Monoclonal Antibodies Against Platelet Glycoproteins

Monoclonal Antibodies Against Platelet Glycoproteins Peter Horsewood, James W. Smith, and John G. Kelton

LATELETS play a pivotal role in hemostasis. They participate in the formation of the hemostatic plug at sites of spontaneous and traumatic damage to the vessel wall, and then release internal components that recruit other platelets to bind to the growing platelet plug. The platelet surface components also serve as a site for activation ofthe coagulation factors. Finally, platelets release substances that cause the vessel wall to repair itself. Consequently, very low platelet counts can result in bleeding, which often requires treatment by platelet transfusions. When damage to the vessel wall is recurring and chronic, which can occur in patients with hypertension, the vessel wall gradually thickens, limiting the flow of blood through the narrowed opening. Often, a layer of platelets cap the damaged vessel wall and, depending on the tissue perfused by this vessel, the embolization of platelet clumps can produce a heart attack or stroke. The recognition of the participation of platelets in hemostasis and thrombosis has led to intense activity investigating the various membrane components of platelets that participate in normal and pathological platelet function. Much of the information gained about platelet structure/ function relationships has been directly attributable to the use of monoclonal antibodies to study platelets. In this artiele we will review some ofthe more important monoclonal antibodies that have been used to study the platelet glycoproteins (GP).

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TECHNIQUES USED TO STUDY PLATELET SURFACE PROTEINS

One of the frrst techniques used to study platelet surface proteins was the gel sieving technique, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE). In this technique the plate-

From the Department oj Pathology, McMaster University Medical Centre, and the Canadian Red Cross Blood Transfusion Service, Hamilton, Ontario, Canada. Supported by a grant from the Medical Research Council oj Canada. Address reprint requests to John G. Kelton, MD, Department oj Pathology, McMaster University, HSC-2N34, 1200 Main St W, Hamilton, Ontario, L8N 3Z5 Canada. Copyright © 1993 by W.B. Saunders Company 0887-79631931070310002$3.0010

156

let lipid membrane is dissolved by using a detergent, and the platelet proteins are separated using a gel to which an electrical voltage is applied. The proteins then can be located by staining with either Coomassie blue or a more sensitive stain such as silver. The early investigators who used this and similar technologies demonstrated that platelets contained a number of different proteins that subsequently were designated from the largest (GP I) to the smallest. A modification of this technique is two-dimensional electrophoresis in which the platelets are electrophoresed under nonreduced conditions in one direction, the electrical field is rotated 900 , and the electrophoresis performed again under reducing conditions. An example of this electrophoretic approach is shown in Fig l. Using more sensitive staining techniques it became apparent that there were a large number of different platelet proteins, and some of the physical characteristics of these proteins could be determined by electrophoretic patterns. For example, the presence of interchain disulfide bonds, which would be broken during electrophoresis under reducing conditions, meant that the protein would be split and would migrate below the diagonal as two separate proteins (eg, GP Ib). However, intrachain disulfide bonds would cause the protein to migrate less quickly in the reduced phase of the electrophoresis, because the breaking of the intrachain disulfide bond allows the protein to unfold and appear larger. An example of this type of protein is GP lIla, which has extensive intrachain disulfide bonds (Fig 1). Antibodies that formed in transfused patients, who lacked either specific platelet GPs (isoantibodies in patients with Glanzmann's thrombasthenia) or a particular epitope (alloantibodies that occur in patients with alloimmune thrombocytopenia) allowed investigators to begin to relate structure and function. However, relating the binding of an antibody to a particular GP required further technical advances in the study of platelet membranes. Radioimmunoprecipitation and immunoblotting (also known as Western blotting) are techniques that have permitted the use of naturally formed antibodies (isoantibodies and alloantibodies) as well as monoclonal antibodies in the study of platelet GPs. These two techniques are similar, Transfusion Medicine Reviews, Vol VII, No 3 (Julyl, 1993: pp 156-172

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wash step to remove unbound radiolabeled platelet proteins, the speeifie platelet protein is then dissoeiated from the antibody and eleetrophoresed using SnS-PAGE. Tbe radiolabe.1ed protein is loeated by autoradiography and its mobility eompared with moleeular weight standards run in paralleI (Fig 2). An example of radioimmunopreeipitation is shown in Fig l. We have found radioimmunoprecipitation to be the most valuable teehnique for studying platelet surfaee proteins: however, it requires that the protein of interest is able to be radiolabeled and also requires the use of radioisotopes. Some laboratories eannot perform this teehnology, so aeeordingly, we reeent1y modified this teehnique to ałlow the use of biotin as a nonradioaetive platelet protein label. 1 Immunoblotting is a similar teehnique exeept that the platelet GPs from the test platelets are Iysed, eleetrophoresed, and then transblotted onto a nitroeellulose strip. Tbe antibody of interest is allowed to reaet with the transblotted GP and, following a wash step, the bound antibody is loeał­ ized using a seeondary antibody that earries a marker (radiolabeled or enzyme labeled). Immu-

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Fig 1. (A) Two-dimensional electrophoresis ot platelet surtace GPs. The 1251-labeled GPs on platelets are separated by SOS-PAGE, first under nonreducing conditions and then under reducing conditions. Reference molecular weight markers are included in the second dimensional, reducing SOS-PAGE. (B) Radioimmunoprecipitation ot platelet surface GPs. Shown are the GPs precipitated by monoclonal antibodies 12F1, antiGP lallla (lane 1); Raj-1, anti-GP IIb/llla (lane 2); 601, anti-GP Ib (lane 3); Beb-1, anti-GP IX (lane 4); and PH-1, anti-GP IX (lane 5). Note thet GP IX does not label well with 1251, and only the GP Ib component ot the GP Ib/IX complex is detected.

but with important differenees. In radioimmunopreeipitation the platelet surfaee GPs are radioIabeled (intaet platelets), and the test platelets are solubilized using a detergent. The antibody of interest is then added and allowed to bind to the radiolabeled platelet GP. The antibodies, plus their bound radiolabeled GPs are immunopreeipitated using either a seeondary antibody or Staphylococcus protein A attaehed to agarose beads. After a

