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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:621-627.)
© 1996 American Heart Association, Inc.

Articles

Agglutination of Isolated Platelet Membranes

Meinrad Gawaz; Ilka Ott; Armin J. Reininger; Ulrich Heinzmann; Franz-Josef Neumann

From the I Medizinische Klinik (M.G., I.O., F.J.N.) and Anatomisches Institut (A.J.R.), Technische Universität München, and Forschungszentrum für Umwelt und Gesundheit, GmbH, Institut für Pathologie, Neuherberg (U.H.), Germany.

Correspondence to Dr Meinrad Gawaz, Medizinische Klinik der Technischen Universität, Ismaningerstraße 22, 81675 München, Germany.

	Abstract

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Abstract Platelet membrane glycoproteins play a central role in platelet aggregation and thus in primary hemostasis. To investigate mechanisms of platelet-platelet interaction in the absence of cellular activation events, we studied immunological and functional aspects of isolated platelet membranes. Platelet membranes contained significant amounts of the inducible fibrinogen receptor, glycoprotein (GP) IIb-IIIa, which exposes conformation-dependent LIBS1 and PMI-1 epitopes in response to fibrinogen-mimetic peptides GRGDSP and HHLGGAKQAGDV. In the presence of soluble fibrinogen, membrane-coated latex beads showed Ca²⁺-dependent agglutination that could be partially inhibited by GRGDSP but not by the biologically inactive peptide GRGESP. Thrombospondin enhanced agglutination of membrane-coated beads, which could be inhibited by polyvalent anti-thrombospondin Fab fragments and anti-thrombospondin monoclonal antibody MA-II. Mg²⁺ inhibited both GPIIb-IIIa– and thrombospondin-mediated agglutination of membranes in a dose-dependent manner. The results of the present study indicate that isolated platelet membranes are a useful tool to study regulation of GPIIb-IIIa– and thrombospondin-mediated platelet-platelet interaction.

Key Words: GPIIb-IIIa • thrombospondin • platelet aggregation • membranes • magnesium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Platelet aggregation plays a pivotal role in acute arterial thrombotic or thromboembolic events. Platelet glycoproteins exposed on the membrane surface mediate platelet aggregation and adhesion to subendothelium, initial events in thrombotic plug formation.^{1 2}

GPIIb-IIIa is the unique platelet receptor for fibrinogen.^{3 4 5 6} Resting platelets bind only small amounts of fibrinogen. When platelets are activated by plasmatic (ADP, thrombin, epinephrine) or subendothelial (collagen) agonists, fibrinogen-binding sites are exposed on GPIIb-IIIa that enable fibrinogen tethering to the platelet surface. This action represents the first, but still reversible, phase of the platelet-aggregation process.^{5 6} The expression of fibrinogen-binding sites on platelets occurs rapidly and results from conversion of the GPIIb-IIIa complex from a low- into a high-affinity state for fibrinogen.^{3 4 5 6} Binding of fibrinogen to GPIIb-IIIa is mediated through amino acid sequences present in regions of the and chains of the fibrinogen macromolecule. Small synthetic peptides, RGD or HHLGGAKQAGDV (H₁₂), that contain these fibrinogen-recognition motifs bind to GPIIb-IIIa and inhibit interaction of fibrinogen with its receptor.^{5 6} Unlike fibrinogen, however, binding of small peptides to GPIIb-IIIa is activation independent.⁷ Peptide binding to GPIIb-IIIa induces exposure of cryptic epitopes on the GPIIb-IIIa complex through conformational changes in the molecule that can be detected by conformation-specific mAbs.⁸

