Cell, Vol. 59, 305-312, October 20, 1999, Copyright 0 1999 ......Barbara C. Furie: John K. Erban,’ Roberta Bonfanti, Denisa D. Wagner,’ and Bruce Furie’ l Center for Hemostasis

Cell, Vol. 59, 305-312, October 20, 1999, Copyright 0 1999 by Cell Press

PADGEM Protein: A Receptor That Mediates the Interaction of Activated Platelets w ith Neutrophils and Monocytes Eric Larsen:t Alessandro Celi: Gary E. Gilbert: Barbara C. Furie: John K. Erban,’ Roberta Bonfanti, Denisa D. Wagner,’ and Bruce Furie’ l Center for Hemostasis and Thrombosis Research Division of Hematology/Oncology New England Medical Center and Departments of Medicine and Biochemistry Tufts University School of Medicine Boston, Massachusetts 02111 t Division of Hematology-Oncology The Children’s Hospital and Dana-Farber Cancer Institute Department of Pediatrics Harvard Medical School Boston, Massachusetts 02115

Summary

PADGEM (platelet activation dependent granule-external membrane protein) is an integral membrane protein of the alpha granules of platelets and Weibel- Palade bodies of endothelial cells that is expressed on the plasma membrane upon cell activation and granule secretion. Activated platelets, but not resting platelets, bind to neutrophils, monocytes, HL90 cells, and U937 cells. This interaction is inhibited by anti-PAD- GEM antibodies, PADGEM, and EDTA; anti-GPllb-Ma, anti-thrombospondin, anti-GPIV, and thtombospondin produce no effect. Neutrophils and U997 cells, in contrast to Jurkatt cells, contain PADGEM recognition sites, as shown by binding of PADGEM contained in phospholipid vesicles. These results indicate that PAD- GEM mediates adhesion of activated platelets to monocytes and neutrophlls. Therefore, PADGEM shares not only structural but also functional homology with ELAM-1 and MEL-14, members of a new family of vascular cell adhesion molecules.

Introduction

PADGEM is an alpha granule membrane protein expressed on the surface of activated platelets upon platelet stimulation and granule secretion (Hsu-Lin et al., 1984; McEver and Martin, 1984; Stenberg et al., 1985; Berman et al., 1988). This integral membrane protein, also known as GMP-140 (Stenberg et al., 1985) has a molecular weight of 140,000 and is composed of a single polypeptide chain. Its primary structure, recently derived from the nucleotide cDNA sequence (Johnston et al., 1989a), indi- cates a domain structure including a lectin domain, an epidermal growth factor-like domain, consensus repeat units, a transmembrane domain, and a short cytoplasmic domain. PADGEM circulates in the blood as a component of the alpha granule membrane of resting, unstimulated platelets (Berman et al., 1988). Upon stimulation by ago- nists that lead to rapid granule exocytosis, the platelet al-

pha granule membrane fuses with the external plasma membrane, and PADGEM is expressed on the platelet membrane surface. Although originally thought to be platelet-specific, PADGEM has since been found in megakaryocytes (Beckstead et al., 1988) in HEL cells (Yeo et al., 1989; Johnston et al., 1989b), and in endothelial cells (McEver et al., 1987) within the Weibel-Palade bodies (Bonfanti et al., 1989).

Blood platelets circulate in a resting, unstimulated form. Upon stimulation and degranulation, activated platelets are recruited into growing thrombi or cleared rapidly from the blood circulation. These activated platelets can be de- tected by in vivo thrombus imaging with radiolabeled anti- PADGEM antibodies (Palabrica et al., 1989). The function of PADGEM is unknown. However, its expression on the surface of activated platelets has led to consideration of this protein as a receptor important in hemostasis. Based upon the marked structural homology of PADGEM with ELAM-1 and MEL-14 (Johnston et al., 1989a; Bevilacqua et al., 1989; Siegelman et al., 1989; Laskey et al., 1989), members of a new family of adhesion proteins, we have evaluated possible functional homology among these proteins. ELAM-1 and MEL-14 are receptors on the surface of endothelial cells and lymphocytes, respectively. ELAM-1 mediates the interaction of IL-1 or TNF-stimulated endothelial cells with neutrophils (Bevilacqua et al., 1989) while MEL-14 functions as a homing receptor (Siegelman et al., 1989; Laskey et al., 1989). Activated platelets bind to phagocytic white cells, including monocytes and neutrophils (Jungi et al., 1988) and this interaction has been observed with HL80 and U987 cell lines (Jungi et al., 1988; Silverstein et al., 1987), cells frequently used as models of monocytes. In the current work, we have evaluated the role of PADGEM in mediating these interactions. We demonstrate that PADGEM is a receptor protein on the stimulated platelet membrane that mediates the adherence of activated platelets with neutrophils and monocytes.

