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Platelet interaction with surface-immobilized IgG induces the secretion of surface bound microparticles and fragmentation of the cells
Colloids and Surfaces B: Biointerfaces 10 (1998) 251–262
Platelet interaction with surface-immobilized IgG induces the secretion of surface bound microparticles and fragmentation of the cells Marita Broberg, Ha˚kan Nygren * Department of Anatomy and Cell Biology, University of Go¨teborg, Go¨teborg, Sweden Received 7 July 1997; accepted 30 December 1997
Abstract The interaction between isolated platelets and surface-immobilized IgG was investigated by immunofluorescence, using albumin and F(ab∞) -fragments as controls. Antibodies directed against platelet membrane antigens (anti-pan 2 platelet) were used to visualize platelet adhesion and the morphology of adhering cells. The number of adhering cells and the relative frequency of different morphologies were counted and analysed after different times of adhesion. Five distinct stages of cell adhesion and activation were detected and defined by different morphologies. Transitions between these states were seen by vital microscopy. Dendritic platelets with long dendrites were seen scattered at low surface densities of adhering cells and microvesicles transported along the dendrites were visualized by anti-pan platelet antibodies. As a last stage before total fragmentation, the platelets were seen to secrete microvesicles and form an area of surface-adhering microparticles on the IgG-coated surface, but not on control surfaces. The adhesion of platelets increased after 10 min, the same time surface bound microparticles appeared at the surface. The results indicate a novel pathway of non-self-recognition of foreign surfaces via IgG-mediated activation of platelets resulting in massive production of microparticles. © 1998 Elsevier Science B.V. Keywords: Platelets; IgG; Adhesion; Spreading; Microparticles
1. Introduction The initial contact of foreign materials with blood induces an array of recognition reactions. Initially, host plasma proteins arrive at the surface [1,2]. The most abundant proteins in human plasma, albumin, fibrinogen and immunoglobulins, will be contained in the initially formed protein film [3,4]. The structure and composition of the adsorbed protein layer are believed to determine the reactions of cells arriving at the * Corresponding author. Fax: +46 31 773 3330. 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 01 1 - 3
surface. Platelets can be detected within a few seconds [5,6 ] and are followed by other cells, such as polymorphonucleated (PMN ) cells [6–8] and monocytes [8]. The fate of an implant, healing or rejection, may depend on the interplay between these cells and between cells and proteins. The adhesion of platelets onto surfaces has been shown to depend critically on the adsorption of fibrinogen [9], whereas IgG-coated surfaces are known to activate complement by the classical pathway [10– 12]. The activation of complement is known to recruit PMN cells to the surface by chemotaxis [13,14].
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Platelets carry receptors for several different proteins. One of the most characterized is glycoprotein IIb/IIIa which binds fibronectin, vitronectin, fibrinogen and von Willebrand factor [15]. Activation of platelets could lead to ligand binding resulting in a shape change. The shape change is accompanied by increased exposure of sialic acid residues at the outer membrane and an increased actin polymerisation inside the cell. New binding sites for adhesive proteins also become accessible on the surface of the platelet. If the shape change is caused by immobilized fibrinogen or collagen, a tyrosine phosphorylation of pp125FAK (a focal adhesion kinase) and of two unknown proteins of 101 kDa and 105 kDa is induced [16 ]. This phosphorylation is accompanied by cytoskeletal reorganisation and spreading of the platelets, but not by secretion of serotonin [16 ]. Aggregated IgG is an agonist known to activate individual platelets through crosslinking of receptors on the platelet surface [17]. The FccRII (subclass A [18]) is the only platelet receptor for immobilized IgG discovered so far [19,20]. The Fc-receptor is not carried on glycoprotein Ib, IIb, IIIa or IX [19], though a topographical association with GPIIb/IIIa has been reported [21]. An adhesion to immobilized IgG also induces a pp125FAK phosphorylation independent of GPIIb/IIIa [22]. This phosphorylation is regulated by protein kinase C, but the phosphorylation of p72SYK is not affected by protein kinase C inhibition [22]. Thrombin activated human platelets have been shown to release one complete and one C-terminal truncated form of FccRIIa2 [23]. FccRIIa2 has been detected in the supernatant of platelets after a-granule release [23] and an addition of purified recombinant FccRIIa2 has been shown to inhibit Fc-mediated aggregation [23]. In order to further illuminate the initial stages in the reaction between blood and foreign surfaces, we have studied the interaction between isolated platelets and surface-immobilized proteins. Here we report that surface-immobilized IgG induces a massive secretion of microvesicles and total fragmentation of the cells.
2. Material and methods 2.1. Chemicals Human gammaglobulin (for injection) was from Kabi-pharmacia, Stockholm, Sweden, while F(ab∞) -fragments from goat antiserum to human 2 IgA was purchased from Kallestad, Chaska, MN, and human thrombin as well as human albumin and fluorescene isothiocyanate (FITC ) labelled phalloidine were purchased from Sigma Chemical Company, St Louis, MO. Percoll was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. All chemicals used for buffer solutions were from Merck, Darmstadt, Germany. 2.2. Preparation of platelets Venous blood from healthy human donors, not on medication, was collected with a 0.6 mm needle into 0.6 ml of CPD (citrate, phosphate, dextrose) anticoagulant to a final volume of 5 ml. The platelets were separated by a Percoll technique. A modified version of Braide and Bjursten’s [24] method for leukocyte preparation was used [25]. In short, the blood was mixed 1:1 with 400 mOsm phosphate buffered saline (PBS) containing 4 mM MgCl . The mixture was transferred to a Percoll 2 gradient. The platelets were collected from the first layer after centrifugation (750g, 5 min, 20°C ), after which they were washed twice with a washing buffer containing albumin, a modified version of Dulbeccos PBS (4 mM MgCl , 0 mM CaCl and 2 2 albumin 1 mg/ml, pH 7.4). After washing they were resuspended and pooled in unmodified Dulbeccos PBS containing albumin (1 mg/ml, pH 7.4). Only polystyrene cups and tubes were used during the isolation. The cell concentration was determined by counting in a Bu¨rker chamber. 2.3. Hydrophobic slides Hydrophobic slides (methylated silica) were prepared by cleaning ordinary glass slides for 30 min in 70% ethanol containing 0.35 M HCl. After rinsing with water (3×10 min) the slides were dried with an air current. To methylize the surface the slides were incubated for 7 min in 0.1%
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1,1,1,3,3,3-hexamethylsilazane in dichloromethane and then allowed to dry in air. 2.4. Protein surfaces The protein surfaces used were prepared on the hydrophobic glass. Dots (40 ml ) with an IgG, albumin or F(ab∞) -fragment concentration of 2 100 mg/ml were added to the hydrophobic glass and incubated at room temperature for 30 min in a humified chamber. The slides were then washed with D-PBS and kept humid until use. All slides were used within 2 h of preparation. The thickness of adsorbed protein layers, prepared on silica, washed and methylized as above, were analysed with an isoscope ellipsometer [26 ] ˚ ) was determined. For IgG and the thickness (A the surface concentration was determined by multi˚ ) with the protein plication of the thickness (A density (1.6 mg/ml for IgG) divided by 100. This factor was modified by multiplication with 0.886 because of the difference in refractive index between silica and proteins [26 ]. In some experiments IgG was adsorbed for 30 min or 4 h. Antibodies, rabbit–anti-human kappa light chain preadsorbed with human IgG; (DAKO A/S, Glostrup, Denmark) and secondary FITC conjugated swine–anti-rabbit antibodies, (DAKO A/S ) were used to detect the adsorbed IgG. Platelets (3×107 cells/ml ) were allowed to adhere to some of the samples while some were fixed directly and the rest incubated with D-PBS for 10 min. The incubation and detection were performed as described below. A comparison was made between the protected zones formed by platelets and visualized by anti-kappa light chain antibodies and the coverage seen by rabbit–antipan platelet antibodies, used as described below.
