British journal of Haernatology. 1991, 79. 263-270

The murine monoclonal antibody, 14A2 .H1, identifies a novel platelet surface antigen LEONIEK . A S H M A N , GABRIELLAW . AYLETT, PIROOZ A . M E H R A B A N I , * LINDA J. B E N D A L L , * S I L V A N A NIUTTA,ANTONY c. C A M B A R E R I , S T E P H E N R. COLE A N D MICHAELc. BERNDT* Department of Microbiology and Immunology, The University of Adelaide, Adelaide, South Australia, and *Department of Medicine, Westmead Hospital, Westmead, N.S. W., Australia Received 25 March 1 9 9 1 ; accepted for publication 25 June 1 9 9 1

Summary. A murine monoclonal antibody 14A2.H11raised against acute myeloid leukaemia cells, identifies a previously undescribed 2 7 kDa platelet surface glycoprotein which is expressed at low copy number ( 1O’/platelet). MAb 14A2.Hl caused aggregation of platelets which was dependent on FcyKII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell

populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.

Platelet surface membranes contain a complex array of glycoproteins (Clemetson, 1985). Some of these have been shown to be directly involved in platelet function and the regulation of haemostasis as cell adhesion molecules and/or as receptors for agonists or antagonists of platelet activation (e.g. Kunicki. 1989). Murine monoclonal antibodies (MAb)have proved invaluable in characterization, molecular cloning and functional studies of the surface proteins of human cells, including platelets (e.g. Knapp et al. 1989). MAb have been used to determine which platelet surface molecules are involved in aggregation and in adhesion to monocytes and to extracellular matrix components such as collagen, fibronectin. fibrinogen and von Willebrand factor (e.g. Coller et al. 1983a, b, 1989: Silverstein et al, 1989). They have also been used to study protein interactions in the platelet membrane (Berndt et al, 1985: Du et al. 1987). conformational changes of membrane proteins on platelet activation (Shattil et al, 1985: Coller. 1985). and the regulation of platelet surface protein expression (O’Tooleet al. 1989). In addition, MAb to platelet surface antigens have been used in assays for mapping functional domains involved in receptor-ligand binding, e.g. the interaction of von Willebrand factor and gp Ib-IX (Berndt et al. 1988), and for the identification of platelet-reactive antibodies in immune thrombocytopenia (Kiefel et al. 1987).

Some MAb. notably those belonging to the CD41 (anti-gpIIbIIIa). CD36 and CD9 clusters have been reported to cause platelet activation (Powling et al, 1989: Ockenhouse et al, 1989: Worthington et al. 1990) while others, CD62 (antiPADGEM) and CD63 (anti-gp53). detect antigens whose cell surface expression is increased on platelet activation (Modderman, 1989). Most anti-platelet MAbs described so far bind to platelet antigens that are expressed at high copy number. MAb 14A2.Hl. raised against peripheral blood blast cells from a patient with acute myeloblastic leukaemia (AML). detects an antigen which is present at low density on human platelets. The antibody and a rnurine L cell transfectant expressing the corresponding antigen (Cole et al, 1989a) were used in studies in the platelet section of the 4th International Leucocyte Typing Workshop and the antibody was shown to be unique (von dem Borne. 1989: von dem Borne et al, 1989: Cole et al, 1989b). In this study we report details of the preparation and properties of MAb 14A2.Hl and demonstrate that it identifies a novel 2 7 kDa platelet surface antigen. MATERIALS A N D METHODS

Monoclonal antibody production. Hybridomas were prepared by fusing myeloma X63-AG8.653 cells with spleen cells from a BALB/c mouse immunized with peripheral blood mononuclear cells (87% blasts) from a patient with M 1 AML. Mice were immunized at 4-weekly intervals with 1 x lo7cells i.p.

Correspondence: Dr L. K. Ashman. Department of Microbiology and Immunology, The University of Adelaide, GPO Box 498. Adelaide 5 0 0 1 . South Australia.