After the initial fibrinogen-dependent step of aggregation, platelets degranulate and release part of their granule contents.^{9 10} Among multiple small molecular compounds, a variety of adhesive glycoproteins that are stored in granules are delivered into the extracellular space and in part bind to the activated platelet surface.^{9 10} Thrombospondin is one of the major glycoproteins of granules and binds to the platelet surface during the degranulation process.^{11 12 13} Thrombospondin has been shown to bind to various sites on the platelet surface and acts as an agglutinin of platelets.^{14 15 16} Thrombospondin specifically interacts with fibrinogen, but the binding sites for thrombospondin appear to be different from the binding sites for GPIIb-IIIa.^{17 18} On the basis of these observations, it has been postulated that the interaction of thrombospondin and fibrinogen on the activated platelet surface may play a role in the aggregation process and promote irreversible stabilization of platelet aggregates.^{13 17 18}

The present work describes the properties of isolated platelet membranes with regard to fibrinogen interaction, thrombospondin-mediated agglutination, and morphological characteristics.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
All reagents, unless specified otherwise, were purchased from Sigma Chemical Co. The peptides GRGDSP, GRGESP, and H₁₂ were obtained from Peninsula Laboratories. Alkaline phosphatase–conjugated anti-mouse IgG was supplied by Bio-Rad. Purified human thrombospondin was purchased from Enzym Research Laboratories.

Antibodies
The mAbs 98DFG and 90BB10 recognize the GPIIb and GPIIIa, respectively, and were kindly provided by Prof Virtanen, University of Helsinki. mAbs anti-LIBS1 and anti-PMI-1 were a generous gift of Dr M. Ginsberg, Scripps Research Institute. Both antibodies are directed against cryptic epitopes on GPIIIa (LIBS1) and GPIIb (PMI-1) that are exposed after conformational changes have occurred in the glycoprotein.⁸ The polyclonal anti-thrombospondin antibodies were obtained from Stago, and Fab fragments were prepared by standard methods. mAbs MA-I and MA-II are specific for human thrombospondin¹⁹ and were kindly provided by Dr J. Lawler (Harvard Medical School, Boston). For flow cytometry, mAbs were conjugated with FITC as previously described.^{20 21} All other mAbs (anti-CD41, anti-CD62P, and anti-CD11b) were purchased as FITC conjugates from Dianova. Anti-CD41 preferentially recognizes the complexed form of GPIIb-IIIa, and anti-CD62P the -granule membrane glycoprotein GMP-140 (PADGEM, P-selectin). Anti-CD11b recognizes the ß₂ integrin MAC1 on leukocytes.

Platelet Membrane Preparation
Platelet membranes were isolated from freshly prepared platelet concentrates according to published methods.²² Immediately after preparation of the platelet concentrate, an antiplatelet cocktail containing PGE1 (200 µg/mL), theophylline (1 mmol/L), indomethacin (1 mmol/L), and adenosine (5 mmol/L)^{20 23} was added to prevent in vitro platelet activation. Platelets were isolated by centrifugation at 700g for 20 minutes and washed twice with Tyrode's buffer (0.1% glucose, 0.1% BSA, 137.5 mmol/L NaCl, 12 mmol/L NaHCO₃, 2.6 mmol/L KCl; pH 7.4), supplemented with antiplatelet cocktail, and resuspended in 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4) supplemented with 0.5 mmol/L PMSF, 10 µg/mL aprotinin, and 0.1 mg/mL leupeptin. The suspension was placed in the pressure chamber of a cell disruption bomb (Parr Instruments Co), and the chamber was filled with nitrogen to a pressure of 10 000 kPa. After 1 hour at room temperature, the suspension was released from the bomb via a needle valve. Thereafter, the disrupted platelets in suspension were centrifuged at 100 000g for 2 hours, the pellet was resuspended in 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), and layered over 27% sucrose in 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4). After centrifugation at 30 000g for 3 hours, the platelet membranes were harvested, resuspended in 150 mmol/L NaCl, 10 mmol/L Tris, homogenized by 10 strokes of a Dounce homogenizer, and passed through a PD 10 column (Pharmacia). The eluate containing the membranes was collected and stored in aliquots at -70°C before use.