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D-PBS and fixed in absolute ethanol at −20°C for 10 min and then rehydrated before antibody staining. Some samples were fixed in 1% formalin at room temperature for 15 min before antibody staining. On some slides the platelets were allowed to spread for an extra time of 15 min after washing before they were fixed. Kinetic studies were also done where slides were washed and fixed after 2.5 up to 30 min. The number of adhering platelets and their morphology were determined. As primary antibody rabbit–anti-pan platelet antibodies, purchased from DAKO A/S, was used. All antibodies were diluted in D-PBS. The samples were incubated for 20 min with the antibodies on a cooling plate, 0°C, from HISTO-LAB, Sweden. They were then rinsed with D-PBS before a second incubation, as above, with secondary FITC-conjugated swine–anti-rabbit antibodies. After a second rinsing the samples were assembled with 1,4-diazabicyclo[2,2,2]octane (DABCO) mixed with glycerol to keep the fluorescence from fading. All photos were taken the same day experiments were performed. To clarify the order of shape changes some adhesion studies of platelets in real-time by vital microscopy were done. The platelet solution was diluted to a concentration of 3×107 platelets/ml. A drop of this solution was transferred to IgGcoated slides with small wells, purchased from Novakemi ab, Stockholm, Sweden. A cover glass was added and the preparate was transferred to a heating plate. The glass surface temperature stabilizes at 37°C according to prior measurements. The adhesion of platelets to the IgG surface was seen and video taped through a Letz Wetzlar microscope at a magnification of 12.5×100. The videotape was analysed and times were recorded for cell adhesion, shape change and spreading.
2.5. Adhesion studies of platelets 2.6. Microfilament staining The platelet solution was diluted to a concentration of 3×107 platelets/ml or 3×108 platelets/ ml and 30 ml dots were put onto each protein spot on the slides. Thrombin was added in some experiments to a final concentration of 1 U/ml. The slides were then incubated for 10 min in a humified chamber at 37°C. The samples were washed with
FITC-labelled phalloidine was used to detect actin filaments in the platelets. The samples were fixed in 1% formalin for 15 min at room temperature and rinsed with D-PBS before treatment with 0.01% Triton 100 for 3 min. After a second rinsing they were incubated for 40 min with FITC-
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labelled phalloidine at a concentration of 0.005 g/l at room temperature. After a final excessive rinsing the samples were assembled with DABCO. All photos were taken the same day experiments were performed. 2.7. Analysis All samples were photographed in a fluorescence microscope Zeiss 3RS. Two to three photos were taken on each protein spot with black and white Kodak T 400 pro-film. Exposure time was kept max constant at 1 min. The photographs for analysis were taken at a magnification of 10×20. Detailed pictures were taken at a magnification of 10×100. After development, the black and white negatives were scanned into Adobe Photoshop on a Power Macintosh 8100/80 desktop with Sprint Scan 35. Some data were transferred to NIH Image on the same computer. The percentage coverage, the mean brightness in the picture as a measurement of amount bound protein, the mean brightness in positive parts of the picture and the number of platelets adhered to the surface and their shapes were analysed in Adobe Photoshop or NIH Image. The area for scanning was kept constant at a size of about 10 000 mm2. 2.8. Statistical evaluation All statistical evaluations were done with the Student’s t-test. The number of observations were between 9 and 18 if not otherwise stated. All comparative analyses were done at the same time and with the same platelet and antibody solutions.
3. Results 3.1. Protein surfaces and stability of the IgG surface The thickness of the IgG surface layer on ˚ with methylized silica was determined to be 67 A ˚ for six measurements, a standard deviation of 5 A corresponding to a surface concentration of 0.94 mg/cm2 or about 6 pmol/cm2. The surface ˚ with a standard layer of albumin was 30 A
Table 1 The mean brightness of the IgG-coated surface after different times for adsorption of IgG and incubation with D-PBS or platelets (mean±S.D.) Treatment of samples
Mean brightness (max 255)
30 min coating 4 h coating 30 min coating+D-PBS 4 h coating+D-PBS 30 min coating+platelets 4 h coating+platelets
107.54±4.61 127.47±4.90 119.53±5.13 118.69±10.57 112.05±5.10 128.00±2.90
Fig. 1. Protected zones formed by platelets adhering to the surface after 10 min, 37°C, on an IgG-coated surface. The protein coating time was 4 h. Staining with rabbit–anti-human kappa light chain and FITC-labelled swine–anti-rabbit IgG antibodies (preadsorbed with human IgG). Original magnification ×1000.