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158

HORSEWOOD, SMITH, AND KELTON

noblotting offers the advantage of simplicity and the potential to store the transblotted proteins for long lengths of time. However, many proteins are not recognized by the corresponding antibodies after they have been denatured and transblotted (Fig 3). The importance of platelet surface GPs in the mediation of hemostatic and thrombotic events is well documented. 2 Duńng the past decade monoclonal antibodies have played a prominent part in identifying and defining the role of these GPs. This review will descńbe those monoclonal antibodies prepared against platelet components involved in the process of platelet activation, adhesion, aggregation, and release. For the most part, these molecules are surface GPs, although some exist in resting platelets within internal granules and become expressed on the surface after platelet activation. Whereas many of these molecules are platelet specific, some, such as thrombospondin (TSP) and GP laJlIa, are descńbed more aptly as platelet associated. These are included because of their significance in platelet hemostasis. The vast majońty of the muńne monoclonal an-

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tibodies described have been prepared by standard fusion techniques following immunization with either whole platelets or puńfied platelet proteins. In some cases, useful antiplatelet antibodies were obtained after immunization with other cell types. As is the case for almost all monoclonal antibodies, specificity is determined by defining the conditions of the screening assay. For example, anti-GP Ib, specific for the von Willebrand factor (vWF) binding site, was prepared by identify that monoclonal antibody capable of inhibiting ństocetin-mediated agglutination of platelets by vWF. 3 Many of the monoclonal antibodies have been useful in structural and functional studies and as diagnostic reagents. Monoclonal antibodies can be grouped together in clusters based on their target when tested under vańous conditions. The molecules identified by the vańous monoclonais have been assigned numbers called cluster designations (CD). Whereas initially applied to leukocytes, this nomenclature system has been extended to include platelets. Findings from the last Workshop on Human Leukocyte Differentiation Antigens (Vienna, Austńa, 1989)4 have resulted in CD designations for 16 different platelet-specific and -associated molecules. The results of this workshop contain a wealth of information on platelet-specific and -associated monoclonal antibodies, particularly on some of the antibodies that previously had not been well descńbed. A complete or even near complete descńption of all the monoclonal antibodies reported would go beyond the scope of this review. Therefore, we will describe those monoclonal antibodies that have a histońcal significance or importance in defining platelet functions. For convenience, the descńption of the monoclonal antibodies is based on the surface molecules they define, and the target molecules will be given their traditional assignments, and when known, the CD number.

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Because of its central importance as the major mediator of platelet adhesion and aggregation, the GP Ub/lIla complex has received more attention than any of the other platelet glycoproteins. Combined with the fact that this large complex is also the most abundant protein on the platelet surface, it is not surpńsing that there have been more mono· clonal antibodies prepared against GP Ub/lIla than

ANTIPLATELET MONOCLONAL ANTIBODIES

159

against other platelet GPs. As a result there is a detailed understanding of the structure and mechanism of action of the GP IIb/IIIa complex. Monoclonal antibodies have been prepared that are specifie for the isolated, individual GP IIb (CD41) and GP IIIa (CD61), and the complexed proteins (CD4l) in either the resting or activated state.

binding to GP IIb/lIla requires additional activation changes to the complex. Similar evidence for the existence of preformed complexes was obtained by another group of investigators, also using a monoclonal antibody specific for GP Ub/lIla. 6 The dependence on calcium for the integrity of the complex was originally suggested by functional studies. Using the monoclonal antibody A2A9, specific for the intact complex, Brass et al? demonstrated that the stability of platelet membrane GP IIb/lIla was dependent on Ca2 + concentration, pH, and temperature. Further, this investigation showed that the Ca2 + stabilizing sites were located on the external surface of the platelet and suggested that cytosolic Ca2+ levels do not regulate the formation of GP Ub/lIla complexes. The regulation, at physiological concentrations of extracellular Ca2 + and Mg2+ , of the surface exposure of GP Ub has been studied by Ginsberg et al. 8 The

STRUCTURE AND DISTRIBUTION

Glycoproteins GP IIb/IIIa form a Ca 2 + dependent, noncovalent1y linked heterodimer complex on the platelet surface (Fig 4). The IIb/lIla complex functions as the fibrinogen receptor following platelet activation and thereby mediates aggregation. Using monoclonal antibodies specifie for GP IIb and the GP IIb/IIIa complex, McEver et aIs showed that the individual subunits exist as a preformed complex on unstimulated platelets. Their findings also suggested that the fibrinogen

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studies were perfoITIled with a monoclonal antibody to the GP Ub protein and showed, in addition to the Ca2+ -dependent orientation of GP Ub, that the regulation correlated with the capacity of platelets to bind fibrinogen. Woods et al9 made use of the Ca2+ dependence and showed that resting platelets contain a substantial, centrally located pool of glycoprotein Ub/lIla complex. They found that after irreversibly dissociating the surface complex with ethylene glycolbis(l3-aminoethyl ether)N,N,N' ,N', -tetraacetic acid (EGTA), a monoelonal anti-GP Ub/lIla antibody no longer bound to platelets. However, after thrombin stimulation, newly reactive GP Ub/lIla, which had originated from a sequestered, internal pool, became exposed on the surface. Similar results were obtained using a panel of monoclonal antibodies and immunofluorescence and immunogold labeling techniques. 1O In this study, both GP Ub/lIla and GP Ib were shown to be present on the platelet surface and in internal membranebound vacuolar structures. GP Ub/IIIa but not GP Ib was shown to be present in the u-granules, and could be translocated to the surface following stimulation. The localization of epitopes for a series of monoelonal antibodies against GP Ub has been used to define several regions of the molecule, ineluding the putative GP IIIa- and fibrinogenbinding regions. II These same antibodies have been used in other studies to help define the interchain and intrachain disulphide bonds of GP Ub. 12 Quantitation of the number of GP Ub/lIla molecules on the surface of nOITIlal donor platelets, using 125I-Iabeled monoelonal antibody and Scatchard analysis, indicates that there are about 40,000 copies of the complex per platelet, l3 with some interindividual variation. 14 The number of complexes increases after thrombin activation, 15 presumably because of exteriorization of the intracellular pooł. 10 The GP Ub/lIla complex is a member of the widely distributed adhesion receptor family teITIled integrins. Other members include the vitronectin receptor, the very late antigens (VLAs), and the leukocyte adhesion receptors. 16 All integrins share certain characteristics: they are noncovalently linked heterodimers of u and 13 subunits, with each subfamily having a common 13 subunit. Using a panel of 28 monoclonal antibodies against platelet GP Ub, GP lIla, and GP Ub/IIIa

