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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:230-235.)
© 1996 American Heart Association, Inc.

Articles

Association of Localized Ca²⁺ Gradients With Redistribution of Glycoprotein IIb-IIIa and F-actin in Activated Human Blood Platelets

Hideo Ariyoshi; Edwin W. Salzman

From the Department of Surgery, Beth Israel Hospital, Harvard Medical School, Boston, Mass.

Correspondence to Edwin W. Salzman, MD, Beth Israel Hospital, 330 Brookline Ave, Boston, MA 02215.

	Abstract

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Abstract We monitored the intracellular distribution of ionized free Ca²⁺ concentration ([Ca²⁺]_i) in individual human platelets by digital imaging fluorescence microscopy with fura 2 during platelet activation induced by surface contact or a soluble platelet agonist (thrombin). Contact of platelets with glass resulted in pseudopod formation and spreading, accompanied by a nonuniform rise in [Ca²⁺]_i. The rise in [Ca²⁺]_i was maximal during pseudopod formation. Locally elevated [Ca²⁺]_i was frequently found in pseudopodia and at the edge and core of spread platelets. This pattern was faithfully duplicated by the local pattern of distribution of the cytoskeletal components F-actin, gelsolin, and surface glycoproteins (GP) IIb-IIIa but not by calmodulin. Platelets stimulated by thrombin also showed an inhomogeneous rise in [Ca²⁺]_i, which was well correlated with the staining of F-actin and GPIIb-IIIa. Cytochalasin D, an inhibitor of actin polymerization, inhibited the inhomogeneous increase or redistribution of F-actin and GPIIb-IIIa but did not inhibit the rise in mean [Ca²⁺]_i. These observations suggest that a localized change in [Ca²⁺]_i may be associated with cytoskeletal reorganization and redistribution of GPIIb-IIIa in activated platelets.

Key Words: fluorescence microscopy • calcium • F-actin • integrins

	Introduction

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An increase in cytoplasmic ionized calcium concentration ([Ca²⁺]_i) is a characteristic feature of platelet activation.^{1 2 3 4} This increase in [Ca²⁺]_i results from inositol-1,4,5-triphosphate (IP₃)–mediated mobilization of Ca²⁺ from internal stores as well as influx of Ca²⁺ from the extracellular medium.⁵ The increase in [Ca²⁺]_i is followed by a return to basal level through an active process of Ca²⁺ extrusion into the extracellular space and restoration of [Ca²⁺]_i into intracellular storage sites.^{6 7} This change in [Ca²⁺]_i is believed to control many platelet responses, such as shape change, secretion, and aggregation.^{1 8} The exact mechanisms by which a rise in [Ca²⁺]_i controls these cellular responses are still unclear.

Using digital imaging microscopy and fura 2 with a calculated fluorescence ratioing technique, several laboratories confirmed the existence of [Ca²⁺]_i gradients within cells and have suggested the possible roles of a local [Ca²⁺]_i rise in function of smooth muscle cells,⁹ amoebas,¹⁰ and eosinophils.¹¹ With digital imaging fluorescence microscopy,^{12 13 14 15} inhomogeneous distribution and oscillation of [Ca²⁺]_i have now been found in single platelets activated by several soluble agonists.

Hartwig⁸ reported the possible involvement of [Ca²⁺]_i in pseudopod formation in quin-2–loaded platelets activated by contact. Stossel¹⁶ suggested the existence of [Ca²⁺]_i and phospholipid-mediated regulation of platelet cytoskeleton through the activation of gelsolin. Several reports indicated dynamic redistribution of surface glycoproteins (GPs) during platelet activation.^{8 17 18 19} It was not unexpected that a localized [Ca²⁺]_i change that mediates cytoskeletal reorganization might cause clustering of surface GPs, because biochemical analysis revealed that GPIIb-IIIa becomes associated with the cytoskeleton in activated platelets.^{19 20 21} A possible role for GP as a Ca²⁺ channel was suggested. The details of interactions between [Ca²⁺]_i and cytoskeletal components or surface GPs have not been fully clarified.

In this report, we discuss the spatial distribution of [Ca²⁺]_i and the cytoskeletal proteins F-actin, gelsolin, or the surface glycoproteins GPIIb-IIIa to test the hypothesis that a local change in [Ca²⁺]_i may be related to the morphological changes or clustering of GPIIb-IIIa in activated platelets through the local modulation of cytoskeletal reorganization.