Results

Effects of Anti-PADGEM Antibodies and Purified PADGEM on the Interaction of Platelets with HL90 Cells Thrombin-activated platelets were incubated with HL80 cells, a human cell line that exhibits monocyte-l ike characteristics. As shown in Figure lA, activated platelets, which present the membrane protein PADGEM on the cell surface, bind to HL80 cells and cause these cells to form rosettes. In contrast, resting unstimulated platelets, which do not express PADGEM on the cell surface, do not bind to HL80 cells or induce rosette formation (Figure 1B). To determine whether or not PADGEM mediates activated platelet-HL80 cell interaction, anti-PADGEM antibodies were evaluated for their ability to block platelet-HL80 cell binding. Monospecific immunoaffinity-purified pOlyClOnal rabbit anti-PADGEM antibodies or rabbit anti-PADGEM antiserum nearly completely inhibited activated platelet-

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Figure 1. Effects of Anti -PADGEM Antibodies and Purified PADGEM on the Interaction of Platelets with HL60 Cells and Neutrophils

(A) Interaction of thrombin-activated platelets (20 nl; 2 x 10s cells/ml) and HL60 cells (20 nl; 2 x lo6 cells/ml). (8) Interaction of unstimulated platelets (20 nl; 2 x 10s cells/ml) and HL60 cells (20 nl; 2 x lo6 cells/ml). (C) Inhibition of adherence of thrombin-activated platelets to HL60 cells with immunoaffinity-purified rabbit ant i-PADGEM antibodies (50 uglml). (D) Inhibition of adherence of thrombin-activated platelets to HL60 cells with purified PADGEM (30 uglml). (E) Interaction of thrombin-activated platelets (20 PI; 2 x 10s cells/ml) and neutrophils (20 nl; 2 x lo6 cells/ml). (F) Inhibition of adherence of thrombin-activated platelets to neutrophils with immunoaffinity-purified rabbit ant i-PADGEM antibodies (50 uglml). Bar = 10 pm.

HL60 cell binding (Figure 1C). However, five monoclonal antibodies directed against PADGEM (KC4, AC1.2, 1-16, 2-15, and 2-17) failed to inhibit the interaction. Polyclonal and monoclonal anti-thrombospondin antibodies, polyclonal anti-GPllb-llla antibodies, polyclonal and monoclonal anti-GPIV antibodies, as well as anti-prothrombin antibodies and preimmune serum, failed to inhibit HL60 cell-activated platelet binding (Figure 2).

If PADGEM is a component of a complex linking activated platelets and HL60 cells, saturation of the PADGEM recognition sites on HL60 cells with soluble PADGEM should inhibit the binding of activated platelets to these cells. As shown in Figure lD, purified PADGEM incubated with HL60 cells prior to the addition of activated platelets inhibited activated platelet-HL60 cell binding by 60%. EDNA also inhibited binding. In contrast, thrombospondin, albumin, mannose bphosphate, and the peptide Arg-Gly- Asp-Ser failed to inhibit activated platelet-HL60 cell binding (Figure 2).

Neutrophils also interacted with activated platelets (Fig- ure 1E) but not with resting platelets. Antibodies to PAD- GEM blocked this interaction (Figure lF), and purified PADGEM inhibited activated platelet binding to neutrophils (data not shown). Equivalent results were obtained with peripheral blood monocytes (data not shown).