Agglutination of Isolated Platelet Membranes
Purified platelet membranes were incubated with small latex beads (10⁵ beads per milliliter; diameter 2.96 µm, Sigma) at a final concentration of 1 mg/mL at room temperature for 2 hours in the presence of 0.5 mmol/L PMSF, 10 µg/mL aprotinin, and 0.1 mg/mL leupeptin. Thereafter, beads were washed once with Tyrode's buffer and incubated with 5% BSA in Tyrode's buffer for a further 1-hour period. The particles were then passed over a PD 10 column (Pharmacia) to separate latex beads from unbound membranes or other small-molecular-weight compounds. The final preparation of membrane-coated particles was resuspended in Tyrode's buffer to obtain a final particle count of 10⁸ per milliliter. In control experiments, latex beads were coated with 5% BSA. Total membrane protein coated on the latex beads was 4 pg of protein per bead, as determined by the protein assay of Bradford (Bio-Rad). Agglutination experiments with membrane-coated beads were performed in a Chronolog aggregometer (Nobis) at a constant stirring rate of 800 rpm at 37°C and in the presence of various compounds as indicated. Agglutination was monitored as an increase in light transmission and recorded for 12 minutes.

Flow Cytometric Analysis (FACS)
FACS analysis of platelet membrane–coated particles was performed as described for intact platelets.^{23 24} In brief, 5-µL aliquots of gel-filtered membrane beads were incubated in a total volume of 50 µL of Tyrode's buffer with saturating concentrations of FITC-labeled mAbs for 15 minutes in the dark at room temperature. Thereafter, 1 mL of 0.2% paraformaldehyde in PBS was added to the suspension and stored at 4°C until FACS analysis. Samples were analyzed on a FACScan cytometer (Becton Dickinson) equipped with an argon laser. Membrane beads were identified in the forward-versus-side scatterplot, and 10 000 particles were analyzed at a rate of less than 100 events per second. Logarithmic amplification was used for the fluorescence and light scatter signals. The mean intensity of immunofluorescence was used as a quantitative measure of antigen exposure. To study the effect of GRGDSP and H₁₂ peptides on LIBS1 or PMI-1 surface expression, membrane beads were incubated with increasing concentrations of peptides.

Scanning Electron Microscopy
For electron-microscopic evaluation of membranes, the coated particles were fixed overnight at 4°C in 2% glutaraldehyde/cacodylate buffer, and electron microscopy was performed as previously described.²⁵

SDS-PAGE and Immunoblotting
For immunoblotting, platelet membranes were solubilized in Tris buffer containing 3% SDS, 0.1 mg/mL leupeptin, and 0.5 mmol/L PMSF. Samples to be run under reducing conditions were treated with 2% mercaptoethanol for 3 minutes at 90°C. Proteins were fractionated by SDS-PAGE on 7.5% resolving slab gels using a 4% stacking gel.²⁶ Each lane was loaded with an equal amount of total protein (50 µg). Proteins were transferred electrophoretically to nitrocellulose membranes at 12 V for 30 minutes (Biometra Fast Blot) in pH-8.3 buffer containing 25 mmol/L Tris and 192 mmol/L glycine. The blots were blocked with 2% Tween 20 in Tris-buffered saline, pH 7.5, then incubated with mAb overnight at 4°C, followed by a horseradish peroxidase–conjugated secondary goat anti-mouse antibody (Bio-Rad).^{26 27} The peroxidase substrate was 4-chloro-1-naphthol (Sigma).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Immunological Characterization of Purified Platelet Membranes
The protein content of the purified membranes was evaluated by SDS-PAGE. Two prominent bands had characteristic electrophoretic mobilities under reducing and nonreducing conditions (Fig 1A). In immunoblot studies, these protein bands reacted with mAbs specific for GPIIb or IIIa, respectively (Fig 1B). mAb PMI-1, which recognizes an epitope present on the NH₂ terminus of GPIIb,⁸ also showed a distinct immunoreactive band in the isolated platelet membrane fraction, indicating that no major degradation of GPIIb had occurred during the isolation procedure (Fig 1B). Weak but significant immunoreactive bands were found when mAbs were used that react with CD62P or TSP, respectively (Fig 1B).

