American Journal ofPathology, Vol. 140, No. 1, January 1992 Copyright © American Association of Pathologists
Dynamic Redistribution of Major Platelet Surface Receptors After Contact-induced Platelet Activation and Spreading An Immunoelectron Microscopy Study
Nelly Kieffer, Josette Guichard, and Janine Breton-Gorius From INSERM U91, Hopital Henri Mondor, Creteil, France
The authors used an immunogold labelingprocedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage ofplatelet adhesion, a typical segregation of receptors occurred Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IlIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIlbIIIa, GPIVandp24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets; whereas GPIb was absentfrom pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized During the early stages of platelet activation; surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranularFg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric ca-granular localization. At later stages of activation and especially after thrombin stimulation; no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were stillpresent inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased
over the granulomere and extended to the cellperiphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton; and 3) during the early stage of platelet activation; a communication e-xists between the alpha granules and the platelet surface. (Am J Pathol 1992, 140:
57-73)
Within seconds after blood vessel injury, platelets adhere to exposed subendothelium, spread, and release the content of their storage organelles. Simultaneously, circulating platelets respond to released agonists by changing shape, extending pseudopodia, and finally aggregating to form the hemostatic plug. Ultrastructural analysis of activated platelets has shown that the morphologic changes that accompany platelet activation correlate with actin polymerization and cytoskeleton reorganization.14 Dendritic stellate type platelets typical of early stages of activation are characterized by the appearance of numerous filaments that become highly organized in bundles and radiate into the pseudopodia, and by microtubules that form concentric circles and centralize the granules to form the granulomere. In addition to the actin filaments that form the cytoskeleton, platelets also contain a submembranous skeleton composed of short actin filaments crosslinked to actin binding protein.§8 Changes in the components of this skeleton at Accepted for publication August 6, 1991. Address reprint requests to Dr. Nelly Kieffer, INSERM U91, H6pital Henri Mondor, F-94010 Cr6teil, France.
57
58
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AJPJanuary 1992, Vol. 140, No. 1
the early stage of platelet activation might contribute to the platelet shape change response, whereas at later stages of activation, when platelets have aggregated, this membrane skeleton becomes disrupted through cleavage of actin binding protein by the Ca2 +-dependent protease.9 Both the platelet membrane skeleton as well as the cytoskeleton have been reported to influence the functional properties of cell surface receptors. Thus the membrane skeleton stabilizes the glycoprotein (GP) Ib-IX complex and regulates its ability to bind von Willebrand factor (vWF).8'10 On the other hand, GPIIb-llla, which becomes attached to the cytoskeleton only after platelet activation, undergoes a conformational change that renders it competent to bind the RGD containing adhesive proteins such as fibrinogen (Fg), fibronectin (Fn), vWF, vitronectin (Vn) and thrombospondin (TSP) (for a review see 1 1). The ultrastructural localization of GPIlb-llla during platelet activation has been extensively studied, and Fg coupled to colloidal gold has been essentially used as an electron-dense probe to visualize the GPIlb-llla receptor. 12-21 Because of the close relationship between the platelet cytoskeleton and the surface receptors and the influence of this skeleton on the functional properties of the receptors during platelet adhesion and aggregation, we have designed a procedure to isolate platelets under conditions of minimal platelet activation. Using this procedure, we have investigated the dynamic redistribution of major platelet receptors and their respective ligands during contact-induced platelet activation.
Materials and Methods Platelet Preparation Blood was drawn by venipuncture from healthy adult volunteers and anticoagulated with 1/10 volume of acid/ citrate/dextrose (71 mM citric acid, 85 mM sodium citrate, 110 mM glucose). Whole blood droplets were dripped on parafilm in a moist chamber and formvar-coated nickle grids were placed directly on top of each drop. Platelets were allowed to adhere and spread on the grid for 5 minutes at 370C. Nonadherent cells were then removed by three successive washes of the grid in Tyrode buffer (buffer A). When required, adherent platelets were further activated for 3 minutes at 370C with 0.1 U/ml of thrombin. The grids were then washed three times for 5 minutes with buffer A and further processed for immunolabeling.