	Methods

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Materials
Fura 2 acetoxymethyl ester (AM) and NBD phallacidin were obtained from Molecular Probes, Inc. Anti-gelsolin and anti-calmodulin (CaM) monoclonal antibodies and cytochalasin D were obtained from Sigma Chemical Co. Monoclonal antibody AP-3, specific for GPIIIa, was obtained from Dr S. Shattil.¹⁸ -Thrombin was the kind gift of Dr John W. Fenton, Jr (New York State Department of Health, Albany). Other chemicals were of the highest analytical grade available.

Platelet Preparation
Human blood was drawn into a 0.1% (wt/vol) volume of 3.8% trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 200g for 20 minutes at room temperature, and the cells were loaded with fura 2 by incubation with fura 2 AM (5 to 10 µmol/L) for 30 minutes at 35°C. The platelet concentration was approximately 3.0x10⁸ cells/mL.

Platelet Immobilization and Activation
Thin glass coverslips with or without poly-L-lysine coating were used to immobilize or activate platelets. Fura 2–loaded platelets in PRP diluted 10 times with HEPES-Tyrode's solution buffer (in mmol/L: NaCl 129, NaHCO₃ 8.9, KH₂PO₄ 0.8, MgCl₂ 0.8, dextrose 5.6, and HEPES 10, pH 7.4) were allowed to settle on the coverslips for 3 minutes at room temperature. After unattached cells were removed by gentle washing with 3 mL of modified HEPES-Tyrode's solution buffer, 0.3 mL of the same buffer containing 1 mmol/L CaCl₂ was added. The chamber was placed on a thermostated stage of an inverted microscope (Carl Zeiss, Inc). After a 2-minute incubation, recording was started. Under the conditions of our assay, it was not possible, of course, to demonstrate the exact time at which each platelet was immobilized. Platelets observed at "time 0" had been immobilized on coverslips and may have become glass activated at some time during the several minutes occupied by washing, adhesion, and incubation.

It was not possible to perform the immobilization and washing steps on the microscope, because at the magnification used in these experiments, the slightest movement of the chamber resulted in loss of field. Floating platelets not yet adherent could not be examined in the detail needed for digitizing and the subsequent ratioing techniques.

Digital Imaging Microscopy
Digital imaging microscopy was carried out as described previously⁷ with some modification. A DC-stabilized xenon lamp (75 W) was the light source. A computer-controlled filter wheel alternately inserted filters of 340 and 380 nm (10 and 13 nm half-bandwidth, respectively) into the light path; emitted light was collected through a 440-nm dichroic long-pass filter and finally through a 505-nm band-pass filter (40 nm half-bandwidth). A Zeiss x100 oil-immersion objective (Neofluar, 100/1.30) was used, giving a total 800x magnification. Images acquired by a silicon-intensified target camera (ISIT 66X, Dage MTI, Inc) were averaged in real time to reduce noise, and the average was digitized to 256 gray levels with an analog to digital converter. We used 256x256 pixel images, and individual wavelength images of fura 2 were divided after subtraction of background on a pixel-to-pixel basis (340/380 nm) to provide a high-resolution ratio image. Mean [Ca²⁺]_i value of individual platelets was calculated as an average of fura 2 fluorescence ratio in each pixel. [Ca²⁺]_i was calibrated as described previously.^{9 10 22}

Detection of F-actin, Gelsolin, Calmodulin, and GPIIb-IIIa
Platelets were fixed by 2% paraformaldehyde immediately after the acquisition of fura 2 ratio images. For detection of F-actin, platelets adhered or immobilized on coverslips were washed 3 times with PBS (pH 7.4) and incubated with PBS containing 10 µmol/L of NBD phallacidin for 30 minutes at room temperature as we described previously.⁴ For the detection of gelsolin or calmodulin, platelets were permeabilized by 0.1% Triton x100 and incubated with PBS containing 2 µg/mL of anti-gelsolin or 5 µg/mL of anti-calmodulin monoclonal antibody (Sigma Chemical Co) followed by incubation with anti-mouse IgG monoclonal antibody labeled by rhodamine B isothiocyanate (RITC). To visualize GPIIb-IIIa, platelets were incubated with 2 µg/mL of AP-3 murine monoclonal antibody against GPIIb-IIIa without permeabilization. Fluorescent images of NBD phallacidin or RITC were obtained by use of digital imaging microscopy with a 488-nm excitation filter and a 530-nm emission filter or a 550-nm excitation filter and a 620-nm emission filter, respectively. Nonspecific binding was negligible; the binding of NBD phallacidin was completely abolished by exogenous F-actin but not by G-actin, and no fluorescence was observed in the platelets incubated with nonimmune control antibody (data not shown).

