British loumal of Hu~mntologg. 1993, 85. 341-347

Anti-fibrinogen antibody mediates fibrinogen binding to platelet membrane glycoprotein IIb-IIIa

HIROSHI MOHRI, JUICHI TANABE, HIROYUKI FUJITA, HEIWA KANAMORI A N D TAKAO OHKUBO The First Department of Internal Medicine, Yokohama City University School of Medicine

Received 3 1 March 1993; accepted for publication 17 May 1993

Summary. The binding of fibrinogen to platelets requires the agonist activation of platelet membrane glycoprotein IIb/IIIa. We have now found an anti-fibrinogen polyclonal antibody (YCU-R3) that increases the fibrinogen affinity of GPIIb/IIIa- binding function (activation) and subsequent platelet aggre- gation. The addition of intact IgG, F(ab)2 fragments or Fab fragments induced platelet aggregation. The antibody- mediated fibrinogen binding was specific and saturable. This

binding was inhibited by native fibrinogen, the RGDS peptide, the peptide of the C-terminus y chain of fibrinogen (1,397- 4 11 ), and the anti-GPIIb/IIIa monoclonal antibody (LJ-CP8). The antibody-dependent fibrinogen binding was similar to that induced by ADP. Moreover, after pretreatment with the anti-fibrinogen antibody and fibrinogen, formalin-fixed plate- lets bound to fibrinogen saturably. These results suggest that this anti-fibrinogen antibody may function as partial agonist.

Integrins are a widely distributed supergene family of heterodimeric membrane proteins that play roles in cell adhesive events (Hynes, 1987). The important function of all integrins is their capacity to recognize macromolecular ligands. Glycoprotein IIb/IIIa (GPIIb/IIIa) was one of the integrins to be biochemically purified (Jennings & Phillips, 1982), to be associated with human disease (Nurden & Caen. 19 74; Phillips & Agin, 1977), and the first to be expressed in completely recombinant form (Bodary e t al, 1989; O’Toole et ul, 1989). Agonists such as thrombin and ADP are required to elicit high affinity binding of macromolecular ligands such as fibrinogen to GPIIb/IIIa (Bennett & Vilaire, 1979: Marguerie et al, 1979). In contrast, small synthetic ligands are capable of binding in an agonist-independent manner (Lam et d. 1987; Pytela et al, 1986). These results lead to the concept that in macromolecular ligands it is possible to bind to the receptors without agonists when the optimal structure of the ligand is obtained.

The specific portion of the fibrinogen molecule known to be involved in the interaction with GPIIb/IIIa corresponds to residues 400-411 of the y chains (Kloczewiak et al, 1982, 1984; Andrieux et aI, 1989), an RGDS sequence (residues 572-575) (Bennett et al, 1988; Plow et al, 1985, 1987; Gartner & Bennett, 1985) and an RGDF sequence (residues 95-98) of the chain (Kloczewiak et al, 1982; Gartner &

Correspondence: Dr Hiroshi Mohri. The First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura. Kanazawa-ku, Yokohama 2 36. Japan.

Bennett, 1985). The binding of KCD peptides to GPIIb/IIIa alters the integrin’s conformation in detergent solution (Parise et d. 1987) and in the platelet membrane (Frelinger rt al. 19 88). Moreover, the binding of macromolecular ligands such as fibrinogen to GPIIb/IIIa needs similar conformational change (Frelinger et a1 1988, 1990). Recently, the agonist- independent binding of RGD sequence to GPIIb/IIIa was demonstrated to lead to changes in GPIIb/IIIa that were associated with acquisition of high affinity fibrinogen-bind- ing function and platelet aggregation (Du et al, 1991).

In the present study, we have developed a new anti- fibrinogen polyclonal antibody, examined the effect of the antibody on the binding of fibrinogen to GPIIb/IIIa and found that the antibody has the ability to induce platelet aggre- gation without addition of a cellular agonist. Thus, this anti- fibrinogen antibody may serve as a trigger that induces a high affinity binding state in ligand and integrins.

