Clin. exp. Immunol. (1975) 21, 284-288.
REACTION OF HUMAN SMOOTH MUSCLE ANTIBODY WITH HUMAN PLATELETS RENI2E NORBERG, ASTRID FAGRAEUS AND K. LIDMAN Department of Immunology, National Bacteriological Laboratory,
Stockholm, Sweden (Received 27 January 1975) SUMMARY
Platelets, prepared from fresh human platelet-rich plasma smeared on slides and stained with human serum containing smooth muscle antibodies (SMA) in indirect IFL, showed a bright cytoplasmic fluorescence with numerous projections extending from the surface. A prerequisite for obtaining a positive reaction with SMA-positive serum was that a chelating agent was present in the suspending medium when preparing the smears. The projections could be demonstrated also by anti-HeLa cell (anti-species) serum. This indicates that the projections had a membraneous cover. Staining of live platelets was always negative. Platelets treated with cytochalasin B for 1 hr were smooth and spherical and did not show any surface projections.
INTRODUCTION The contractile substance of platelets, thrombosthenin, constitutes 15-20% of the total platelet protein (Bettex-Galland & Liischer, 1961) and has been shown to be directly involved in platelet aggregation (Booyse & Rafelson, 1969) and clot retraction (Nachman, Marcus & Safier, 1967). Thrombosthenin has been localized on the external surface of human platelets fixed in 400 glutaraldehyde using the antibody-peroxidase (PAP) staining method (Booyse et al., 1971). Surface thrombosthenin (S-thrombosthenin) was found to comprise only about 6-10% of the total platelet thrombosthenin, the remaining amount, cytoplasmic thrombosthenin (C-thrombosthenin) was extracted from lysed platelets (Booyse & Rafelson, 1971). It has been proposed that platelets attach to one another by the formation of interplatelet S-thrombosthenin bridges (Booyse & Rafelson, 1969). When activated, platelets should extrude their thrombosthenin-rich cytoplasm through the cell membrane (Booyse et al., 1972). Gabbiani et al. (1973) noted partial absorption of smooth muscle antibodies (SMA) by platelets damaged prior to incubation with SMA-positive serum but they were unable to absorb out SMA with intact platelets. SMA also react with several other 'non-muscular' structures. These include, e.g. lymphocytes (Fagraeus, The & Biberfeld, 1973; Gabbiani et al., 1973), brush border of intestinal epithelial cells and renal tubules Correspondence: Dr Rende Norberg, Department of Immunology, National Bacteriological Laboratory, 105 21 Stockholm, Sweden.
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(Gabbiani et al., 1973; Fagraeus, Lidman & Norberg, 1975) and liver cells (Farrow, Holborow & Brighton, 1971). SMA did not stain live suspended lymphoid cells (Fagraeus et al., 1975) but revealed on smeared lymphoblastoid cells, as well as a moderate cytoplasmic staining, strongly fluorescent long-hairy microvilli, extending from the surface of single cells (Fagraeus et al., 1974). A prerequisite for staining of smeared lymphoblastoid cells with SMA is that a chelating agent is present in the suspending medium when preparing the smears (Fagraeus et al., 1975). The reaction of human platelets with SMA was investigated under the same conditions as those for lymphoblastoid cells. Indirect immunofluorescence using SMA-positive serum and smeared platelets showed brightly stained cells with numerous projections, whereas intact suspended platelets were not stained with SMA. On the other hand anti-HeLa cell (anti-species) serum stained live platelets, also revealing the projections.
MATERIALS AND METHODS Platelets Blood, obtained from healthy blood donors, was collected into Fenwal J2A-25 double Platelet-Pack (Travenol Laboratories S.A. Brussels, Belgium). Platelet-rich plasma was obtained by sedimenting the red cells at 400 g for 15 min at 21°C. Platelets were sedimented at 1500 g for 15 min at 21'C. They were washed twice with saline and twice with a buffered salt solution (NaCl, 7-56 g; KCl, 0-20 g; Na2HPO4.2H20, 0-065 g; KH2PO4, 0-15 g; NaHCO3, 0 70 g; ascorbic acid, 0 003 g; Aq. dest. ad 1000 ml) and finally suspended and kept in the salt solution. SMA-containing sera These were collected from patients with chronic active hepatitis. Anti-HeLa cell serum This was produced in rabbits as described earlier (Fagraeus, Espmark & Jonsson, 1965). The serum was absorbed once with an extract of pig stomach (Biberfeld, Fagraeus & Lenkei, 1974).