Immunogold Labeling For the immunogold labeling procedure, all the steps were performed at 40C. Since the major aim of the label-
ing procedure was to achieve specific labeling of unfixed living cells, experimental conditions were selected that gave the lowest background to the detriment of labeling efficiency. Before immunogold labeling, platelets spread on formvar-coated grids were incubated for 20 minutes in buffer A containing 0.1% bovine serum albumin (BSA) and 20% normal goat serum to block nonspecific antibody binding. All the antibodies used in this study are listed in Table 1, with their species origin and subclass type indicated.2242 The specific antibody was applied at an appropriate dilution for 20 minutes (Table 2). After three washes of 15 minutes each with buffer A containing 0.1% BSA, platelets were treated for 20 minutes with Janssen auroprobes (Janssen Pharmaceutica, Beerse, Belgium) used at a 1/5 dilution in buffer A containing 1% BSA. For double labeling procedures combining rabbit and mouse antibodies, the second stage auroprobes were goat anti-rabbit IgG (GAR G5) together with goat anti-mouse (GAM G15); for double labeling using mouse IgG and IgM, the auroprobes were goat anti-mouse IgG (GAM G1 5) together with goat anti-mouse IgM (GAM G5) (Table 2). On occasion, platelets were fixed with 0.005% of glutaraldehyde in phosphate buffer before the immunogold labeling procedure. After the labeling procedure, the grids were washed three times for 5 minutes with buffer A containing 0.1% BSA and three times with buffer A alone. Then, a 3-minute fixation by 1 % glutaraldehyde in phosphate buffer, washes in distilled water, and progressive dehydration in alcohol at 20, 50, 70, 90, and 100% were performed. The grids were dried, stained with uranyl acetate and lead nitrate, and the nonembedded whole-mount platelet preparations were observed at 100 KV using a Philips CM10 electron microscope. For GPla-lla (CDw49b) localization, platelets spread on formvar-coated glass coverslips were treated as whole-mount preparations but after glutaraldehyde fixation, were postfixed in 1% osmic acid, dehydrated, and embedded open-faced in epon as previously described.33 Platelets were then sectioned in series parallel to the plane of attachment through the entire cell thickness.
Results Isolation of platelets by differential centrifugation or gel filtration causes platelet activation and cytoskeleton reorganization, unless the isolation procedures are performed in the presence of platelet activation inhibitors, such as prostacyclin or PGE1.7 In the present report, we have investigated the redistribution of platelet receptors during contact-induced platelet activation. With the procedure described, platelet activation during the isolation procedure could almost completely be prevented by simply eliminating the centrifugation steps. Thus, antico-
Surface Receptors on Adherent Platelets
59
AJPJanuay 1992, Vol. 140, No. 1
Table 1. Primary Antibodies Antigen
FA6-152
CD36
IgG class IgG (mouse) IgG (mouse) IgG (mouse) IgG (rabbit) IgG (mouse) IgM (mouse) IgG (mouse) IgG (mouse) IgG (rabbit) IgG (mouse)
VLA2-GPIa (a2) VLA2-GPIa (@2) VLA2-GPIa-lla (a2 P)
Gi 14 CLB/thromb 4 Gi 9
CDw49b CDw49b CDw49b
IgG (mouse) IgG (mouse) IgG (mouse)
p24 p24 GP49
FMC 56 Lt 9.4 T6 (control)
CD9 CD9 CD1a
IgG (mouse) IgM (mouse) IgG (mouse)
von Willebrand Factor (vWF)
Polyclonal
IgG (rabbit)
Thrombospondin (TSP) Fibrinogen (Fg) Fibrinogen
Polyclonal F8 Cll Polyclonal
IgG (rabbit) IgG (mouse) IgG (rabbit)
GPlb GPlb GPlb Glycocalicin GPIlIla GPIlb-lIla GPIlb-Ilia GPIlb-illa GPIlb-lila GPIV
Antibody AN51 6DI API Polyclonal C17 J15 PBM 6.4 CLB/thromb 1
Polyclonal
CD design CD42b CD42b CD42b
CD61 CD41a CD41a CD41a
agulated whole blood was directly brought into contact with formvar-coated grids and platelets were allowed to adhere for 5 minutes at 370C. Since platelet interaction with the grid was not synchronized, all stages of platelet
Reference or source McMichael et al. Coller et al. Montgomery et al. Cramer et al. Tetteroo et al. Vainchenker et al. Von dem Borne et al. Von dem Borne et al. Cramer et al. Edelman et al. Kieffer et al. Von dem Borne et al. Von dem Borne et al. Immunotech (Marseille, France) Von dem Borne et al. Von dem Borne et al. Immunotech (Marseille, France)
(22) (23) (24) (25) (26) (27) (28) (28) (29) (30) (31)
(28) (28)
(28) (28)
Dakopatts (Copenhagen,
Denmark) Hourdille et al. Soria (unpublished) Cappel Laboratory (Cochranville, PA)
(32)
activation could be observed on a single preparation: dendritic stellate platelets typical for early stages of platelet activation as well as flattened spread platelets representing later stages of activation.
