Platelet membrane glycoproteins: functions in cellular interactions.

Annu. Rev. Cell Bioi. 1990.6: 329-57 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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PLATELET MEMBRANE Annu. Rev. Cell. Biol. 1990.6:329-357. Downloaded from www.annualreviews.org by Syracuse University Library on 05/19/13. For personal use only.

GLYCOPROTEINS: FUNCTIONS IN CELLULAR INTERACTIONS N.

Kieffer and D. R. Phillips

Cor Therapeutics Inc., South San Francisco, California 94080 KEY

WORDS:

platelet membrane glycoprotein, cell adhesion, adhesive protein, integrin, thrombosis

CONTENTS INTRODUCTION..............................................................................................................

329

PLATELET MEMBRANE GENE FAMILIES . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . .... ... . . . . . . . .......... . . . . . . . . . .........

331

THE INTEGRIN GENE FAMILY .. .. .. . . ............. . ............ . . . .............. . ... ........ . . . . ......... . . .............

332

Glycoprotein lIb-IlIa . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . The Vitronectin Receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycoprotein Ia-/Ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Fibronectin Receptor . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . ... . . . . . . . . . . . The Laminin Receptor . . . . . . . . . . . . ....... . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(

) GENE FAMILy...................................................

THE LEUCINE-RICH GLYCOPROTEIN LRG

The Glycoprotein Ib-IX Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycoprotein V . . . . .. . . . . . . . . . ... . . . . . . . . . . . . . .. ... . . . . . . . .......... . . . . . . . .......... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . THE SELECTIN GENE F AMILY ..... . .... . . . ........ . . . . . .. . ....... . . . ............. ... . . . .......... . . . . .................. .

333 341 342 342 343 343 344 347

Granule Membrane Protein 140 . . . . . ..... . . . .... . . . . . . . . . . ... . . . . . . . . . . ..... . . . . . . . . . . ........ . . . . . . . . .........

347 348

THE IMMUNOGLOBULIN GENE SUPERFAMILY . . ......... . . . . . . ........ . . . . .... . . .......... . . . . . . . ... ..... . . . . . . . .

348

PECA M-1... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

OTHER PLATELET PROTEINS ............................................................................................

349

SUMMARY AND CONCLUSION ..............................•.............................•.............................

350

INTRODUCTION

In most mammals blood platelets initiate and provide the structural basis for the hemostatic plug responsible for the primary arrest of bleeding. In 329 0743-4634/90/1115-0329$02.00

330

KIEFFER & PHILLIPS

avian species this function is performed by thrombocytes, which can be viewed as nucleated platelets; in still lower animals (e.g. Iimulus) by cells termed amoebocytes, which are multifunctional and not only prevent blood loss, but also carry oxygen and eliminate foreign substances. Inherited defects in platelet function have been described that cause several different bleeding syndromes. Platelets in man can, in addition, be involved in the formation of thrombi within arteries that generate high shear, thereby initiating such life-threatening conditions as acute myocardial

Annu. Rev. Cell. Biol. 1990.6:329-357. Downloaded from www.annualreviews.org by Syracuse University Library on 05/19/13. For personal use only.

infarction, unstable angina, and thrombotic stroke. Thus, while platelets are essential for the maintenance of life, they are also involved in important genetic and pathological disorders. A variety of platelet interactions are required for hemostasis and throm bosis processes to occur. These include (a) adhesions to extracellular matrix proteins (e.g. von Willebrand factor, collagen, fibronectin, vitronectin, laminin), (b) aggregation with each other to make a platelet plug, and

(c)

heterologous adhesions to other cells. Because platelet functions depend primarily on adhesive intcractions, it is not surprising that most of the glycoproteins on the platelet membrane surface are receptors for adhesive proteins, or otherwise mediate cellular interactions. Many of these recep tors have been identified; most of these have been cloned and sequenced and can now be classified within larger gene families, which mediate a wide variety of cellular interactions. Thus interactions of platelets are specialized adaptations of adhesive mechanisms used by a wide variety of cells. An understanding of these platelet membrane adhesion receptors and their mechanisms of action is applicable not only to an understanding of normal platelet function, to the characterization of genetically abnormal platelet function where adhesive functions are altered, and to the design of thera peutic agents to control life threatening platelet-initiated thrombosis, but also to an understanding of the adhesive mechanisms used by many cells to form physical interactions with their environment. This review summarizes recent information on the structure of platelet membrane glycoproteins, the role of these glycoproteins in mediating platelet interactions, and the relationship of these glycoproteins to larger gene families of membrane receptors involved in cellular adhesions. Unless otherwise indicated, this information will refer to human glycoproteins, as this has been the species most studied. Where understood, genetic defects in these proteins in platelets will be identified. Characterization of these defects will become increasingly relevant to understanding the structure function relationships of gene families of adhesion receptors. Finally, reagents currently evaluated for their therapeutic regulation of platelet interactions will be discussed. These reagents may constitute a new class of antithrombotic agents.