˚ while the surface layer of deviation of 7 A ˚ with a standard deviaF(ab∞) -fragments was 28 A 2 ˚ tion of 6 A, both for 10 measurements. The protein (IgG) was adsorbed for 30 min or 4 h before rinsing with D-PBS. Samples were fixed directly or incubated for 10 min at 37°C with or without addition of platelets. From the mean brightness of the surface, areas covered by platelets excluded, a slightly higher amount of IgG was found after 4 h adsorption time ( Table 1). When a platelet-covered IgG surface was stained with anti-IgG antibodies, protected zones were seen ( Fig. 1) where the platelets prevented antibodies from reaching the surface protein coating. From a comparison between the area of the protected zones and the coverage area for platelets stained
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Table 2 Coverage area for protected zones and the coverage of platelets adhered to an IgG-coated surface for 10 min (mean±S.D.) Coating time and antibody
Coverage (%)
30 min anti-pan platelet 30 min anti-kappa light chain 4 h anti-pan platelet 4 h anti-kappa light chain
7.14±0.91 4.19±0.85 7.09±0.73 3.58±1.00
with anti-platelet antibodies, a significantly ( p<0.001) higher coverage was seen than the protected zones formed ( Table 2). 3.2. Shapes of platelets On samples with platelets added to IgG surfaces at least five different shapes of platelets were seen [Fig. 2(a)] while only three different shapes were found on albumin- [Fig. 2(b)] and F(ab∞) -coated 2 surfaces [Fig. 2(c)]. The shapes of the platelets found on the IgG coated surface were: the normal compact platelet [Fig. 3(a)], the spread platelet with a smooth edge [Figs. 3(b) and 3(c)], and dendritic platelets either with a compact central body with many long dendrites [Fig. 3(d )], or with smaller dendrites [Fig. 3(e)]. Finally [Fig. 3(f )], remnants of platelets after secretion of microparticles and fragmentation. Less than 0.3% platelets with long dendrites were seen (Fig. 4) on the albumin- and the F(ab∞) -coated surfaces, and no microparticles 2 were found at these surfaces. From the percentage of platelets in a certain shape, a significantly ( p<0.001) higher amount of dendritic platelets could be seen on the IgG-coated surface than on the control surfaces. Differences were also seen between the F(ab∞) -coated surface 2 and the albumin surface. A significantly ( p<0.001) higher amount of smoothly spread platelets were seen on the albumin-coated surfaces than on the F(ab∞) - and the IgG-coated. In summary the 2 Fig 2. (a) Platelets adhering to an IgG-coated surface. (b) Platelets adhering to an albumin-coated surface. (c) Platelets adhering to a F(ab∞) -fragment-coated surface. The incubation 2 was done for 10 min at 37°C. Staining with rabbit–anti-human platelet antibodies and FITC-labelled swine–anti-rabbit IgG
(a)
(b)
(c) antibodies (preadsorbed with human IgG). Platelet concentration used was 3×107 platelets/ml. Original magnification ×1000.
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(a)
(b)
(c)
(d)
(e)
(f)
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Fig. 4. The distribution of the different shapes for platelets adhering to albumin-, IgG- or F(ab∞) -coated surfaces (compact, smoothly 2 spread, short dendrites, long dendrites, microparticle and fragmented ). Platelet concentration used was 3×107 platelets/ml. For significant differences see results (mean±S.D.).
platelets on the F(ab∞) -coated surface were quite 2 compact and did not spread whereas the platelets adhering to the albumin-coated surface spread smoothly to a high extent. On the IgG-coated surface many of the platelets formed long or short dendrites and a fragmentation of platelets was seen (Fig. 4). 3.3. Actin filament staining The staining of actin filament with phalloidine [Fig. 5(a)–(c)] showed a high amount of filamentous actin in the central body. Actin filaments can also be seen in the dendrites and along the edge of smoothly spread platelets. In some smoothly spread platelets focal attachment points can be observed. The cell morphology seen after staining with FITC-phalloidin correlated with the morphology seen after staining with the anti-pan platelet antibody. No F-actin was found in microparticles or cell fragments.
3.4. Kinetics of platelet adhesion The number of adhering platelets increased during the 30 min of incubation studied on the IgG-coated surface ( Fig. 6). The number of new adhering compact platelets seemed to stabilize after 20 min while the number of smoothly spread and fragmented platelets continued to increase. The number of platelets with long dendrites seemed quite constant. At most platelets with long dendrites [Fig. 3(d )] it was possible to see microvesicles along the dendrites. Microparticles and fragmentized platelets were often seen as lonely cells without interaction with other platelets. This was confirmed by the lack of fragmented and dendritic platelets at a higher (3×108 platelets/ml ) platelet concentration. From the real-time studies with vital microscopy it was possible to see that platelets adhering to a surface seemed to change shape either very rapidly or remain almost the same shape with only minor
Fig. 3. The different shapes seen for platelets adhering to an IgG-coated surface at a low platelet concentration (3×107 platelets/ml ). Staining with rabbit–anti-human platelet antibodies and FITC-labelled swine–anti-rabbit IgG antibodies (preadsorbed with human IgG). Original magnification ×5000. (a) A compact platelet retaining its original shape as seen in circulating blood. (b, c) The smoothly spread platelet in two different shapes. (d) A dendritic platelet with long dendrites. (e) A dendritic platelet with short dendrites. (f ) Remnants of a platelet after secretion of microparticles and fragmentation.
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(a)
(b)
(c)
Fig. 5. The actin filament distribution in adhering platelets. (a) On an IgG-coated surface, (b) on an albumin-coated surface, and (c) on a F(ab∞) -fragment-coated surface. The incubation time was 10 min at 37°C and the platelet concentration used was 2 3×107 platelets/ml. Original magnification ×3000.
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Fig. 6. The distribution of different shapes for platelets adhering to an IgG-coated surface after different times of incubation at 37°C. Platelet concentration used was 3×107 platelets/ml. Samples were taken after 2.5, 5, 10, 15, 20 and 30 min. The different shapes of the cells are: compact, smoothly spread, with short dendrites, with long dendrites and microparticle and fragmented.
Fig. 7. The number of platelets with different shapes (compact, smoothly spread, short dendrites, long dendrites, microparticle and fragmented) adhering to an IgG-coated surface after 10 min of incubation at 37°C and after 15 min of extra incubation after rinsing. Platelet concentration used was 3×107 platelets/ml. ** p<0.001.
changes for a very long time. A change in diameter from about 3 mm to more than 9 mm in 40 s was seen. A dendritic platelet changed shape to a smoothly spread form in 3 min and continued into fragmentation and was eroded 5 min later. Many
smoothly spread platelets continued to change shape, after a longer or shorter lag phase, and turned into a more and more fragmented appearance until the cell body disappeared and only a rough area could be seen, corresponding to a
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microparticle covered surface. No cell parts could be observed to leave the surface during this change. 3.5. Extra incubation after rinsing When the platelets were incubated for an extra 15 min after rinsing before they were fixed, a 10% lower number of platelets was seen on the surface (98±7 compared to 110±15). For the different shapes significant ( p<0.01) differences could be seen for all shapes, except the cells with short dendrites, between normally incubated samples and samples incubated for an extra 15 min after rinsing ( Fig. 7). The smoothly spread platelets were at a lower concentration after the extra incubation, indicating that they may leave the surface or fragmentize.