MATERIALS AN-D METHODS

Antibodies. Polyclonal antibodies to fibrinogen were pro- duced by immunizing rabbits. Two antibodies, YCU-R1 and YCU-R3, were derived, YCU-R1 was used as an irrelevant antibody. IgGs were purified by passing them through a column of fibrinogen linked to cyanogen bromide-activated sepharose 4B (Sigma, St Louis, Mo.). The IgG molecules bound to fibrinogen were eluted with 0.1 M glycine at pH 2.4 and dialysed against a Tris buffer composed of 0.02 M Tris- HCI and 0.1 5 M NaCl adjusted to pH 7.3 5 .

341

342 Hiroshi Mohri et a1 Anti-GPIIb/IIIa monoclonal antibody (LJ-CP8) and anti-

GPlb monoclonal antibody (LJ-Ibl ) were prepared and characterized as described (Niiya et al, 1987; Handa et al, 1986). LJ-CP8 inhibits the binding of fibrinogen, von Wille- brand factor (vWF) and fibronectin to platelets. LJ-IB1 inhibits ristocetin-dependent vWF binding to platelets and asialo-vWF binding. These antibodies were generous gifts of Dr Zaverio M. Ruggeri (Scripps Clinic, La Jolla, Calif.). IgG was purified by affinity chromatography on Protein A-Sepharose CL-4B (Ey et al, 1978). Anti-fibrinogen monoclonal antibody (MAB-12 1) was purchased from Chemico International Inc.

F(ab’)? fragments were prepared by the digestion of IgGs with 10% (wt/wt) pepsin (Sigma Chemical Co.. St Louis, Mo.) in a 0.1 16 M acetate buffer, pH 3.8, containing 0.05 M NaCI, for 5 h a t 37°C (Nisonoff & Dixon, 1964; Parham, 1983). The optimum conditions for digestion (pH, time for incubation, pepsin concentration) were determined experimentally in each case. For preparation of Fab fragments, purified IgG was digested with mercuripapain (Sigma Chemical Co., St Louis, Mo.) at 1 : 99 ratio of papain to protein for 4 h at 3 7°C in a 0.01 M phosphate-buffered saline (PBS) (pH 7.4). The reaction was terminated by adding iodoacetamide to a final concentration of 10 mg/ml (Tomiyama et aI, 1992). Both F(ab’)2 and Fab fragments were separated from Fc fragments and undigested IgG by chromatography on Protein A- Sepharose CL-4B. The IgG concentrations was calculated from the optical density of the purified solutions at 280 nm using an extinction coefficient of 1.4.

Preparation of platelets. Human platelets were obtained from fresh ACD-anticoagulated blood as described elsewhere (Walsh et al, 1977). Washed platelets were resuspended in modified Tyrode’s buffer (5 mM HEPES, 0.1 5 M NaCI, 2.5 mM KCI. 1 2 mM NaHCO>, 5.5 mM glucose, 0.2% [v/v] bovine serum albumin [BSA]).

Adhesion proteins. Human fibrinogen was purified from plasma by the method of glycine precipitation (Kazal et al, 1963). Fibronectin was isolated from plasma by affinity chromatography on gelatin-Sepharose (Pharmacia Fine Chem, Piscataway, N.J.) (Engvall & Ruoslahti, 1977). vWF was a generous gift of Dr Zaverio M. Kuggeri (Scripps Clinic, La Jolla, Calif.). Protein concentration was measured by the method of Bradford (1976) using purified BSA (Sigma, St Louis, Mo.) as a reference standard.

Synthetic peptides. Synthetic peptides containing the tetra- peptide (RGDS), RGES peptide and the carboxy-terminal 1 5 residues of the y chain (GQQHHLGGAKQAGDV: G15) were purchased from Bachem Fine Chemicals Inc. (Torrence, Calif.). All peptides were analysed and stored as previously described (Mohri et al, 1991).

