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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3154-3158.)
© 1997 American Heart Association, Inc.

Articles

Binding Properties and Inhibition of Platelet Aggregation by a Monoclonal Antibody to CD31 (PECAM-1)

Xiao-Wei Wu; ; Eric C.-Y. Lian

From the Hemophilia and Thrombosis Center/Sylvester Cancer Center, University of Miami and Veterans Affairs Medical Center, Miami, Fla.

Correspondence to Eric C.-Y. Lian, MD, Veterans Affairs Medical Center, 1201 NW 16th St, Miami, FL 33125. E-mail elian{at}mednet.med.miami.edu

	Abstract

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Abstract CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1) is expressed on platelets, endothelial cells, myeloid cells, monocytes, and certain lymphocyte subsets. It has been shown that CD31 is involved in the homophilic and heterophilic cellular adhesion and leukocyte transendothelial migration, but little is known about the role of CD31 in platelet functions. Previously we have shown that monoclonal antibody (MAb) AAP2 produced in our laboratory bound to a 110-kD platelet antigen and gave an enhanced binding to activated platelet membrane. In this study we demonstrated that platelet lysate depleted of the antigen through adsorption by an AAP2-solidified affinity column was bound by MAbs against CD62 and CD42 but not by MAb 5.6E against CD31 or AAP2 on the immunoblot. Rabbit antibodies against CD31 completely inhibited the binding of AAP2 to platelets in the flow cytometry analysis. This indicates that AAP2 is specifically against CD31. ¹²⁵I-labeled AAP2 bound to resting platelets with 5587±1765 sites/platelet and a K_d of 1 nmol/L and to thrombin-activated platelets with 17 625±4865 sites/platelet and a K_d of 0.24 nmol/L. Addition of 10 µg/mL AAP2 inhibited the aggregation induced by 4 µmol/L ADP by 78.6%, 6 µmol/L epinephrine by 79.4%, 1 µg/mL collagen by 78.7%, and 0.25 U/mL thrombin by 29%. The platelet aggregation was completely restored when higher concentration of agonists were used. MAb 5.6E did not have any effect on platelet aggregation. These results suggest that AAP2 binds to a special epitope of CD31 on platelets and that CD31 is involved in platelet aggregation.

Key Words: CD31 (PECAM-1) • platelet aggregation • monoclonal antibody

	Introduction

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The involvement of platelet membrane proteins in platelet physiologic functions has been illustrated by study of the effect of MAbs against these membrane proteins on platelets. It has been shown that the effects of MAbs to CD41/CD61 and CD42 on platelet aggregation were heterogeneous, reflecting the different molecular basis of these MAbs, including their ligand selectivity and mechanism of platelet functions.¹

CD31/ PECAM-1 is a member of the immunoglobulin gene superfamily found on platelets, most leukocytes, and endothelial cells.² Although the precise function of CD31/PECAM-1 is not known in all the cell types that express it, recent investigations have demonstrated its ability to function as a cellular adhesive molecule.^3–6 It has both homophilic and heterophilic adhesive properties that potentially play significant roles in a variety of important processes such as leukocyte recruitment at inflammatory sites,^7,8 regulation of release of bone marrow leukocytes,⁹ and cardiovascular development.¹⁰ Rosenblum et al^11,12 also found that anti-CD31 antibody delays platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles.

To analyze directly the role of CD31 in platelet function, we produced a MAb, AAP2, which was found to recognize CD31 antigen. Using it to study its interaction with platelets, we found that AAP2 had increased binding to activated platelets and inhibited platelet aggregation induced by agonists.

	Materials and Methods

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Materials
ADP, epinephrine, FITC isomer I, 3,3'-diaminobenzidine tetrahydrochloride, and a control polyclonal antibody against human C4 were purchased from Sigma Chemical Co. Collagen was obtained from Chronolog Co. Thrombin (Fibrindex) was from Ortho Diagnostics Inc. GammaBind-Plus column was purchased from Pharmacia BioTech. Megakaryocytic cell line Meg-01 was a gift from Dr H. Saito, Japan. MAb AAP2 was produced from a hybridoma derived from BALB/c mice spleen cells after immunization with thrombin-activated human platelets and fusion with Sp2/0 Ag14 myeloma cells.¹³ MAb 5.6E (against CD31) was obtained from Immunotech. MAb WM-59 (against CD31) was bought from PharMingen. MAb S12 (against CD62p) was obtained from Centocor Inc. MAbs H4A3 and H4B4 (against LAMP1 and LAMP2, respectively) were obtained from American Type Culture Collection. MAb Apt5 (P019) (against glycoprotein Ib) was produced in our laboratory.¹⁴ SEW 16 (polyclonal antibodies against PECAM-1) was kindly provided by Dr Peter J. Newman. Skin endothelial cells were kindly provided by Dr J.P. Cai at the University of Miami.