HORSEWOOD, SMITH, AND KELTON

complex, Kieffer et al 17 demonstrated that GP Ub is a megakaryocyte/platelet restricted antigen. These investigators found that the antibodies reacted with platelets and the erythroleukemic cell line, HEL, but were negative with peripheral blood mononuclear cells ineluding granulocytes, monocytes, and rosette-foITIling T cells. Endothelial cells were shown to react with anti-GP IIIa monoelonal antibodies but not with anti-GP Ub or complex-dependent anti-GP Ub/lIla antibodies. These observations are in keeping with the monoelonal antibody studies that indicate that the GP Ub/lIla reactivity previously reported to be associated with monocytes is attributable to platelet contamination. 18 Additionally, the observations are consistent with GP IIIa being present on endothelial cells associated with a v (CD51), which together comprise the vitronectin receptor. The presence of GP IIIa molecules complexed with a non-GP Ub u subunit would explain the observation of immunologically related GP IIb/lIla-like molecules on endothelial cells detected with anti-GP IIIa monoelonal antibodies. 19 Related studies using monoelonal antibodies specific for the vitronectin receptor complex have shown that platelets express both ajIIIa and GP IIb/IIIa complexes on their cell surfaces. 20 A further study using monoelonal antibodies showed both complexes on nOITIlal platelets, but found that Glanzmann's thrombasthenic platelets lacked GP Ub/lIla, whereas they had nOITIlal amounts of the vitronectin receptor (uj lIla). 21 Recent investigations have cast new doubt on the presence of GP Ub- or GP IIIa-like molecules on cells other than platelets. In characterizing monoclonal antibodies specific for fragments of GP Ub, two reports have found immun
The interaction of the GP Ub/IIIa complexes and those adhesive proteins important for hemostasis was established before the advent of plateletspecific monoclonal antibodies. However, mono-

ANTIPLATELET MONOCLONAL ANTIBODIES

clonal antibodies have furthered our understanding of the role of adhesive proteins binding to platelets. Initial studies with Glanzmann's thrombasthenic platelets showed that the GP Ub/lIla complex was involved in fibrinogen binding and aggregation. Further confirmation for the role of the complex as the fibrinogen receptor came from inhibition studies using monoclonal antibodies. A GP Ub/lIlaspecific antibody, lOE5, completely inhibited binding of fibrinogen to platelets, blocked aggregation induced by adenosine diphosphate (ADP), thrombin, and epinephrine, and inhibited both platelet adhesion to glass and clot retraction. J4 Similar inhibitory monoclonal antibodies have been produced by several other groups of investigators, including Heinrich et al. 24 One of these antibodies, Gi6, inhibited aggregation and release initiated by various agonists using PIA1_positive but not PI AJ -negative platelets. Presumably, this antibody binds to an epitope that contains the PIAl alloantigen site or to one that is sterically dependent on the alloantigen. Fibrinogen binding to GP Ub/lIla can be competitively inhibited by a tripeptide sequence of amino acids, Arg-Gly-Asp (RGD), a common recognition sequence for many adhesive proteins. J6 PAC-I is an IgM monoclonal antibody that competes with both fibrinogen and RGD-containing peptides for binding to the GP Ub/lIla complex on activated platelets. This monoclonal antibody has been shown to contain an Arg-Tyr-Asp (RYD) peptide sequence within its heavy chain third complementary determining region, thus mimicking the RGD sequence. 25 Synthetic peptides that encompass the antibody-binding sequence can inhibit PAC-l and fibrinogen binding . Comparative sequence analysis of this kind may be useful in identifying other platelet-specific, ligand-binding domains. The binding of platelets to exposed subendothelium through glycoprotein GP Ib is initiated by vWF (discussed in a subsequent section). It also can bind to GP Ub/lIla. Binding of vWF can be inhibited by monoclonal antibodies to the GP Ub/ lIla complex, and studies indicate that GP Ub/lIla is a major binding site for released platelet vWF. 26 Because fibrinogen and vWF are mutually competitive in their binding to GP Ub/lIla, and because both are inhibited by RGD-containing peptides, it might be assumed that there is a common binding

161

site for both ligands. Inhibition of the binding of vWF, fibrinogen, fibronectin, and thrombospondin by anti-Ub/llla monoclonal antibodies would support this view. 27 However, the interaction may be more complex: one monoclonal antibody (LJP5) has been shown to inhibit the binding of vWF but not fibrinogen to GP Ub/lIla. 28 The same monoclonal antibody was used to demonstrate that adhesive proteins other than fibrinogen are involved in GP I1b/IIIa-mediated adhesion and thrombus formation, but only under conditions of high shear rate. 29 It also remains uncertain whether vWF will bind to GP Ub/lIla in the presence of physiological concentrations of plasma fibrinogen. Recent studies using a series of monoclonal antibodies to the various components involved in the interactions indicate that vWF and fibrinogen compete for binding to GP Ub/lIla on activated platelets. 30 In this study it was proposed that high molecular weight multimers of vWF, released on platelet activation, may bind with sufficient affinity to mediate platelet adhesion. GP Ub/lIla exists on the resting platelet surface as a preformed heterodimer, but it is not normally occupied by either fibrinogen or vWF. Presumably activation results in the exposure of a vWF/ fibrinogen-binding site on platelet GP Ub/lIla. To investigate the expression of this cryptic binding site, Coller31 used a monoclonal antibody, 7E3, directed against an epitope formed during the activation-dependent change of GP Ub/lIla. Using multimers and fragments of the 7E3 antibody, the effect of ligand size on accessibility to the binding site was studied. Results indicated that activation involves a conformation and!or microenvironmental change in the complex that permits large molecules, such as fibrinogen, to bind more rapidly. This result explains the previous observation that small RGD-containing peptides of the fibrinogen 'Y chain bind equally well to resting and activated platelets. Further insight into the nature of the changes in the GP Ub/lIla complex that accompanies platelet activation and fibrinogen binding have come from studies with the monoclonal antibody PAC-1. 32 This immunoglobulin M (IgM) monoclonal antibody binds to the surface of platelets activated by agonists such as ADP, epinephrine, and thrombin, but does not bind to resting platelets. The binding is not dependent on platelet secretion and differentiates it from activationspecific monoclonal antibodies against platelet