Similar results were obtained for U937 cells, another human cell line considered to be a model for monocytes. The inhibition of activated platelet adherence to U937 cells by affinity-purified polyclonal rabbit anti-PADGEM antibodies is shown in Figure 3A. Half-maximal inhibition was observed with about 7 pglml anti-PADGEM antibodies, and complete inhibition was observed with antibody in excess of 20 ug/ml. PADGEM, including preparations of the most highly purified material, also inhibited activated platelet-U937 cell adherence. As shown in Figure 38, half- maximal inhibition of binding was observed at 2 f.tg/ml PADGEM, and maximal inhibition was observed at PAD- GEM concentrations in excess of 30 ug/ml. PADGEM in-

corporated into phospholipid vesicles also inhibited the binding of activated platelets to U937 cells.

The expression of PADGEM on the activated platelet has been shown to be agonist-independent (Hsu-Lin et

+Antiz2~; w

+Anti-PADGEM Serum

0 20 40 60 100

Adherence (%)

Figure 2. Effect of Different Agents of HL60 Cell Adherence to Acti- vated Platelets

The percentage of HL60 cells bound to two or more platelets was determined under phase microscopy. Antibodies include anti-PADGEM antibody (50 pglml; an affinity-purified rabbit polyclonal antibody), anti- PADGEM antiserum (1:lOO dilution), anti-prothrombin antiserum (1:lOO dilution; anti-PT), anti-thrombospondin (1:lOO dilution; anti-TSP). anti- GPllb-llla antibody (33 @ml; anti-GP Ilblllla), and the anti-PADGEM monoclonal antibodies AC1.2 (130 nglml), KC4 (42 &ml), l-16, 2-15, and 2-17 (1:lOO dilution of ascites). Purified PADGEM (30 r(g/ml). thrombospondin (100 &ml; TSP), bovine serum albumin (10 &ml), EDTA (5 mM), Arg-Gly-Asp&r (3 mM; RGDS), and mannose Sphos- phate (10 mfvl; M-6-P) were also tested. Only PADGEM, anti-PADGEM antibodies, and EDTA inhibited activated platelet binding to HL60 cells. Resting platelets, open bars; activated platelets, solid bars.

PADGEM Mediates Adhesion to Monocytes and Neutrophils 307

.; : I 1 [Anti-PADGEM kiboies] (Kg/ml)

:o

Figure 3. Quantitative Inhibition of the Adherence of Activated Plate- lets to U937 Cells by PADGEM and Anti-PADGEM Antibodies

(A)Affinity-purified anti-PADGEM antibodies were incubated for 20 min with thrombin-activated platelets, and then the activated platelets (20 al; 2 x 10s cells/ml) were incubated with U937 cells (20 al; 2 x 10s cells/ml) for 20 min. The interaction of activated platelets with U937 cells was monitored by the formation of rosettes. (9) PADGEM (as indicated) was incubated for 20 min with U937 cells (3 x 10s cells/ml), and then these cells were added to activated platelets (2 x lo8 cells/ml) for 20 min at 22%. The interaction of activated platelets with U937 cells was monitored by the formation of rosettes.

al., 1984). In addition to thrombin-stimulated platelets, ADP-, collagen-, and epinephrine-stimulated platelets bind to HL60 and U937 cells. These interactions are inhibited by PADGEM and anti-PADGEM antibodies.

Distribution of the PADGEM Recognition Site Activated platelets, but not resting platelets, bind to HL60 cells, U937 cells, monocytes, and neutrophils. Other vascular cells were evaluated for their ability to interact specifically with resting and activated platelets (Figure 4). Mono- cytes and neutrophils both bound activated platelets, confirming earlier results (Jungi et al., 1986). This interaction was inhibited by purified PADGEM or by polyclonal anti-PADGEM antibodies. Normal lymphocytes, red blood cells, CEM cells (a human cell line expressing T cell characteristics), Jurkatt cells (a human cell line expressing T cell characteristics), and Daudi cells (a human cell line expressing B cell characteristics) failed to bind to either resting or activated platelets. These results indicate that

0 20 40 60 80 1

% Adherence

0

Figure 4. Interaction of Resting and Activated Platelets with Various Cell Types

The percentage of cells observed to bind two or more platelets was determined under phase microscopy. Cells tested included neutrophils, monocytes, lymphocytes, red cells (RBC), HL90 cells, U937 cells, Jur- katt cells, and Daudi cells at a final concentration of 1 x 10s cells/ml; the platelet concentration was 1.5 x 108 cells/ml. Anti -PADGEM antibodies were added as indicated. Resting platelets, open bars; activated platelets, hatched bars; activated platelets incubated with anti- PADGEM antibodies, solid bars.

phagocytic cells, including monocytes and neutrophils, contain a recognition site on the cell membrane that binds to PADGEM expressed on the surface of activated platelets.