Platelet Preparation
Normal human blood was collected into a plastic tube containing 3.8% sodium citrate in a 9:1 proportion and platelet rich plasma was obtained after centrifugation at 180g for 10 minutes at 22°C. Platelets were isolated and washed as previously described.¹³

Purification of AAP2 IgG and Preparation of AAP2 F(ab')₂ and FITC-Conjugated AAP2 IgG
AAP2 IgG was purified from ascitic fluid by GammaBind-Plus affinity chromatography according to the manufacturer's instruction. F(ab')₂ was prepared as described previously.¹⁵ AAP2 IgG was coupled with FITC in 0.025 mol/L carbonate buffer, pH 9.2, at 4°C for 16 hours with gentle stirring. The protein solution was then centrifuged at 12 000g for 10 minutes. The supernatant was applied onto a Sephadex G-25 column to separate free FITC from labeled protein.

Indirect Immunofluorescence and Flow Cytometry Studies
The expression of the AAP2 antigen on resting and activated platelets or leukocytes was determined as previously described by Michelson et al.¹⁶ Briefly, 10 µL of platelet-rich plasma or washed platelets (2 to 4x10⁸/mL) was incubated with 100 µL of AAP2 hybridoma supernatant or control IgG at 22°C for 10 minutes followed by stimulation of platelets with 10 µmol/L ADP, 10 µmol/L epinephrine, 2.0 µg/mL collagen, 1.0 mg/mL ristocetin, or 0.2 U/mL thrombin at 22°C for 10 minutes, then fixed with an equal volume of 1% paraformaldehyde in phosphate-buffered saline at 22°C for 10 minutes, and washed with buffer before addition of FITC-conjugated F(ab')₂ of goat anti-mouse Fc. After 20 minutes, the platelets were resuspended in 1 mL of buffer for flow cytometry analysis or resuspended in no-fade mounting medium (90% glycerine and 0.1% p-phenylenediamine in 0.01 mol/L phosphate-buffered saline, pH 8.0) and examined under a fluorescence microscope.

In another study, washed platelets were fixed with a equal volume of 1% paraformaldehyde for 15 minutes at room temperature, and then incubated with 0.1% saponin in buffer for permeabilization for 30 minutes at room temperature. Afterward, platelets were processed for the indirect immunofluorescence study.

Iodination of AAP2
Purified AAP2 IgG was iodinated using the lactoperoxidase technique, according to the procedure recommended by the manufacturer (Bio-Rad Laboratories). Unincorporated isotope was removed by gel filtration through a Sephadex G-25 column. About 90% of the radioactivity precipitated in 10% trichloroacetic acid. Specific activity of the iodinated AAP2 was 120 000 cpm/µg of protein.

Binding Studies With ¹²⁵I-AAP2
Binding of ¹²⁵I-AAP2 to fixed resting platelets or platelets activated with 0.2 U/mL of thrombin was studied by incubating varying amounts of ¹²⁵I-AAP2 with platelet suspension. After incubation for 1 hour at room temperature, platelets and unbound labels were separated by layering 100 µL of platelet suspension aliquot on top of 900 µL of 30% sucrose and centrifuging for 5 minutes at 12 000g at room temperature. Then 500 µL of supernatant was sampled for measuring free ¹²⁵I-MAb AAP2, the tips that contained platelet pellet were cut off, and platelet-bound ¹²⁵I-MAb AAP2 radioactivity was determined in a gamma counter. Nonspecific binding was determined by adding a 40-fold excess of unlabeled MAb to the incubation mixture. Specific binding was calculated by subtracting nonspecific binding from the total binding.