PLATELET MEMBRANE GLYCOPROTEINS

331

PLATELET MEMBRANE GENE FAMILIES Surface labeling of intact platelets has shown that the platelet membrane glycocalyx is composed of numerous glycoproteins (Phillips & Agin 1977). Most of these proteins have now been cloned and sequenced and can be classified within known gene families (Figure

1). Perhaps the most abun

dant is the integrin family, which includes, in order of decreasing amounts, glycoproteins GP lIb-IlIa, GP Ta-ITa, GP Tc-ITa, the fibronectin receptor, and the vitronectin receptor. As shown in Table I, these integrins on


platelets serve as receptors for a variety of extracellular matrix and adhesive proteins and mediate such f undamental processes as aggregation, activation by collagen, and adhesion to fibronectin. Another gene family present in platelet membranes is the leucine-rich glycoprotein (LRG) fam ily represented by the GP Ib-TX complex and GP V. The GPlb-IX complex is a receptor for von Willebrand factor on unstimulated platelets and mediates adhesion of platelets to the subendothelium. Yet other gene families with less defined functions include the family of selectins rep-

Integrins

GP lib-lila

Vttronectm receptor Fibronectln receptor

--_ - -o:::D

Laminln receptor

----

GPla-lia

Leucine rich glycoproteins (LRG)

GP IX GP Ib

�

GP Ib"

Immunoglobulin domain molecules

PECAM-I HL A class 1

� 2 mJcroglobulin

Selectins

GMP 140

CD 36

GP IV

� Cysteln rich repeats

n •

-1J Figure I

Immunoglobin-Ilke domain LRG repeat

Cd

Lectin-hke domain

_

I domain

#

EGF-Ilke domain

IIDIDDI Cystein rich domain

Complement regulatory protein repeal

Schematic representation of platelet cell surface glycoproteins belonging to

ditTerent gene families.

3 32

KIEFFER & PHILLIPS

Table 1

Platelet membrane glycoproteins and their receptor function

Glycoprotein GP lIb-IlIa (rx"bp J)

Receptor

Platelet function

Fibrinogen, von Wille brand factor

Aggregation

Fibronectin, thrombospondin and

Adhesion at high shear rales

vilronectin

Vitronectin receptor (rx'P,) Vitronectin, von Wille brand factor

Adhesion

Fibronectin, fibrinogen and thrombospondin


GP la-lia (rx2P,) GP Ic-I1a

(rx5fJ ,)

Collagen

Adhesion

Fibronectin

Adhesion

GP Ic'- l l a (rx6f3,)

Laminin receptor

Adhesion

GP Ib-IX

von Willebrand factor and

Adhesion

thrombin GPV

Thrombin substrate

GP IV (GP IIIb)

Thrombospondin and collagen

Adhesion

GMP-140 (PADGEM)

?

Platelet-leukocyte

PECA M - l (GP ITa)

?

?

inte ractio n

resented by G MP-140, the immunoglobulin gene family with the HLA class r antigen, and the cell adhesion molecule (CAM ) related PECAM- l . The major platelet glycoprotein, which appears unrelated to any other known gene family, is GP IV (IlIa). A comparison of these glycoproteins to the total platelet glycoproteins characterized by surface labeling indi cates that the glycoproteins identified in Figure I constitute more than 75% of the glycoprotein mass on the platelet membrane surface. THE INTEGRIN GENE FAMILY

Integrins constitute a large family of related ap heterodimers, which are widely distributed on many cells and are involved in cell-matrix or cell cell interactions. Distinct but homologous P subunits termed PI, P2, P , 3 P4, and P5 have been identified, each of which is capable of associating individually with one or more a subunits (Hynes 1987 ). Integrins with the p, subunit were initially described on activated lymphocytes and were termed the very late activation antigens (VLA) (reviewed in Hemler 1990). They include seven known proteins that can serve as receptors for fibronectin (rt.,3p" a4p" a5p" rt.,vPI), collagen (a'p" rt.,2p" rt.,3PI), laminin (alp" a2p" rt.,6PI), and vitronectin (aVPI) (reviewed in Hemler 1990; Ruoslahti &