162

granule-secreted components such as multimerin and GMP 140. PAC-l binds only to undissociated GP IIb/IIIa, and the binding is Ca2+ dependent. The epitope recognized by both PAC-l and fibrinogen, whose binding are mutually exclusive, is represented by an agonist-induced change in the GP IIb/IIIa complex. This epitope is likely different from that of the 7E3 monoclonal antibody, because the latter binds to inactivated platelets, albeit at asiower binding rate than to activated platelets. It is likely that the PAC-l epitope lies deeper in the fibrinogen-binding pocket of GP IIb/IIIa than the 7E3 epitope, becoming exposed only on activation-dependent conformation changes. Additional studies of the GP IIb/IIIa conformation changes that accompany platelet activation and fibrinogen receptor exposure were made using monoclonal antibody D3GP3. 33 This antibody recognizes a conformation-dependent epitope on glycoprotein GP IIIa that exists in low numbers on resting platelets, but which increases after dissociation or receptor occupancy of the GP IIb/IIIa complex. Platelet activation resulting in aggregation can be detected by the monoclonal antibody PMI_1. 34 This antibody inhibits platelet adhesion and spreading, binds minimally to platelets in plasma, but shows good binding to ligand-occupied GP Ub/ IIIa. 35 The antibody is specific for a neoantigen site at the carboxy terminus of the GP IIb «-chain. The epitope is induced by conformational changes resulting from GP Ub/IIIa occupancy by either fibrinogen or even small RGD-containing peptides. Neoantigens of this type are called ligand-induced binding sites (LIBS) and monoclonal antibodies specific for several of these sites on GP IIb/IIIa have been prepared. 36 MONOCLONAl ANTIBODIES TO GPIB AND GPIX (CD428 AND CD42A)

A useful maneuver that allows the preparation of several monoclonal antibodies against a whole molecule is exemplified by the technique used to produce antibodies to components of GP Ib. An initial anti-Ib monoclonal antibody, derived from an immunization with whole platelets, was selected by testing for inhibitory activity to ristocetin-induced platelet aggregation. The initial monoclonal antibody was then used to isolate and purify glycocalicin, a large proteolytic fragment of GP Ib. The glycocalicin was next used for immunization and, in this manner, many further monoclonal

HORSEWOOD, SMITH, AND KELTON

antibodies specific for multiple sites on GP Ib were prepared. 37 STRUCTURAL AND DISTRIBUTION STUDlES

The glycoprotein Ib/IX complex plays a critical role in early hemostasis. GP Ib is the receptor for the initial binding of platelets to exposed subendothelium at sites of vascular injury. GP Ib is a carbohydrate-rich, two-chain integral membrane protein composed of a larger l45-kD «-chain disulphide linked to a 24-kD f3 chain (Fig 5). This heterodimer is complexed in a l: l ratio with the 17-kD GP IX. The transmembrane association of the complex with cytoskeletal submembrane actin filaments has been shown using a monoclonal antibody probe specific for GP Ib. Both GP Ib and a protein identified as actin-binding protein were isolated when platelet lysate, prepared in the presence of inhibitors of calcium-dependent proteases, was added to immobilized antibody to GP Ib. 38 The actin-binding protein was not bound in the absence of the protease inhibitors and subsequently was shown to be internally derived. Similar studies using anti-glycocalicin monoclonal antibody, AK2, showed that actin-binding protein interacts with the cytoplasmic domain of the GP Ib-IX complex. 39 Binding studies performed with radiolabeled monoclonal antibodies directed against either the individual GP Ib and GP IX glycoproteins or the intact complex have shown that there are about 24,000 copies of GP Ib/IX per platelet. 40 In this study the complex specific antibodies were unreactive with the separated GP Ib and GP IX glycoproteins but did react on reconstitution. Taken together, these observations suggest that aU GP Ib and GP IX present in the platelet membrane exist in a complexed form. Furthermore, the availability of a monoclonal antibody to the complex, which does not react with either component alone, suggests a close physical proximity. The nature of the noncovalent interactions that bind the two glycoproteins is unknown, but because the GP Ib molecule is highly glycosylated and possesses a negative charge, it is unlikely that the interaction involves this large extracellular region. Evidence using monoclonal antibody immunoprecipitation analysis has shown that both GP Ib and GP IX are acylated with palmitic acid through thioester linkages. 41 It is possible that these fatty acids may be

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involved in hydrophobic interactions between the proteins, which helps keep the molecules together. Limited proteolysis used in conjunction with monoclonal antibody recognition of the resultant fragments has localized the vWF binding site of GP Ib to the amino terminus. 37 Other studies have shown that the carbohydrate-rich region of GP Ib is in the central part of the molecule. As is the case for GP IIb/IIIa, there is evidence for an intraplatelet pool of GP Ib molecules. Immunof1uorescent localization studies with monoclonal antibodies to GP Ib indicate an intracellular pool of GP Ib within vacuolar structures that are probably part of the platelet open canalicular system. 1O However, unlike GP IIb/IIIa, no GP Ib is present in the n-granules. GP Ib-like molecules have been identified in human umbilical vein endothelial cells and in bovine aortic smooth muscle cells. 42 The GP Ib-like material in these cells is functional as measured by

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ristocetin-dependent agglutination and vWF binding. However, the protein in endothelial cells seems to be a single-chain molecule, unlike the two-chain disulphide-linked GP Ibn and GP Ibl3 subunits found on platelets. FUNCTIONAL STUDIES