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(2 x ), then i.v. (1 x ), and spleens taken 4 d later for fusion. Procedures for fusion and cloning were as previously described (Gadd & Ashman. 1985). except that supernatants were screened on the immunizing cells and an autologous Epstein-Barr virus (El3V)-transformed B cell line using the ‘Rose Bengal’ assay (Lyons & Ashman. 1985). Specijcity studies. Screening of the antibody for binding to cells and tissue sections was carried out, as indicated, by indirect immunofluorescence and flow cytometry or immunohistochemistry using the alkaline phosphatase-anti alkaline phosphatase (APAAP)technique as described by Lyons et a1 (1988). To determine whether the antigen identified by MAb 14A2.Hl is expressed on normal haemopoietic progenitor cells (colony-formingcells, culture: CFU-C). bone marrow mononuclear cells were separated into ‘positive’and ‘negative’ fractions by an immune rosetting technique as described elsewhere (Cambareri et al, 1988) prior to plating in the colony assay. Cell lines and leukaeniic specimens. BALM-1, MOLT-4 and Daudi were obtained from Dr H. Zola, Flinders Medical Centre, South Australia. RC-2A. THP-1 and K562 were obtained from Mr G. Pilkington. The Cancer Institute, Melbourne, Australia. HL-60 and HEL were supplied by the American Type Culture Collection, Rockville. Mass. A transfected murine L cell line expressing the 14A2.Hl antigen was prepared in this laboratory (Cole et al, 1989a). Leukaemic specimens were collected and handled as described previously (Ashman et a!, 1987). Antigen characterizationand purification Pronase treatment. RC-2A cells were incubated at 10h/mlin serum-free medium for 1 h at 37°C with pronase (Calbiochem) at a final concentration of 0.5 mg/ml. To terminate the reaction, fetal calf serum was added to 10% and the cells washed twice. An aliquot was removed for assay and the remaining cells were cultured with or without tunicamycin (Boehringer-Mannheim) at 1 pglml. After 24 h culture the cells were harvested and assayed for binding of 14A2.H 1 and control MAbs 5A2.G5 (CD31) and Sal-2 (negative control). Neuraminidase treatment. RC-2A cells and human peripheral blood neutrophils (prepared by density gradient centrifugation by the method of Ferrante & Thong, 1980) were incubated in serum-free medium at 37OC with neuraminidase (Boehringer-Mannheim) at a final concentration of 0.1 mU/ml for 1 h. Fetal calf serum was added to 10% and the cells washed twice prior to assay for antibody binding by indirect immunofluorescence and flow cytometry. Antigen purijcation. Freshly prepared platelet concentrates (10 units) were obtained from the Sydney Blood Bank Regional Centre at Parramatta. Sydney, N.S.W. The platelets were washed and crude platelet membranes prepared as previously described in detail elsewhere (Berndt et al. 1985). Monocyte contamination of the platelet preparation was less than 0.1%. The membranes from 50 units of platelet concentrate were suspended in 150 ml of 0.02 M Tris buffer, 0.15 ~ N a C I . 0 . 0 0 1MCaC12,l%(w/v)TritonX-lOO,pH 7.4. After 30 min at 4°C. the suspension was made 100 pg/ml in leupeptin. 0.2 mM in phenylmethanesulfonyl fluoride. and 1 0 U/ml in aprotinin. and then ultracentrifuged at 100000 g