PLATELET MEMBRANE GL YCOPROTEINS

333


Giancotti 1990). Integrins with the f32 subunit are found only on leukocytes and comprise three receptors, LFA-I, Mac- I, and p 150,95, which mediate many adhesive interactions of these cells (reviewed in Arnaout 1990). Integrins with the f33 subunit are GP IIb-lIla (If.lIbf33) and the vitronectin receptor (If.vf33) (reviewed in Phillips et aI 1988). GP lIb-IlIa is restricted to platelets and other cells of the megakaryocytic lineage, while the vitronectin receptor is expressed in several hematopoietic cells such as platelets and macrophages as welI as in a broad range of nonhematopoietic cells. One each of the /34 and f3 5 integrins is known. Glycoprotein lIb-lIla

Normal platelets contain about 50,000 molecules of GP lIb-IlIa ( I to 2% of the total platelet protein), which are randomly distributed on the surface of resting platelets (Isenberg 1987). The GP JIb-rna complex is composed of one molecule of GP lIb (Mr 140,000), which consists of a large ( Mr 125,000) chain disulfide linked to a light chain (Mr 22,000), and one molecule of GP TIIa (Mr 150,000), which is a single polypeptide chain (Jennings & Phillips 1982). The GP Ilb-IlIa complex is present as a Ca 2+ -dependent heterodimer, noncovalently associated on the platelet surface (Fitzgerald et al 1985). Figure 2h shows the structure of purified GP lIb-IlIa as viewed by rotary shadowing electron microscopy (CarrelI et al 1985). The heterodimer consists of two domains, a globular head of 8 x 10 nm, and two rod-like tails extending 15 nm from one side of the globular domain. The diagram of GP lIb-lIIa in Figure 3 depicts how the primary amino acid sequence can be accommodated within this structure. The mature GP IlIa polypeptide consists of 762 amino acids and contains a 29-residue transmembrane domain and a 4 1-residue cytoplasmic tail (Fitzgerald et al 1987b). Because the tips of the tails contain the membrane inserting hydrophobic sites, it would appear that one of them contains the carboxy terminus of GP IlIa. The extracellular domain consists of amino acid residues I through 692 and contains 56 cysteines, all of which appear to be disulphide-linked. Thirty-one cysteine residues are clustered in four tandemly repeated segments of about 40 amino acids each. The amino acid sequence of GP IlIa is 45 and 39% homologous to the f31 and f32 integrin subunits, respectively. Regions of strong identity include 12 resi dues on either side of the transmembrane domain, the cytoplasmic domain, and a region towards the N-terminus extending from residue 110 to 350. Within this latter region, six segments of 14 to 38 amino acids are highly conserved, which suggests that these regions are of functional importance. The two chains of GP lIb are post-translationally cleaved from a single precursor, which remains disulfide linked. The large chain of mature GP IIb is formed of 871 amino acids and the light chain of 137 (Poncz et al =

=

=

=


334

KIEFFER & PHILLIPS

J Figure 2

,

Electron micrographs of rotary shadowed molecules of human fibrinogen, von

Willebrand factor, GP IIb-ITTa and GP lb-IX. Selected examples are shown with the inter pretive drawings of the micrographs beneath. A: fibrinogen

(x 200,000);

(x 200,000);

B: GP lib-lIla

C: von Willebrand factor: an internal section with four subunits of the von

Willebrand factor multimer is shown

(x 1 50,000); D: GP lb-IX (x 1 25,000).

1987). Only the light chain has a transmembrane domain, 26 residues long, and would appear to constitute the second tail on the complex. The large chain contains four repeating segments of about 65 amino acids, each of which contains a l 2-amino acid sequence characteristic of the Ca 2 + -bind ing f3 turns of calmodulin. Ca 2+ is required for maintenance of GP lIb and GP IlIa in the heterodimeric complex and for binding of adhesive proteins. Each cysteine residue of GP lIb (beginning at residue 56) is disulfide-bonded to its nearest neighbor in the amino acid sequence and forms relatively small disulfide loops. Since the cysteine residues of integrin IX subunits are conserved, it would appear that the 4isulfide bond patterns are most probably also conserved (Calvete et al 1989; Beer & Coller 1989). RECEPTOR FUNCTION OF GP lIb-IlIa On activated platelets, the GP lIb-IlIa complex functions as a receptor for fibrinogen, fibronectin, von Willebrand factor, vitronectin, and thrombospondin (reviewed in Plow et aI 1986). GP

PLATELET MEMBRANE OLYCOPROTEINS

FIBRINOGEN

G P lib-IlIa

f)� C\ ·

/

RGD(I09.171) ......,

Ca

Ca

Fibrinogen (211·222)

N

I

C

\


c� DOOecapepti.de (294-314)

N

GPIIIa

GPIIb

,WF (251-279)

� X, ;I

--- I NA

+Aa.Chain C

�ll ..