View larger version (43K): [in this window] [in a new window]   Figure 1. SDS-PAGE and immunoblotting of whole platelet homogenates and isolated platelet membranes. Proteins of platelet lysates (Plt, 50 µg) and of purified platelet membranes (M, 50 µg) were separated by slab gel electrophoresis using a 10% acrylamide separating gel and a 4% stacking gel. Samples were run under reducing and nonreducing conditions. After separation, the gel was stained with Coomassie blue (A). For immunoblotting, proteins separated under reducing conditions were transferred to nitrocellulose membranes and stained with (2) anti-GPIIb (98DFG and PMI-1), (1) anti-GPIIIa (90BB10), (4) anti-CD62P (CLB-Thromb/6), and (3) anti-thrombospondin (MA-II) mAbs, respectively (B), followed by peroxidase-conjugated goat anti-mouse second antibody and 4-chloro-1-naphthol. TSP indicates thrombospondin.

To characterize further the glycoprotein content of the isolated platelet membranes, flow cytometry was used to measure binding of mAbs specific for platelet glycoproteins. Small latex particles comparable in size with intact platelets were coated with isolated membranes. Beads coated with platelet membranes showed significant GPIIb and GPIIIa immunofluorescence signals, indicating that substantial amounts of both glycoproteins are present (Fig 2). Moreover, significant binding of complex-specific anti–GPIIb-IIIa mAb to isolated membranes showed that both glycoproteins are structurally intact and complexed in isolated membranes (Fig 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Immunofluorescence histograms of platelet membranes. Small latex beads (0. 29 µm in diameter) were coated with purified platelet membranes, and glycoprotein content was evaluated by flow cytometry. Representative immunofluorescence histograms of mAb (anti-CD41 [GPIIb-IIIa], anti-CD61 [GPIIIa], anti-CD62P [P-selectin], anti-thrombospondin [TSP], and anti-CD11b) binding to BSA-coated beads (dashed line), membrane-coated beads (bold solid line), and intact platelets (solid line) are shown.

To investigate granule-stored glycoproteins that are present only on the activated platelet surface, isolated membranes were probed with antibodies to CD62P and TSP. Representative histograms showed weak immunofluorescence signals, suggesting that only small amounts of both glycoproteins are present in isolated membranes (Fig 2). In control experiments, virtually no binding of the irrelevant leukocyte-specific mAb anti-CD11b was found (Fig 2). Fig 3 shows isolated platelet membranes that are coated homogenously on small latex particles.

View larger version (128K): [in this window] [in a new window]   Figure 3. Scanning electron microscopy of platelet membrane–coated latex particles. Shown are two latex beads coated with purified platelet membranes. Magnification x50 000.

Functional Characterization of Purified Platelet Membranes
To assess whether the glycoprotein complex GPIIb-IIIa is functional, membranes were incubated with increasing concentrations of GRGDSP or H₁₂ peptides. Peptide-induced changes in receptor conformation were assayed by binding of conformation-specific mAbs anti-LIBS1 and anti–PMI-1. Peptide-induced LIBS1 and PMI-1 expression in isolated platelet membranes was dose dependent (Fig 4). In contrast, platelet agonists (eg, ADP, thrombin, PMA, and collagen), which induce conformational change of GPIIb-IIIa on intact platelets, did not effect LIBS1 or PMI-1 expression on the isolated membranes (data not shown). These results suggest that signal-transduction mechanisms required for GPIIb-IIIa activation are absent and that physiological agonists do not regulate GPIIb-IIIa activity in isolated membranes.