Table 2. Dilutions of Ptimary Antibodies (PA) and Nature of Secondary Anti-immunoglobulin Antibodies (SA) Single labeling Double labeling PA SA (dilution 1:5) Dilution PA Dilution SA dilution 1:5 Anti GPIlb-llla C17 1:100 PBM 6.4 GAM* IgG G5 1:100 GAM IgG G15 J15 1:50 GAM IgM G5 polyclonal anti Fg 1:100 GAR G5 Polyclonal 1:50 G15 GARt Anti Fg F8C11 1:2 GAM IgG G15 polyclonal anti GPIlb-ltla GAR 1:50 G5 F8C11 1:2 GAM IgG G15 Anti GPIV FA6-152 1:50 and 1:200 GAM IgG G15 J15 GAM IgM G5 1:50 FA6-152 1:50 GAM IgG G15 Anti TSP Polyclonal 1:25 GAR G15 LI 9.4 1:100 GAM IgM G5 PBM 6.4 1:100 GAM IgG G15 Anti VLA2 Gi14 1:100 GAM IgG G15 FA6-152 1:50 GAM IgG G15 CLB/thromb 4 1:100 GAM IgG G15 anti TSP 1:25 GAR G5 Anti p24 FMC56 1:100 GAM IgG G15 F8C11 1:2 GAM IgG G15 anti TSP GAR 1:25 G5 Anti GP49 T6 (control) 1:100 GAM IgG G15 Anti GPlb AN51 1:20 6D1 1:20 mixed GAM IgG G15 AP1 1:20 Polyclonal anti-glycocalicin 1:100 GAR G10 GAM G5 goat anti-mouse immunoglobulin gold: 5 nm.
t GAR G15 goat anti-rabbit immunoglobulin gold: 15 nm.
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Kieffer, Guichard, and Breton-Gorius
AJPJanuary 1992, Vol. 140, No. 1
Visualization of Receptors and Ligands Involved in Platelet Adhesion Immunogold labeling of GPIb was performed using monoclonal antibodies directed against the GPlb a chain. On dendritic platelets, GPlb was present all over the platelet surface with the exception of pseudopodia (Figure 1). On fully spread platelets, the peripheral web was free of label, whereas the gold particles over the cell body tended to align with the electron-dense amorphous material corresponding most likely to microfilament bundles (Figure 1). A similar alignment of GPlb molecules with platelet skeletal structures has been reported by Fox et al.10 and Hartwig and DeSisto.8 Prolonged adhesion of platelets to the formvar-coated grids for a period of more than 30 minutes before immunogold labeling did not alter the GPlb distribution, nor did it change when platelets were fixed before the labeling procedure. In contrast, when adherent platelets were treated with thrombin, a centripetal redistribution of GPlb to the granulomere was observed (Figure 2). To compare the topologic distribution of GPlb with other cell surface receptors involved in platelet adhesion, two we studied the distribution of GPIV and GPla-Ila receptors that have been reported to function as cell adhesion receptors involved in collagen binding.34'35 As illustrated in Figure 3A, the labeling observed with the antiGPIV antibody FA6-152 showed a distribution different from that of GPlb. The labeling was markedly increased on the granulomere as compared with the hyalomere, and this increase was even more apparent when a higher dilution of the antibody was used (Figure 3B). The intensity of labeling on the granulomere was not an artifact caused by the gold particles, since identical results were obtained with platelets that had been fixed before the immunogold labeling procedure (Figure 4A). In contrast to GPlb, a strong labeling was also observed on the pseudopods as well as at sites of contact of two platelets. This distinct receptor segregation was further demonstrated by double labeling experiments using antibodies to GPIlb-llla (J15) and GPIV (FA6-152) (Figure 4B). Despite the poor labeling efficiency with the IgM MoAb J15, used here for the purpose of double labeling, it is apparent that GPIV underwent a centripetal movement, whereas GPIlb-llla remained randomly distributed. The localization of GPla-lla was studied using three different monoclonal antibodies. Similar to anti-GPIV MoAb, all three anti-GPla-Ila antibodies showed an increased labeling over the granulomere region (Figure 5). To investigate the precise localization of GPla-lla at the level of the granulomere in whole-mount preparations, adherent platelets that had been immunolabeled with MoAb Gi9 and GAM 15 nm gold particles were fixed, and