GP lIb-lIla Bmdmg

C

+BaChain

'-fl.,�.

. {+ (RGD) Sites @ (Dodecapeptide)

VON WILLEBRAND FACTOR

GP Ib - IX Thrombin�

335

GPlba

�� !�Il

GP Ib Binding Sile

GPIIb-JIla Binding Site

� (449·728)

• (RGD)

'-

OLeucineltklJRepeat

c

Figure 3

Schematic representations of the structures and ligand binding sites of OP IIb

nIa and GP Ib-IX, and the structures and receptor binding sites of fibrinogen and von Willebrand factor. GP lIb-lIla is depicted with the typical head and tail morphology illus trated in Figure 2: the transmembrane domains are near the carboxyl termini of GP lIb and GP lIla. The three chains of the GP Ib-IX complex are depicted, each with a transmembrane domain. The structure of fibrinogen depicts its trinodular structure with the amino termini of the six chains in the central nodule. A portion of the filamentous polymeric von Willebrand factor is shown. Fibrinogen has three GP lIb-IlIa binding sites: two RGD sequences on the ct-chain and the dodecapeptide at the carboxy terminus of the y chain. Von Willebrand factor also has an ROD sequence that is used to bind to GP lIb-IlIa, plus a sequence included within amino acids 449-728 that mediates binding to GP lb-IX. Cross-linking studies show that RGD peptides cross-link to a domain within amino acids 1 09- 1 7 1 of GP IlIa, whereas the dodecapeptide from the y chain of fibrinogen cross-links to a domain within residues 244-3 1 4 of GP IIbct. Another region involved in fibrinogen-binding activity of GP lIb-IlIa corresponds to residues 2 1 1-222 of GP IlIa. The von Willebrand factor binding domain of the GP Ib-IX complex encompasses amino acids 251 -279 of GP lbct, whereas thrombin binds to the N-terminal 45-kd fragment of GP Ibct, which contains the 7 leucine-rich repeats.

lIb-IlIa binds the trinodular fibrinogen (Figure 2a), or the filamentous von Willebrand factor (Figure 2c) to mediate platelet aggregation; the binding of the other proteins may additionally allow platelet adhesion and spreading. Recent studies have characterized molecular properties of ligand interaction with GP lIb-IlIa. These include identification of sites in fb i rinogen


336

KIEFFER & PHILLIPS

sites for fibrinogen thrombasthenic patients with abnormal receptor function of GP lIb-lIla. Because of the large size of the adhesive proteins, it was not anticipated that the receptor recognition sequences could be localized to small peptide sequences. Pierschbacher & Ruoslahti (1984), however, identified one of these sequences when they found that the Arg-Gly-Asp (RGD) sequence in fibronectin mediates cellular adhesion. This integrin recognition sequence is now known to be present on numerous adhesive proteins including fibrinogen, von Willebrand factor, thrombospondin, laminin, vitronectin, and collagen (Ruoslahti & Pierschbacher 1987). The pro miscuous capability of GP IIb-Illa to bind to many adhesive proteins is due to its ability to bind to the RGD sequence contained within them. Equilibrium binding studies have shown that GP lIb-IlIa contains one RGD-binding site which reversibly binds RGD-containing peptides with many ofthe properties of fibrinogen et al 1989). Fibrinogen contains two RGD sequences in its \I. chain, one near the N-terminus (residues 95-97) and a second near the C-terminus (residues 572- 574) (Doolittle et al 1979) (Figure 3). lmmunoinhibition experiments show that fibrinogen primarily uses the C-terminal RGD sequence to bind GP IIb-Illa (Cheresh et al 1989). A second site on fibrinogen that binds to GP lIb-IlIa has also been identified. This is the 12-amino acid sequence located at the carboxyl-terminus of the y-chain of fibrinogen (Kloczewiak et al 1984) (Figure 3). This dodecapeptide is not found in other adhesive proteins and does not contain the RGD sequence, but competes with RGD-containing peptides for binding to GP lIb-IlIa (Lam et al 1987). Peptides containing either RGD or dodecapeptide sequences inhibit the binding of fibrinogen, von Willebrand factor, and fibronectin to the GP lIb-lIla complex on activated platelets and therefore block platelet aggregation (Plow et al 1985; Gartner & Bennett 1 985; Kloczewiak et al 1984, 1 989). Several approaches have been used to identify the fibrinogen-binding domains on GP lIb-IlIa (Figure 3). In one approach, a monoclonal anti body that inhibited fibrinogen binding and platelet aggregation was shown to bind to an epitope on the N-terminus of GP IlIa, which suggests that this region ofGP IlIa contains a fibrinogen-binding domain (Calvete et al 1988). This domain was further localized by a second approach, which directly cross-linked labeled RGD-containing peptides to GP IlIa in the complex (Santoro & Lawing 1987); proteolytic digests further localized this site within a peptide bounded by residues 109 through 171 (D'Souza et al 1988). This segment is highly conserved among the f3 subunits of the integrin gene family (76% sequence identity), which indicates that integrins containing other f3 subunits might use a homologous site to bind the RGD