Immunofluorescence (IFL) About 30 x 106 platelets were suspended in 0-025 ml of saline, buffered salt solution, or 0-034 M sodium citrate. The cells were immediately smeared on glass slides, dried before a fan and fixed in dry acetone at -20°C for 15'min. The slides were then used for indirect IFL, utilizing FITC-conjugated sheep anti-human immunoglobulin (SBL 4832) or sheep anti-rabbit immunoglobulin (SBL 6410"'). The optic system was Zeiss microscope equipped with a HBO 200 mercury lamp and oil condenser. Primary filter BG3 and secondary filters 44 or 44-47 were used. Staining of live platelets This was performed by mixing 100 x 106 cells with 0-2 ml of test serum appropriately diluted in saline buffered salt solution, or 0-128 M (isotonic) sodium citrate. After 30 min at 21°C the cells were washed three times. Thereafter, 0-2 ml of the appropriately diluted FITC conjugate was added. After 30 min the cells were washed three times and placed on slides. Glutaraldehyde fixation of intact platelets was performed as described by Booyse et al. (1971). Staining of fixed cells was done as described above for live platelets.
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Cytochalasin B treatment ofplatelets 100 X 106 cells were suspended in 1 ml of buffered salt solution supplemented with 10 jug of cytochalasin B. They were kept for 1 hr at 370C, then centrifuged, suspended in 0 05 ml of 0 034 M sodium citrate, smeared on slides and subjected to indirect IFL. RESULTS Intact platelets suspended in saline, 0 13 M sodium citrate or buffered salt solution were not stained with SMA-positive serum in indirect IFL. Rabbit anti-HeLa cell serum, on the other hand, gave a bright membrane fluorescence under the same conditions and revealed many cells with numerous projections. Platelets fixed in 400 glutaraldehyde failed to react with SMA-positive serum or with anti-HeLa cell serum.
FIG. 1. Human platelets suspended in 0 034 M sodium citrate, smeared and indiiectly stained with SMApositive serum. The platelets show a strong cytoplasmic fluorescence with numerous projections. (Magnification x 840.)
FIG. 2. Human platelets suspended in 0 34 M sodium citrate, smeared and indirectly stained with anti-HeLa cell (anti-species) serum that reveals, besides a cytoplasmic fluorescence, numerous projections. (Magnification x 615.)
FIG. 3. Human platelets suspended in buffer solution supplemented with 10 jig of cytochalasin B per millilitre, smeared and indirectly stained with SMA-positive serum. The smooth and spherical platelets show a faint cytoplasmic fluorescence but no projections. (Magnification x615.)
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Platelets suspended in 0 034 M sodium citrate, smeared on slides, fixed in acetone and stained in indirect IFL with SMA-positive serum showed a bright cytoplasmic fluorescence and numerous projections extending from the surface (Fig. 1). In contrast, platelets suspended in saline but otherwise treated as above did not show any fluorescence. Anti-HeLa cell serum gave a cytoplasmic fluorescence and exhibited projections whether saline, buffered salt solution, or 0034 M sodium citrate was used as suspending medium (Fig. 2). Platelets treated with cytochalasin B for 1 hr, then suspended in 0034 M sodium citrate and immediately smeared on slides, were spherical. The cytoplasm was stained with antiHeLa cells serum, but no projections were visible. There were weak reactions of cytochalasin B-treated platelets with SMA-positive serum (Fig. 3). DISCUSSION Live platelets were not stained in indirect IFL by SMA-positive serum. On the other hand, anti-HeLa cell serum gave a bright fluorescence of live platelets and of projections extending from the cell surface. Platelets fixed in 400 glutaraldehyde were not stained by SMA nor by anti-HeLa cell serum. According to Booyse & Rafelson (1971) a small amount of Sthrombosthenin was found in the exterior 'fluffy' coat of the platelets. This might have been undetectable by the suspension-staining technique used by us. However, a large number of SMA-positive sera from patients with chronic active hepatitis have been tested in the platelet aggregation test and no serum gave a positive result (Biberfeld & Norberg, 1974), which would be anticipated if antigens reacting with SMA were localized on the platelet surface. The specificity of SMA is not fully clarified and it might well be that SMA fails to react with the antigenic determinants of S-thrombosthenin that have been described as identical with myosin (Booyse et al., 1972). If so, S-thrombosthenin must be resistant to fixation with 40/a glutaraldehyde. We have found (Gatti, Ostborn & Fagraeus, 1974) that glutaraldehyde usually impairs various types of cell membrane antigens and we have not been able to preserve antigens reacting with SMA after glutaraldehyde fixation. Projections extending from the platelets were revealed by SMA-positive serum under the same conditions as those earlier described for lymphoblastoid cells or intestinal epithelial cells (Fagraeus, Lidman & Norberg, 1975). Thus, the platelets must be suspended before smearing in a medium containing a chelating agent. The mechanism by which the chelating