embedded in epon; serial sections were performed in the entire thickness of the cell parallel to the plane of spreading. Interestingly, all the sections close to the site of adhesion were devoid of label (Figure 6A), whereas all gold particles were concentrated at the level of the hump that contained centrally concentrated organelles (Figure 6B). At this level, the cell membrane and few segments of the open canalicular system (OCS) were labeled; the last section at the top of the hump also revealed membrane labeling (Figure 6C). Since vWF binds to GPlb as well as GPIlb-llla and plays an important role in platelet adhesion to the subendothelium, we investigated the redistribution of the endogenous platelet a-granule vWF during platelet activation induced by cell attachment and spreading. As shown in Figure 7A, labeling of adherent platelets with an anti-vWF antibody was either negative or weak and restricted to the zone of the granulomere. Interestingly, often this labeling was present in individual a-granules that appeared less electron dense than the unlabeled a-granules. Also, the labeling was eccentric and similar to that previously reported by our laboratory for intragranular vWF of nonactivated platelets in suspension,?6 thus providing evidence that intragranular proteins were accessible to gold-coupled antibodies. After thrombin treatment of adherent platelets, the number of labeled a-granules increased; in addition, a strong labeling was seen in large, clear vacuoles resulting probably from the fusion of several a-granules (Figure 7B). However, no labeling was present at the surface of the cell, the periphery or on pseudopods (not shown).
Visualization of Receptors and Ligands Involved in Platelet Aggregation Immunogold particles identifying GPIlb-llla were spread over the entire platelet surface, including hyalomere, granulomere, and the cell periphery, although a greater particle density was observed on pseudopods (Figure 8A). Fixation of adherent platelets before the immunogold labeling procedure did not alter the distribution of gold particles although the intensity of labeling decreased, probably due to partial denaturation of the epitope by the fixative. When adherent platelets were activated by thrombin to induce complete release reaction, the granulomere appeared completely empty and thus provided morphologic evidence that the release reaction had occurred. Under these conditions, no change in the localization of gold particles occurred, although the pseudopods, which had a tendency to form an enlarged terminal plaque, were strongly labeled (Figure 8B). Translocation of adhesive proteins from the a-granule to the platelet surface during platelet attachment and
Surface Receptors on Adherent Platelets 61 AJPJanuary 1992, Vol. 140, No. 1
Figure 1. Spreadplatelets that werefixed and incubated with a mixture ofANi51, 6D1 and APi (dilution 1:20) and then with GAM G15. In a dendritic platelet, the labeling is uniformly distributed over the cell body but is absentfrom the pseudopods. In thefully spreadplatelet, the labeling aligns with electron dense material. Note that the peripheral zone is devoid of labeling, x 10,300.
62
Kieffer, Guichard, and Breton-Gorius
AJP January 1992, Vol. 140, No. 1
Figure 2. Livtng spread platelet activated by thrombin and labeled with anti-glycocalicin (dilution 1:100) and then with GAR Glo. 7he labeling is mainly concentrated on the central region, X24,920.
spreading was investigated using antibodies to the adhesive proteins Fg and TSP. Immunogold staining of Fg revealed minimal labeling of the platelet surface. This labeling was restricted exclusively to the granulomere, pro-
viding evidence for the beginning of the contact induced a-granule release reaction. In some adherent platelets, the weak labeling was associated with the matrix of identifiable a-granules (Figure 8C) After thrombin activation,
Surface Receptors on Adherent Platelets
63
AJP January 1992, Vol. 140, No. 1
N.,.
A
I
Figure 3. A: Spreadplatelets treated with FA6-152 monoclonal antibody 0.50) and then with GA CG15. The labeling is heavily present over the granulomere, and at the cell periphery at the level of contact between two platelets (arrows). A less intense labeling is present over the byalomere, x 11,800. B: Platelet treated as in (A) except that a higher antibody dilution 01200) was used. The labeling is mainly concentrated over the granulomere (arrows) and at the cell periphery, at the level ofpseudopod, particularly the terminal plaque (double arrows), X26,500.
64
._Sr. Ipt.j~ ~ -W,. . ,
Kieffer, Guichard, and Breton-Gorius
AJPJanuary 1992, Vol. 140, No. 1
* ~ ~~~~~~~~~~~~~~~~~ _,rj;it
i 39!