(a)
3.6. Higher platelet concentrations At a high concentration of platelets (3×108 platelets/ml ) at the IgG-coated surface no dendritic or fragmented platelets [Fig. 8(a)] were seen. In this case all cells spread smoothly or formed aggregates. An extra incubation of these samples for 15 min resulted only in more spread platelets [Fig. 8(b)]. There were no platelets seen alone without interactions with other platelets. 3.7. Adhesion with thrombin present When thrombin was present at a concentration of 1 U/ml at the time of adhering, 25% more platelets adhered to the surface (35±7 compared to 27±2). These extra adhering platelets seemed to take the shape of compact or smoothly spread platelets ( Fig. 9).
4. Discussion Platelets may become activated during separation, for example by contact with air. In order to isolate platelets without activation and shape change we have used Mg2+ ions at a concentration of 4 mM as activation inhibitor [27]. Magnesium is tried as a platelet inhibitor in very different experimental models, both in vivo and in vitro
(b) Fig. 8. Platelets at a concentration of 3×108 cells/ml on an IgGcoated surface. (a) After 10 min of incubation at 37°C. (b) After 10 min of incubation and 15 min of additional incubation at 37°C after rinsing. Original magnification ×1000.
[28–31]. The interchange between magnesium and calcium ions at the GPIIb/IIIa [32] gives the possibility of a reversible inhibition. Platelet activation by non-aggregated IgG through Fcc-receptors is inhibited by high viscosity, which prevents cell–cell interactions between platelets [33]. The experimental procedure used in our study with a plasma free solution prevents platelet adhesion through complement activation. In our studies we have used surface-immobilized IgG and most often a low concentration of platelets which minimizes the cell–cell interactions. The thickness of the IgG surface in our experiments ˚ , which equals was determined to be 67 A
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Fig. 9. The number of platelets with different shapes (compact, smoothly spread, short dendrites, long dendrites, microparticle and fragmented) adhering to an IgG-coated surface after 10 min of incubation at 37°C. The platelets were either incubated alone or with a thrombin concentration of 1 U/ml. Platelet concentration used was 3×107 platelets/ml. ** p<0.001 (mean±S.D.).
6 pmol/cm2. A tightly packed monolayer of IgG is, according to Werthe´n et al. [34], around 8 pmol/cm2. Our surfaces would then be about 75% covered by a monolayer of IgG, which may be distributed in 2D-aggregates, but probably not in 3D-aggregates. In our immunofluorescence experiments with an IgG-coated surface with platelets adhered to the surface we could see protected zones, indicating that antibodies do not pass through the adhering cells. These protected zones have been described by Loike et al. [35]. However, since the area covered by cells was larger than the protected area, some cells leak antibodies through their membranes, which is consistent with the finding that the cells end up as fragments at the IgG-coated surface. Because the number of platelets with long dendrites were quite constant over time, the most probable progress for compact platelets was to first develop a few short dendrites before starting to spread to a smoothly spread platelet. Some cells with short dendrites continue the dendrite formation and form quite stable long dendrites. The microparticles along the dendrites are probably the first step of fragmentation.
The platelets with long dendrites seen in our experiments were always scattered cells interacting only with the surface and not with other cells. Dendritic platelet shape has earlier been recorded at platelet interactions with 14% SO Pellethane 3 surfaces [36 ]. This surface could in some way stimulate the Fc-receptor or the following events. Waples et al. [36 ] also conclude that the spreading of platelets is Ca2+ dependent. In all our experiments Ca2+ concentration (1 mM ) is lower than physiological conditions due to problems with stability of the buffer at higher CaCl concen2 trations. Waples et al. [36 ] used up to 1 mM, making these results comparable. Crawford [37] observed terminal swellings on platelet pseudopodia and suggested that microparticles were a result of these swellings. A close relation between pseudopodia and microparticle formation has been confirmed [38]. In our experiments we have detected microparticles along the dendritic pseudopodia [Fig. 3(d )] and not only as terminal swellings. This is still agreeable with the observations reported by Yano et al. [38], also reporting a few filamentous structures in the microparticles but not determining their nature. According to our studies filamentous actin was
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seen in dendrites and along the edges of the cell membrane as well as in the central body but was not detected in the microparticles. The microparticles might have an attracting effect on other platelets which would explain the big increase in adhesion seen after 10 min incubation time. At the same time the first fragmented platelets appeared. These fragments, or platelet microparticles, are known to be potent activators of PMN cells [39,40], suggesting a possible pathway of inflammatory reactions at the surface [6 ]. In conclusion, the results of the present study show that platelets undergo dramatic morphological changes in contact with surface-immobilized IgG. The cells secrete microvesicles, recognized by anti-pan platelet antibodies.
Acknowledgment Supported by Grant from the Swedish Medical Research Council (06235). We are very grateful to Ulf Bagge for helping us with the vital microscopy.