337

sequence on adhesive proteins. An additional study identifies this domain through analyzing sequences of naturally occurring integrin mutants. Glanzmann's thrombasthenia is a heritable bleeding disorder with an autosomal recessive mode of inheritance caused by a deficit of platelets to bind to adhesive proteins. Ginsberg et al (1 986) have identified where the GP lIb-IlIa complex in their platelets has a specific the binding of RGD-containing peptides. Characterization of this gene revealed a single point mutation, resulting in an aspartic acid to tyrosine substitution in residue 11 9 of GP lIla; a finding idea that GP IlIa contains the RGD-binding site ( Loftus et al 1 989). In contrast to RGD peptides, the y chain dodecapeptide crosslinks primarily to GP lIb (D'Souza et al 1990). The crosslinking site has been localized to a sequence bounded by residues 294-314 located within the second putative calcium-binding domain of GP IIb, a highly conserved sequence in integrin IX subunits. The third approach used to pinpoint fibrinogen contact sites on GP lIb-IlIa measured the abilities of synthetic peptides based on the sequences of GP IlIa to inhibit fibrinogen IlIa (Charo et al 1 989). These studies found that the linear sequence representing residues 211 through 222 of GP IlIa inhibits fb i rinogen ing as do antibodies generated against this sequence, thus indicating that this sequence is critically involved in the ligand-binding activity of GP Ub IlIa. Since many integrins bind adhesive proteins through their RGD sequences (e.g. the vitronectin receptor, the fb i ronectin receptor, integrin, Mac-I, and GP lIb-IlIa), it would appear that additional sites, such as those identified integrin interactions. Platelet aggregates are not only responsible for normal hemostasis; they also initiate a variety of life-threatening thrombotic complications. The primary work of Coller & colleagues using 7E3, a monoclonal antibody against GP lIb-IlIa that blocks fb i rinogen that blocking GP lIb-IlIa-mediated platelet aggregation inhibits thrombus formation in the comparatively high shear coronary and carotid arteries (Coller et al 1989) and facilitates the activities of fibrinolytic agents in opening occluded arteries (Gold et aI 1988). Recent studies have identified new classes of inhibitors of GP lIb-IlIa function that have provided potential alternative strategies for developing antithrombotic agents (Pier schbacher & Ruoslahti 1987). One class of inhibitors comprises synthetic peptides that are 7 to 10 amino acids long and contain variations in the RGD sequence. Another class of inhibitors is found in many snake venom toxins (see Shebuski et al 1 989). These are highly disulfide-cross-linked pep tides of 48 to 83 amino acids long that appear to have high activity because of the proper display of the RGD sequence at their active sites.