PADGEM-Phospholipid Vesicle Binding to Vascular Cells Since PADGEM is an integral membrane protein (Berman et al., 1986) and appears to contain a transmembrane domain (Johnston et al., 1989a), purified PADGEM was incorporated into phospholipid vesicles composed of phosphatidylcholine and a fluorescent phosphatidylcholine analog, NBD-phosphatidylcholine (2-(6-(N+itrobenzy- 2-oxa-l,3-diazol-4-yl)amino)caproyl-3-palmitoyl-L-a-phosphatidylcholine)). The interaction of these fluorescent vesicles with neutrophils, U937 cells, and Jurkatt cells was studied by f luorescence microscopy and radioimmunoas- say. Figure 5a demonstrates the binding of PADGEM-containing phospholipid vesicles to the surface of U937 cells by f luorescence microscopy. In contrast, PADGEM-containing phospholipid vesicles did not bind to the Jurkatt cells, and phospholipid vesicles lacking PADGEM did not bind to U937 cells (Figure 5a). Phospholipid vesicles containing glycoprotein Ilb-llla did not interact with U937 cells (data not shown).

A similar series of experiments was performed that demonstrated the binding of PADGEM-containing phospholipid vesicles to neutrophils (Figure 5b). Contrary to this, phospholipid vesicles lacking PADGEM or containing glycoprotein Ilb-llla did not interact with neutrophils.

The binding of PADGEM-containing phospholipid vesicles to neutrophils or U937 cells was mediated by PAD-

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a A B C

GEM. Anti-PADGEM antibodies inhibited the interaction of PADGEM-containing phospholipid vesicles with neutrophils and with U937 cells (Figure 5~).

The interaction of PADGEM-containing phospholipid vesicles with U937 cells was quantitated using 1251-labeled AC1.2 antibody. The 1251-labeled AC1.2 antibody, which does not inhibit activated platelet-U937 cell interaction, was preincubated with the PADGEM-containing phospholipid vesicles, and the antibody-PADGEM-vesicle complex was added to cell suspensions and incubated for 30 min at 37%. The unbound vesicles were separated from

Figure 5. Binding of Phospholipid Vesicles Containing PADGEM to Neutrophils and U937 Cells Top, phase-contrast micrographs; Bottom, fluorescence micrographs of the identical fields. Bar = 10 urn. (a) Binding of PADGEM-containing phospholipid vesicles to U937 cells. Fluorescent phospholipid vesicles composed of phosphatidylcholine and NBD-labeled phosphatidylcholine were incubated with U937or Jurkatt cells. Lane A, U937 cells and PADGEM-containing phospholipid vesicles; lane B, Jurkatt cells and PADGEM-containing phospholipid vesicles; lane C, U937 cells and phospholipid vesicles without PADGEM. (b) Binding of PADGEM-containing phospholipid vesicles to neutrophils. Fluorescent phospholipid vesicles, as above, were incubated with neutrophils. Lane A, neutrophils and phospholipid vesicles containing PADGEM; lane B. neutrophils and phospholipid vesicles without PADGEM; lane C, neutrophils and phospholipid vesicles containing GPllb-llla. (c)Inhibition of binding of PADGEMcontaining phospholipid vesicles to U937 cells and neutrophils by anti-PADGEM antibodies. Fluorescent phospholipid vesicles, as above, were incubated with neutrophils or U937 cells. Lane A, U937 cells and PADGEM-containing phospholipid vesicles; lane B, U937 cells, PADGEM- containing phospholipid vesicles and anti- PADGEM antibodies; lane C. neutrophils and PADGEMcontaining phospholipid vesicles; lane D, neutrophils. PADGEM-containing phospholipid vesicles, and anti-PADGEM antibodies.

cell-bound vesicles by centrifugation through oil, and the cell pellet was assayed for 1251. As shown in Figure 6, the interaction of PADGEMcontaining vesicles with U937 cells is specific and saturable; minimal binding of vesicles was noted with Jurkatt cells employed as a control.