for 60 min a t 4°C. The supernatant was immediately loaded at 40 ml/h onto a 1.5 x 25 cm column of concanavalin ASepharose 4B equilibrated with 0.02 M Tris buffer, 0.15 M NaCI, 0.001 M CaCh 0.1%(w/v) Triton X-100,0-02%(w/v) NaN3, 0.2 mM phenylmethanesulfonyl fluoride, 10 U/ml aprotinin. pH 7.4 (Buffer A). After thorough washing, the platelet membrane glycoproteins were eluted with 0.2 M methyl-a-D-mannopyranoside in buffer A. After dialysis against buffer A. the platelet glycoprotein fraction was loaded at 20 ml/h onto a 1 x 5 cm column of 14A2-Affigel-10. This column was prepared by coupling 14A2.Hl IgG (purified as previously described (Ruan et al, 1987))to Affigel-10 (Biorad) according to the manufacturer’s instructions. After brief washing with buffer A, the 14A2 antigen was eluted with 0.1 M glycine, 0.1%(w/v) Triton X-100, pH 2.4. The eluted fractions were immediately neutralized by the addition of one-fifth volume of 1 M Tris. pH 8.0, and then dialysed against buffer A. Molecular characterization ofthe purifiedantigen. The material eluted from the immuno-affinity column was subjected to SDS-polyacrylamide gel electrophoresis, and Western blotting as previously described (Ruan at al, 1987). Platelet binding studies. Purified 14A2.Hl antibody was labelled with 1251 using Chloramine T and its binding to purified human platelets measured using previously published methods (Berndt et al. 1985; Du et al, 1987). Briefly. platelets were prepared from venous blood collected into 3.2% sodium citrate as anticoagulant. Increasing amounts of radiolabelled antibody, specific activity 360-450 cpm/ng, were added to the washed platelets at 2 x 108/ml and incubated at 22°C for 60 min to allow equilibration. Platelets were pelleted through a sucrose cushion and the associated radioactivity determined. Non-specific binding was determined by carrying out the experiment in the presence of 100fold excess unlabelled antibody and accounted for 1-274 of the total platelet-associated radioactivity. Platelet activation and aggregation studies. These were carried out as previously described (Ruan et al, 1987). Briefly, platelet aggregation induced by 14A2.Hl (10-50 pg/rnl, final concentration) was followed in citrated platelet-rich plasma (PRP) ( 3 x l o K platelets/ml, final concentration) stirred at 900 rpm at 37OC using a Payton (Scarborough. Ontario) lumiaggregometer (model 1000). In experiments addressing the role of Fc-receptor in 14A2.Hl-induced platelet aggregation, the PRP was incubated with either buffer, WM15 IgG or IV.3 IgG (10pg/ml, final concentration) for 5 min prior to the addition of 14A2.Hl IgG (50 pg/rnl. final concentration). The anti FcyRII MAb IV.3 was supplied by Medarex Inc., West Lebanon, N.H.. and the control IgCl MAb (WM15. CD13) was a gift from Dr K. Bradstock, Westmead Hospital. RESULTS Hybridoma production Hybridoma 14A2 was selected for cloning and further study since it was positive on the immunizing cells, but negative on the autologous B cell line. Cloning was carried out at limiting dilution and. of 24 wells plated at 1 ce11/2 wells, six produced

A N e w Platelet Surface Antigen

NEG

CTRL

265

14A2.H 1

HLA- ABC

I I

1

Fig 1. Binding of MAh 14A2.HI to peripheral hlood leucocytes and the immunizing leukaemic cells. Leucocytes were isolated from heparinkzed peripheral blood by density gradient centrifugation (Ferrante & Thong. 1980).Binding of 14AL.HI and subclass matched control MAbs specific for an irrelevant antigen or HLA-ABC antigens was assessed by indirect immunofluorescence and Bow cytometry using a FACS analyser. Lymphocytes, monocytes and granulocytes were separately gated based on two-dimensional light scatter characteristics.

colonies of which five yielded positive supernatants. Clone H1 was selected for expansion as a good positive and morphologically a single clone. The heavy chain isotype was shown to be IgC 1 by immunodiffusion using subclass-specific antisera (Meloy, Springfield, V a . ) . Sprcificitg stridips

MAb 14A2.HY was screened for binding to normal peripheral blood leucocytes. bone marrow cells and haemopoietic cell lines by indirect immunofluorescence and flow cytometry. As shown in Fig 1, the antibody bound weakly to monocytes but not to other peripheral blood leucocytes. Fig 1 also illustrates the weak to moderate level of binding to the immunizing AML cells. Binding to normal bone marrow leucocytes followed a similar pattern, with detectable binding only to cells in the

monocyte gate. However. immunohistochernical analysis of normal marrow smears using the APAAP technique demonstrated positivity only on platelets (weak) and megakaryocytes (not shown). Binding of the antibody to haemopoietic progenitor cells (colony forming cells-culture: CFU-C) which make up only about 0.1% of bone marrow cells was assessed indirectly by separating bone marrow mononuclear cells into positive and negative fractions using an immune rosetting technique prior to culture with colony-stimulating factors in soft agar. As shown in Table I. CFU-C were found in the 14AZ.Hl -negative fraction. The antibody did not bind to the lymphoblastoid cell lines BALM-I, MOLT4 and Daudi. but bound to cells of several myeloid lines (KC-2A, HL-60. K562. THP-1 and HEL) as assessed by indirect immunofluorescence and flow cyto-