Western blot analysis. Adhesion proteins were analysed on a 4-20% gradient SDS-polyacrylamide gel. After transfer to an immobilon membrane (Millipore, Bedford, Mass.), nonspecific binding was blocked in PBS/5% non-fat dry milk (BLOTTO). The sheets were incubated with each antibody at 300 pg/ml for 1 h at 22°C. After washing three times with PBS/O.O 5% Tween-20, the membranes were then incubated with biotinylated anti-rabbit IgG (Tago Inc.) and strepto- avidin alkaline phosphatase (BRL) and stained by the technique as described elsewhere (Beardley et al, 1984).

Protein radioiodination. Radioiodination was performed with carrier-free NaLZ5I (Radiochemical Centre, Amersham, U.K.) using iodogen (Pierce Chemical Co.. Rockford, Ill.) according to the method of Fracker & Speck (1978). The specific activity of the radiolabelled molecules ranged from 0.5 to 2.0 mCi/mg.

Binding assay. The binding of ‘”I-fibrinogen to platelets was measured according to the method as described (Niiya et al. 198 7). Briefly, lZ51-fibrinogen was incubated with platelets at a final concentration of 1 x 108/ml, the antibody, and 1 .0 mM CaClz for 30 min at 22°C. Bound fibrinogens were separated from free fibrinogens by centrifuging through a 20% sucrose layer. Nonspecific binding was estimated as fibrinogen binding in the presence of 0.5 mM RGDS peptide.

Platelet aggregation. Blood was collected in 3.8% sodium citrate (9: 1, vol/vol). PRP was obtained after centrifugation at 200 g for 10 min and the platelet count was adjusted to a final concentration of 3 x 10x/ml. Aggregation was studied with a dual channel aggregometer (Nikko Bioscience, Tokyo, Japan) with PRP or FFP added to siliconized cuvettes and stirred at 1000 rpm at 37°C. The instrument was calibrated to 100% light transmission with platelet-poor plasma or PBS.

RESULTS

Characterization of anti-fibrinogen antibodies The new anti-fibrinogen polyclonal antibody used in this study was designated YCU-R3. YCU-R1 was used as a control antibody. As shown in Fig 1, YCU-R3 bound only fibrinogen and did not recognize any other adhesion protein including fibronectin, vWF or vitronectin under reducing conditions. YCU-R1 recognized all three polypeptide chains of fibrinogen. Although YCU-R3 is a polyclonal antibody, it reacted with only the CI chain of fibrinogen.

Platelet aggregation studies The addition of polyclonal antibody, YCU-R3, to fibrinogen induced platelet aggregation (Fig 2A). Increasing amounts of the antibody caused increasing aggregation and decreasing lag time to aggregation. The addition of F(ab’)? or Fab fragments of this antibody also induced aggregation of PRP similar to intact IgG, indicating that Fc receptor was not required for aggegation. The control polyclonal antibody, YCU-R1, did not induce any platelet aggregation (Fig 2A).

Spec$city offibrinogen binding to platelets The specificity of the effect of the antibody (YCU-R3) on fibrinogen binding to platelets was examined. As shown in Fig 3, when fibrinogen was pretreated with increasing concentrations of YCU-R3, the binding of fibrinogen to platelets increased, approaching a maximum at a concen- tration of 150 pg/ml. Similar results were obtained when F(ab’)2 or Fab fragments of YCU-R3 were used instead of native YCU-R3 (data not shown). In contrast, after treatment with increasing concentrations of YCU-R1, no increase in fibrinogen binding was observed.

We also examined the effects of inhibitors of fibrinogen binding to GPIIb/IIIa on fibrinogen binding to platelets mediated by YCU-R3. The binding of fibrinogen was inhibited

Antibody Mediates Fibrinogen Binding to GPIlbllIZa 343

Fig 1 . Western blot analysis of the epitope recognized by antifibrinogen antibodies. Adhesion proteins were run on a 4-20% gradient SDS- polyacrylamide gel under reducing conditions and blotted onto an immobilon membrane. Lane 1 : Amido Black staining of the reduced fibrinogen. Lane 2: immunoblot of reduced fibrinogen probed with YCU-K3. Lane 3: immunoblot of reduced fibrinogen probed with YCU-RI. Lane 4: immunoblot of reduced fibronectin probed with YCU-R3. Lane 5: immunoblot of reduced vWF probed with YCU-R3. Lane 6: immunoblot of reduced vitronectin probed with YCU-R3. The positions of each of the three chains of fibrinogen (Aa, Bfi and 11) are indicated.