S
.4
1
i,~~~~~~~~~~~~~~~P
i
Dynamic Redistribution of Major Platelet Surface Receptors After Contact-induced Platelet Activation and Spreading An Immunoelectron Microscopy Study
Nelly Kieffer, Josette Guichard, and Janine Breton-Gorius From INSERM U91, Hopital Henri Mondor, Creteil, France
The authors used an immunogold labelingprocedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage ofplatelet adhesion, a typical segregation of receptors occurred Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IlIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIlbIIIa, GPIVandp24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets; whereas GPIb was absentfrom pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized During the early stages of platelet activation; surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranularFg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric ca-granular localization. At later stages of activation and especially after thrombin stimulation; no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were stillpresent inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased
over the granulomere and extended to the cellperiphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton; and 3) during the early stage of platelet activation; a communication e-xists between the alpha granules and the platelet surface. (Am J Pathol 1992, 140:
57-73)
Within seconds after blood vessel injury, platelets adhere to exposed subendothelium, spread, and release the content of their storage organelles. Simultaneously, circulating platelets respond to released agonists by changing shape, extending pseudopodia, and finally aggregating to form the hemostatic plug. Ultrastructural analysis of activated platelets has shown that the morphologic changes that accompany platelet activation correlate with actin polymerization and cytoskeleton reorganization.14 Dendritic stellate type platelets typical of early stages of activation are characterized by the appearance of numerous filaments that become highly organized in bundles and radiate into the pseudopodia, and by microtubules that form concentric circles and centralize the granules to form the granulomere. In addition to the actin filaments that form the cytoskeleton, platelets also contain a submembranous skeleton composed of short actin filaments crosslinked to actin binding protein.§8 Changes in the components of this skeleton at Accepted for publication August 6, 1991. Address reprint requests to Dr. Nelly Kieffer, INSERM U91, H6pital Henri Mondor, F-94010 Cr6teil, France.
57
58
Kieffer, Guichard, and Breton-Gorius
AJPJanuary 1992, Vol. 140, No. 1
the early stage of platelet activation might contribute to the platelet shape change response, whereas at later stages of activation, when platelets have aggregated, this membrane skeleton becomes disrupted through cleavage of actin binding protein by the Ca2 +-dependent protease.9 Both the platelet membrane skeleton as well as the cytoskeleton have been reported to influence the functional properties of cell surface receptors. Thus the membrane skeleton stabilizes the glycoprotein (GP) Ib-IX complex and regulates its ability to bind von Willebrand factor (vWF).8'10 On the other hand, GPIIb-llla, which becomes attached to the cytoskeleton only after platelet activation, undergoes a conformational change that renders it competent to bind the RGD containing adhesive proteins such as fibrinogen (Fg), fibronectin (Fn), vWF, vitronectin (Vn) and thrombospondin (TSP) (for a review see 1 1). The ultrastructural localization of GPIlb-llla during platelet activation has been extensively studied, and Fg coupled to colloidal gold has been essentially used as an electron-dense probe to visualize the GPIlb-llla receptor. 12-21 Because of the close relationship between the platelet cytoskeleton and the surface receptors and the influence of this skeleton on the functional properties of the receptors during platelet adhesion and aggregation, we have designed a procedure to isolate platelets under conditions of minimal platelet activation. Using this procedure, we have investigated the dynamic redistribution of major platelet receptors and their respective ligands during contact-induced platelet activation.
Materials and Methods Platelet Preparation Blood was drawn by venipuncture from healthy adult volunteers and anticoagulated with 1/10 volume of acid/ citrate/dextrose (71 mM citric acid, 85 mM sodium citrate, 110 mM glucose). Whole blood droplets were dripped on parafilm in a moist chamber and formvar-coated nickle grids were placed directly on top of each drop. Platelets were allowed to adhere and spread on the grid for 5 minutes at 370C. Nonadherent cells were then removed by three successive washes of the grid in Tyrode buffer (buffer A). When required, adherent platelets were further activated for 3 minutes at 370C with 0.1 U/ml of thrombin. The grids were then washed three times for 5 minutes with buffer A and further processed for immunolabeling.