View larger version (24K): [in this window] [in a new window]   Figure 4. LIBS1 and PMI-1 expression induced by GRGDSP and H12 in isolated membranes and intact platelets. Platelet membranes coated onto latex beads or intact platelets were incubated with increasing concentrations of GRGDSP or H12 peptides as indicated. Conformational changes in the GPIIb-IIIa complex were assessed by binding of conformation-specific anti-LIBS1 (anti-GPIIIa) or anti-PMI-1 (anti-GPIIb) mAb by flow cytometry. Results are depicted as (F-Fo)/(Fmax-Fo), where F is mean fluorescence intensity, Fo is mean fluorescence intensity in absence of peptide, and Fmax is mean intensity of peptide. Representative results of three independent experiments are shown.

Having established that GPIIb-IIIa preserves its immunological and adhesive characteristics in isolated membranes, we examined whether the soluble form of fibrinogen can interact with membranes. Small latex particles were coated with membranes, and agglutination was monitored by light-transmission aggregometry in the presence of fibrinogen and Ca²⁺. As shown by representative tracings, significant agglutination occurred at a maximum within 10 minutes (Fig 5). Membrane agglutination was fibrinogen dependent, since agglutination was significantly reduced when fibrinogen was omitted (Fig 5). Virtually no agglutination occurred in the presence of 2 mmol/L EGTA, indicating that Ca²⁺ is an absolute requirement for membrane agglutination (Fig 5). Agglutination was also verified by scanning electron microscopy, as shown in Fig 6.

View larger version (23K): [in this window] [in a new window]   Figure 5. Fibrinogen-dependent agglutination of membranes. Agglutination experiments were performed in a platelet aggregometer. Platelet membrane–coated latex particles (see Fig 3) were stirred at 37°C in the presence of fibrinogen (50 µg/mL) and Ca2+ (Fg+Ca2+, 4 mmol/L), Ca2+ (4 mmol/L), or fibrinogen and EGTA (Fg+EGTA, 4 mmol/L) as indicated. Agglutination was monitored by increase of light transmittance. Representative curves are shown (left). The histobars (right) depict the effect of fibrinogen or control peptides GRGDSP (2 mmol/L), H12 (2 mmol/L), GRGESP (2 mmol/L), and Fab fragments of the mAb anti-TSP polyclonal on agglutination.

View larger version (71K): [in this window] [in a new window]   Figure 6. Scanning electron microscopy of agglutination of membrane-coated latex particles. Agglutination was performed under constant stirring at 37°C in the presence of fibrinogen (50 µg/mL) and Ca2+ (4 mmol/L) (A) or fibrinogen (50 µg/mL) and EGTA (4 mmol/L) (B).

Fibrinogen peptides GRGDSP and H₁₂, which antagonize fibrinogen binding to GPIIb-IIIa, significantly inhibited fibrinogen-dependent agglutination of membranes (Fig 5), whereas the biologically inactive GRGESP peptide elicited no significant effect. This implies that agglutination of membranes in the presence of fibrinogen is specific for GPIIb-IIIa.

To study the effect of thrombospondin on membrane agglutination, purified human thrombospondin was added to the membrane suspension instead of fibrinogen. Thrombospondin enhanced membrane agglutination significantly, albeit to a lesser degree than fibrinogen (Fig 7). Again, thrombospondin-induced agglutination was strongly dependent on the presence of Ca²⁺, since EGTA inhibited membrane agglutination (Fig 7). In the presence of polyclonal anti-thrombospondin Fab fragments, thrombospondin-mediated agglutination was reduced significantly (Fig 7). mAb MA-II directed against the heparin-binding domain of thrombospondin also inhibited thrombospondin-mediated membrane agglutination, whereas anti-thrombospondin mAb MA-I did not (Fig 7).