Antibody Cross-Blocking Studies
Washed platelets (10 µL; 2 to 4x10⁸/mL) were preincubated with 30 µL of anti-LAMP1 or anti-LAMP2 MAbs (500 µg/mL), 5 µL of anti-CD31 polyclonal antibodies (12 mg/mL), or appropriate control antibody for 20 minutes at room temperature before addition of 30 µL of AAP2-FITC conjugates (2.8 µg/mL) at room temperature for another 20 minutes. Fluorescence intensity was determined in a Profile II flow cytometer (Coulter Co).

Immunoblot Studies
Whole platelet lysate was used to pass through an MAb AAP2 affinity column three times. The unbound fractions were shown to be devoid of AAP2 antigen. These fractions and whole cell lysates were separated on 6% sodium dodecyl sulfate-polyacrylamide gels followed by transferring onto nitrocellulose membrane. The membrane was blocked with Tris-balanced saline buffer containing 10% calf bovine serum at 4°C for overnight before addition of MAb. It was subsequently incubated with MAb for 2 hours, then incubated with horseradish peroxidase-conjugated anti-mouse IgG for 1 hour and developed using 3,3'-diaminobenzidine tetrahydrochloride substrate.

Platelet Aggregation
Platelet aggregation was measured by using a Chronolog aggregometer. 500 µL of platelet-rich plasma was preincubated with 10 µg/mL of MAb AAP2 IgG [or F(ab')₂], 5.6E, or WM59 for 10 minutes at room temperature and 10 minutes at 37^oC. Different concentrations of ADP, epinephrine, collagen, and thrombin were used. Aggregation was expressed as percent change in optical density. The percentage of aggregation at 6 minutes after addition of ADP, epinephrine, or collagen was used for calculation of inhibition by AAP2 compared with the aggregation by the lowest concentration of agonist in the absence of AAP2.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Distribution of MAb AAP2 Target Antigens in Various Cell Types
Resting platelets incubated with MAb AAP2 showed weak fluorescence, but platelets activated by ADP, epinephrine, collagen. and thrombin showed much stronger intensity of fluorescence. Platelets treated with 0.1% saponin, which allow permeabilization of antibody, showed strong intracellular granular fluorescence staining. The distribution of AAP2 antigen in blood cells was carried out using citrated peripheral blood samples from healthy donors. It was shown that MAb AAP2 reacted with almost all of monocytes (94.1±4.8%), neutrophiles (98.2±2.1%), and 29.1±11.6 of lymphocytes but not red blood cells.

Platelet Binding Sites as Determined by MAb AAP2
Binding studies with ¹²⁵I-AAP2 IgG were performed on freshly fixed resting platelets and fixed thrombin-activated platelets. The binding of MAb AAP2 to resting and thrombin-activated platelets was evaluated using a Scatchard analysis. These results indicate a single class of antibody-binding sites on the platelet surface. The number of binding sites on freshly fixed resting platelets was 5587±1765 with a dissociation constant of 1.0 nmol/L and that on the fixed thrombin-activated platelets was upregulated to 17 625±4865 with a dissociation constant of 0.24 nmol/L.

Competitive Binding Studies of MAb AAP2 With Known MAbs
To differentiate MAb AAP2 from MAbs to lysosomal proteins LAMP1 and LAMP2, which recognize the equivalent molecular weight proteins in the platelet lysate as MAb AAP2 does, MAbs against LAMP1 and LAMP2 were used for competitive binding study by flow cytometer. Neither LAMP1 nor LAMP2 antibodies (500 µg/mL) competitively inhibited AAP2 (2.8 µg/mL) binding to thrombin-activated platelets. In contrast, SEW 16 (a polyclonal antibody against CD31) completely inhibited AAP2 binding (Fig 1). These results suggest that MAb AAP2 recognizes CD31 antigen.

View larger version (22K): [in this window] [in a new window]   Figure 1. Competitive binding of MAb AAP2 and polyclonal antibody SEW 16 against CD31 to platelets. Platelets were preincubated with SEW 16 or isotypic IgG or buffer at room temperature for 15 minutes before addition of FITC-conjugated MAb AAP2.

Immunoblot Studies
Immunoblot studies on lysates of several cell types were carried out with AAP2. Meg-01 megakaryocytic cell line, skin endothelial cells, peripheral blood mononuclear cells and platelets were used. AAP2 bound a molecule with the average apparent molecular mass varying from 110 to 140 kD (Fig 2).