Immunogold Labeling For the immunogold labeling procedure, all the steps were performed at 40C. Since the major aim of the label-
ing procedure was to achieve specific labeling of unfixed living cells, experimental conditions were selected that gave the lowest background to the detriment of labeling efficiency. Before immunogold labeling, platelets spread on formvar-coated grids were incubated for 20 minutes in buffer A containing 0.1% bovine serum albumin (BSA) and 20% normal goat serum to block nonspecific antibody binding. All the antibodies used in this study are listed in Table 1, with their species origin and subclass type indicated.2242 The specific antibody was applied at an appropriate dilution for 20 minutes (Table 2). After three washes of 15 minutes each with buffer A containing 0.1% BSA, platelets were treated for 20 minutes with Janssen auroprobes (Janssen Pharmaceutica, Beerse, Belgium) used at a 1/5 dilution in buffer A containing 1% BSA. For double labeling procedures combining rabbit and mouse antibodies, the second stage auroprobes were goat anti-rabbit IgG (GAR G5) together with goat anti-mouse (GAM G15); for double labeling using mouse IgG and IgM, the auroprobes were goat anti-mouse IgG (GAM G1 5) together with goat anti-mouse IgM (GAM G5) (Table 2). On occasion, platelets were fixed with 0.005% of glutaraldehyde in phosphate buffer before the immunogold labeling procedure. After the labeling procedure, the grids were washed three times for 5 minutes with buffer A containing 0.1% BSA and three times with buffer A alone. Then, a 3-minute fixation by 1 % glutaraldehyde in phosphate buffer, washes in distilled water, and progressive dehydration in alcohol at 20, 50, 70, 90, and 100% were performed. The grids were dried, stained with uranyl acetate and lead nitrate, and the nonembedded whole-mount platelet preparations were observed at 100 KV using a Philips CM10 electron microscope. For GPla-lla (CDw49b) localization, platelets spread on formvar-coated glass coverslips were treated as whole-mount preparations but after glutaraldehyde fixation, were postfixed in 1% osmic acid, dehydrated, and embedded open-faced in epon as previously described.33 Platelets were then sectioned in series parallel to the plane of attachment through the entire cell thickness.
Results Isolation of platelets by differential centrifugation or gel filtration causes platelet activation and cytoskeleton reorganization, unless the isolation procedures are performed in the presence of platelet activation inhibitors, such as prostacyclin or PGE1.7 In the present report, we have investigated the redistribution of platelet receptors during contact-induced platelet activation. With the procedure described, platelet activation during the isolation procedure could almost completely be prevented by simply eliminating the centrifugation steps. Thus, antico-
Surface Receptors on Adherent Platelets
59
AJPJanuay 1992, Vol. 140, No. 1
Table 1. Primary Antibodies Antigen
FA6-152
CD36
IgG class IgG (mouse) IgG (mouse) IgG (mouse) IgG (rabbit) IgG (mouse) IgM (mouse) IgG (mouse) IgG (mouse) IgG (rabbit) IgG (mouse)
VLA2-GPIa (a2) VLA2-GPIa (@2) VLA2-GPIa-lla (a2 P)
Gi 14 CLB/thromb 4 Gi 9
CDw49b CDw49b CDw49b
IgG (mouse) IgG (mouse) IgG (mouse)
p24 p24 GP49
FMC 56 Lt 9.4 T6 (control)
CD9 CD9 CD1a
IgG (mouse) IgM (mouse) IgG (mouse)
von Willebrand Factor (vWF)
Polyclonal
IgG (rabbit)
Thrombospondin (TSP) Fibrinogen (Fg) Fibrinogen
Polyclonal F8 Cll Polyclonal
IgG (rabbit) IgG (mouse) IgG (rabbit)
GPlb GPlb GPlb Glycocalicin GPIlIla GPIlb-lIla GPIlb-Ilia GPIlb-illa GPIlb-lila GPIV
Antibody AN51 6DI API Polyclonal C17 J15 PBM 6.4 CLB/thromb 1
Polyclonal
CD design CD42b CD42b CD42b
CD61 CD41a CD41a CD41a
agulated whole blood was directly brought into contact with formvar-coated grids and platelets were allowed to adhere for 5 minutes at 370C. Since platelet interaction with the grid was not synchronized, all stages of platelet
Reference or source McMichael et al. Coller et al. Montgomery et al. Cramer et al. Tetteroo et al. Vainchenker et al. Von dem Borne et al. Von dem Borne et al. Cramer et al. Edelman et al. Kieffer et al. Von dem Borne et al. Von dem Borne et al. Immunotech (Marseille, France) Von dem Borne et al. Von dem Borne et al. Immunotech (Marseille, France)
(22) (23) (24) (25) (26) (27) (28) (28) (29) (30) (31)
(28) (28)
(28) (28)
Dakopatts (Copenhagen,
Denmark) Hourdille et al. Soria (unpublished) Cappel Laboratory (Cochranville, PA)
(32)
activation could be observed on a single preparation: dendritic stellate platelets typical for early stages of platelet activation as well as flattened spread platelets representing later stages of activation.