Fab ( 15O~xg/m I 1

Ab .1

A

J

Time

Fibrinogen B ' 4

( 1 ) )

> Time

Fig 2. Aggregation induced by anti-fibrinogen antibodies. (A) PRP was incubated in a cuvette at a concentration of 3 x 10'/ml. Antibodies (intact YCU-R3, F (ab')2 fragments or Fab fragments of YCU-R3. native YCII-Rl) were the added at the times indicated. The instrument was calibrated to 0% and 100% light transmission (100% aggregation) with PRP and PPP, respectively. (B) Fixed platelets pretreated with (2, 3 and 4) or without (1 ) 1 50 pg/ml YCU-R3 and 50 nM fibrinogen were resuspended in the modified Tyrode's buffer at a final concentration of 3 x 10'/ml. Fibrinogen (400 nM) was added and aggregation monitored. (1) and (4), no inhibitors were added: (2 ) , 200 p~ RGDS: ( 3 ) . 200 pg/ml LJ-CP8.

344 Hiroshi Mohri et a1

r uYCU-R,

Antibody Concentration(pg/ml)

Fig 3. Dose effect of the binding of fibrinogen to platelets by YCU-R3. Platelets (1 x 1O8/ml). 0 .5 ~ L M 'L51-fibrinogen and 1 mM calcium were incubated with YCU-R3 (closed circles) or YCU-Rl (open circles) at concentrations from 1.0 to 1000 pg/ml at 22OC for 30 min. and bound radioactivity was estimated. The results were expressed as the percentage of the maximal fibrinogen binding and are the average of triplicates.

by RGDS peptide, a peptide derived from the C-terminus of fibrinogen y chain (y39 7-41 1). a anti-GPIIb/IIIa monoclonal antibody, LJ-CP8, known to inhibit fibrinogen binding to platelets (Fig 4). Thus, the antibody-mediated fibrinogen binding was similar to fibrinogen binding to GPIIb/IIIa induced by agonists such as ADP.

We considered the possibility that the enhanced affinity of

+ Y 397-41 1

~

1 10 100 1000 Peptide Concentration( pM)

fibrinogen to receptor might occur after the antibody- dependent binding. To test this, receptor-bound fibrinogen was assessed by a monoclonal antibody, MAB-121 (Chemi- con International Inc.), directed against fibrinogen. Labelled MAB-121 failed to bind when SO nM fibrinogen was incubated with washed platelets in the absence of YCU-R3. In contrast, when SO mM fibrinogen was incubated with washed platelets in the presence of YCU-R3, specific binding of labelled monoclonal antibody occurred (Fig 5).

Activation of GPIlbIlZla by anti-jbrinogen antibody in intact platelets We exploited the observation that platelets may be fixed with formaldehyde, resulting in GPIIb/IIIa that is in either an active or inactive state depending on events prior to fixation (Peerschke & Zucker, 198 1; Plow & Marguerie, 1982). When intact resting platelets were incubated with 50 nM fibrinogen and 150 pg/ml YCU-R3 in the presence of inhibitors of platelet activation (PGE' and apyrase) prior to fixation and washout of fibrinogen and antibody, these fixed platelets bound fibrinogen saturably. On the other hand, the fixed platelets did not bind fibrinogen when YCU-R1 was used instead of YCU-R3 (Fig 6). In contrast, when intact resting platelets were incubated only with fibrinogen and then fixed with formaldehyde, the fixed platelets did not bind fibrinogen (data not shown). This binding was specific with respect to preincubated antibody in that YCU-R3 but not YCU-Rl had this property. Also, the binding of fibrinogen to these fixed platelets was inhibited by RGDS peptide, cold fibrinogen and the monoclonal antibody, LJ-CP8 (data not shown). More- over, the active receptor in paraformaldehyde-fixed cells was also capable of supporting platelet aggregation as shown in Fig 2B. This aggregation also inhibited by inhibitors of the