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Leonie K. Ashman et a1

Table 1. The 14A2.HI antigen is not expressed by haemopoietic progenitor cells: Normal human bone marrow mononuclear cells were separated into antibody-bindingand non-binding fractions by immune rosetting and plated in 5-fold replicate in the CFU-C assay with human placental-conditioned medium as the source of colonystimulatory factors as described previously (Cambareriet al. 1988). The number of colonies in the positive and negative fractions combined for each antibody represent the average yield from 5 x 1O4 bone marrow MNC. Control antibodieswere theirrelevant IgCl MAb Sal-2 and the progenitor cell specific IgCl CD34 MAb BI-3C5 (Tindle rt al. 1987).kindly provided by Dr R. Tindle. No. of colonies/plate (average of five replicates)

Colony type:

Neutrophil

Experiment 1 Sal-2 + Sal-2 CD34 + CD34 14A2.HI + 14A2.HI Experiment 2 Sal-2 + Sal-2 CD34 + CD34 14A2.HI + 14A2.HI ~~

Monocyte

1

1

9

30

7

27 5 4 24

1

1

8

Eosinophil

Mixed

0 1

0

5

1

4

0

0

0

0

1

3

0

1

0

1

9

17

1

4

10 0

I6 1

1 0

2 0

1

0

0

12

2

3

1 11

_____~

~______

metry. A limited study of the binding of 14A2.Hl to leukaemic specimens from patients was also carried out. Specimens in which > 20% of cells were positive (compared with IgCl negative control) with a peak shift of > 1 0

channels were considered positive. Using these criteria, 12/ 48 AML specimens were scored positive, compared with 1/7 acute lymphoblastic leukaemia specimens. Both of two chronic granulocytic leukaemia specimens in myeloid blast crisis were positive. Immunohistochemical studies of MAb 14A2.Hl on buffered formal acetone-fixed frozen sections of tonsil by the APAAP technique showed strong binding to epithelial cells in crypts and to microvascular endothelium (Fig 2). The pattern ofendothelial cell staining was similar to that observed with a CD9 MAb 1AA2.H9 (von dem Borne et al. 1989) although weaker, but differed from that obtained with CD31 MAbs which stained larger vessels. Thus, expression of the antigen is not confined to haemopoietic cells.

Antigen characterization and purification Preliminary studies involving treatment of whole KC-2A cells with pronase and neuraminidase indicated that the antigen identified by 14A2.Hl is a glycoprotein. (Results of a representative experiment are shown in Table 11.) Antibody binding was lost on preincubation of the cells with pronase. but enhanced by treatment with neuraminidase. Culture of pronase-treated cells for 2 4 h resulted in re-expression of the 14A2.Hl antigen. In contrast to the binding of the CD31 MAb SA2.GS which appears to identify a carbohydrate epitope, the level of 14A2.H9 binding was not inhibited, but actually enhanced by culture in the presence of tunicamycin which blocks N-linked glycosylation. These results suggest that 14AZ.Hl binds to a peptide epitope which is partially blocked by glycosylation on RC-2A cells. However, neuraminidase treatment of normal peripheral blood neutrophils showed that the lack of binding of 14A2.Hl to neutrophils was not due to masking of the epitope by sialic acid. As a control, the CD15 MAb FMCl0 showed increased binding to neuraminidase-treated neutrophils as expected (Tetteroo K t al, 1984). Further evidence that the 14A2.Hl antigen is

Table 11. Effects of enzyme treatments of the binding of MAb 14A2.HI to RC-2A cells and neutrophils Peak fluorescence intensity (arbitrary units) Antibody:

Treatment

Control (Sal-2)

14A2.HI

0.83

2.80

0.90

1.00 6.89

CD31 (5A2)

CD15 (FMC10)

27.1 1.32

~

RC-2A cells None Pronase Neuraminidase Control, 24 h culture Pronase. 24 h culture Pronase. 24 h culture+ tunicamycin Neutrophils Control Neuraminidase

0.92

3.12 3.12

43.2 41.7

ND ND ND ND ND

0.89

6.65

6.19

ND

2.20

0.77 1.52

ND ND

85.5 226

2.03 1.10

1.58

30.1

RC-2A cells and fresh peripheral blood neutrophils were treated with pronase or neuraminidase as described in the text prior to assay of the binding of 14A2.HI culture supernatant or control MAb culture supernatants by indirect immunofluorescence. Labelled cells were analysed on a Coulter Profile Flow Cytometer.