Discussion

PADGEM is an integral membrane protein located in the alpha granules of resting platelets and the Weibel-Palade bodies of endothelial cells. PADGEM is translocated to the


[PADGEM-PLV] f~g/ml)

Figure 6. Quantitative Analysis of Binding of PADGEM-Containing Phospholipid Vesicles to U937 Cells

PADGEMcontaining phosphdipid vesicles were incubated with 1251-labeled AC1.2 antibody. The PADGEM-phospholipid vesicle-antibody complex, at indicated PADGEM concentrations, was incubated with U967 cells and Jurkatt cells for 36 min at 67%. Phospholipid vesicles bound to the cell surface were quantitated by assay of 1251 in the pellet after centrifugation of the cell-vesicle incubation mixture. The concentration given on the x-axis [PADGEM-PLV] is the total concentration of PADGEM. 0, U967 cells; 0, Jurkatt cells.

plasma membrane upon platelet degranulation and fusion of the granule membrane with the plasma membrane. The kinetics of this process are rapid, occurring within sec- onds of the initiation of platelet activation. Although platelet activation is temporally associated with the develop- ment of critical biological activities of the membrane surface, the specific function of PADGEM remains to be elucidated. The predicted primary structure of this protein (Johnston et al., 1989a) shows structural homology with ELAM-1 and MEL-14, two proteins involved in vascular cell-cell interaction (Bevilacqua et al., 1989; Seigelman et al., 1989; Laskey et al., 1989). On this basis, we tested the hypothesis that PADGEM is a receptor protein that mediates the binding of activated platelets to neutrophils and monocytes. Our results demonstrate that the adherence of activated platelets to HL60 cells, U987 cells, monocytes, and neutrophils involves PADGEM as a receptor protein on platelets. Anti-PADGEM antibodies inhibit activated platelet-leukocyte interaction by binding to the protein surface that interacts directly with the PADGEM recognition site on leukocytes. The available anti-PADGEM monoclonal antibodies are not directed against determinants in this region. Saturation of the PADGEM recognition site on leukocytes with purified PADGEM or PADGEM contained within phospholipid vesicles inhibits activated platelet- leukocyte interaction. Using phospholipid vesicles containing PADGEM, we directly observe the specific binding of these vesicles to cells that express the PADGEM recognition site. These results indicate that PADGEM can function as a heterotypic intercellular adhesion molecule mediating the binding of activated platelets to certain classes of leukocytes that express a PADGEM recognition site.

In addition to platelet alpha granules, PADGEM is also

a component of Weibel-Palade bodies of endothelial cells (Bonfanti et al., 1989). Stimulation of endothelial cells with the secretagogue thrombin (Levine et al., 1982) results in an increased adhesion of neutrophils to endothelial cells (Zimmerman et al., 1985). We treated human umbilical vein endothelial cells with thrombin and observed a mod- est increase in binding of HL60 cells or neutrophils to the stimulated cells. This increase was inhibited with polyclonal antibodies to PADGEM. Since the numbers of cells bound in our experiments were small, we cannot yet con- clude that PADGEM also mediates binding of phagocytic cells to the injured endothelium. However, such a hypothesis is attractive and under further study.

The chemical nature of the PADGEM recognition site and the specific requirements for the PADGEM-PADGEM recognition site interaction are unknown. The PADGEM recognition site appears to be present only on monocytes, neutrophils, and tumor cell lines with monocyte-l ike properties. One critical unanswered question lies in the rela- tionship of the PADGEM recognition site and the ELAM-1 recognition site on neutrophils and monocytes. These structures may be homologous or even identical.

The interaction of activated platelets, but not resting platelets, with certain classes of leukocytes was previously described (Jungi et al., 1986). Silverstein and Nach- man (1987) have ascribed a role for thrombospondin in this interaction. Thrombospondin is a soluble component of the alpha granule that, following platelet activation and secretion, binds noncovalently to the activated platelet membrane (Lawler, 1986). In the experiments of Silver- stein and Nachman (1987) inhibition of fixed activated platelet interaction with U987 cells or human monocytes was observed with the addition of Fab fragments of polyclonal anti-thrombospondin antibodies, monoclonal anti- thrombospondin antibodies, or thrombospondin. We have not observed an effect of these agents in our system. Sev- eral differences in experimental design, including intact polyclonal anti-thrombospondin antibodies instead of Fab fragments, fresh platelets instead of fixed platelets, and an assay temperature of 25% instead of 4%, may be critical. In our experiments, PADGEM was determined to be free of thrombospondin contamination, and the anti-PADGEM antibodies did not contain anti-thrombospondin. Further study of the role of thrombospondin and glycoprotein IV in platelet-leukocyte interaction will probably clarify these questions. It would appear that, using the current experimental system, the role of thrombospondin in activated platelet-leukocyte interaction is independent of that of PADGEM.