100

50

c

t LJ-CP8 -G- LJ-lbl

1 10 100 1000 Antibody Concentration( pg/rnl)

Fig 4. Dose-dependent inhibition by the synthetic peptides and the monoclonal antibodies of 1251-fibrinogen binding to platelets mediated by YCU- R3. Washed platelets (1 x 108/ml) were mixed with Tris buffer (control mixture), synthetic peptides or monoclonal antibodies at the concentrations indicated on the abscissa. '"I-fibrinogen (0.5 ~ L M ) and 1 m M CaClz were then added, followed by the antibody (1 50 btg/ml), and the mixture was incubated for 30 min at 22OC. The results were expressed as the percentage of maximal fibrinogen binding in the absence of inhibitors.

Antibody Mediates Fibrinogen Binding to GPlIb/lIla 345

None

0 Fibrinogen

Fibrinogen +cold MAE-1 21

h

N rn a 5 -

0 0 0 % ‘ X 4 ,

v h k 2 U

- + -

n ” Control YCU-R,-treated

Fig 5. Antibody-mediated fibrinogen binding to platelets assayed with monoclonal anti-fibrinogen antibody (MAB-121). Washed platelets (1 x 1 O8/m1) were preincubated with 150 &ml YCU-R3 or buffer (control). Then the mixture was incubated with 30 pg/ml IriI- labelled MAB-121 only or with labelled antibody with addition of 100 IIM fibrinogen of fibrinogen+ 1 500 pg/ml cold MAE-121. After the incubation for 30 min at 2 2 T , bound 1’51-labelled anti- fibrinogen antibody was measured.

h 3.0 c a, 0 m 0.

- c - m 0 2 2.0

E.

m : 1.0

v U c 0

M

C ._ L

0 k jY 2

0

I -C- YCU-R, -pretreated

too 200 300 400 Fibrinogen added(nM)

Fig 6. GPIIb/IIIa activation by pretreatment of fibrinogen and YCI R3. Washed platelets (in modified Tyrode’s buffer with 300 ng/ml PGEl and 1 50 pg/ml apyrase) were incubated for 10 min with 50 nM fibrinogen and 150 pg/ml YCII-R3 (closed circles) or with 50 nM fibrinogen and 1 50 pg/ml YCU-RI (open circles), and then fixed with 0.50/, paraformaldehyde at 22*C for 1 h. After being washed with and resuspended in the modified Tyrode’s buffer, the platelets at a concentration of 1 x 10X/ml were incubated with various concentra- tions of labelled fibrinogen. and the binding was determined.

binding of fibrinogen to GPII/IIIa such as RGDS and LJ-CP8 (data not shown).

DISCUSSION The present study of polyclonal antibody against fibrinogen indicates that (1) the exposure of GPIIb/IIIa to fibrinogen with polyclonal antibody (YCU-R3) results in the acquisition of high affinity of fibrinogen binding; (2) the binding of fibrinogen to GPIIb/IIIa mediated by the antibody is specific in that it is inhibited by peptides and a monoclonal antibody which inhibit the binding of fibrinogen to GPIIb/IIIa induced by an agonist such as ADP or thrombin: ( 3 ) the structural specificity and dose response of GPIIb/IIIa activated by fibrinogen-antibody complex are similar to those for inhibi- tion of fibrinogen binding, suggesting that both events are a consequence of occupancy of the same ligand-binding pocket; (4) the activation does not require a signal transduc- tion event.