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[14] J. Nusbacher, S.I. Rosenfeld, J.L. MacPherson, P.A. Thiem, J.P. Hleddy, Blood 51 (1978) 359. [15] C.A. Buck, A.F. Horwitz, Ann. Rev. Cell Biol. 3 (1987) 179. [16 ] B. Haimovich, L. Lipfert, J.S. Brugge, S.J. Shattil, J. Biol. Chem. 268 (1993) 15868. [17] J.G.J. Van de Winkel, W.J. Tax, C.W. Jacobs, T.W. Huizinga, P.H. Willems, Scand. J. Immunol. 31 (1990) 315. [18] G.W. Chacko, A.-M. Duchemin, K.M. Coggeshall, J.M. Osborne, J.T. Brandt, C.L. Anderson, J. Biol. Chem. 269 (1994) 32435. [19] J.G. Kelton, J.W. Smith, A.V. Santos, W.G. Murphy, P. Horsewood, Am. J. Hematol. 25 (1987) 299. [20] S.I. Rosenfeld, R.J. Looney, J.B. Leddy, D.C. Phipps, G.N. Abrahem, C.L. Andersson, J. Clin. Invest. 76 (1985) 2317. [21] M.C. Berndt, A.V. Mazurov, D.V. Vinogradov, G.F. Burns, C.N. Chesterman, Platelets 4 (1993) 190. [22] B. Haimovich, C. Regan, L. DiFazio, E. Ginalis, P. Ji, U. Purohit, R.B. Rowley, J. Bolen, R. Greco, J. Biol. Chem. 271 (1996) 16332. [23] C. Gachet, A. Astier, H. de la Salle, C. de la Salle, W.H. Fridman, J.P. Cazenave, D. Hanau, J.L. Teillaud, Blood 85 (1995) 698. [24] M. Braide, L.M. Bjursten, J. Immunol. Meth. 93 (1986) 183. [25] H. Nygren, M. Broberg, accepted for publishing by J. Biomat. Sci. Polym. Ed. [26 ] M. Stenberg, H. Nygren, J. de Phys. C10 (suppl. 2) (1983) 83. [27] M. Gawas, I. Ott, A.J. Reininger, F.J. Neumann, Throm. Haemo. 72 (1994) 912. [28] E. Hardy, S. Heptinstall, P.C. Rubin, E.H. Horn, Platelets 6 (1995) 346. [29] M. Gawas, A. Reininger, F.J. Neumann, Throm. Res. 83 (1996) 341. [30] H. Berg-Ravn, H. Vissinger, S.D. Kristensen, A. Wennmalm, K. Thygesen, S.E. Husted, Throm. Haemo. 75 (1996) 939. [31] H.B. Ravn, H. Vissinger, S.D. Kristensen, S.E. Husted, Throm. Haemo. 76 (1996) 88. [32] D. Gulino, C. Boudignon, L. Zhang, E. Concord, M.J. Rabiet, G. Marguerie, J. Biol. Chem. 267 (1992) 11001. [33] G.P. Anderson, J.G.J. Van de Winkel, C.L. Anderson, Br. J. Haematol. 79 (1991) 75. [34] M. Werthe´n, M. Stenberg, H. Nygren, Progr. Colloid Polym. Sci. 82 (1990) 349. [35] J.D. Loike, R. Silverstein, L. Cao, L. Salomon, J. Weitz, E. Haber, G.R. Matsueda, M.S. Bernatowicz, C. Silverstein, J. Cell. Biol. 121 (1993) 945. [36 ] L.M. Waples, O.E. Olorundare, S.L. Goodman, Q.J. Lai, R.M. Albrecht, J. Biomed. Mater. Res. 32 (1996) 65. [37] N. Crawford, Br. J. Haematol. 21 (1971) 53. [38] Y. Yano, J.I. Kambayashi, E. Shiba, E. Oiki, K. Fukunda, T. Kawasaki, T. Mori, Biochem. J. 299 (1994) 303. [39] C.H. Gemmel, M.R. Sotero, E.L. Yeo, M.W. Sefton, J. Lab. Clin. Med. 125 (1995) 276. [40] W. Jy, W.W. Mao, L.L. Horstman, J. Tao, Y.S. Ahn, Blood Cells, Mol. Dis. 21 (1995) 217.
Platelet interaction with surface-immobilized IgG induces the secretion of surface bound microparticles and fragmentation of the cells Marita Broberg, Ha˚kan Nygren * Department of Anatomy and Cell Biology, University of Go¨teborg, Go¨teborg, Sweden Received 7 July 1997; accepted 30 December 1997
Abstract The interaction between isolated platelets and surface-immobilized IgG was investigated by immunofluorescence, using albumin and F(ab∞) -fragments as controls. Antibodies directed against platelet membrane antigens (anti-pan 2 platelet) were used to visualize platelet adhesion and the morphology of adhering cells. The number of adhering cells and the relative frequency of different morphologies were counted and analysed after different times of adhesion. Five distinct stages of cell adhesion and activation were detected and defined by different morphologies. Transitions between these states were seen by vital microscopy. Dendritic platelets with long dendrites were seen scattered at low surface densities of adhering cells and microvesicles transported along the dendrites were visualized by anti-pan platelet antibodies. As a last stage before total fragmentation, the platelets were seen to secrete microvesicles and form an area of surface-adhering microparticles on the IgG-coated surface, but not on control surfaces. The adhesion of platelets increased after 10 min, the same time surface bound microparticles appeared at the surface. The results indicate a novel pathway of non-self-recognition of foreign surfaces via IgG-mediated activation of platelets resulting in massive production of microparticles. © 1998 Elsevier Science B.V. Keywords: Platelets; IgG; Adhesion; Spreading; Microparticles
1. Introduction The initial contact of foreign materials with blood induces an array of recognition reactions. Initially, host plasma proteins arrive at the surface [1,2]. The most abundant proteins in human plasma, albumin, fibrinogen and immunoglobulins, will be contained in the initially formed protein film [3,4]. The structure and composition of the adsorbed protein layer are believed to determine the reactions of cells arriving at the * Corresponding author. Fax: +46 31 773 3330. 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 01 1 - 3
surface. Platelets can be detected within a few seconds [5,6 ] and are followed by other cells, such as polymorphonucleated (PMN ) cells [6–8] and monocytes [8]. The fate of an implant, healing or rejection, may depend on the interplay between these cells and between cells and proteins. The adhesion of platelets onto surfaces has been shown to depend critically on the adsorption of fibrinogen [9], whereas IgG-coated surfaces are known to activate complement by the classical pathway [10– 12]. The activation of complement is known to recruit PMN cells to the surface by chemotaxis [13,14].
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Platelets carry receptors for several different proteins. One of the most characterized is glycoprotein IIb/IIIa which binds fibronectin, vitronectin, fibrinogen and von Willebrand factor [15]. Activation of platelets could lead to ligand binding resulting in a shape change. The shape change is accompanied by increased exposure of sialic acid residues at the outer membrane and an increased actin polymerisation inside the cell. New binding sites for adhesive proteins also become accessible on the surface of the platelet. If the shape change is caused by immobilized fibrinogen or collagen, a tyrosine phosphorylation of pp125FAK (a focal adhesion kinase) and of two unknown proteins of 101 kDa and 105 kDa is induced [16 ]. This phosphorylation is accompanied by cytoskeletal reorganisation and spreading of the platelets, but not by secretion of serotonin [16 ]. Aggregated IgG is an agonist known to activate individual platelets through crosslinking of receptors on the platelet surface [17]. The FccRII (subclass A [18]) is the only platelet receptor for immobilized IgG discovered so far [19,20]. The Fc-receptor is not carried on glycoprotein Ib, IIb, IIIa or IX [19], though a topographical association with GPIIb/IIIa has been reported [21]. An adhesion to immobilized IgG also induces a pp125FAK phosphorylation independent of GPIIb/IIIa [22]. This phosphorylation is regulated by protein kinase C, but the phosphorylation of p72SYK is not affected by protein kinase C inhibition [22]. Thrombin activated human platelets have been shown to release one complete and one C-terminal truncated form of FccRIIa2 [23]. FccRIIa2 has been detected in the supernatant of platelets after a-granule release [23] and an addition of purified recombinant FccRIIa2 has been shown to inhibit Fc-mediated aggregation [23]. In order to further illuminate the initial stages in the reaction between blood and foreign surfaces, we have studied the interaction between isolated platelets and surface-immobilized proteins. Here we report that surface-immobilized IgG induces a massive secretion of microvesicles and total fragmentation of the cells.