A New Platelet Surface Antigen

267

Fig 2. Staining of epithelium and endothelium on tonsil sections by MAb 14A2.Hl. Frozen sections (4 prn) of human tonsil were fixed with buffered forrnol-acetone and stained with MAbs. as indicated. by the APAAP technique then lightly counterstained with haematoxylin. ( A ) Epithelial crypt stained with 14A2.H I , (B-1)) Staining ofinterfollicular tissue by 14AZ.Hl (B). CD31 MAb (C)and CD9 MAb (D)showing staining of vascular endotheliurn. All photographs were taken with a 1 0 x objective. No specific staining of follicles was observed except with the CD9 antibody which weakly labelled the B cell region.

glycosylated comes from its isolation in the Con A-binding fraction of platelet lysates (see below). Attempts to determine the molecular weight of the antigen identified by MAb 14A2.H 1 by immunoprecipitation from lysates of surface biotinylated or iodinated RC-2A cells or platelets and gel electrophoresis were unsuccessful. However, the antigen could be purified from the membrane glycoprotein (Con A binding) fraction of platelet lysates by affinity chromatography on 14A2.HI coupled to Affigel-10. The yield was 1 0 0 p g per 5 0 units of platelet concentrate. The eluted material gave a single 2 7 kDa protein band on SDSpolyacrylamide gel electrophoresis (Fig 3A). This material bound MAb 14A2.Hl after Western blotting (Fig 3B). but could not be iodinated by standard procedures, suggesting that it either lacks tyrosine or that tyriosine residues are inaccessible.

-

Binding to transfwtants Although there was a marked similarity in the pattern of binding of 14A2.Hl and CD9 MAbs to peripheral blood and bone marrow cells and tonsil sections, and the molecular weights of the identified antigens are similar Mr ( 2 7 kDa versus 24 kDa), 14A2.HI and CD9 MAbs clearly identify

distinct antigens. Murine L-cell transfectants isolated using 14A2.Hl and CD9 antibodies were assayed for their binding of these two MAbs as well as the negative control IgGl MAb Sal-2. Transfectant T33.8. which was isolated with 14A2.Hl. bound this antibody strongly but failed to bind the CD9 MAb lAA2.Hl (shift in mean fluorescence intensity of 8 5 and - 5 arbitrary units, respectively, relative to the negative control: Fig 4A). Similarly, the CD9 transfectant T8.9 bound the homologous MAb. lAA2.HY. 60-fold more strongly than 14A2.Hl (shifts of mean fluorescence intensity of 740 and 12 arbitrary units respectively: Fig 4B). The results confirm that these two antibodies are directed against different antigens.

Platelet binding studies Equilibrium binding studies were carried out using platelets from five normal donors. In each case saturation was observed at approximately 2 pg/ml of 14A2.Hl. The number of molecules of 14H2.Hl bound/platelet was 1000%300. Platelets from two donors were also examined after activation by the phorbol ester phorbol myristate acetate (PMA). No significant change in antibody binding was observed (900 f200 copies/cell). Platelets from one donor were

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Leonie K. Ashman et a1

g

300

-

Fig 3. SDS-polyacrylamide gel electrophoresis of the affinity-purified 14A2.Hl antigen. (A) Analysis of protein fractions during purification of the 14A2.Hl antigen: lane 1, concanavalin A-purified platelet membrane glycoproteins: lane 2. flow-through of the 14A2.HIAffigel 10 column: lane 3 , purified 14A2.Hl antigen eluted from the 14A2.Hl-AKigel 10 column by 0.1 M glycine buffer, pH 2.4. containing 0.1%(w/v)Triton X-100. Samples were analysed on a 520% exponential SDS-polyacrylamidegel under non-reducing conditions. The positions of markers of known molecular weight (kDa)are indicated by arrows on the left. The markers are, in order of decreasing molecular weight, myosin, /j'-galactosidase. phosphorylase b. bovine serum albumin, ovalbumin. carbonic anhydrase. soybean trypsin inhibitor and lysozyme. (B) Western blot of SDSpolyacrylamide-gel-separated 14A2.Hl antigen probed with WMl5 (lane 1 ) or 14A2.Hl (lane 2). Electrophoresis conditions and molecular weight standards are the same as in (A).