In the resting state of GPTIb/IIIa there is generally believed to be reduced accessibility of recognition sequences in macromolecular ligands to the binding site of GPIIb/IIIa (Coller, 1986). Platelet activation by an agonist may change the conformation of GPIIb/IIIa to permit the access of the recognition sequences (Shattil et al. 1985: O’Toole et al. 1990). Small synthetic peptides bind to GPIIb/IIIa without agonist and their binding may then induce the active conformation of GPIIb/IIIa (Lam et al, 1987). This implies that it may directly activate GPIIb/IIIa and initiate the binding of the intact ligand without platelet activation when fibrinogen changes its conformation and exposes its recogni- tion to ligand-binding sites in unactivated GPIIb/IIIa. Indeed, fibrinogen changes its conformation on the plastic surface (Zamarron et a/. 1990) and binds to unactivated GPIIb/IIIa in resting platelets or in transfected CHO cells (O’Toole et al, 1990; Coller, 1980). Although we have no direct evidence. our results may suggest that the antibody changes the conformation of ligand to enhance the affinity to GPIIb/IIIa. The binding of small RGDS-peptide to GPIIb/IIIa requires no agonist and leads to changes in GPIIb/IIIa that are associated with acquisition of high affinity fibrinogen-binding function and subsequent platelet aggregation (Du ef al, 1991). After the binding of fibrinogen to GPIIb/IIIa by the antibody (YCU- K3), fibrinogen has the possibility to activate GPIIb/IIIa through the binding ofRGDS and/or i’397-411 to GPIIb/IIIa.

Occupancy of integrins leads to intracellular signal trans- duction events (Banga et al, 1986: Werb et al, 1989). However, a recent paper showed that RGDS peptide-induced platelet activation is not inhibited by the inhibitors of platelet activation (Du et al, 1991). The activation effects by the antibody observed here do not depend on such events. This conclusion is based on the failure of the inhibitors (PGE1, apyrase) of platelet activation to block the activation mediated by this antibody. The conformational changes in GPIIb/IIIa by peptide binding to GPIIb/IIIa have been observed by immunochemical techniques (Frelinger et al. 1988. 1990). Thus, the conformational changes caused by the antibody-mediated binding of a sequence of RGDS or 739 7-41 1 to GPIIb/IIIa are probably responsible for increas- ing the affinity of fibrinogen binding to GPIIb/IIIa.

346 Hiroshi Mohri et a1 Agonist is required for the binding of fibrinogen to GPIIb/

IIIa. The present studies indicate that anti-fibrinogen anti- body also functions as partial agonists, and their capacity to generate a high affinity of fibrinogen binding to GPIIb/IIIa may depend on conformational change.

REFERENCES Andrieux, A., Hudry-Clergeon. G.. Ryckewaert. J.-J., Chapel, A,,

Ginsberg. M.H., Plow. E.F. & Marguerie. G. (1989) Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIb/IIIa. Journal of Biological Chemistry. 264, 92 58- 9265.

Banga, H.S., Simons, EX., Brass, L.P. & Rittenhouse, S.E. (1986) Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoprotein IIb/IIIa and dual role of epinephrine. Proceedings of the National Academy of Sciences of the United States of America. 83, 9197-9201.

Beardley. D.S.. Spuegel. J.E.. Jacobs, M.M., Handin, R.I. & Lux, S.E. (1984) Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombo- cytopenia. lournd of Clinical Investigation, 74, 1701-1 707.

Bennett, J.S., Shattil, S.J., Power, J.W. & Gartner. T.K. (1988) Interaction of fibrinogen and its platelet receptor. Joirrnal of Biological Chemistry, 263, 12948-12953.

Bennett. J.S. & Vilaire. G. (1979) Exposure of fibrinogen receptors by ADP and epinephrine. Journal of Clinical Investigation. 64, 1393- 1401.

Bodary. S.C.. Napier, M.A. & McLean, J.W. (1989) Expression of recombinant platelet glycoprotein IIb/IIIa results in a functional fibrinogen-binding complex. Journal of Biological Chemistry, 264,

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle ofprotein-dye binding. Analytical Biochemistry, 72,248- 2 54.

Coller. B.S. (1980) Interaction of normal, thrombasthenic, and Bernard-Soulier platelets with immobilized fibrinogen: defective platelet-fibrinogen interaction in thrombasthenia. Blood, 55, 169-176.