View larger version (19K): [in this window] [in a new window]   Figure 7. Thrombospondin-dependent agglutination of membranes. Agglutination experiments were performed in a platelet aggregometer. Platelet membrane–coated latex particles (see Fig 3) were stirred at 37°C in the presence of thrombospondin (TSP, 50 µg/mL) and Ca2+ (4 mmol/L) as indicated. Agglutination was monitored by increase of light transmittance. Representative curves are shown (left). The histobars depict the effect of polyvalent (anti-thrombospondin Fab, 50 µg/mL) and monoclonal anti-thrombospondin (MA-I and MA-II, 50 µg/mL) antibodies on agglutination in the presence of thrombospondin. In addition, the effect of fibrinogen peptide GRGDSP (2 mmol/L) and EGTA (4 mmol/L) is shown.

Effect of Divalent Cations on Agglutination
Fibrinogen-mediated agglutination of platelet membranes required Ca²⁺ and was maximal when Ca²⁺ was present in concentrations >4 mmol/L (not shown). Similarly, thrombospondin-mediated agglutination was also strongly Ca²⁺ dependent (Fig 7). Mg²⁺ inhibited both fibrinogen- and thrombospondin-mediated membrane agglutination (Fig 8). The inhibitory effect of Mg²⁺ was dose dependent (Fig 8), with half-maximal effects at 3 mmol/L. Agglutination was virtually absent at Mg²⁺ concentrations above 8 mmol/L (Fig 8).

View larger version (17K): [in this window] [in a new window]   Figure 8. Effect of Mg2+ on platelet membrane agglutination. Agglutination experiments were performed as described in "Methods." All experiments were performed in the presence of Ca2+ (4 mmol/L) and Ca2+ and fibrinogen (Fg+Ca2+, 50 µg/mL) or Ca2+ and thrombospondin (TSP+Ca2+, 50 µg/mL), respectively. As indicated, Mg2+ at final concentrations of 1, 2, 4, 8, and 16 mmol/L was added to the suspension before start of agglutination.

The inhibitory effect of Mg²⁺ was found to be dependent on the Ca²⁺ concentration present in the suspension medium. Significantly higher concentrations of Mg²⁺ were required to inhibit membrane agglutination when Ca²⁺ concentrations were elevated (Fig 9).

View larger version (18K): [in this window] [in a new window]   Figure 9. Effect of Ca2+ on Mg2+-dependent inhibition of membrane agglutination. Agglutination experiments were performed in the presence of fibrinogen (50 µg/mL) and varying concentrations of Ca2+ (4, 10, and 20 mmol/L). Before the start of agglutination, Mg2+ was added in the final concentrations as indicated.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of the present study are (1) In isolated platelet membranes, the GPIIb-IIIa preserves its immunological and adhesive characteristics and mediates fibrinogen-dependent agglutination. (2) Thrombospondin enhances the membrane agglutination, which in turn can be inhibited by anti-thrombospondin antibodies. (3) Both fibrinogen- and thrombospondin-mediated membrane agglutination is dependent on Ca²⁺; Mg²⁺ inhibits membrane agglutination in a dose-dependent manner.

Isolated platelet membranes have been a useful tool for providing information on the regulation of activation-dependent processes in platelets, including regulation of the adenylate cyclase,²⁸ phospholipase C,²⁹ and platelet fibrinogen receptor on GPIIb-IIIa.²² Our present data show that purified platelet membranes bear significant amounts of GPIIb-IIIa complexes. The facts that isolated membranes (1) reveal an increased LIBS1 and PMI-1 surface expression in the presence of GRGDSP or H₁₂ peptides and (2) agglutinate in the presence of soluble fibrinogen (in a GRGDSP-dependent manner) show that GPIIb-IIIa is functional in membranes. In our studies, isolated platelet membranes did not respond to agonists, including thrombin, ADP, PMA, and collagen, that activate GPIIb-IIIa in intact platelets.⁹ This indicates that agonist-induced signal transduction mechanisms involved in GPIIb-IIIa activation are absent in membranes, thereby enabling investigation of platelet-platelet interaction independent of cellular activation.

