and staining of sections Cryostat sections were cut at 6/~ inteivals at --20~ and air dried. Sections to be

used for platelet staining were fixed by a two stage ethanol/ether and ethanol/water technique 11. All sections were washed before staining in 3 changes of phosphate buffered saline (0.01 M, pH 7.1), dried and placed in a moist chamber at room tempe- rature. The gamma globulin fractions prepared from antifibrin serum (AFG) or antiplatelet serum (APG) were applied to the sections for 60 min and the sections were then washed in 4 changes of phosphate-buffered saline, dried, and covered with conjugated anti-rabbit gamma globulin for 45 min. Finally, the sections were washed in 4 changes of phosphate-buffered saline, dried, and mounted in buffered glycerol (pH 8.9). The material was examined with a Leitz fluorescence microscope using an HB200 mercury vapour lamp, a dark field condenser, UGI and BG38 primary filters, and a K430 barrier filter. Cryostat sections adjacent to those treated with antisera were stained by haematoxylin and eosin (HE), Sudan IV, and a modified picro-Mal- lory techniquOL

Specificity of antisera Specificity was tested on sections of human blood clot, thrombus and spleen.

These showed that APG reacted specifically with platelets but that AFG gave a weak cross-reaction with platelets. APG was also tested against smears of whole human blood and well-washed platelet and leucocyte suspensions. Intense staining of platelets


was found under these circumstances but the white cells also gave a slight reaction. These cross reactions have been reported previously lz. When test sections and smears were treated with APG that had been absorbed with an excess of fibrinogen or a well- washed finely ground suspension of fibrin (prepared from fibrinogen using bovine thrombin), similar staining patterns were seen to that given by unadsorbed APG. On testing APG against human plasma and serum by immuno-electrophoresis (using thinner agar than normal) and the Ouchterlony technique, a very faint thin precipitation band was sometimes formed with plasma. When APG was adsorbed twice with kidney homogenate (as was done for all routine staining) no precipitation band occurred.

When the first stage of the immunological system was replaced by (a) phosphate- buffered saline, (b) non-immune rabbit gamma globulin, (c) APG that had been absorbed with an excess of washed human platelets, or (d) A F G absorbed with an excess of washed ground fibrin or fibrinogen, specific staining was abolished. Blocking with non-conjugated goat antirabbit gamma globulin prior to the application of conjugated goat antirabbit gamma globulin was found either to eliminate specific fluorescence or to reduce it to a minimum.

RESULTS

Coronary arteries In most sections of coronary artery treated with A F G a line of intense specific

yellow-green fluorescence was observed on the inner surface of the intima (Fig. 1) and a similar though thinner line of specific fluorescence was also frequently noted after staining with APG. Identical reactions were sometimes seen on the endothelial surface of small blood vessels in the adventitia and have been noted by us in small blood vessels in other tissues.

Using AF G specific apple-green fluorescence was found within the intima in 50 of 58 sections where intimal thickening was slight (intima thinner than media) and in 112 of 114 sections with more conspicuous intimal thickening of diffuse or eccentric type. In slight intimal thickenings fluorescence was usually more conspicuous in the inner half of the intima and often took the form of fine flecks (Fig. 2). Fine threads or strands of fluorescence were also sometimes seen (Fig. 1) and occasionally coarser bands or clumps. With greater degrees of intimal thickening the reaction with A F G was usually much more prominent though sometimes largely localised in certain areas. Some of the reacting deposits were also coarser and often took the form of meshworks (Fig. 3), bands, or clumps (Fig. 4). Bands of fluorescence were usually most conspicuous in the inner intima and were found to be deposited in layers (Fig. 5) in at least some part of the inner intima of around 90 ~ of fibrous or fibrofatty plaques and just under 60 ~ of diffusely thickened intimas. Meshworks of fluorescence were particularly common in fibrous or fibrofatty plaques and together with clumps of specific fluorescence showed a tendency to occur in areas of lipid deposition. Aside from this relationship, and a few cases where HE sections showed small deposits of fibrin on the surface or in the substance of the intima, HE and Sudan-treated sections


Fig. 1. Coronary artery treated with AFG. A line of fluorescence is present on the inner surface of the intima and threads and strands of fluorescence in the substance of the intima. • 65. Fig. 2. Coronary artery treated with AFG. Flecks of specific fluorescence are scattered through the intima. Autofluorescence of elastic tissue is present at bottom. • 65.