2. Material and methods 2.1. Chemicals Human gammaglobulin (for injection) was from Kabi-pharmacia, Stockholm, Sweden, while F(ab∞) -fragments from goat antiserum to human 2 IgA was purchased from Kallestad, Chaska, MN, and human thrombin as well as human albumin and fluorescene isothiocyanate (FITC ) labelled phalloidine were purchased from Sigma Chemical Company, St Louis, MO. Percoll was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. All chemicals used for buffer solutions were from Merck, Darmstadt, Germany. 2.2. Preparation of platelets Venous blood from healthy human donors, not on medication, was collected with a 0.6 mm needle into 0.6 ml of CPD (citrate, phosphate, dextrose) anticoagulant to a final volume of 5 ml. The platelets were separated by a Percoll technique. A modified version of Braide and Bjursten’s [24] method for leukocyte preparation was used [25]. In short, the blood was mixed 1:1 with 400 mOsm phosphate buffered saline (PBS) containing 4 mM MgCl . The mixture was transferred to a Percoll 2 gradient. The platelets were collected from the first layer after centrifugation (750g, 5 min, 20°C ), after which they were washed twice with a washing buffer containing albumin, a modified version of Dulbeccos PBS (4 mM MgCl , 0 mM CaCl and 2 2 albumin 1 mg/ml, pH 7.4). After washing they were resuspended and pooled in unmodified Dulbeccos PBS containing albumin (1 mg/ml, pH 7.4). Only polystyrene cups and tubes were used during the isolation. The cell concentration was determined by counting in a Bu¨rker chamber. 2.3. Hydrophobic slides Hydrophobic slides (methylated silica) were prepared by cleaning ordinary glass slides for 30 min in 70% ethanol containing 0.35 M HCl. After rinsing with water (3×10 min) the slides were dried with an air current. To methylize the surface the slides were incubated for 7 min in 0.1%
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1,1,1,3,3,3-hexamethylsilazane in dichloromethane and then allowed to dry in air. 2.4. Protein surfaces The protein surfaces used were prepared on the hydrophobic glass. Dots (40 ml ) with an IgG, albumin or F(ab∞) -fragment concentration of 2 100 mg/ml were added to the hydrophobic glass and incubated at room temperature for 30 min in a humified chamber. The slides were then washed with D-PBS and kept humid until use. All slides were used within 2 h of preparation. The thickness of adsorbed protein layers, prepared on silica, washed and methylized as above, were analysed with an isoscope ellipsometer [26 ] ˚ ) was determined. For IgG and the thickness (A the surface concentration was determined by multi˚ ) with the protein plication of the thickness (A density (1.6 mg/ml for IgG) divided by 100. This factor was modified by multiplication with 0.886 because of the difference in refractive index between silica and proteins [26 ]. In some experiments IgG was adsorbed for 30 min or 4 h. Antibodies, rabbit–anti-human kappa light chain preadsorbed with human IgG; (DAKO A/S, Glostrup, Denmark) and secondary FITC conjugated swine–anti-rabbit antibodies, (DAKO A/S ) were used to detect the adsorbed IgG. Platelets (3×107 cells/ml ) were allowed to adhere to some of the samples while some were fixed directly and the rest incubated with D-PBS for 10 min. The incubation and detection were performed as described below. A comparison was made between the protected zones formed by platelets and visualized by anti-kappa light chain antibodies and the coverage seen by rabbit–antipan platelet antibodies, used as described below.
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D-PBS and fixed in absolute ethanol at −20°C for 10 min and then rehydrated before antibody staining. Some samples were fixed in 1% formalin at room temperature for 15 min before antibody staining. On some slides the platelets were allowed to spread for an extra time of 15 min after washing before they were fixed. Kinetic studies were also done where slides were washed and fixed after 2.5 up to 30 min. The number of adhering platelets and their morphology were determined. As primary antibody rabbit–anti-pan platelet antibodies, purchased from DAKO A/S, was used. All antibodies were diluted in D-PBS. The samples were incubated for 20 min with the antibodies on a cooling plate, 0°C, from HISTO-LAB, Sweden. They were then rinsed with D-PBS before a second incubation, as above, with secondary FITC-conjugated swine–anti-rabbit antibodies. After a second rinsing the samples were assembled with 1,4-diazabicyclo[2,2,2]octane (DABCO) mixed with glycerol to keep the fluorescence from fading. All photos were taken the same day experiments were performed. To clarify the order of shape changes some adhesion studies of platelets in real-time by vital microscopy were done. The platelet solution was diluted to a concentration of 3×107 platelets/ml. A drop of this solution was transferred to IgGcoated slides with small wells, purchased from Novakemi ab, Stockholm, Sweden. A cover glass was added and the preparate was transferred to a heating plate. The glass surface temperature stabilizes at 37°C according to prior measurements. The adhesion of platelets to the IgG surface was seen and video taped through a Letz Wetzlar microscope at a magnification of 12.5×100. The videotape was analysed and times were recorded for cell adhesion, shape change and spreading.
2.5. Adhesion studies of platelets 2.6. Microfilament staining The platelet solution was diluted to a concentration of 3×107 platelets/ml or 3×108 platelets/ ml and 30 ml dots were put onto each protein spot on the slides. Thrombin was added in some experiments to a final concentration of 1 U/ml. The slides were then incubated for 10 min in a humified chamber at 37°C. The samples were washed with
FITC-labelled phalloidine was used to detect actin filaments in the platelets. The samples were fixed in 1% formalin for 15 min at room temperature and rinsed with D-PBS before treatment with 0.01% Triton 100 for 3 min. After a second rinsing they were incubated for 40 min with FITC-
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labelled phalloidine at a concentration of 0.005 g/l at room temperature. After a final excessive rinsing the samples were assembled with DABCO. All photos were taken the same day experiments were performed. 2.7. Analysis All samples were photographed in a fluorescence microscope Zeiss 3RS. Two to three photos were taken on each protein spot with black and white Kodak T 400 pro-film. Exposure time was kept max constant at 1 min. The photographs for analysis were taken at a magnification of 10×20. Detailed pictures were taken at a magnification of 10×100. After development, the black and white negatives were scanned into Adobe Photoshop on a Power Macintosh 8100/80 desktop with Sprint Scan 35. Some data were transferred to NIH Image on the same computer. The percentage coverage, the mean brightness in the picture as a measurement of amount bound protein, the mean brightness in positive parts of the picture and the number of platelets adhered to the surface and their shapes were analysed in Adobe Photoshop or NIH Image. The area for scanning was kept constant at a size of about 10 000 mm2. 2.8. Statistical evaluation All statistical evaluations were done with the Student’s t-test. The number of observations were between 9 and 18 if not otherwise stated. All comparative analyses were done at the same time and with the same platelet and antibody solutions.