	Results

Top Abstract Introduction Methods Results Discussion References

 
We studied the time course and spatial distribution of [Ca²⁺]_i in human platelets during changes in platelet cytoskeleton to test the hypothesis derived from observations in other cells that localized [Ca²⁺]_i gradients are involved in cell motility and related processes.^{9 10 11} Platelets change their shape and form pseudopodia in response to various agonists, such as ADP or thrombin, or in response to contact with active surfaces. However, most pseudopodia that are formed in response to soluble agonists are too small in diameter and contain too few fluorescent molecules to be detected by digitizing fluorescent microscopy with fura 2 (H. Ariyoshi, unpublished data, 1995). The limited spatial resolution that is imposed by the emission wavelength of fura 2 (505 nm) and the limited capability of the detector (silicon-intensified target camera)²³ result in a practical limit of resolution at about 250 nm.⁸ We did not demonstrate thrombin-induced small pseudopod formation. However, we were able to study platelets activated by contact with glass, which is known to induce a visible change of shape, including pseudopod formation and spreading.²⁰ As shown in Fig 1, platelets in contact with glass underwent pseudopod formation followed by spreading, which was accompanied by a variable rise of [Ca²⁺]_i. The distribution of [Ca²⁺]_i was inhomogeneous, and a localized [Ca²⁺]_i rise was frequently observed at a site of pseudopod formation, at the edge of a lamella, or in the core of the spread platelets during platelet activation.

View larger version (21K): [in this window] [in a new window]   Figure 1. Fluorescent images show spatial distribution of ionized free calcium concentration ([Ca2+]i) in platelets activated by contact with glass. Fura 2–loaded platelets were deposited on glass coverslips and allowed to become activated. Fura 2 ratio images were obtained every 5 seconds as described in "Methods." Pseudocolored images of typical glass-activated platelets are shown 0 minutes (a), 2 minutes (b), and 4 minutes (c) after observation was begun. Localized high [Ca2+]i spots around pseudopodia are indicated by arrows.

As shown in Fig 2, we found oscillation of [Ca²⁺]_i in the platelets activated by contact with glass. The pseudopod presented in Fig 2b became visible at 150 seconds and stopped linear growth at 210 seconds. The amplitude of oscillation of mean [Ca²⁺]_i in whole platelets was maximal during pseudopod formation and decreased after pseudopod formation was completed. Although it is difficult to estimate exactly when platelets are activated in this system because of the uncertainty of the duration of the lag period before the platelets settle onto the observation platform, these observations suggested an association of [Ca²⁺]_i rise with regulation of cytoskeletal reorganization in contact-activated platelets.

View larger version (29K): [in this window] [in a new window]   Figure 2. Temporal change in mean ionized free calcium concentration ([Ca2+]i) in a platelet activated by contact with glass. Fura 2–loaded platelets were deposited on coverslips and allowed to become activated by contact with glass. Fura 2 ratio images were obtained every 5 seconds as described in "Methods." Typical response of mean [Ca2+]i in a representative platelet is presented. Arrows indicate the time at which the fluorescent images shown were obtained. The edge of a cell was determined by eye with use of pseudocolored ratio images, since the margin of the platelets examined in pseudocolored images corresponded to that observed in phase contrast image in our preliminary study. The magnification in 2a is shown (bar); the same magnification was used in 2b and 2c. Differences in cross-sectional area reflected the effects of spreading on the glass surface. Morphological change was typical of the process in the more than 100 platelets examined. In preliminary experiments (eg, in Fig 4), phase micrography was found to correspond satisfactorily to fluorescence microscopy for determination of the platelet perimeter.