On the basis of its primary structure as derived from its nucleotide cDNA sequence (Johnston et al., 1989a), PADGEM is structurally homologous to a new family of adhesion proteins, including the endothelial leukocyte adhesion molecule (ELAM-1) and the murine lymphocyte homing receptor (MEL-14; Siegelman et al., 1989). This new family of adhesive proteins is characterized by a unique structure consisting of a lectin domain, an EGF- like domain, a series of concensus repeats homologous to complement regulatory proteins, a transmembrane region, and a small cytoplasmic domain (Johnston et al.,

Cell 310

1989a; Bevilacqua et al., 1989; Stoolman, 1989). ELAM-1 functions as an inducible receptor on cytokine-stimulated endothelial cells mediating the adhesion of neutrophils (Bevilacqua et al., 1987). The ELAM-1 recognition site on leukocytes has not been identified. MEL-14 is expressed on the surface of circulating lymphocytes and mediates the binding of these cells to high endothelial venules in or- ganized lymphoid tissue (Siegelman et al., 1989). The MEL-14 recognition site on endothelial cells has not been identified but may involve a saccharide structure (Stool- man et al., 1987; Rosen et al., 1985). Mannose 6-phosphate, fructose l-phosphate, fucoidin, and PPME (poly- phosphomonoester core polysaccharide derived from the yeast Hansenula holstii) inhibit MEL-14-directed attach- ment of lymphocytes to high endothelial venules (Stool- man et al., 1987). As ELAM-1 is critical for endothelial cell-leukocyte binding, and MEL-14 is critical for lymphocyte-endothelial ceil binding, our results demonstrate that PADGEM shares functional similarities with ELAM-1 and MEL-14 in that it mediates the interaction of activated platelets with neutrophils and monocytes.

The physiologic role of PADGEM in activated platelets remains speculative. Activated platelets in the circulation are thrombogenic and must be cleared rapidly as a means of localizing hemostasis to the sites of tissue injury. It is possible that monocytes-macrophages in the reticuloen- dothelial system, expressing a PADGEM recognition site, recognize PADGEM specifically on the activated platelet and remove the platelet from the circulating blood into a sequestered site in tissue. Thus, PADGEM could play a dominant role in the normal process of the removal of activated platelets from blood. Furthermore, microparticles derived from platelets have been found in platelet preparations (Sims et al., 1988). These particles, although of un- certain physiologic significance, are rich in procoagulant activity and contain PADGEM. Clearance of these particles from the circulation may involve a PADGEM-mediated defense mechanism. To initiate the generation of a fibrin clot in the area of vascular injury, PADGEM may play a primary role in the assembly of cellular components necessary for hemostasis. Activated platelets bind to injured endothelial and subendothelial surfaces through well-studied mechanisms involving glycoprotein lb and von Willebrand factor. The expression of platelet PADGEM at these sites may lead to binding of monocytes and neutrophils, cells capable of initiating the tissue factor-mediated extrinsic pathway of blood coagulation (Furie and Furie, 1988). Thus, if leukocytes play a role in the inflam- matory and hemostatic responses associated with vascular injury, PADGEM may be responsible for the localization of certain classes of leukocytes in this region. Further studies are likely to test these hypotheses and to identify the structural relationships of the PADGEM, ELAM-1, and MEL-14 recognition sites on blood cells and cells of the blood vessel wall.

Experimental Procedures

isoietion of Cells Platelets were isolated by gel filtration from fresh anticoagulated blood obtained from normal human donors (Hsu-Lin et al., 1984). Activated

platelets were prepared by incubating ceils without stirring for 20 min at 220 with thrombin at a final concentration of 0.25 U/ml. Fresh piate- lets were used in cell adhesion assays within 30 min of preparation.