The adhesive glycoprotein thrombospondin is the major -granule glycoprotein released during platelet degranulation.^{10 11 12 13} Thrombospondin has been shown to possess agglutinating activity and plays a role in platelet aggregation by formation of a specific divalent cation–dependent complex with the macromolecule fibrinogen.^{10 11 12 13 14 15 16 17} It has been postulated that the interaction of thrombospondin and fibrinogen on the activated platelet surface may be an important step in determining the size and reversibility of platelet aggregates.^{16 17} In addition, surface-associated thrombospondin may bind to specific receptors on the opposing platelet membrane to support platelet-platelet interaction.^{10 11 12 13} We found that agglutination of purified platelet membranes is supported by addition of thrombospondin and that this agglutination can be blocked by both polyvalent anti-thrombospondin antibodies and MA-II, a mAb directed at the NH₂-terminal heparin-binding domain of thrombospondin.^{16 19} This observation is in support of findings previously reported^{16 18 19} and implies that thrombospondin contributes to the stabilization of platelet aggregation by promoting formation of platelet macroaggregates.¹⁸ Currently, we are not able to provide conclusive data on the counterreceptor present in the platelet membranes involved in thrombospondin-mediated agglutination. Whether other ß₃ integrins such as the vitronectin receptor a_vß₃ or surface-bound fibrinogen act as thrombospondin counterreceptor in membrane agglutination remains to be elucidated.

In agreement with previous studies on intact platelets,^{24 30 31 32 33} we found that high concentrations of Mg²⁺ inhibit platelet membrane agglutination in a dose-dependent manner. Physiological agonists did not activate GPIIb-IIIa receptors, indicating the absence of signal-transduction mechanisms. Accordingly, we conclude that Mg²⁺ directly modulates receptors on the platelet surface. It has been shown that fibrinogen binding to its receptor on GPIIb-IIIa requires the presence of Ca²⁺.^{3 4 5 6 7} Four Ca²⁺-binding sites are present on GPIIb.³⁴ Occupancy of these Ca²⁺-binding sites is required to allow binding of soluble fibrinogen to its receptor, which is mediated by the GRGDSP and -chain H₁₂ recognition sites on GPIIb-IIIa.^{3 4 5 6 7} In addition, Mg²⁺ has been shown to modulate conformation of GPIIb-IIIa.³⁵ Mg²⁺ inhibits the interaction of Ca²⁺ with GPIIb, possibly by competition for the same sites on the GPIIb molecule.³⁴ Thus, we suggest that high concentrations of extracellular Mg²⁺ displace Ca²⁺ from its binding sites on GPIIb, resulting in decreased binding activity of the receptor for soluble fibrinogen.

Similarly, Mg²⁺ could inhibit thrombospondin-mediated agglutination by replacing the Ca²⁺ that is required to stabilize the trimeric conformation of thrombospondin.¹⁹

Understanding the molecular mechanisms that regulate platelet function and development of pharmacological means to inhibit platelet function requires experimental approaches for evaluating function of platelet-membrane glycoproteins. The data presented here show that isolated platelet membranes preserve important aspects of platelet functions that allow platelet-platelet interactions to be studied. Moreover, since platelets are involved in a variety of cellular cross-talk events, the method described here may also be useful in evaluating adhesion mechanisms involved in platelet-leukocyte or platelet-endothelium interactions.

Moreover, the antiplatelet effect of extracellular Mg²⁺ described here extends our knowledge of how platelet function is modulated by Mg²⁺. This could explain in part the beneficial effect of intravenous Mg²⁺ reported in patients with prothrombotic diseases such as acute coronary syndromes³⁶ and preeclampsia.³⁷

	Selected Abbreviations and Acronyms

 

FACS = fluorescence-activated cell sorter GPIIb-IIIa = glycoprotein complex IIb-IIIa mAb(s) = monoclonal antibody(ies) SDS-PAGE = SDS–polyacrylamide gel electrophoresis

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (grant Ga 381/2-1). The authors are indebted to Caroline Bogner and Stefan Mehringer for their excellent technical assistance.

Received October 26, 1995; accepted December 15, 1995.

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