338

KIEFFER & PHILLIPS


These and other GP lIb-IlIa antagonists may provide the foundation for a new class of potent anti-thrombotic agents. ROLE OF GP JIh-IIJa IN SIGNAL TRANSDUCTION Although it has been known for several years that the receptor function of GP lIb-IlIa is activated by stimulus-response mechanisms occurring during platelet activation, recent data indicate that the binding of adhesive proteins to GP lIb-lIla initiates additional events. RGD peptides and the dodecapeptide induce marked conformational changes with detergent solubilized GP I1b-IIIa (Parise et al 1987). For GP IIb-IIIa in intact platelets, this change in conformation upon ligand binding can be demonstrated by the expression of new epitopes that have been referred to as ligand-induced binding sites or LIBS. LIBS epitopes exist on both GP IIb and GP IlIa, which indicates that the conformation of both glycoproteins in the complex is affected by ligand occupancy (Frelinger et a1 1988, 1990). Ligand-receptor interactions also induce clustering of GP lIb-IlIa (Isenberg et al 1987). Other effects of ligand binding on GP lIb-IlIa include an increase in tyrosine-specific protein phosphorylation (Ferrell & Martin 1989) and an increase in Na + /H+ exchange in epinephrine-stimulated platelets (Banga et al 1986). Aggregation additionally causes an increase in the phosphorylation of threonine residues ofGP IlIa (Parise et al 1990) and the association ofGP lIb-IlIa with the platelet cytoskeleton (Phillips et aI 1 980). Taken together, these data indicate that the conformational changes induced by the binding of adhesive proteins to GP lIb-lIla generate signals affecting metabolic activities within the cell. BIOSYNTHESIS AND PROCESSING OF GP IIb-IIla The pathway leading to the surface expression of the GP lIb-IlIa complex is characteristic for integrin biosynthesis (Bray et al 1986; Duperray et a11989; Rosa & McEver 1989). GP lIb is synthesized as a single polypeptide precursor. This pro-GP JIb, which carries high mannose oligosaccharides, associates with GP IlIa and is cleaved to yield the small and large chain ofGP lIb. The oligosaccharides ofGP lIb are subsequently converted to complex-type carbohydrates, and the complex is expressed on the membrane surface. No precursor form of GP IlIa has been identified, and mature GP IlIa seems to carry non processed high mannose-type oligosaccharides. Transient expression of recombinant GP lIb and GP IlIa in C OS cells (O'Toole et al 1989), or . human embryonic kidney cells (Bodary et aI1989), and stable transfection ofGP JIb in M-21L cells (Kieffer et al ]990) also showed that the GP IIb lIla complex must assemble and be processed in the endoplasmic reticulum before it is expressed on the plasma membrane. Understanding the pathway of GP lIb-IlIa biosynthesis is essential to the understanding of Glanzmann's thrombasthenia. Although the Glanz-



339

mann phenotype is most likely caused by a heterogenous array of genetic defects, the requirement for assembly of both subunits for surface expres sion suggests that a defect in only one subunit would prevent the for mation of the complex and its expression at the membrane surface. In several thrombasthenic patients characterized by a complete absence of surface expressed GP lIb-IlIa, trace amounts of GP lIb and GP IlIa or GP IlIa alone have been detected by immunoblotting (Nurden et al 1985; Coller et al 1987). In an additional patient, normal expression of the vitronectin receptor could be detected in endothelial cells even though platelets lacked GP lIb-lIla (Giltay et al 1987). In the latter patient this expression suggests a defective synthesis of GP lIb. A cellular model for this type of Glanzmann's thombasthenia is a human melanoma cell line M21-L, which has an intracellular pool of free GP IlIa in the absence of both GP lIb and the vitronectin receptor IJ. chain (Cheresh & Spiro 1987). Whe n these cells are transfected with GP IIb eDNA, the endogenous f33 subunit is rescued and expressed on the cell surface as a functional GP lIb-IlIa complex (Figure 4) (Kieffer et aI1990). These results demonstrate that gene therapy is feasible in treating Glanzmann's thrombasthenia and other related genetic disorders (Hibbs et al 1990) caused by a low level of integrin expression. GP IIb·IIIa GENE STRUCTURE The genes encoding GP lIb and GP IlIa are contained within a single 260 kb segment in the q21-23 band on chro mosome 17 (Bray et al 1988). The entire coding unit for the mature GP IlIa protein spans 60 kb of DNA sequence and contains 14 exons (Lanza et aI1990). Several structural domains of the GP IlIa protein are contained within individual exons, including the transmembrane domain, the cyto plasmic region with the potential phosphorylation site, and the six domains in the N-terminal half of GP IlIa that are highly conserved between the two other integrin p subunits. Other domains, such as the four cysteine rich repeats, are interrupted by introns. The gene for the P chain of the fibronectin receptor was found to have a similar exon distribution thus providing genetic evidence for the common evolutionary origin of the integrin subunits. An alternative spliced form of GP Ilia has been isolated from a placental cDNA library and differs from the initially reported P 3 subunit in the cytoplasmic domain by having an alternative 1 3-amino acid C-terminal peptide, which has no sequence homology with P3 or the other published P subunits (van Kuppevelt et aI1989). These 13 amino acids are encoded by an intron sequence contiguous to exon M, while the OP Ilia sequence is generated through the use of a splice site between nucleotides 2270 and 2271 of the published sequence. The potential functional import ance of the alternative spliced form of GP IlIa is underlined by the identi-

340

KIEFFER & PHILLIPS 100

A.