Neutrophils were prepared by the method of English and Anderson (1974). The neutrophil preparations were greater than 95% pure by light microscropy. Monocytes were prepared by washing the mononuclear leukocyte fraction with human serum, 5 mM EDTA twice and incubating the cells in RPMI, 10% fetal calf serum in sterile plastic dishes for 2 hr at 3PC. The dishes were washed three times with PBS at 3PC to remove nonadherent ce!ls. PBS at OOC was added, and the ceils were incubated at 4°C for 1 hr. Adherent cells were gently de- tached with a rubber policeman, washed in PBS, and resuspended in RPMI, 1% fetal calf serum. Lymphocytes were obtained by washing the nonadherent cells with PBS and resuspending these cells in RPMI, 1% fetal calf serum. The purity of these preparations was established to be greater than 90% by light microscopy using Wright, specific esterase, and nonspecific esterase stains.

Ceil lines HL60, U937, Jurkatt, CEM. and Daudi were maintained in culture in RPM1 1640 medium supplemented with penicillin G sodium (100 U/ml), streptomycin sulfate (100 pg/mi). HEPES (10 mM), sodium pyruvate (1 mM), L-glutamine (2 mM), Dmercaptoethanol (0.0004%), and 10% fetal calf serum.

Preparation of Monoclonal Antl -PADGEM Antlbody AC1.2 Monocional antibodies directed against PADGEM were prepared using the same strategy originally employed to produce KC4 (Hsu-Lin et al., 1964). BALB/c mice were immunized with thrombin-activated platelets. Splenocytes were fused with NSl ceils using standard methods (Kohler and Miistein, 1975). A hybridoma. designated AC1.2, had characteristics similar to KC4 (Hsu-Lin et al., 1964). This antibody, an IgG,, binds activated platelets but not resting platelets, reacts with purified PADGEM, reacts with a 140,000 molecular weight band after SDS gel electrophoresis and Western blotting of detergent-solubilized platelets, and localizes specifically to the Weibel-Palade bodies of fixed permeabilized unstimulated endotheiial ceils (Bonfanti et al., 1989). AC1.2 was iodinated by the lactoperoxidase-glucose oxidase method (Enzymobead, BioRad), according to the manufacturer’s protocol.

isolation of Antibodies and Proteins PADGEM was purified from platelets by immunoaffinity chromatography as previously described with minor modifications (Hsu-Lin et al., 1984). Briefly, 50 U of frozen platelets was thawed, washed, and sonicated. The platelet lysate was sedimented by centrifugation at 100,000 x g for 30 min at 4%, and then the pellet was sonicated in 1% Lubrol and subjected to centrifugation at 100,000 x g for 30 min at 4OC. The supernatant was applied to an AC1.2-Sepharose column. After exten- sive washing of the column in buffer without detergent, the bound protein was eiuted with diethyiamine, exhaustively dialyzed, concen- trated, and redialyzed against Tris-buffered saline (TBS; pH 7.5). This preparation was applied to a nonimmune IgG-Sepharose column equilibrated with TBS (pH 7.5). In some preparations, the PADGEM was further purified by SDS gel electrophoresis and electroeiution. Polycionai antibodies were raised in rabbits using a standard immuni- zation schedule, and anti-PADGEM antibodies were isolated by affinity chromatography on PADGEM-Sepharose (Berman et al., 1986). These antibodies were previously demonstrated to bind only to activated platelets (Berman et al., 1986; Palabrica et al., 1989) and to interact solely with PADGEM upon Western blotting of detergent-soiubi- iized platelets (Berman et al., 1988).

Dr. Jack Lawler provided thrombospondin and poiycionai rabbit anti- thrombospondin antibodies, Dr. David Phillips provided rabbit anti- GPilb-iiia, Dr. Marie Josephe Rabiet provided GPiib-iiia. Dr. Jan Sixma provided monocional antibodies to PADGEM (1-18, 2-15, 2-17). and Dr. Roy Silverstein provided monoclonai and polyclonai antibodies to GPIV and thrombospondin.