3. Results 3.1. Protein surfaces and stability of the IgG surface The thickness of the IgG surface layer on ˚ with methylized silica was determined to be 67 A ˚ for six measurements, a standard deviation of 5 A corresponding to a surface concentration of 0.94 mg/cm2 or about 6 pmol/cm2. The surface ˚ with a standard layer of albumin was 30 A
Table 1 The mean brightness of the IgG-coated surface after different times for adsorption of IgG and incubation with D-PBS or platelets (mean±S.D.) Treatment of samples
Mean brightness (max 255)
30 min coating 4 h coating 30 min coating+D-PBS 4 h coating+D-PBS 30 min coating+platelets 4 h coating+platelets
107.54±4.61 127.47±4.90 119.53±5.13 118.69±10.57 112.05±5.10 128.00±2.90
Fig. 1. Protected zones formed by platelets adhering to the surface after 10 min, 37°C, on an IgG-coated surface. The protein coating time was 4 h. Staining with rabbit–anti-human kappa light chain and FITC-labelled swine–anti-rabbit IgG antibodies (preadsorbed with human IgG). Original magnification ×1000.
˚ while the surface layer of deviation of 7 A ˚ with a standard deviaF(ab∞) -fragments was 28 A 2 ˚ tion of 6 A, both for 10 measurements. The protein (IgG) was adsorbed for 30 min or 4 h before rinsing with D-PBS. Samples were fixed directly or incubated for 10 min at 37°C with or without addition of platelets. From the mean brightness of the surface, areas covered by platelets excluded, a slightly higher amount of IgG was found after 4 h adsorption time ( Table 1). When a platelet-covered IgG surface was stained with anti-IgG antibodies, protected zones were seen ( Fig. 1) where the platelets prevented antibodies from reaching the surface protein coating. From a comparison between the area of the protected zones and the coverage area for platelets stained
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Table 2 Coverage area for protected zones and the coverage of platelets adhered to an IgG-coated surface for 10 min (mean±S.D.) Coating time and antibody
Coverage (%)
30 min anti-pan platelet 30 min anti-kappa light chain 4 h anti-pan platelet 4 h anti-kappa light chain
7.14±0.91 4.19±0.85 7.09±0.73 3.58±1.00
with anti-platelet antibodies, a significantly ( p<0.001) higher coverage was seen than the protected zones formed ( Table 2). 3.2. Shapes of platelets On samples with platelets added to IgG surfaces at least five different shapes of platelets were seen [Fig. 2(a)] while only three different shapes were found on albumin- [Fig. 2(b)] and F(ab∞) -coated 2 surfaces [Fig. 2(c)]. The shapes of the platelets found on the IgG coated surface were: the normal compact platelet [Fig. 3(a)], the spread platelet with a smooth edge [Figs. 3(b) and 3(c)], and dendritic platelets either with a compact central body with many long dendrites [Fig. 3(d )], or with smaller dendrites [Fig. 3(e)]. Finally [Fig. 3(f )], remnants of platelets after secretion of microparticles and fragmentation. Less than 0.3% platelets with long dendrites were seen (Fig. 4) on the albumin- and the F(ab∞) -coated surfaces, and no microparticles 2 were found at these surfaces. From the percentage of platelets in a certain shape, a significantly ( p<0.001) higher amount of dendritic platelets could be seen on the IgG-coated surface than on the control surfaces. Differences were also seen between the F(ab∞) -coated surface 2 and the albumin surface. A significantly ( p<0.001) higher amount of smoothly spread platelets were seen on the albumin-coated surfaces than on the F(ab∞) - and the IgG-coated. In summary the 2 Fig 2. (a) Platelets adhering to an IgG-coated surface. (b) Platelets adhering to an albumin-coated surface. (c) Platelets adhering to a F(ab∞) -fragment-coated surface. The incubation 2 was done for 10 min at 37°C. Staining with rabbit–anti-human platelet antibodies and FITC-labelled swine–anti-rabbit IgG
(a)
(b)
(c) antibodies (preadsorbed with human IgG). Platelet concentration used was 3×107 platelets/ml. Original magnification ×1000.
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(a)
(b)
(c)
(d)
(e)
(f)
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Fig. 4. The distribution of the different shapes for platelets adhering to albumin-, IgG- or F(ab∞) -coated surfaces (compact, smoothly 2 spread, short dendrites, long dendrites, microparticle and fragmented ). Platelet concentration used was 3×107 platelets/ml. For significant differences see results (mean±S.D.).
platelets on the F(ab∞) -coated surface were quite 2 compact and did not spread whereas the platelets adhering to the albumin-coated surface spread smoothly to a high extent. On the IgG-coated surface many of the platelets formed long or short dendrites and a fragmentation of platelets was seen (Fig. 4). 3.3. Actin filament staining The staining of actin filament with phalloidine [Fig. 5(a)–(c)] showed a high amount of filamentous actin in the central body. Actin filaments can also be seen in the dendrites and along the edge of smoothly spread platelets. In some smoothly spread platelets focal attachment points can be observed. The cell morphology seen after staining with FITC-phalloidin correlated with the morphology seen after staining with the anti-pan platelet antibody. No F-actin was found in microparticles or cell fragments.
3.4. Kinetics of platelet adhesion The number of adhering platelets increased during the 30 min of incubation studied on the IgG-coated surface ( Fig. 6). The number of new adhering compact platelets seemed to stabilize after 20 min while the number of smoothly spread and fragmented platelets continued to increase. The number of platelets with long dendrites seemed quite constant. At most platelets with long dendrites [Fig. 3(d )] it was possible to see microvesicles along the dendrites. Microparticles and fragmentized platelets were often seen as lonely cells without interaction with other platelets. This was confirmed by the lack of fragmented and dendritic platelets at a higher (3×108 platelets/ml ) platelet concentration. From the real-time studies with vital microscopy it was possible to see that platelets adhering to a surface seemed to change shape either very rapidly or remain almost the same shape with only minor
Fig. 3. The different shapes seen for platelets adhering to an IgG-coated surface at a low platelet concentration (3×107 platelets/ml ). Staining with rabbit–anti-human platelet antibodies and FITC-labelled swine–anti-rabbit IgG antibodies (preadsorbed with human IgG). Original magnification ×5000. (a) A compact platelet retaining its original shape as seen in circulating blood. (b, c) The smoothly spread platelet in two different shapes. (d) A dendritic platelet with long dendrites. (e) A dendritic platelet with short dendrites. (f ) Remnants of a platelet after secretion of microparticles and fragmentation.