B.

c.

D.

75 50


�(.)

0 � .Q E � t: '"

25

0 100

.�

�

&!

75

50 25

0 0.1 10 100 Fluorescence intensity [Log] Figure 4

Phase-contrast micrographs of mock-transfected and GP IIb-transfected M21-l

melanoma cells plated on fibrinogen-coated microtiter plates. Panel B shows mock-trans fected M21-L cells that are unable to attach and spread on fibrinogen-coated surfaces; panel

D shows GP IIb-transfected M21-L cells that have acquired the ability to attach and spread on fibrinogen. F[ow cytometry analysis of cell surface expression of GP IIb-nla as monitored with monoclonal antibody A2A9 is displayed in panel A and C. Panel A: mock-transfected

M21-L cells; panel C: M21-L cells transfected with GP IIb eDNA.

fication of a similar splicing variation in the integrin fil subunit (van Kuppevelt et al 1989). The gene encoding GP lIb spans 17 kb and contains 30 exons (Hei denreich et aI 1990). Interestingly, the exon demarcations do not correlate with potential functional domains, in contrast to the gene encoding the pJ50,95 rJ. integrin subunit, where each of the three divalent cation-binding repeats is encoded by a separate exon (Corbi et aI1990). The 5/ end of the GP lIb gene is devoid of canonical TATA and CAAT boxes, which suggests that for GP lIb gene expression, transcription is initiated by another cis-acting sequence. Recent data have shown that a core sequence 5'-


341

TGATAA-3/, located at - 55 in the GP lIb promoter and recognized by GF-l (NF-El ), the principal DNA-binding protein of the erythroid lineage, is involved in transcription of the GP lIb gene. GF-l, which is expressed in erythroid cells as well as in megakaryocytic cells, may thus function as a transcriptional regulator of both erythroid- and mega karyocytic-specific genes (Romeo et al 1990; Martin et aI 1990).


The Vitronectin Receptor The vitronectin receptor (VnR, aVfJ 3) is a minor platelet integrin (Lam et al 1989). The fJ subunit of the vitronectin receptor is identical to GP lIla; the a subunit has 36% sequence identity to GP lIb and is predicted to have a similar structure (Fitzgerald et al 1987a). Like GP lIb-lIla, the vitronectin receptor is a promiscuous receptor that binds a variety of RGD-containing, adhesive proteins such as vitronectin, fibronectin, fibrinogen, von Willebrand factor (Cheresh & Spiro 1987), and throm

bospondin (Lawler & Hynes 1989). The RGD-binding sites appear similar; photoaffinity cross-linking studies of a \ 25I-RGD-containing peptide to the virtonectin receptor identified one domain on the fJ subunit (residue 61-203), which overlaps with the 109-171 sequence identified on the fJ subunit of GP lIb-lIla (Smith & Cheresh 1988). Two distinct sites on the ex subunit of the VnR have been identified as ligand-binding sites that encompass residues 139 to 349 of the highly conserved putative divalent cation-binding sites (Smith & Cheresh 1990) and include the sequence identified by D'Souza et al (1990) as a ligand-binding site. Despite the structural similarities and the apparent identity of the RGD-binding site of the vitronectin receptor and GP lIb-IlIa, differences in ligand-receptor interactions individualize the two receptors and thus reflect specialized functions. The VnR receptor function is constitutive and allows it to function essentially as a cell adhesion receptor, while the receptor function of GP IIb-I1Ia in platelets is only manifest following cell activation. Although the binding of fibrinogen to the vitronectin receptor is also mediated by the C-terminal RGD sequence of the fibrinogen ex chain, the y chain dodecapeptide is apparently not used (Cheresh et al 1989). Differences in ligand affinity of both receptors have also been observed. Soluble vitronectin binds with high affinity to the vitronectin receptor, but poorly to GP IIb-I1Ia immobilized on microtiter plates (I. Charo, personal communication). These differences are reflective of cellular adhesions. HEL cells normally express GP IIb-lIla, but not the vitronectin receptor, and are therefore unable to attach to immobilized vitronectin. A variant of this cell line, however, which also expresses the vitronectin receptor, termed HEL-ADI cells, attaches and spreads on vitronectin and adopts

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a fibroblastoid-type morphology (Kieffer & Phillips 1990). On human platelets, the vitronectin receptor may thus function as an activation independent receptor for platelet attachment, spreading on vitronectin and possibly other RGD-containing adhesive proteins.