agent exerts its effect is not fully understood. It has been demonstrated, however, that a variety of inhibitors of platelet aggregation i.a. EDTA has no effect on the surface projections formed at platelet activation but prevent further interaction and contraction of the platelets (Booyse & Rafelson, 1971). The chelating agents might in some way immobilize the projections and render them stainable by SM, e.g. by causing a depolymerization of the contractile proteins. According to Booyse et al. (1971) the platelet membrane does not directly participate in the early events preceeding and leading to platelet aggregation. It was postulated that at activation the platelet membrane permeability was changed sufficiently to allow the thrombosthenin-rich cytoplasm to be extruded through the membrane. But the platelet projections could be demonstrated by anti-HeLa cells serum in indirect immunofluorescence both on undamaged cells and on cells smeared on slides. This anti-HeLa cell serum had been absorbed with a crude extract of smooth muscle from pig stomach to avoid reactions with contractile proteins. This might argue against the theory which proposed that the projections were formed solely by extruded thrombosthenin, and it indicates that the projections
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have a membraneous cover. If the platelet membrane will change to allow cytogel extrusion later on in the sequence of events in platelet aggregation has not been investigated. Clot retraction is mediated by the contraction of blood platelets. This retraction is abolished by cytochalasin B (Wessels et al., 1971). The way in which cytochalasins act is not clear but the view that cytochalasin B acts by disrupting the function of the contractile microfilament system has been much promoted (Wessels et al., 1971). Platelets treated with cytochalasin B did not show any surface projections. The cells were smooth and spherical. Under the present experimental conditions SMA-positive serum showed a faint staining of smeared cytochalasin-treated cells in indirect immunofluorescence, whereas anti-HeLa cells serum gave a bright cytoplasmic fluorescence. The effect of cytochalasin B on platelets was of the same kind as that on lymphoblastoid cells (Fagraeus et al., to be published). The hairy projections ofthese cells disappeared within a few minutes and staining with SMApositive serum gave negative results. Thus cytochalasin B seemed to cause not only a relaxation of the contractile microfilaments but also an alteration in the contractile antigens rendering them less reactive with SMA. REFERENCES
BETTEX-GALLAND, M. & LJSCHER, E.F. (1961) Thrombosthenin, a contractile protein from thrombocytes. Its extraction from human blood platelets and some of its properties. Biochem. biophys. Acta (Amst.), 49, 536. BIBERFELD, G., FAGRAEUS, A. & LENKEI, R. (1974) Reaction of human smooth muscle antibody with thyroid cells. Clin. exp. Immunol. 18, 371. BIBERFELD, G. & NORBERG, R. (1974) Circulating immune complexes in Mycoplasma pneumoniae infection. J. Immunol. 112,413. BOOYSE, M.F., HOVEKE, T.P., KIsIELESKI, D. & RAFELSON, M.E., JR (1972) Mechanism and control of platelet-platelet interaction. Microvasc. Res. 4, 179. BOOYSE, F.M. & RAFELSON, M.E., JR (1969) Hypothesis: studies on human platelets. II. A contractile protein model for platelet aggregation. Blood, 33, 100. BOOYSE, F.M. & RAFELSON, M.E., JR (1971) Human platelet contractile proteins: location, properties, and function. Ser. Haemat. 4, 52. BOOYSE, F.M., STERNBERGER, L.A., ZSCHOCKE, D. & RAFELSON, M.E., JR (1971) Ultrastructural localization of contractile protein (thrombosthenin) in human platelets using an unlabeled antibody-peroxidase staining technique. J. Histochem. Cytochem. 19, 540. FAGRAEUS, A., ESPMARK, A. & JONSSON, J. (1965) Mixed haemadsorption: a mixed antiglobulin reaction applied to antigens on glass surface. Immunology, 9, 161. FAGRAEUS, A., LIDMAN, K. & BIBERFELD, G. (1974) Indirect immunofluorescence staining of contractile proteins in smeared cells by smooth muscle antibodies. Nature (Lond.), 252, 246. FAGRAEUS, A., LIDMAN, K. & NORBERG, R. (1975) Indirect immunofluorescence staining of contractile proteins in smeared cells by smooth muscle antibodies. Clin. exp. Immunol. (In press.) FAGRAEUS, A., THE, H. & BIBERFELD, G. (1973) Reaction of human smooth muscle antibody with thymus medullary cells. Nature: New Biology, 246, 113. FARROW, L.J., HOLBOROW, E.J. & BRIGHTON, W.D. (1971) Reaction of human smooth muscle antibody with liver cells. Nature: New Biology, 232, 186. GABBIANI, G., RYAN, G., LAMELIN, J.-P., VASALLI, P., MAJNO, G., BOUVIER, C.A., CRUCHAUD, A. & LUSCHER, E.F. (1973) Human smooth muscle antibody. Amer. J. Path. 72, 473. GATTI, R.A., OSTBORN, A. & FAGRAEUS, A. (1974) Selective impairment of cell antigenicity by fixation. J. Immunol. 113, 1361. NACHMAN, R.L., MARCUS, A.J. & SAFIER, L.B. (1967) Platelet thrombosthenin: subcellular localization and function. J. clin. Invest. 46, 1380. WESSELLS, N.K., SPOONER, B.S., ASH, J.F., BRADLEY, M.O., LUDVENA, M.A., TAYLOR, E.L., WRENN, J.T. & YAMADA, K.M. (1971) Microfilaments in cellular and developmental processes. Science, 171, 135.