Table 2. Dilutions of Ptimary Antibodies (PA) and Nature of Secondary Anti-immunoglobulin Antibodies (SA) Single labeling Double labeling PA SA (dilution 1:5) Dilution PA Dilution SA dilution 1:5 Anti GPIlb-llla C17 1:100 PBM 6.4 GAM* IgG G5 1:100 GAM IgG G15 J15 1:50 GAM IgM G5 polyclonal anti Fg 1:100 GAR G5 Polyclonal 1:50 G15 GARt Anti Fg F8C11 1:2 GAM IgG G15 polyclonal anti GPIlb-ltla GAR 1:50 G5 F8C11 1:2 GAM IgG G15 Anti GPIV FA6-152 1:50 and 1:200 GAM IgG G15 J15 GAM IgM G5 1:50 FA6-152 1:50 GAM IgG G15 Anti TSP Polyclonal 1:25 GAR G15 LI 9.4 1:100 GAM IgM G5 PBM 6.4 1:100 GAM IgG G15 Anti VLA2 Gi14 1:100 GAM IgG G15 FA6-152 1:50 GAM IgG G15 CLB/thromb 4 1:100 GAM IgG G15 anti TSP 1:25 GAR G5 Anti p24 FMC56 1:100 GAM IgG G15 F8C11 1:2 GAM IgG G15 anti TSP GAR 1:25 G5 Anti GP49 T6 (control) 1:100 GAM IgG G15 Anti GPlb AN51 1:20 6D1 1:20 mixed GAM IgG G15 AP1 1:20 Polyclonal anti-glycocalicin 1:100 GAR G10 GAM G5 goat anti-mouse immunoglobulin gold: 5 nm.
t GAR G15 goat anti-rabbit immunoglobulin gold: 15 nm.
60
Kieffer, Guichard, and Breton-Gorius
AJPJanuary 1992, Vol. 140, No. 1
Visualization of Receptors and Ligands Involved in Platelet Adhesion Immunogold labeling of GPIb was performed using monoclonal antibodies directed against the GPlb a chain. On dendritic platelets, GPlb was present all over the platelet surface with the exception of pseudopodia (Figure 1). On fully spread platelets, the peripheral web was free of label, whereas the gold particles over the cell body tended to align with the electron-dense amorphous material corresponding most likely to microfilament bundles (Figure 1). A similar alignment of GPlb molecules with platelet skeletal structures has been reported by Fox et al.10 and Hartwig and DeSisto.8 Prolonged adhesion of platelets to the formvar-coated grids for a period of more than 30 minutes before immunogold labeling did not alter the GPlb distribution, nor did it change when platelets were fixed before the labeling procedure. In contrast, when adherent platelets were treated with thrombin, a centripetal redistribution of GPlb to the granulomere was observed (Figure 2). To compare the topologic distribution of GPlb with other cell surface receptors involved in platelet adhesion, two we studied the distribution of GPIV and GPla-Ila receptors that have been reported to function as cell adhesion receptors involved in collagen binding.34'35 As illustrated in Figure 3A, the labeling observed with the antiGPIV antibody FA6-152 showed a distribution different from that of GPlb. The labeling was markedly increased on the granulomere as compared with the hyalomere, and this increase was even more apparent when a higher dilution of the antibody was used (Figure 3B). The intensity of labeling on the granulomere was not an artifact caused by the gold particles, since identical results were obtained with platelets that had been fixed before the immunogold labeling procedure (Figure 4A). In contrast to GPlb, a strong labeling was also observed on the pseudopods as well as at sites of contact of two platelets. This distinct receptor segregation was further demonstrated by double labeling experiments using antibodies to GPIlb-llla (J15) and GPIV (FA6-152) (Figure 4B). Despite the poor labeling efficiency with the IgM MoAb J15, used here for the purpose of double labeling, it is apparent that GPIV underwent a centripetal movement, whereas GPIlb-llla remained randomly distributed. The localization of GPla-lla was studied using three different monoclonal antibodies. Similar to anti-GPIV MoAb, all three anti-GPla-Ila antibodies showed an increased labeling over the granulomere region (Figure 5). To investigate the precise localization of GPla-lla at the level of the granulomere in whole-mount preparations, adherent platelets that had been immunolabeled with MoAb Gi9 and GAM 15 nm gold particles were fixed, and