examined before and after thrombin activation and once again no difference was observed (respectively 800 and 700 copies/platelet) (data not shown). Incubation of platelets in platelet-rich plasma with 14A2.Hl in the range 10-50 pg/ml resulted in their aggregation. Pre-incubation of the platelets with a MAb (IV. 3) directed against the major platelet Fc-receptor (FCyRII) but not with an irrelevant IgGl MAb (WM15: CDl3) blocked subsequent platelet aggregation by 14A2.Hl (Fig 5). suggesting that 14A2.Hl induced platelet aggregation was dependent on both binding of 14A2.H9 by the specific combining site as well as the Fc region. DISCUSSION Most of the monoclonal anti-platelet antibodies that have been reported bind to major platelet antigens, i.e. those expressed at high copy number (von dem Borne et al, 1989). In this paper we describe the purification and characterization of a new minor platelet antigen using a unique MAb 14A2.Hl. The antigen is expressed at approximately 1000 copies per platelet and this was not altered by activation with

B

0 100 FL 1

fluorescence intensity Fig4. BindingofMAb 14A2.Hl totransfectantT33.8. but not toCD9 transfectant T8.9. Murine L cell transfectants T33.8 and T8.9 were harvested from confluent cultures using FDTA. Binding of MAbs was assessed by indirect imrnunofluorescence and flow cytometry on a FACS analyser. (A) T33.8. transfectant isolated using MAb 14A2.Hl: (B)T8.9. transfectant isolated using CD9 MAb lAAZ.H9. (-) Sal-2, negative control IgG1. MAb; (----) MAb 14A2.Hl: (. . . .) CD9 IgGl MAb 1AA2.H9.

PMA or thrombin. It is a Con A-binding glycoprotein with a M, of 2 7 kDa and apparently lacks tyrosine residues based on its inability to be iodinated by the chloramine T method. In view of the similarities between the antigens identified by 14A2.Hl and CD9 antibodies in molecular weight and cell distribution it was necessary to exclude the possibility that 14A2.Hl is a weak CD9 antibody. This was done in several ways. Firstly the 14A2.Hl antigen was present in the ConAbinding fraction of platelet lysates whereas the CD9 antigen is not (unpublished observation). Secondly, the 14A2.Hl antigen is present a t much lower copy number o n platelets than the CD9 antigen (1000 compared with 6 5 000; Miller et (11, 1986). Thirdly, 14A2.Hl displayed negligible binding to a murine L cell transfectant expressing the CD9 antigen. and CD9 MAbs failed to bind to a transfected cell line isolated with 14A2.Hl. Finally, 14A2.Hl did not cluster with CD9 antibodies in the Fourth International Workshop (von dem Borne, 1989: von dem Borne et al. 1989). MAb 14A2.Hl was capable of causing platelet aggregation. As was recently reported by Worthington et al(1990).

A N e w Platelet Surface Antigen

H

Time ( m i d Fig 5. Aggregation of platelets by MAb 14A2.H 1. The figure shows theeffect ofbuffer ( A ) . WM 15 (B) and IV.3 (C) on 14A2.HI-induced platelet aggregation. Platelet rich-plasma ( 3 x 1Ox platelets/ml. final concentration) was incubated with buffer control, WM 1 5 or IV.3 ( 1 0 pg/ml. final concentration) for 5 min prior to the addition of 14A2.H 1 ( 5 0 pg/ml) as indicated by the arrows in traces A. Rand C.