3 4

Fig. 3. Coronary artery treated with AFG. Meshwork of fluorescence in substance of intima. • 290. Fig. 4. Coronary artery treated with AFG. Bands and strands of fluorescence lying parallel to the intimal surface with clumps of fluorescence in the deeper intima. • 65.

gave little indica t ion of the extent and form of fluorescence in the int ima. Similar

pat terns of fluorescence were found in int imal tissues of greatly varying cellularity and

density. Evidence of fibrin deposi t ion was sometimes seen in picro-Mallory prepara-

t ions but never in the quanti t ies revealed by immunofluorescence.

With A P G specific in t imal fluorescence was seen in 9 of 58 sections (15 ~ ) with

slight in t imal thickening, in 24 of 41 sections (59 ~ ) with diffuse in t imal thickening,


a n d in all ( I 0 0 ~ ) o f t he f ib rous o r fa t ty p l a q u e s w h i c h were p r e sen t in 73 sect ions .

T h e r e a c t i o n was a l m o s t i n v a r i a b l y m u c h m o r e res t r i c ted t h a n in sec t ions t r ea t ed

wi th A F G . L o c a l i s a t i o n o c c u r r e d m o s t f r e q u e n t l y in the a rea o f the m e s h w o r k s

5 6

Fig. 5. Coronary artery treated with AFG. Parallel bands of fluorescence lying close to the intimal surface. • 65. Fig. 6. Coronary artery treated with APG. A meshwork of reacting material is present in the substance of the intima (centre) and there is also a line of reaction on the intimal surface. Autoftuorescence of hyaline material is present at right margin. • 290.

Fig. 7. Media of coronary artery treated with AFG. Numerous small clumps of specific fluorescence are present. The internal and external elastic laminas are autofluorescent. There are a few specks of specific fluorescence in the intima at top. • 65.

M U R A L THROMBOSIS A N D ATHEROGENESIS 549

of fluorescent material seen in sections treated with A F G (Fig. 6) and in a number of sections virtually all such areas seemed to give at least a patchy reaction. I t was also commonly noted in areas where the banded or clumped patterns of fluorescence had been seen in A F G preparations. In places where only flecks or strands of reaction had been observed in AFG-treated sections there was usually no localisation of APG.

Though the preliminary specificity tests carried out with A P G seemed to exclude the possibility of a significant closs-reaction with fibrinogen/fibrin it was decided to stain further sections with A P G that had been absorbed with plasma rendered free of platelets and platelet fragments v. In the 40 sections so treated the reaction with the plasma-absorbed A P G was in all cases similar to that observed in adjacent sections treated with unabsorbed APG, though in some instance the intensity of the reaction was slightly reduced. The latter finding could be attributed to dilution.

Reactions in the media o f coronary arteries In most sections treated with A F G specific fluorescence was observed in the

media and especially in its outer half (Fig. 7). The reaction was unrelated to the amount of fluorescence seen in the intima and the extent or character o f intimal thickening. Some of the reacting material was clearly located in the interstices between muscle fibres. Similar reactions were observed in sections treated with two commercial anti-fibrinogen antisera (Hyland, Behringwerke AG). Additional washings with phosphate buffered saline did not reduce the reactions. When APG was used foci of fluorescence were occasionally noted in capillaries or close to torn adventitia.

Reactions o f thrombi in coronary arteries In 10 recent thrombi treated with A F G coarse and fine meshworks of fluoresc-

ence were prominent. With A P G irregular clumps of fluorescence were the dominant feature (Fig. 8). These features were generally maintained in 11 organising thrombi. In 20 completely organised thrombi the reaction with A F G was usually still conspicuous and sometimes meshworks, bands, clumps and flecks of fluorescence were noted similar to those observed in atheromatous lesions (Fig. 9). The vascular spaces within the thrombi frequently showed intense fluorescence in their walls (Fig. 10). A P G gave a positive though sometimes weak reaction in all cases and generally occurring in areas where coarser forms of fibrin had been observed with AFG.

None of these coronary thrombi showed evidence of significant lipid deposition in Sudan preparations though sudanophilic lipid was often prominent in adjacent atherosclerotic plaques.

Aorta In 68 sections showing a wide range of intimal thickening A F G gave a specific

intimal staining in all instances and APG in all but 5. The patterns of staining were very similar to those seen in coronary material with similar degrees of intimal thickening. The media reacted with A F G in most cases but the reaction was nearly always less marked than in coronary arteries:

550 J. HUDSON, W. T. E. McCAUGHEY

Fig. 8. Recent coronary artery thrombus treated with APG. Clumps of platelets show intense fluorescence. • 290.

9 10

Fig. 9. Organised coronary artery thrombus treated with AFG. Meshworks, strands and clumps of fluorescence are present, x 65. Fig. 10. Organised coronary artery thrombus treated with AFG. Vascular spaces within the thrombus show prominent fluorescence of their walls, x 65.