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(a)
(b)
(c)
Fig. 5. The actin filament distribution in adhering platelets. (a) On an IgG-coated surface, (b) on an albumin-coated surface, and (c) on a F(ab∞) -fragment-coated surface. The incubation time was 10 min at 37°C and the platelet concentration used was 2 3×107 platelets/ml. Original magnification ×3000.
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Fig. 6. The distribution of different shapes for platelets adhering to an IgG-coated surface after different times of incubation at 37°C. Platelet concentration used was 3×107 platelets/ml. Samples were taken after 2.5, 5, 10, 15, 20 and 30 min. The different shapes of the cells are: compact, smoothly spread, with short dendrites, with long dendrites and microparticle and fragmented.
Fig. 7. The number of platelets with different shapes (compact, smoothly spread, short dendrites, long dendrites, microparticle and fragmented) adhering to an IgG-coated surface after 10 min of incubation at 37°C and after 15 min of extra incubation after rinsing. Platelet concentration used was 3×107 platelets/ml. ** p<0.001.
changes for a very long time. A change in diameter from about 3 mm to more than 9 mm in 40 s was seen. A dendritic platelet changed shape to a smoothly spread form in 3 min and continued into fragmentation and was eroded 5 min later. Many
smoothly spread platelets continued to change shape, after a longer or shorter lag phase, and turned into a more and more fragmented appearance until the cell body disappeared and only a rough area could be seen, corresponding to a
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microparticle covered surface. No cell parts could be observed to leave the surface during this change. 3.5. Extra incubation after rinsing When the platelets were incubated for an extra 15 min after rinsing before they were fixed, a 10% lower number of platelets was seen on the surface (98±7 compared to 110±15). For the different shapes significant ( p<0.01) differences could be seen for all shapes, except the cells with short dendrites, between normally incubated samples and samples incubated for an extra 15 min after rinsing ( Fig. 7). The smoothly spread platelets were at a lower concentration after the extra incubation, indicating that they may leave the surface or fragmentize.
(a)
3.6. Higher platelet concentrations At a high concentration of platelets (3×108 platelets/ml ) at the IgG-coated surface no dendritic or fragmented platelets [Fig. 8(a)] were seen. In this case all cells spread smoothly or formed aggregates. An extra incubation of these samples for 15 min resulted only in more spread platelets [Fig. 8(b)]. There were no platelets seen alone without interactions with other platelets. 3.7. Adhesion with thrombin present When thrombin was present at a concentration of 1 U/ml at the time of adhering, 25% more platelets adhered to the surface (35±7 compared to 27±2). These extra adhering platelets seemed to take the shape of compact or smoothly spread platelets ( Fig. 9).
4. Discussion Platelets may become activated during separation, for example by contact with air. In order to isolate platelets without activation and shape change we have used Mg2+ ions at a concentration of 4 mM as activation inhibitor [27]. Magnesium is tried as a platelet inhibitor in very different experimental models, both in vivo and in vitro
(b) Fig. 8. Platelets at a concentration of 3×108 cells/ml on an IgGcoated surface. (a) After 10 min of incubation at 37°C. (b) After 10 min of incubation and 15 min of additional incubation at 37°C after rinsing. Original magnification ×1000.
[28–31]. The interchange between magnesium and calcium ions at the GPIIb/IIIa [32] gives the possibility of a reversible inhibition. Platelet activation by non-aggregated IgG through Fcc-receptors is inhibited by high viscosity, which prevents cell–cell interactions between platelets [33]. The experimental procedure used in our study with a plasma free solution prevents platelet adhesion through complement activation. In our studies we have used surface-immobilized IgG and most often a low concentration of platelets which minimizes the cell–cell interactions. The thickness of the IgG surface in our experiments ˚ , which equals was determined to be 67 A
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Fig. 9. The number of platelets with different shapes (compact, smoothly spread, short dendrites, long dendrites, microparticle and fragmented) adhering to an IgG-coated surface after 10 min of incubation at 37°C. The platelets were either incubated alone or with a thrombin concentration of 1 U/ml. Platelet concentration used was 3×107 platelets/ml. ** p<0.001 (mean±S.D.).
6 pmol/cm2. A tightly packed monolayer of IgG is, according to Werthe´n et al. [34], around 8 pmol/cm2. Our surfaces would then be about 75% covered by a monolayer of IgG, which may be distributed in 2D-aggregates, but probably not in 3D-aggregates. In our immunofluorescence experiments with an IgG-coated surface with platelets adhered to the surface we could see protected zones, indicating that antibodies do not pass through the adhering cells. These protected zones have been described by Loike et al. [35]. However, since the area covered by cells was larger than the protected area, some cells leak antibodies through their membranes, which is consistent with the finding that the cells end up as fragments at the IgG-coated surface. Because the number of platelets with long dendrites were quite constant over time, the most probable progress for compact platelets was to first develop a few short dendrites before starting to spread to a smoothly spread platelet. Some cells with short dendrites continue the dendrite formation and form quite stable long dendrites. The microparticles along the dendrites are probably the first step of fragmentation.
The platelets with long dendrites seen in our experiments were always scattered cells interacting only with the surface and not with other cells. Dendritic platelet shape has earlier been recorded at platelet interactions with 14% SO Pellethane 3 surfaces [36 ]. This surface could in some way stimulate the Fc-receptor or the following events. Waples et al. [36 ] also conclude that the spreading of platelets is Ca2+ dependent. In all our experiments Ca2+ concentration (1 mM ) is lower than physiological conditions due to problems with stability of the buffer at higher CaCl concen2 trations. Waples et al. [36 ] used up to 1 mM, making these results comparable. Crawford [37] observed terminal swellings on platelet pseudopodia and suggested that microparticles were a result of these swellings. A close relation between pseudopodia and microparticle formation has been confirmed [38]. In our experiments we have detected microparticles along the dendritic pseudopodia [Fig. 3(d )] and not only as terminal swellings. This is still agreeable with the observations reported by Yano et al. [38], also reporting a few filamentous structures in the microparticles but not determining their nature. According to our studies filamentous actin was
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seen in dendrites and along the edges of the cell membrane as well as in the central body but was not detected in the microparticles. The microparticles might have an attracting effect on other platelets which would explain the big increase in adhesion seen after 10 min incubation time. At the same time the first fragmented platelets appeared. These fragments, or platelet microparticles, are known to be potent activators of PMN cells [39,40], suggesting a possible pathway of inflammatory reactions at the surface [6 ]. In conclusion, the results of the present study show that platelets undergo dramatic morphological changes in contact with surface-immobilized IgG. The cells secrete microvesicles, recognized by anti-pan platelet antibodies.
Acknowledgment Supported by Grant from the Swedish Medical Research Council (06235). We are very grateful to Ulf Bagge for helping us with the vital microscopy.
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