The importance of GP Ib as the primary receptor linking platelets to subendothelium via vWF has been well documented. Monoclonal antibodies have played an important part in these studies. Anti-GP Ib monoclonal antibody, 6D I, inhibits bovine vWF-induced platelet aggregation and the binding of vWF to platelets induced by ristocetin, and produces a reduction in platelet retention by glass beads. 3 The vWF binding to thrombinstimulated platelets is not inhibited by the GP Ibspecific antibody API, an observation consistent with the role of GP IIb/IIIa as a vWF receptor after thrombin activation. 43

164

Studies of whole blood perfusion of deendothelialized human umbilical arteries have shown that platelet adhesion can be strongly inhibited by a monoclonal antibody against GP Ib. 44 This result is consistent with the abnormal adhesion observed with Bernard-Soulier platelets, which lack the GP Ib/lX proteins. In addition to the role of vWF binding to GP Ib at sites of endothelial damage, similar interactions can take place under conditions of high shear, as might occur in stenosed or obstructed arterial vessels. Monoclonal antibody to GP Ib inhibits shearinduced platelet aggregation in a process requiring VWF. 45 The findings are compatible with vWF binding to GP Ib as the initiating step in highshear-induced aggregation. A monoelonal antibody, TM60, has been used to identify a thrombin-binding site on GP Ib. 46 A 43-kD tryptic fragment from GP Ib bound to both TM60 and thrombin affinity columns. The monoelonal antibody inhibited binding to the immobilized thrombin, and also inhibited thrombin- and ristocetin-induced aggregation. These results are in agreement with other functional studies that show thrombin binds to GP Ib. Whereas it is important to emphasize that GP Ib is not the primary receptor for thrombin, recent studies would indicate that GP Ib is involved in thrombin-induced platelet aggregation. 47 Whether a collagen-binding site also exists on GP Ib is less certain: however, two monoelonal antibodies against GP Ib have been shown to inhibit collagen-induced aggregation. 48 ,49 Because Bemard-Soulier platelets, which lack GP Ib, aggregate normally to collagen, the studies were interpreted to indicate a elose proximity of GP Ib to the collagen receptor. Thrombocytopenia caused by quinine/quinidine drug-dependent antibody interactions has been studied using monoelonal antibodies against GP Ib and GP IX. 50 Anti-GP łba antibodies did not block drug-dependent platelet aggregation: however, an antibody against GP IX blocked aggregation, and further investigation suggested that interaction between the GP Ib and GP IX in the intact complex was necessary for drug-dependent antibody recognition. MONOClONAl ANTIBODIES AGAINST VERY LATE ANTIGENS

Members of the VLA family of cell surface heterodimers, like those of the GP I1b/I1Ia complex,

HORSEWOOD, SMITH, AND KELTON

are composed of a large a subunit noncovalentIy associated with a smaller 13 subunit. Ali members share a common 131 subunit, which is expressed on many tissues and cells. Individual members are characterized by a distinct a subunit. Platelet express three VLA complexes that function as receptors mediating adhesion to extracellular matrix proteins. Using radioimmunoprecipitation techniques and a specific monoelonal antibody, 12Fl, the a and 13 subunits of VLA-2 were found to correspond to the platelet surface glycoproteins GP la and GP Ha, respectively (CDw49b and CD29).51 Further studies with the same monoelonal antibody implicated the GP IblIla complex as a platelet collagen receptor,52 an observation consistent with descriptions of defective collagen responses in patients lacking GP la. 53 Further support for this specificity has come from studies in which binding to collagen was inhibited by monoclonal antibodies to GP la. 54 There still remain unresolved issues regarding the identity of the platelet collagen receptor(s), because at least six different platelet surface proteins have been implicated as sucho However, recent evidence would suggest that glycoprotein GPIV and the GP Ib/I1a complex are the important platelet collagen receptors. Fibronectin and laminin together with collagen serve as the major adhesive components of the subendothelium matrix. Presumably all participate in the formation of the hemostatic plug at sites of vessel damage, and each may have its own platelet receptor. VLA-5 has been identified as the fibronectin receptor,55 and VLA-6 is the laminin receptor. 56 The a subunits of the fibronectin (VLA-5) and laminin receptors (VLA-6) both resemble a platelet glycoprotein previously designated as Ic. Hence, it is likely that there are two platelet Ic proteins. 57 These two proteins are not readily separabie by electrophoretic methods, and therefore are better described as a 5 and a6' MONOCLONAL ANTIBODIES TO GPIV (CD36)

Platelet glycoprotein GPIV, also known as GP I1Ib, is a single-chain, carbohydrate-rich glycoprotein having an apparent molecular weight of approximately 90 kD (Fig 6). It is widely distributed among different celi types and plays a major role in platelet adhesion functions. Monoelonal antibodies have helped in elucidating the function of GPIV, particularly studies performed with DKM-S. TSP is a mediator of cell-cell and cell-substrate

ANTIPLATELET MONOCLONAL ANTIBODIES

165 Cysteine ~ich Region

/ .....

TSP Binding Region ....-/

l Fig 6. Platelet GP IV (CD361. The approximate locations are shown for the T8P-binding re· gion and the cysteine-rich re· gion.

interactions and it also binds to platelets. This binding is inhibited by some monoclonal antibodies to GPIV, suggesting that GPIV is the platelet TSP receptor. 58 Similar studies have shown that monoclonal anti-GPIV antibody inhibits the TSPdependent binding of platelets to monocytes. 59 Further evidence for a platelet receptor for TSP has come from studies showing specific binding of TSP to GPIV captured with a monoclonal antibody.60 Other investigators have been unable to show inhibition of platelet adhesion to a TSPcoated surface using an anti-GPIV monoclonal antibody.61 GPIV also may serve as the receptor for malariainfected red blood cells. Monoclonal antibody OKM-5 inhibits binding of infected red blood cells to target cells. 62 In other studies, binding of malaria-infected red blood cells to GPIV derived from platelets was inhibited by OKM_5. 63 Individuals who lack GPIV (Naka negative) were identified originally through studies with the serum from a patient who became refractory to platelet transfusions. Subsequent investigations showed the serum reactivity was caused by isoantibodies directed against a protein (GPIV) absent from the patient's own platelets rather than against a platelet alloantigen. Naka-negative platelets are unreactive with OKM-5 monoclonal antibody, and it seems that the isoantisera and OKM-5 are directed against the same or spatially proximate epitopes. 64 The Naka-negative phenotype occurs with a much higher frequency in the J apanese and Eastern than in the Western population. MONOCLONAL ANTIBODIES AGAINST PLATELET ACTIVATION ANTIGENS