View larger version (64K): [in this window] [in a new window]   Figure 2. Molecular weight of the AAP2 antigen from different cell types. Peripheral blood mononuclear cells, platelets, skin endothelial cells, and Meg-01 cells were lysed with 1% of Triton X-100. Cell lysates were separated on 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with MAb AAP2. Lane S, molecular weight markers; lane 1, platelets; lane 2, Meg-01 cells; lane 3, skin endothelial cells; lane 4, peripheral blood mononuclear cells.

To further prove that MAb AAP2 recognizes CD31, a immunoabsorption by an AAP2-solidified affinity column and an immunoblot study were performed. As shown in Fig 3, both MAbs AAP2 and 5.6E (against CD31) bound to a protein from whole platelet lysate with a molecular mass of approximately 110 kD under nonreducing conditions. However, the unbound fraction from the AAP2-solidified affinity column failed to show the component reactive with MAb 5.6E or AAP2, but it reacted with MAbs S12 (against CD62P) and Apt5 (against CD42b). Thus, these results confirm that the MAb AAP2 target antigen is CD31.

View larger version (48K): [in this window] [in a new window]   Figure 3. Immunoblot studies of platelet lysates and those devoid of AAP2 antigen by passing through AAP2 solidified affinity column using MAbs against different platelet antigens. Lane S, molecular weight standards; lanes 1 to 2, MAb AAP2; lanes 3 to 4, MAb S12 (to CD62P); lanes 5 to 6, MAb 5.6E (to CD31); lanes 7 to 8, MAb Apt5 (to CD42b). Lanes 1, 3, 5, and 7 are platelet lysates devoid of AAP2 antigen; lanes 2,4,6, and 8 are untreated platelet lysates.

Effects of MAb AAP2 on Platelet Aggregation
To evaluate the functional aspects of CD31 in platelets, a platelet aggregation study was performed. The ability of MAb AAP2 to block aggregation was dependent on the concentrations of agonists. One example of 15 platelet aggregation inhibition experiments is shown in Fig 4. Addition of 10 µg/mL AAP2 IgG inhibited the aggregation induced with 4 µmol/L ADP by 78.6%, 6 µmol/L epinephrine by 79.4%, 1 µg/mL collagen by 78.7%, and 0.25 U/mL thrombin by 29%. The inhibitory effect was overcome when higher concentration of agonists (12 µmol/L ADP, 48 µmol/L epinephrine, and 2 µg/mL collagen) were used. MAb AAP2 F(ab')₂ showed the same effects as whole IgG. MAb 5.6E did not have any effect on platelet aggregation. We examined the effect of another MAb, WM59, against CD31 on platelet aggregation and found it had an effect similar to that of AAP2 (Table). These results suggest that AAP2 binds to a special epitope of CD31 on platelets and that CD31 is involved in platelet aggregation.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effects of MAb AAP2 on platelet aggregation. Platelet aggregation was performed in the presence (curves 1, 2, and 3) or absence (curve 4) of 10 µg/mL MAb AAP2. In A1, A2, and A3, 4, 6, and 12 µmol/L, respectively, of ADP were added. In B1, B2, and B3, 6, 24, and 48 µmol/L, respectively, of epinephrine were added. In C1, C2, and C3, 1, 1.5, and 2 µg/mL, respectively, of collagen were added. In D1 and D4, 0.25 U/mL thrombin was added. The changes in optical density were monitored using an aggregometer.

View this table: [in this window] [in a new window]   Table 1. Inhibition of 3 µmol/L Epinephrine-Induced Platelet Aggregation by CD31 Antibodies

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The wide distribution of CD31 on leukocytes, platelets, and endothelial cells suggests that it may play a number of important roles in vascular biology.¹⁷ Previous studies demonstrated that it is involved in homophilic and heterophilic cellular adhesion and leukocyte transendothelial migration. However, the function of CD31 on platelets still remains to be determined. In the present study, we describe the effects of CD31 on platelets aggregation by using MAb AAP2. That MAb AAP2 recognizes CD31 was shown by the following facts: (1) polyclonal antibodies against CD31 completely blocked the binding of AAP2 to platelets; and (2) an immunoblot study using the platelet lysate depleted of AAP2 antigen by passing through AAP2-solidified affinity column and various MAbs against platelet membrane proteins showed that the platelet lysate devoid of AAP2 antigen did not react with MAb 5.6E (which is against CD31), but reacted with MAbs to CD62P or CD42b.