Platelet-shape change results from reorganization of cytoskeletal proteins, including polymerization and depolymerization of actin, modulated by several other proteins, including gelsolin and calmodulin.^{8 16} The effects of gelsolin on actin polymerization have been reported to be controlled mainly by [Ca²⁺]_i and phosphatidylinositol 4,5-biphosphate,¹⁶ which is a source of IP₃, an intracellular messenger that causes Ca²⁺ mobilization. Activation of calmodulin-dependent kinases has been reported to play some role in cytoskeletal reorganization, resulting in phosphorylation of proteins such as actin-binding protein and myosin light chain kinase.¹ GPIIb-IIIa is reported to be associated with cytoskeletal proteins as well as fibrinogen during its participation in platelet signal transduction.¹⁷ We studied the relation of [Ca²⁺]_i to F-actin, gelsolin, calmodulin, and GPIIb-IIIa in platelets activated by contact with glass (Fig 3). Platelets observed in the act of changing their shape displayed heterogeneous areas of [Ca²⁺]_i rise, which faithfully corresponded to hot spots of GPIIb-IIIa, F-actin bundles, and gelsolin. Local [Ca²⁺]_i changes corresponded to zones of actin polymerization, gelsolin activation, and redistribution of GPIIb-IIIa. In platelets activated by contact with glass, F-actin bundles were concentrated in at least three different locations: at the edge of the platelets, in rings at the core of the platelets, or in bridges of F-actin connecting the other two structures. [Ca²⁺]_i and GPIIb-IIIa were distributed in the same regions, suggesting structural interactions between F-actin and GPIIb-IIIa in association with localized [Ca²⁺]_i gradients. In contrast to the distribution of F-actin, gelsolin, and GPIIb-IIIa, the distribution of calmodulin (Fig 3) was more homogeneous and was not particularly correlated with sites of morphological change. Earlier biochemical study revealed an association of calmodulin with modulation of actin-myosin interactions in the generation of force required for cell motility.²⁴

View larger version (36K): [in this window] [in a new window]   Figure 3. Relation between the distribution of ionized free calcium concentration ([Ca2+]i) and F-actin, gelsolin, calmodulin, and GPIIb-IIIa. Fura 2–loaded platelets were deposited on coverslips and fura 2 ratio images were obtained as described in "Methods." After fixation by 2% paraformaldehyde immediately after the acquisition of fura 2 ratio images, platelets were stained with NBD phallacidin and anti-gelsolin, calmodulin (CaM), or GPIIb-IIIa monoclonal antibody as described in "Methods." Fluorescent images of NBD phallacidin or rhodamine B isothiocyanate were obtained by digital imaging microscopy. Fluorescence from fura 2 was not detectable after the immunostaining procedures. Images presented here were representative of five different preparations. Across each row arrowheads indicate the same platelet.

Observation of platelets activated by contact with glass suggested an association of localized [Ca²⁺]_i gradients, cytoskeletal reorganization, and redistribution of GPIIb-IIIa, but the sequence of these events during platelet activation is not clear. To understand the possible relation of a localized rise in [Ca²⁺]_i to these phenomena, we used cytochalasin D, an inhibitor of actin polymerization,²⁵ which decreased platelet adhesion to glass coverslips. Platelets adherent to poly-L-lysine were stimulated by thrombin. As shown in Fig 4, platelets adhering to a poly-L-lysine–coated coverslip did not show a remarkable morphological change or a rise in mean [Ca²⁺]_i on stimulation. Thrombin increased the content of F-actin and GPIIb-IIIa, an action that was abolished by cytochalasin D. In platelets before stimulation or in platelets treated with cytochalasin D, staining of F-actin and GPIIb-IIIa was relatively weak and evenly distributed, which may be a reflection of the small content of F-actin and GPIIb-IIIa. In this system, spatial resolution is limited by the emission wavelength for each dye (approximately one-half wavelength of emission fluorescence) and the intensity of emission fluorescence, making it impossible to detect homogeneously distributed surface antigens such as GPIIb-IIIa in resting platelets. As shown in Figs 4 and 5, the increase and oscillation of mean [Ca²⁺]_i was not affected by cytochalasin D. In the absence of extracellular Ca²⁺, the initial rise of [Ca²⁺]_i was comparable to that in the presence of extracellular Ca²⁺ and was not affected by cytochalasin D (data not shown). These findings strongly suggested that actin polymerization and redistribution of GPIIb-IIIa were not necessary for the [Ca²⁺]_i rise.