The studies using monoclonal antibodies 7E3 and PAC-l, which detect activation-specific

ceOH changes in the GP Ub/lIla complex, have been described previously. In addition, several other monoclonal antibodies have been prepared that identify activation-dependent proteins on platelets. Some monoclonais recognize conformationall environmental changes on preexisting surface antigens, ie, GP Ub/lIla: however, other monoclonais recognize antigens expressed during the activation process. The prototypical activation marker in this category is the 140-kD a-granule membrane protein, P-Selectin (CD62) (Fig 7). Identified initially by monoclonal antibodies and known variously as GMP-14065 (granule membrane protein, 140 kD) or platelet activationdependent, granule-external membrane (PADGEM),66 these antibodies have provided novel information in several areas. For example, patients with gray platelet syndrorne, who have a deficiency of a-granules and many of the associated a-granule proteins, nonetheless have normai ameunts of P-selectin. 67 This study suggested that the gray platelet defect may be in targeting endogenously synthesized secretory proteins into developing a-granules in megakaryocytes. Recent studies in which monoclonal antibodies inhibited binding of platelets to neutrophils indicate that P-selectin plays a role in mediating adhesion of stimulated platelets to neutrophils and monocytes, and thereby provides a link between hemostatic and inflammatory responses. 68 The exposure of a-granule proteins on the platelet surface following platelet activation results in the surface expression of proteins that previously were absent or present only in very low numbers. These surface-bound proteins are important physiologically, and also can be used as markers of platelet activation. TSP is present only at very low levels in plasma, and the large amount on the surface of activated platelets originates from internal

HORSEWOOD, SMITH, AND KELTON

166

9 Tandem Repea!s - 62 Amino Acids - Each with 6 Cysteine Residues Complement Regulatory Repeats

lun

a-granule stores. A monoclonal antibody to TSP has been used as an activation-specific marker to monitor platelet changes during in vitro secretion and also in patients with suspected platelet abnormalities. 69 Binding of monoclonal antibodies to TSP correlated with binding of antibodies to P-selectin for both in vitro- and in vivo-activated platelets. Nieuwhenhuis et al 70 have described a monoclonal antibody against a lysosomelike granule protein that is secreted and expressed on the surface of activated platelets. This protein is expressed in low numbers on resting platelets but can be detected in large numbers after activation. In contrast to the negative resuIts found with anti-TSP and P-selectin,69 the anti-53-kD lysosomelike protein (CD63) was reported to be expressed in vivo in patients undergoing cardiopulmonary bypass surgery. Monoclonal antibodies have recently been described against Iysosomal-associated membrane proteins (LAMPs) that become expressed on the platelet surface membrane after activation. 71 •72 Unlike the P53 Iysosomal protein, which is released and bound, the LAMP proteins are integral

Fig 7. Platelet GP P-Selectin ICD621. The approximate loeations are shown for the epidermai growth factorlike IEGFI domain, the Ca 2 + -dependent, lectinlike domain, and the nine tandem repeets releted to those found in complement-reguletory proteins.

membrane proteins that directly translocate to the surface after activation. A monoclonal antibody, JS-I, has been shown to identify a large muItimeric platelet activationspecific protein having a subunit size of 155 kD. 73 The protein, multimerin, is released after thrombin stimulation and binds to the surface of the activated platelets. The function of the protein is presently unknown but its multimeric nature, like that of vWF, suggests that it is involved in adhesion/ aggregation events. A protein or protein complex on the surface of activated human T cells and platelets has been detected with the monoclonal antibody 1B3. 74 The antibody does not react with resting platelets but does after thrombin stimulation, and immunoprecipitates molecules of 180 and 150 kD. Other monoclonal antibodies showing similar reactivity pattems also have recently been described. 75 PLATELET-ACTIVATING MONOCLONAL ANTIBODIES

Certain monoc1onal antibodies by themselves initiate platelet activation. Several reports de-

ANTIPLATELET MONOCLONAL ANTIBODłES

scribed antibodies that bind to a 22- to 24-kD surface protein (CD9) , which results in platelet aggregation and release. One of several such antibodies is Alb-6, and studies have indicated that activation involves the platelet Fc receptor (CDw32).76 Excluding those antibodies that activate platelets via complement fixation we believe that most (perhaps alI) monoclonal antibodies activate platelets via the Fc receptor. The one exception is the platelet Fc receptor itself, which initiates platelet aetivation when it is clustered. We have shown that platelet activation is independent of the number of antigen sites on the platelet surface but dependent on antigen mobility.77 Monoclonal antibodies against GP Ub78 and GP lIla,33 whose Fab fragments can induee platelet aggregation, may not belong to this general class of activators but represent antibodies that merely expose fibrinogenbinding sites without concomitant activation proeesses. Studies with activating monoclonal antibodies could prove useful in determining if human autoantibodies and alIoantibodies aetivate platelets by similar mechanisms. DIAGNOSTIC AND THERAPEUTIC STUDlES

Because of their defined specificity, monoclonal antibodies have found widespread use as diagnostic reagents. Today they play an important role in the diagnosis and investigation of congenital and acquired disorders of platelet function. The use of monoclonal antibodies as therapeutic agents has been slower. This slowness is not a reflection of the lack of suitable antibody speeificity but a consequence of predictable in vivo limitations. Glanzmann's thrombasthenia is a hereditary platelet disorder eharaeterized by a deficiency of GP Ub and GP lIla. The genetie defeet is a result of altered protein biosynthesis and processing at the megakaryocyte level. Immunocytochemical analysis using anti-GP Ub/lIla monoclonal antibodies combined with electron microscopy has shown that normal megakaryocytes stain strongly, but megakaryocytes from thrombasthenic patients are negative. 79 An assay for diagnosing Glanzmann's thrombasthenia using monoclonal antibodies has been developed and requires only very smalI volumes of whole blood. 80 Other antibodybased studies have been used to detect heterozygotes for Glanzmann's thrombasthenia; obligate carriers have levels of the GP Ub/lila glycoproteins that are intermediate between normal and homozygote-defieient levels. 81 Flow cytometric