Comparison of coronary thrombosis cases with controls The intensi ty and character of the in t imal reactions with A F G and A P G was

graded in all sections wi thout prior knowledge of their oiigin. A compar ison of the


results obtained in the coronary thrombosis and control cases revealed no significant difference between the 2 groups in respect of any degree of intimal thickening in the coronary arteries and aorta.

DISCUSSION

This investigation has shown that fibrinogen/fibrin deposits are present in a high proportion of atherosclerotic lesions of the coronary arteries and that their frequency and character is similar to that in the aorta. Evidence of platelet residues has also been found in the area of a substantial proportion of the coarser forms of deposit, such as were almost invariably noted in intimal thickenings which were more than slight. As fibrinogen is known to occur in human plateletsla, 14 and the APG used sometimes contained a weak antibody to fibrinogen when tested by gel methods before absorption with kidney homogenate, the possibility exists of a cross-reaction between APG and the fibrinogen/fibrin. However, the reactions of the APG on test sections, and following absorption with platelet-free plasma, clearly exclude this factor as having any significant bearing on our results.

The present study has confrmed the observation of Woolf and Carstairs 4 that there is a high incidence of the banded or laminated pattern of fluorescence in raised intimal lesions treated with AFG and that a positive reaction with APG frequently occurs in the same areas. We have also been impressed, however, by the frequency with which AF G reveals meshwork-type deposits in raised intimal lesions in both the coronary arteries and aorta and by the fact that such deposits react even more frequently with APG than the banded forms. Further evidence that these and other deposits are of thrombotic origin is our finding that A F G reveals similar deposits in organised thrombi in the coronary arteries.

In macroscopically normal coronary arteries where only slight degrees of intimal thickening were noted histologically, fibrinogen/fibrin deposits occurred in a high proportion of cases and mostly in the form of small flecks or strands. These findings are similar to those of Woolf 1 in macroscopically normal aorta. Our observation that the intima reacts with APG in a small proportion (15 ~ ) of cases suggests that thrombus deposition may sometimes be involved in even the early stages of intimal thickening.

The present study has shown that fibrin and platelet antigen are consistently present in organised coronary thrombi. This indicates that both antigens may persist for long periods of time and in turn that the immunoftuolescent findings in atheromatous lesions may at a minimum reflect the extent of thrombotic episodes over several years prior to death.

The findings in this study clearly support the view that repeated episodes of thrombosis play some part in the development of the atherosclerotic lesion, but whether thrombosis is the main pathogenetic factor is a matter for speculation. Our observation that sudanophilic lipid is not present in significant amounts in occlusive coronary thrombi of various ages is further evidence against the view that the accumu-


lations o f lipid in a theroma derive f rom constituents o f thrombi 15. It is o f interest,

however, that aortic intimal plaques induced by thrombosis in hypercholesterolaemic rabbits accumulate much more cholesteiol than immediately adjacent aor ta 16. This

preferential accumulat ion o f lipid has been attr ibuted to the increased permeabili ty o f newly formed intimal capillaries iv. Also significant in relation to the " th rombogen ic"

concept is the recent observation that aortic thrombi induced in normol ipaemic rabbits develop into lesions remarkably similar to fibrofatty a theromatous lesions in man is.

Woolf et al. s have found that banded fibrin deposits occur more frequently in white fibrous aortic plaques from patients with coronary artery occlusions than in those from patients without significant coronary artery stenosis and have equated this finding with a greater incidence of incorporated and incompletely organised residue of mural thrombosis in the aortic plaques of the coronary occlusion group. We have found no significant difference in the intimal reactions of coronary thrombosis and control cases with AFG and APG. Our comparison, however, was largely based on coronary artery material and some of our control cases had moderate degrees of coronary artery stenosis.

Previous workers in general do not appear to have observed a reaction with antifibrin or antifibrinogen serum in the media of the aorta, though it has been noted that fluoresceinated antifibrinogen gamma globulin occasionally stains the media of the inner aorta a. Our AFG as well as commercial antifibrinogen sera consistently stained the media of both the coronary arteries and aolta, especially in their outer halves. This finding is in keeping with histochemical studies which have demonstrated tryptophan-rich protein deposits in human aortic media and have shown by means of solubility studies that they contain fibrinogen as well as variable amounts of globulin 19.