Flow cytometry studies demonstrated that AAP2 recognized an antigen that is enhanced on activation of platelets. The increase in AAP2 binding may be explained by the presence of additional binding sites translocated from the -granule membrane to the platelet plasma membrane after thrombin stimulation as previously described.^13,18

Immunoblot analysis of AAP2 antigen in several cell types revealed a remarkable heterogeneity. The average apparent molecular mass varied from 110 kD in platelet lysate to 140 kD in endothelial cells and megakaryocytic cell line Meg-01, which may reflect in the difference of glycosylation or the effect of proteolysis on CD31 antigen.

Platelet activations by ADP, epinephrine, collagen, and thrombin have common pathways that produce shape change, the redistribution of cytoskeletal proteins, the formation of the fibrinogen receptor and binding of fibrinogen, primary reversible aggregation, and the release of the contents of dense and -granules, which were associated with secondary irreversible aggregation.^19–22 AAP2 specifically inhibited secondary aggregation of stirred platelet-rich plasma after activation by ADP, epinephrine, and collagen. It did not interfere with the ADP-, epinephrine-, or collagen-induced initial wave of platelet aggregation. This phenomenon may be interpreted by one of the following mechanisms: (1) the binding of AAP2 to CD31 on the platelet surface blocks the activated or released CD31 molecules on the platelet membrane, which have different steric epitope to exert homophilic or heterophilic adhesion of CD31 molecules among platelets; and (2) the binding of AAP2 to CD31 on the platelet surface blocks the release reaction of platelets after first-wave aggregation or modulates the conformational change of glycoprotein IIb/IIIa receptors through interference of outside-in or inside-out signal transduction of CD31.

The inhibition of AAP2 in platelet aggregation was dependent on the concentrations of agonists. When higher concentrations of agonists were used, the inhibition of AAP2 in platelet aggregation was surmounted. It is possible that AAP2 interferes with initial aggregation-induced protein phosphorylation reactions, which are part of a cascade of events that control platelet reactivity.²³ CD31 becomes phosphorylated on serine and tyrosine residues on cellular activation.²² Protein tyrosine phosphatase, SHP-2, binds phosphorylated CD31 and forms a complex during platelet aggregation, suggesting a link between CD31 and integrin-mediated signaling pathways.²⁴ A constructed integrin chimeric protein containing the cytoplasmic domain of PECAM-1 is able to cause phosphorylation of focal adhesion kinase, suggesting that the cytoplasmic domain of CD31 can substitute for the ß₂ cytoplasmic domain in relaying signals to focal adhesion kinase.²⁵ The protein serine/threonine phosphatase inhibitor calyculin A inhibits Ca²⁺ fluxes, phosphatidylinositol metabolism, dense granule secretion, cytoskeletal rearrangement, and aggregation by 60% to 80% induced by low concentrations of thrombin.²⁶ Mazurov et al²⁷ has demonstrated the colocalization of CD31 with F-actin in human aortic endothelial cells. Binding of CD31 to the platelet surface inhibits the second-wave aggregation induced by agonists that may be achieved through blocking the cytoskeletal rearrangement.

Studies of the effect of monoclonal antibodies against CD31 on platelet aggregation have been reported. It has been reported that MAb VM 64²⁷ and MAb RUU-PL 7E8²⁸ against CD31 did not inhibit the platelet aggregation induced by various agonists. We observed that 5.6E, a MAb to CD31, did not have any effect. This is different from AAP2 and WM59 (against CD31), which inhibit platelet aggregation. The different effect of MAbs against CD31 is probably due to the different bioactive epitopes within the CD31 molecule involved in platelet aggregation and/or signal transduction. Consequently, CD31 on platelets is involved not only in platelet adhesion to injured endothelium^11,12 but also in platelet aggregation.

	Selected Abbreviations and Acronyms

 

FITC = fluorescene isothiocyanate MAb = monoclonal antibody PECAM-1 = platelet endothelial cell adhesion molecule 1

	Acknowledgments

 
The work was supported in part by the Department of Veterans Affairs.

Received March 12, 1997; accepted August 18, 1997.

	References

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