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analysis of GP levels using monoclonal antibody probes has been used to study the distribution of the GP IIb/lIla complex in norma! and thrombasthenic platelets.82 The studies indicate a functional heterogeneity in Glanzmann's thrombasthenia. Type I thrombasthenia is eharaeterized by the eomplete absence of GP Ub/lIla, and the rare type II is characterlzed by low but not absent levels of GP IIb/lIla. These findings exclude the possibility of a gene deletion in these patients. In a preliminary step to the antenatal diagnosis of both Glanzmann's thrombasthenia and Bernard-Soulier syndrome, surface GPs were quantified on normal fetal platelets by flow cytometry using monoelonal antibodies. 83 The GP IIb/llla complex levels were in the same range in fetuses and adults, whereas the GP Ib levels were slightly higher in fetuses than adults. The results suggest the potential for the antenatal detection of certain congenital platelet disorders. Quantifieation analysis cannot be applied to the diagnosis of thrombasthenia variant patients who present with the usual clinica! and funetional abnormalities. These patients have been shown to have normalor near norma! levels of the GP IIb/ lila complex but the complex has defective receptor funetion. In one investigation of a thrombasthenic variant patient using monoclonal antibodies direeted against GP IIb/lIla epitopes, it was shown that the GPs on the patient's platelets were very sensitive to divalent eation ehelation.84 This defeet resulted in unstable GP IIb/lIla eomplexes, whieh were unable to support fibrinogen binding. One patient has been identified who exhibited platelet defeets typical of Glanzmann's thrombasthenia but who had normallevels of GP Ub/lila eomplexes as determined by the monoclonal antibodies 115 and C17.85 Immunoglobulins eluted from the patient's platelets bound to GP Ub/lIla eomplex and inhibited both ADP- and eollagen-indueed aggregation. It is possible that the defeet was caused by anti-GP Ub/lIla autoantibodies, resulting in aequired thrombasthenia. Bernard-Soulier syndrome, like Glanzmann's thrombasthenia, is an autosomaI reeessive disorder eharaeterized by thromboeytopenia, with very large platelets and absent GP Ib/IX. The major funetional abnormality is an absent (or severely impaired) platelet interaetion with ristoeetin and vWF. Platelet glyeoprotein analysis has shown absent or deereased amounts of the surface GP V in addition to the absent or severely reduced amounts

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of the OP Ib/IX complex. The absence of both OP Ib and OP IX glycoproteins has been confirmed with monoclonal antibodies. 86 ,87 These observations suggest either a genetic absence of multiple proteins or, more likely, a genetic absence of a single protein necessary for the expression of the protein complex. The syndrome can be diagnosed using a monoclonal antibody-binding assay. 80 This assay uses whole blood, which avoids the difficulty of isolating platelets from these patients. Plow cytometry using the GP Ib/IX complexspecific monoclonal antibody SZ2 has been used to identify homozygous Bernard-Soulier patients, and presumably this technique could be used to identify heterozygote carriers. 49 Michelson 88 has shown the absence of OP Ib on Bernard-Soulier platelets and of OP IIb/lIla on thrombasthenic platelets. In all the cases studied, no subpopulations of platelets were detected. In contrast, this same study showed that the platelets from six children with chronic myeloid leukemia had distinct subpopulations of platelets that either did or did not carry OP Ib and OP IIb/lIla. Application of this technology may be useful for investigating heterogeneity of congenital and acquired disorders of platelet function. Pollowing activation, platelets undergo morphological and chemical changes critical to their role in hemostasis. It would be useful clinically to identify the surface molecular changes in those abnormalities where platelet activation occurs. Not surprisingly, there has been considerable effort in studying activation antigens on platelets with activation-dependent monoelonal antibodies. Using flow cytometry, as little as 0.8% activated platelets in a sample can be detected, and only microlitre volumes of blood are required. 89 The method possesses many advantages over the more conventional assays used for investigating platelet activation in patients with disorders such as adult respiratory distress syndrome or those undergoing cardiopulmonary bypass. 69 In a study to investigate platelet activation in patients who have had cardiopulmonary bypass, a significant reduction in the binding of a monoclonal antibody to OP IIb/ lIla complex was observed. 90 However, there was no increase in the expression of TSP and the n-granule membrane protein OMP-l40, suggesting insufficient activation to initiate degranulation. Activation-specific monoclonal antibodies may be useful in assessing changes in platelet conditions

HORSEWOOD, SMITH, AND KELTON

during storage and therefore their suitability for transfusion. 91 Thrombus imaging with radiolabeled monoelonal antibodies to platelet-specific markers has potential for the diagnosis of thrombotic vascular disease. Preliminary work in this direction has been reported using an anti-GP I1b/lIIa monoelonal antibody labeled with 122In in dogs. 92 Experimentally induced thrombi were visualized by radioimmunoscintigraphy, and the method offered advantages over alternative techniques. In another study using a 112ln-bound anti-OP IIb-specific monoclonal antibody, in vivo labeling of platelets was performed on six patients. 93 In two patients with known recent thrombi, positive scintigraphy imaging was observed, whereas a third patient, who had an old thrombus, was negative. Subsequent studies by the same group of investigators have further substantiated the clinical utility of this approach. 94 The detection and identification of antiplatelet autoantibodies and alloantibodies will lead to an improved understanding of the pathogenesis of immunologically mediated platelet disorders. In this respect, the development of capture assays to detect pathogenic antibodies directed against specific platelet proteins offers advantages over older assays that can only measure IgO on or inside the platelets. Capture assays use immobilized monoelonal antibodies to specifically bind platelet surface proteins. The bound protein is then used as a target antigen to look for the presence of autoantibodies of interesL The first assay of this type was used by WOodS et al 95 to detect autoantibodies against the OP IIblllIa complex in patients with idiopathic immune thrombocytopenia (ITP). Subsequently, Kiefel et al96 have modified the technique, which is termed monoelonal antibodyspecific immobilization of platelet antigens (MAIPA). In this method, platelets are sensitized simultaneously with the human serum of interest and a specific monoclonal antibody. The sensitized platelets are washed and solubilized and the resulting complexes bound to microtiter plates via immobilized anti-mouse immunoglobulin. Bound complexes are detected with an enzyme-Iabeled anti-human immunoglobulin. Using monoclonal antibodies specific for platelet antigens and platelets of known phenotype, antibodies of known epitope and serological specificity can be identified. A potentially simpler and more direct com-