The line of specific fluorescence that we regularly observed on the luminal surface of sections of coronary artery and aorta treated with AFG, is of the same type as that described by Woolf I in sections of macroscopically normal aorta from infants and children treated with antihuman fibrin serum. It does not appear to have been noted by other workers using immunofluorescent techniques, but it is of interest that a distinct band of tryptophan-rich protein which appears to contain fibrinogen or fibrin has been observed in the subendothelial zone of"'normal" human aorta 2~ That these reactions may relate at least in part to deposition of a fine layer of postmortem clot is suggested by their widespread disposition and our finding that APG frequently reacts in the same area.

REFERENCES

1 WOOLF, N., The distribution of fibrin within the aortic intima, Amer. J. Pathol., 39 (1961) 521. 2 HAUST, M. D., WYLLm, J. C. AND MORE, R. H., Atherosclerosis and plasma constituents, Part 1

(Demonstration of fibrin in the white plaque by the fluorescent antibody technique), Amer. J. Pathol., 44 (1964) 255.

3 KAO, V. C. Y. AND WISSLER, R. W., A study of the immunohistochemical localisation of serum lipoproteins and other plasma proteins in human atherosclerotic lesions, Exp. Mol. Pathol., 4 (1965) 465.


4 WOOLF, N. AND CARSTAIRS, K. C., Infiltration and thrombosis in atherogenesis. A study using immunofluorescent techniques, Amer. J. PathoL, 51 (1967) 373.

5 WOOLF, N. AND CRAWFORD, T., Fatty streaks in the aortic intima studied by an immuno-histochemical technique, J. PathoL BacterioL, 80 (1960) 405.

6 WYLL1E, J. C., MORE, R. H. AND HAUST, M. O., Demonstration of fibrin in yellow aortic streaks by the fluorescent antibody technique, J. Pathol. Bacteriol., 88 (1964) 335.

7 WALTON, K. W. AND WILLIAMSON, N., Histological and immunofluorescence studies in the evolution of the human atherosclerotic plaque, J. Atheroscler. Res., 8 (1968) 599.

8 WOOLF, N., SACKS, M. I. AND DAVIES, M. J., Aortic plaque morphology in relation to coronary artery disease, Amer. J. PathoL, 57 (1969) 187.

9 CRAWFORD, T. AND WOOLF, N., Hyaline arteriolosclerosis in the spleen. An immunohistochemical study, d. Pathol. Bacteriol., 79 (1960) 221.

10 DAUSSET, J., COLIN, M. AND COLUMBANI, J., Immune platelet isoantibodies, J. Vox Sang., 5 (1960) 4.

11 VAZQUEZ, J. J. AND DIXON, F. J., Immunohistochemical analysis of amyloid by the fluorescence technique, J. Exp. Ned., 104 (1956) 727.

12 CARSTAIRS, K. C., The identification of platelets and platelet antigens in histological sections, J. Pathol. Bacteriol., 90 (1965) 225.

13 SELIGMANN, M., COUDEMAND, B., JANIN, A., BERNARD, J. AND GRABAR, P., Etudes immunochimi- ques sur la pr6sence de fibrinogen dans des extraits de plaquettes humaines lavees et dans certains extraits leucocytaires, Rev. HaematoL, 12 (1951) 302.

14 CASTALDI, P. A,, AND CAEN, J., Platelet fibrinogen, J. Clin. Pathol., 18 (1965) 579. 15 DUGUID, J. B., Thrombosis as a factor in the pathogenesis of coronary atherosclerosis, J. Pathol.

Bacteriol., 58 (1946) 207. 16 FRIEDMAN, M. AND BYERS, S. O., Experimental thrombo-atherosclerosis, J. Clin. Invest., 40 (1961)

1139. 17 FRIEDMAN, M., BYERS, S. A. AND ST. GEORGE, S., Origin of lipid and cholesterol in experimental

thrombo-atherosclerosis, J. Clin. Invest., 41 (1962) 828. 18 HUANT, S.-M., SUMIYOSHI, A., WEIGENSBERG, B. E. AND MORE, R. H., Morphologic evolution of

aortic thrombus to fibrofatty atherosclerotic lesions in normolipaemic rabbits, Circulation, 46 Suppl. 2 (1972) 1034.

19 DAHL, A. A., ADAMS, C. W. M. AND BAYLISS, O. B,, Histochemical identification of plasma proteins in human aortic media, J. Pathol. Bacteriol., 90 (1965) 233.

20 MACMILLEN, R., ADAMS, C. W. M. AND IBRAHIM, M. Z. M., Histochemical identification of plasma proteins in the human aortic intima, J. Pathol. BacterioL, 89 (1965) 225.

Documents

Mural thrombosis and atherogenesis in coronary arteries and aorta: An investigation using antifibrin and antiplatelet sera