for CD9 MAbs of IgGl class, aggregation was inhibited by MAb to the platelet FcyRlI receptor. These workers concluded that the CD9 antigen itself does not necessarily have signal transducing function but that FcyKII immobilization by MAb-mediated crosslinking to the CD9 antigen may lead to activation via that receptor. Although the mode of anchoring of the 14A2.H 1 antigen in the membrane is not known, its low M, suggests that a similar mechanism may apply. It is interesting to note that, despite its very low abundance in the platelet membrane, aggregation induced via the 14A2.H 1 antigen was rapid, and comparable with that induced by MAbs to the high copy number CD9 antigen (Gorman et a / , 1985: Jennings rt d,1990). This suggests a possible physical association between the 14A2.HI antigen and FcyRII in the platelet membrane leading to rapid crosslinking on ligand binding. FcyRII. like the 14A2.Hl antigen, is present at approximately 10’ copies per platelet (Rosenfeld & Anderson, 1989). In contrast to results reported in the 4th International Workshop (von dem Borne et a/. 1989). we found that the level of expression of the antigen was not altered on platelet activation by thrombin or by PMA. Apart from platelets. the only peripheral blood cells to bind 14A2.H 1 were monocytes. This binding was weak and may be attributable to adhering platelets since heparinized blood was used. Similarly, by flow cytometry, monocytic cells in normal bone marrow appeared to bind the antibody weakly, but this was not observed by immunohistochemistry on bone marrow smears which showed specificity for platelets and megakaryocytes. Studies reported in the Fourth Inter-

269

national Leucocyte Typing Workshop (see von dem Borne. 1989)showed similar specificity for platelets and megakaryocytes in normal bone marrow smears. Nevertheless, MAb 14A2.Hl is not completely specific for cells of megakaryocyte/platelet lineage. The antibody bound to cells of several myeloid cell lines which do not show megakaryocytic characteristics (although K562 and HEL cells can be induced to display some megakaryocytic attributes (Hoffmans, 1989)) and to endothelial and epithelial cells in tonsil sections. MAb 14A2.Hl did not bind to haemopoietic progenitor cells which give rise to colonies of monocytes. neutrophils and eosinophils in vitro. but whether it binds to megakaryocytic colony forming cells is not known. Despite its lack of demonstrable binding to haemopoietic progenitor cells, 14A2.Hl was positive on blast cells from 12/48 acute myeloid leukaemia specimens. While the possibility that binding was due to adherent platelets has not been formally excluded in these cases, the results with myeloid leukaemic cell lines support the view that AML cells express the antigen. Thus, as in the case of the CD9 antigen (Ashman et al. 1987) the phenotype of leukaemic cells may differ from that of the corresponding normal progenitors with respect to the expression of this antigen. ACKNOWLEDGMENTS This work was supported by grants from the National Health and Medical Research Council of Australia and The Rotary Peter Nelson Leukaemia Research Fund. REFERENCES Ashman. L.K.. White, D.. Zola, H. & Dart. G.W. (19871 Expression of the non-T ALL.-associated p24 antigen on leukaemic blasts from patients with ANLL. Leukemia Resmrrh. 1 1 . 97-101. Berndt. M.C.. Du. X. & Booth, W.J. (1988) Ristocetin-dependent reconstitution of binding of von Willebrand Factor to purified human platelet membrane glycoprotein Ib-IX complex. Biocheinistry, 27, 633-640. Berndt. M.C.. Gregory, G.. Kabral. A,. Zola, H.. Fournier. D. & Castaldi. P A . ( 1985) Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane. European /ournal of Biochernistrg. 1 5 1 , 637-649. Cdmbareri. A.C.. Ashman. L.K.. Cole. S.R. & Lyons, A.B. (1988) A monoclonal antibody to a human mast cell/myeloid leukaemiaspecific antigen binds to normal haemopoietic progenitor cells and inhibits colony formation iri vitro. Leukemia Reseurch. 12. 929939. Clemetson. K.J. (1985) Glycoproteins of the platelet plasma membrane. Platelet Membrane Glycoproteiris (ed. by J. ri.George. A. T. Nurden and D. R. Phillips). pp. 51-85. Plenum Press, New York. Cole. S.R.. Kriek. G.W.. Hope. R.M. 6; Ashman. L.K. (1989a) Transfection ofgenes for human cell surface antigens identified by monoclonal antibodies. Immunology and Cell Biology. 67, 377384. Cole. S.R.. Kriek. G.W. & Ashman. L.K. (1989b) Binding of platelet panel antibodies to murine L-cell transfectants expressing human leucocyte antigens. kiiroryte Typirig IV. White cdl diflerentiation aritigeiis (ed. by W. Knapp et al), pp. 991-992. Oxford University Press. Coller. B.S. (1985) A new monoclonal antibody reports an activation-dependent change in the conformation and/or micro-

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