Ceil Adhesion Asseys Twenty microliters of platelet suspension (2 x 10s/ml) was mixed with 20 PI of cell suspension (2 x 106/ml) and incubated for 20 min at 2X in a microfuge tube. An aiiquot of the cell suspension was then placed in a Neubauer chamber and evaluated by light microscropy using an Olympus model BH-2 microscope. Three samples from each assay were evaluated by counting 200 cells and scoring the percentage of


cells with two or more adherent platelets. Antibody inhibition studies were performed by preincubating 20 pl of platelet suspension (2 x 10s/ml) with 20 ul of antibody solution for 20 min at 2X. Cells (20 ul; 3 x lb cells/ml) were added to the incubation mixture for 20 min at 2s. In some experiments, 20 pl of cells (3 x 10s cells/ml) was preincubated with 20 gl of purified PADGEM, thrombospondin, or albumin for 20 min at 2X. Subsequently, 20 ul of platelet suspension was added for 20 min at 2X.

Cell Blndlng of Phosphollpld \Ibslcleo Conteining PADGEM PADGEM was incorporated into phospholipid vesicles using the method of Rivnay and Metgzer (1982). Briefly, 5 mg of phosphatidylcholine (egg yolk; Avanti Polar Lipids) and 0.25 mg of NBD-labeled phosphatidylcholine (Avanti Polar Lipids) in chloroform were mixed, and the chloroform was removed by evaporation at 3X. The dried lipids were resuspended in methylene chloride, and the solvent was removed by evaporation twice. PADGEM (1 ml; 200 ug/ml TBS) or GPllb-llla (480 uglml TBS) was added to the dried phospholipids, CHAPS was added to a final concentration of 10 mM, and the lipids were resuspended. The preparations were dialyzed under argon against TBS, 0.02% NaNs for 2 hr, the sedimented material was resuspended, and dialysis continued for 24 hr. Vesicles were separated from unincorporated protein by gel filtration on a Sepharose 48 column. The incorporation of PADGEM into vesicles was confirmed by a dot blot technique using the antibody AC1.2; the incorporation of GPllb-llla into vesicles was confirmed by the same technique using anti-GPllb-llla. Vesicles were stored at 4% under nitrogen in the dark. The vesicle preparation was diluted 15 with cells (1 x 10s/ml) sus- pended in RPM1 1840 with 1% fetal calf serum and 2% bovine serum albumin. After a 10 min incubation at 23oC the cells were sedimented at 18,,ooO x g for 15 s. Cells were washed once with TBS and resuspended in the same buffer. Observation by fluorescence and phase contrast microscopy was performed using a Zeiss Axioscope microscope.

For quantitative evaluation of vesicle binding to cells, the vesicle preparation was diluted 1:l in RPM1 1840 containing 1% fetal calf serum and 2% bovine serum albumin. After incubation with a fixed amount of lssl-labeled AC1.2 antibody for 45 min at 3X, vesicles were added to the cell suspension at varying concentrations and incubated for 30 min at 3pc. Cells were then sedimented by centrifugation at 10,000 x g for 5 min through a layer of oil (n-butyl phtha- late:Apiezon oil, 93:7 v/v [Aldrich]), and the sediment was assayed for tssl. The concentration of PADGEM exposed on vesicles was esti- mated based upon random orientation of PADGEM on the inner and outer aspect of the phospholipid bilayer.

Acknowledgments

This work was supported by grant HL42443 from the National Insti- tutes of Health. E. L. and J. E. were supported by Institutional National Research Service Awards HLOi’574 and HLO7437 G. G. is the recipient of an Individual NASA HLO7440. D. W. is an Established Investigator of the American Heart Association. We thank Dr. John Lawler for thrombospondin and rabbit anti-thrombospondin antiserum, Dr. David Phillips for rabbit anti-GPllb-llla, Drs. Jan Sixma and Marcel Metzelaar for monoclonal antibodies to PADGEM. Dr. Roy Silverstein for polyclonal and monoclonal anti-GPIV and anti-thrombospondin antibodies, Dr. Marie Josephe Rabiet for GPllb-llla, and Dr. Martha Furie, Dr. San- ford Shattil, and Dr. Antonio Puccetti for helpful discussions.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734.

Received May 30, 1989; revised July 28, 1989.

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Cell, Vol. 59, 305-312, October 20, 1999, Copyright 0 1999 ......Barbara C. Furie: John K. Erban,’ Roberta Bonfanti, Denisa D. Wagner,’ and Bruce Furie’ l Center for Hemostasis

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