Coller. B.S. (1986) Activation affects access to the platelet receptor for adhesive glycoproteins. Journal ojCell Biology, 103,451-462.

Du, X., Plow, E.F., Frelinger, A.L., O’Toole, T.E., Loftus. J.C. & Ginsberg, M.H. (1991) Ligands ‘activate’ integrin aI1bB3 (platelet GPIIb-IIIa). Cell, 65, 409-416.

Ey, P.L., Prowse. S.J. & Jenkin. C.R. (1978) Isolation of pure IgGl, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry, 15, 429-436.

Engvall, E. & Ruoslahti, E. (1 977) Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. International Journal of Cancer, 20, 1-9.

Fracker, D.J. & Speck, J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloramide 1,3,4.6 tetra- chloro 3a,6a diphenyl-glycoluril. Biochemical and Biophysical Research Communications, 80, 849-857.

Frelinger. A.L., 111, Lam, S.C.-T.. Plow, E.F.. Smith, M.A.. Loftus, J. & Ginsberg. M.H. (1988) Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion. Journal of Biological Chemistry, 263, 12397-12402.

Frelinger, A.L., 111, Cohen, I., Plow, E.F., Smith, M.A.. Roberts, J., Lam, S.C.-T. & Ginsberg, M.H. (1990) Selective inhibiton of integrin function by antibodies specific for ligand-occupied recep- tor conformers. Journal of Biological Chemistry, 265, 6346-6354.

Gartner, T.K. & Bennett, J.S. (1985)The tetrapeptide analogueof the

18859-18862.

cell attachment site of fibrinogen inhibits platelet aggregation and fibrinogen binding to activated platelets. loirrnal of Biological Chemistry, 260. 11891-11894.

Handa, M.. Titani. K., Holland, L.Z., Roberts, J.R. & Ruggeri. Z.M. (1986) The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal anti- bodies and partial amino acid sequence analysis of proteolytic fragments. Journal of Biological Chemistry, 261, 12479-12585.

Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell,

Jennings, L.K. &Phillips. D.R. (1982) Purification ofglycoproteins IIb and III from human platelet plasma membanes and characterixa- tion of a calcium-dependent glycoprotein Ilb-IIla complex. Journal of Biological Chemistry, 257, 10458-10466.

Kazal, L.A., Amsel. S., Miller, O.P. & Tocantins. L.M. (1963) The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proceedings of the Society for Experi- mental Biology and Medicine, 113, 989-994.

Kloczewiak, M., Timmons, S. & Hawiger, J. (1982) Localization of a site interacting with human platelet receptor on the carboxy- terminal segment of human fibrinogen a chain. Biochemical and Biophysical Research Communications, 107, 181-1 86.

Kloczewiak, M.. Timmons. S.. Lukas. T.J. & Hawiger. J , (1984) Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the 1) chain. Biochemistrg. 23, 1767-1774.

Lam, S.C.-T., Plow, E.F., Smith. M.A., Andrieux, A., Ryckwaert, I.-]., Marguerie, G. & Ginsberg, M.H. (1987) Evidence that arginyl- glycylaspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets. Journal of Biological Chemistry, 282, 947-950.

Marguerie, G.A., Plow, E.F. & Edgington, T.S. (1979) Human platelets possess an inducible and saturable receptor specific for fibrinogen. Journal of Biological Chemistry. 254, 5357-5363.

Mohri. H., Hashimoto. Y. . Ohba, M., Kumagal, H. & Ohkubo, T. (1 99 1 ) Novel effect of cyclicization of the Arg-Gly-Asp-containing peptide on vitronectin binding to platelets. American Journal of’ Hematology. 37, 14-19.

Niiya. K., Hodson. E.. Bader. R.. Byers-Ward. V.. Koziol. J.A.. Plow, E.F. & Ruggeri. Z.M. (1987) Increased surface expression of the membrane glycoproteinIIb/IIIa complex induced by platelet acti- vation. Relationship to the binding of fibrinogen and platelet aggregation. Blood. 70, 475-483.