169

ANTIPLATELET MONOCLONAL ANTIBODIES

petitive assay for serological typing could be performed using monoclonal antibodies with specificity for an alloantigen-dependent epitope. To date, only one monoclonal antibody of this type has been reported, with specificity for the native form of PIAl (Zwa ) on platelets. 24 More recently, monoclonal antibodies have been prepared that are able to differentiate PLA allotypes.97,98 Using either enzyme immunoassay or immunoblot assay, one antibody, termed LK4, was able to differentiate PLAl homozygous platelet extracts from PI A2 homozygous and PLAl/PLA2 heterozygous extracts. However, using intact platelets and flow cytometry, the antibody was unable to differentiate between allotypes, reacting equally with both PLAl and PLA2 homozygous platelets. 97 The antithrombotic therapeutic potential of monoclonal antibodies has been studied by two groups of investigators in animal systems. Early studies by Coller,99,IOO using antibody 7E3 in dog and monkey models, had shown inhibition of both platelet aggregation and thrombus formation, with-

out adverse effects such as bleeding or thrombocytopenia. Hanson et al lOl infused anti-GP Ub/lila complex-specific antibodies into baboons and assessed various platelet-dependent functions in addition to measuring platelet deposition on implanted Dacron vascular grafts. After antibody infusion, bleeding times were prolonged, platelet aggregation responses were absent or reduced, and graft-associated platelet thrombus formation was reduced. As a preliminary step to the use of monoclonal reagents in humans, a study was performed on a corpse maintained on a respirator. 102 Low doses of the F(ab')z fragment of an anti-GP Ub/lIla complex monoclonal antibody caused inhibition of aggregatory responses with no evidence of hemorrhage. Currently, studies are in progress to assess the use of "humanized" chimeric monoclonal antibodies as therapeutic anti-thrombotic reagents. These antibodies retain the specificity of the parent murine monoclonal antibody and incorporate human framework sequences in hopes of avoiding antimurine antibody formation in recipients.

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HORSEWOOD, SMITH, AND KELTON

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ANTIPLATELET MONOCLONAL ANTIBODIES

51. pisciieł KD, Bluestein HG, Woods VL: Platelet glycoproteins la, Ic and IIa are physiochemically indistinguishable from the very late activation antigens adhesion-related proteins of Iymphocytes and other celltypes. J Clin Invest 81:505-513, 1988 52. Santoro SA, Rajpara SM, Staatz WD, et al: Isolalion and characteńzation of a plate1et surface collagen binding complex re1ated to VLA-2. Biochem Biophys Res Commun 153: 217-223, 1988 53. Nieuwenhuis HK, Akkermann JWN, Houdijk WPM, et al: Human blood platelets showing no response to collagen fail to express surface glycoprotein la. Nature 318:470-472, 1985 54. Kunicki TJ, Nugent DJ, Staats SJ, et al: The human fibroblast c1ass II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein laJlla complex. J Biol Chem 263:4516-4519, 1988 55. Piotrowicz RS, Orchekowski RP, Nugent DJ, et al: Glycoprotein lellla functions as an activation-independent fibrone...tin receptor on human platelets. J Celi Biol 106:13591364, 1988 56. Sonnenberg A, Moddermann PW, Hogervorst F: Laminin receptor on platelets is the integńn VLA-6. Nature 336: 487-489, 1988 57. Hemler ME, Crouse C, Takada Y, et al: Multiple very late anligen (VLA) heterodimers on platelets. J Biol Chem 263: 7660-7665, 1988 58. Asch AS, Barnwell J, Silverstein RL, et al: Isolalion of the thrombospondin membrane receptor. J Clin Invest 79:10541061, 1987 59. Silverstein RL, Asch AS, Nachman RL: G1ycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 celi adhesion. J Clin Invest 84:54-552, 1989 60. Legrand C, Thibert V, Dubemard V, et al: Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: Modulation of platelet aggregalion. Blood 79:1995-2003, 1992 61. Tuszynski GP, Kowalska MA: Thrombospondininduced adhesion of human platelets. J Clin Invest 87:13871394, 1991 62. Barnwell JW, Ockenhouse CF, Knowles II DM: Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endotheIial, and C32 melanoma cells. J ImmunoI135:3494-3497, 1985 63. Ockenhouse CF, Tandon NN, Magowan C, et al: Idenlification of a platelet membrane glycoprotein as a falciparum malańa sequestration receptor. Science 243:1469-1471, 1989 64. Shibata Y, Kim N, MońtaS, et al: Monoclonal anlibody OKMS and platelet alloantibody anti-Nak(a) have the same specificity. Proc Japan Acad 66:41-46, 1990 65. McEver RP, Martin MN: A monoclonal anlibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 259:9799-9804, 1984 66. Hsu-Lin S, Berman CL, Fuńe BC, et al: A platelet membrane protein expressed duńng platelet activalion and secretion. J Biol Chem 259:9121-9126, 1984 67. Rosa JP, George JN, Bainton DF, et al: Gray platelet syndrome. J Clin Invest 80:1138-1146, 1987 68. Larsen E, Celi A, Gilbert GE, et al: PADGEM protein: A receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 59:305-312, 1989 69. George JN, Pickett EB, Saucerman S, et al: Studies on

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