View larger version (39K): [in this window] [in a new window]   Figure 4. Fluorescent images show the change in the distribution of ionized free calcium concentration ([Ca2+]i), F-actin, and GPIIb-IIIa induced by thrombin (THR) and the effects of cytochalasin D (cyto D). Fura 2–loaded platelets preincubated with or without 10 µmol/L cytochalasin D for 10 minutes at room temperature were deposited on poly-L-lysine–coated coverslips by incubation in 10x diluted plasma-rich platelets for 3 minutes at room temperature. After washing out unattached cells, adherent platelets were stimulated by 1.0 U/mL thrombin in the presence of 1 mmol/L extracellular Ca2+. Fura 2 ratio images were acquired before and 5 seconds after stimulation of the platelets. Platelets were fixed by 2% paraformaldehyde immediately after acquisition of the images. F-actin or GPIIb-IIIa was detected by NBD phallacidin or AP-3 monoclonal antibody as described in Fig 3. Images presented here were representative of five different preparations. Arrowheads in the middle row indicate the same platelet.

View larger version (23K): [in this window] [in a new window]   Figure 5. The effect of cytochalasin D (cyto D) on the temporal change in [Ca2+]i induced by thrombin. Fura 2–loaded platelets preincubated with or without 10 µmol/L cytochalasin D for 10 minutes at room temperature were deposited on poly-L-lysine–coated coverslips by incubating with 10x diluted plasma-rich platelets for 3 minutes at room temperature. After washing out unattached platelets, platelets on coverslips were stimulated by 1.0 U/mL thrombin in the presence of 1 mmol/L extracellular Ca2+. Fura 2 ratio images were acquired every 5 seconds. Typical results from five different preparations are shown.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
A change in [Ca²⁺]_i has been believed to play an important role as a second messenger in a variety of cells, including human blood platelets.¹ Digital imaging fluorescence microscopy revealed the existence of localized [Ca²⁺]_i gradients and allowed observation of the local [Ca²⁺]_i rise in conjunction with local cellular responses in single cells.^{9 10 11} Taylor et al¹⁰ reported localized linear [Ca²⁺]_i gradients associated with cellular motion in amoebas. Since cellular motion and change in cell shape require activation of the actomyosin system and cytoskeletal reorganization,^{1 20} we hypothesized that local changes in intracellular [Ca²⁺]_i might regulate some platelet reactions. For example, aggregation of membrane surface GPIIb-IIIa induced by several agonists and referred to as clustering has been observed by immunohistochemical analysis with electron microscopy.^{17 19 21} Several reports suggested an association of GPIIb-IIIa with cytoskeletal proteins in activated platelets,^{17 19 21} and it is likely that clustering of GPIIb-IIIa in activated platelets is a phenomenon related to cytoskeletal reorganization. On the other hand, there have been reports that suggested a potential role of GPIIb-IIIa as a mediator of Ca²⁺ influx in activated platelets.²⁶ It is likely that there is some truth in both of these hypotheses and that local gradients in [Ca²⁺]_i are linked to both cytoskeletal reorganization and clustering of GPIIb-IIIa. The present study appears to be the first demonstration of the similarity of distribution of localized [Ca²⁺]_i gradients with F-actin, gelsolin, and GPIIb-IIIa in single platelets. We found that under the circumstances of these experiments, clustering of GPIIb-IIIa and F-actin bundles occurred at the site of local elevation of [Ca²⁺]_i, which was observed in the neighborhood of pseudopod formation. Colocalization of gelsolin suggests that reorganization of cytoskeletal F-actin takes place at the same loci. These observations strongly support hypotheses concerning intertwining roles of localized [Ca²⁺]_i change in controlling cell motility and in the possible functions of GPIIb-IIIa.

Since cytochalasin D, an inhibitor of actin polymerization, inhibited redistribution of GPIIb-IIIa without inhibiting the [Ca²⁺]_i rise in thrombin-stimulated platelets, the clustering of GPIIb-IIIa does not seem to be required for a rise in [Ca²⁺]_i. We suggest that local changes in [Ca²⁺]_i regulate platelet shape change and redistribution of GPIIb-IIIa through cytoskeletal reorganization, possibly through the local activation of several Ca²⁺-dependent pathways, such as the kinase-phosphatase system, calpain-mediated proteolysis, the actin-myosin system, or Ca²⁺-binding regulating proteins like gelsolin or calmodulin.

Because of the limited spatial resolution of the system used in the present study, it could not be determined for certain that clustered GPIIb-IIIa receptors contribute to maintenance of localized [Ca²⁺]_i gradients in activated platelets.

	Acknowledgments

 
This work was supported by grants (37610 and 33014) from the National Heart, Lung, and Blood Institute.

Received February 21, 1995; accepted October 18, 1995.

	References

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