Nisonoff, A. & Dixon, D.J. (1964) Evidence for linkage of univalent fragments or half-molecules of rabbit y-globulin by the same disulfide bond. Biochemistry, 3, 1338-1342.

Nurden, A.T. & Caen. J.P. (1974) An abnormal platelet glycoprotein pattern in three cases of Glanzmann’s thrombasthenia. British Journal ofHaernatology. 28, 253-258.

O’Toole. T.E.. Loftus, J.C., Du. X . , Glass, A.A., Ruggeri. Z.M.. Shattil, S.J.. Plow, E.F. & Ginsberg, M.H. (1990) Affinity modulation of the alpha IIb beta 3 integrin (platelet HPIIb-IIIa) is an intrinsic property of the receptor. Cell Regulation. 1, 883-893.

O’Toole, T.E., Loftus, J.C., Plow, E.F., Glass, A., Harper, J.R. & Ginsberg, M.H. (1 989) Efficient surface expression of platelet GPIIb-IIIa requires both subunits. Blood, 74, 14-20.

Parham, P. (1983) On the fragmentation of monoclonal IgG1, IgGZa, and IgGZb from BALB/c mice. Journal of Imnztmology, 131,

Parise, L.V., Helgerson, S.L.. Steiner. B.. Nannizzi. L. & Phillips. D.R. (1987) Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa. Journal of Biological Chemistry, 262, 12597-12602.

48, 549-554.

2895-2902.

Antibodg Mediates Fibrinogen Binding to GPZlb/Illa 347 Peerschke. E.I. & Zucker, M.B. (1981) Fibrinogen receptor exposure

and aggregation of human blood platelets produced by ADP and chilling. Blood. 57, 663-669.

Phillips, U.R. & Agin. P.P. (1977) Platelet membrane defects in Glanzmann’s thrombasthenia. Journal of Clinical Investigation. 60, 535-545.

Plow, E.F. & Marguerie, G. (1982) Inhibition of fibrinogen binding to human platelets by the tetrapeptide glycyl-L-prolyl-L-arginyl-L- proline. Proceedings of the National Acaderng of Sciences of the United States ofAmerica. 79, 3711-3715.

Plow, E.F., Pierschbacher. M.D.. Ruoslahti. E.. Marguerie, G.A. & Ginsberg. M.H. (1 985) The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proreedings of’tlre Nafiorial Acndeiny ofSrierices ofthe United Stutrs ofilmericn. 82, 8057-8061.

Plow, E.F., Pierschbacher, M.D., Ruoslahti, E.. Marguerie, G. & Ginsberg, M.H. (1987) Arginyl-glycyl-aspartic acid sequences and fibrinogen binding to platelets. Blood. 70, 110-1 15.

Pytela. R.P.. Pierachbacher, M.D., Ginsberg. W.H., Plow, E.F. & Ruoslahti. E. ( 1 986) Platelet membrane glycoprotein IIb/IIIa:

member of a family of Arg-Gly-Asp-specific adhesion receptors. Science, 231, 1559-1562.

Shattil, S.J., Hoxie. J.A.. Cunningham. M. & Brass, L.F. (1985) Changes in the platelet membrane glycoprotein Ilb-IIIa complex during platelet activation. Journal of Biological Chemistry. 260, 11 107-11114.

Tomiyama. Y.. Taubakio. T., Pitrowicz. R.S., Kurata. Y., Loftus. J. & Kunicki. T.J. (1992) The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules. Blood, 79, 2303-2 31 2.

Walsh. P.N.. Mills, D.C.B. & White, J.G. (1977) Metabolism and function of human platelets washed by albumin density gradient separation. British Journal of Haetnatolug3, 36, 281-298.

Werb, Z., Tremble, P.M., Behrendtsen, 0.. Crowley, E. & Damsky. C. ( 1 989) Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. Journnl of Cell Biology, 109, 877-889.

Zamarron, C.. Ginsberg, M.H. & Plow, E.F. (1990) Monoclonal antibodies specific for conformationally altered state of fibrinogen. Thrombosis and Hnernostasis. 64, 41-46.