embedded in epon; serial sections were performed in the entire thickness of the cell parallel to the plane of spreading. Interestingly, all the sections close to the site of adhesion were devoid of label (Figure 6A), whereas all gold particles were concentrated at the level of the hump that contained centrally concentrated organelles (Figure 6B). At this level, the cell membrane and few segments of the open canalicular system (OCS) were labeled; the last section at the top of the hump also revealed membrane labeling (Figure 6C). Since vWF binds to GPlb as well as GPIlb-llla and plays an important role in platelet adhesion to the subendothelium, we investigated the redistribution of the endogenous platelet a-granule vWF during platelet activation induced by cell attachment and spreading. As shown in Figure 7A, labeling of adherent platelets with an anti-vWF antibody was either negative or weak and restricted to the zone of the granulomere. Interestingly, often this labeling was present in individual a-granules that appeared less electron dense than the unlabeled a-granules. Also, the labeling was eccentric and similar to that previously reported by our laboratory for intragranular vWF of nonactivated platelets in suspension,?6 thus providing evidence that intragranular proteins were accessible to gold-coupled antibodies. After thrombin treatment of adherent platelets, the number of labeled a-granules increased; in addition, a strong labeling was seen in large, clear vacuoles resulting probably from the fusion of several a-granules (Figure 7B). However, no labeling was present at the surface of the cell, the periphery or on pseudopods (not shown).
Visualization of Receptors and Ligands Involved in Platelet Aggregation Immunogold particles identifying GPIlb-llla were spread over the entire platelet surface, including hyalomere, granulomere, and the cell periphery, although a greater particle density was observed on pseudopods (Figure 8A). Fixation of adherent platelets before the immunogold labeling procedure did not alter the distribution of gold particles although the intensity of labeling decreased, probably due to partial denaturation of the epitope by the fixative. When adherent platelets were activated by thrombin to induce complete release reaction, the granulomere appeared completely empty and thus provided morphologic evidence that the release reaction had occurred. Under these conditions, no change in the localization of gold particles occurred, although the pseudopods, which had a tendency to form an enlarged terminal plaque, were strongly labeled (Figure 8B). Translocation of adhesive proteins from the a-granule to the platelet surface during platelet attachment and
Surface Receptors on Adherent Platelets 61 AJPJanuary 1992, Vol. 140, No. 1
Figure 1. Spreadplatelets that werefixed and incubated with a mixture ofANi51, 6D1 and APi (dilution 1:20) and then with GAM G15. In a dendritic platelet, the labeling is uniformly distributed over the cell body but is absentfrom the pseudopods. In thefully spreadplatelet, the labeling aligns with electron dense material. Note that the peripheral zone is devoid of labeling, x 10,300.
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Kieffer, Guichard, and Breton-Gorius
AJP January 1992, Vol. 140, No. 1
Figure 2. Livtng spread platelet activated by thrombin and labeled with anti-glycocalicin (dilution 1:100) and then with GAR Glo. 7he labeling is mainly concentrated on the central region, X24,920.
spreading was investigated using antibodies to the adhesive proteins Fg and TSP. Immunogold staining of Fg revealed minimal labeling of the platelet surface. This labeling was restricted exclusively to the granulomere, pro-
viding evidence for the beginning of the contact induced a-granule release reaction. In some adherent platelets, the weak labeling was associated with the matrix of identifiable a-granules (Figure 8C) After thrombin activation,
Surface Receptors on Adherent Platelets
63
AJP January 1992, Vol. 140, No. 1
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Figure 3. A: Spreadplatelets treated with FA6-152 monoclonal antibody 0.50) and then with GA CG15. The labeling is heavily present over the granulomere, and at the cell periphery at the level of contact between two platelets (arrows). A less intense labeling is present over the byalomere, x 11,800. B: Platelet treated as in (A) except that a higher antibody dilution 01200) was used. The labeling is mainly concentrated over the granulomere (arrows) and at the cell periphery, at the level ofpseudopod, particularly the terminal plaque (double arrows), X26,500.
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Kieffer, Guichard, and Breton-Gorius
AJPJanuary 1992, Vol. 140, No. 1
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