Glycoprotein Ia-Ila

Three {J 1 integrins have been identified on human platelets. One is GP la lla (a2{J I ) , which is identical to VLA-2 and exists at about 2000 copies per platelet (Pischel et al 1988; Hemler et al 1988). The primary structure of GP Ia has recently been established (Takada & Hemler 1989). The overall sequence homology of GP la with other integrin a subunits is 18-25%, but contains a similar distribution of cysteine residues, metal-binding domains, and transmembrane domain. A major characteristic of GP Ia is the presence of the 191 residue I domain insert also found in the a subunits of leukocyte integrins of the {J 2 family (see Larson et al 1989). Although GP Ia-lla is only a minor component of the platelet membrane, its role in platelet function became apparent when platelets from a patient with a mild bleeding disorder were found to be totally refractory to collagen-induced activation, defective in the adhesion to collagen , and deficient in GP Ia (Nieuvenhuis et al 1985). Subsequently, a

monoclonal antibody to GP Ia-lla was found to inhibit platelet adhesion to collagen type I and III (Kunicki et al 1988), and purified GP la-I1a was shown to bind to collagen in a Mg2+ -dependent manner (Staatz et aI1989). The GP Ia-lla binding domain on collagen lacks an RGD sequence (Staatz et al 1990). The data indicate that GP Ia-lla can account for most of the physiologic aspects of collagen binding despite observations showing that many collagen-binding proteins exist in platelets (reviewed in Santoro 1988). Although it is not known how collagen binding to GP Ia-IIa induces platelet activation, the finding that another integrin, GP lIb-IlIa (see above) affects metabolic activities of platelets following receptor occu pancy is a precedent for an integrin functioning as a signal transducing receptor. Since the I domain is 23-41 % similar to domains in cartilage matrix protein and von Willebrand factor, respectively, this sequence may be responsible for collagen-binding activity. Although GP la-lIa functions exclusively as a collagen receptor in human platelets and on fibroblasts, in other cell types it functions as a laminin receptor (Languino et al 1989; Elices & Hemler 1989). The Fibronectin Receptor

Another minor integrin in platelets with {J 1 as the {J subunit is GP Ic-IIa, which has been shown to be identical to the fibronectin receptor (a5{J a (Giancotti et al 1987; Wayner et al 1988; Piotrowicz et al 1988). The



343

functional activity of this integrin in platelets is apparent from observations showing that platelets from patients with Glanzmann's thrombasthenia, which lack functional GP lIb-IlIa, retain the ability to interact with fibronectin-coated surfaces (Piotrowicz et al 1988). This adhesion of plate lets to fibronectin does not require platelet activation and is inhibited by soluble fibronectin, antibodies to fibronectin, antibodies to the fibronectin receptor, and RGD-containing peptides. It thus appears that platelets possess two distinct fibronectin receptors, one dependent upon platelet activation (GP lIb-IlIa) (Plow & Ginsberg 1981) and another constitutive and present on unstimulated platelets (GP Ic-IIa). The Laminin Receptor

The existence of a laminin receptor on human platelets was initially sug gested by III et al (1984), who found that platelets adhered to substrate bound laminin. Interestingly, the adhesion of platelets to laminin differed from adhesion to fibronectin: platelets did not spread on laminin, whereas platelets completely flattened on fibronectin. Also the adherence of plate lets to laminin was completely independent of RGD-mediated attach ment, whereas platelet adherence to fibronectin was inhibited by RGD containing peptides. The identity of the laminin receptor on platelets was made by Sonnenberg et al (1987, 1988), who found that the monoclonal antibody GoH3, which immunoprecipitated a new heterodimeric complex from platelet lysates termed VLA6 (Hemler et al 1988), completely inhibited platelet adhesion to laminin. One feature of the VLA6 complex is that the subunits seem to be only weakly associated (Hemler et aI1988). This apparent weak a.f3 association seen forVLA6 is reminiscent ofVLA4, the other VLA integrin whose subunits are weakly associated (Hemler 1990). Mn2+, Co2+, and Mg2+ support the laminin-binding activity of this integrin, while Ca 2+ , Zn 2+ and Cu 2+ do not (Sonnenberg et al 1988). This selective cation dependency is similar to that of the GP Ia-IIa-mediated adhesion of platelets to collagen (Santoro, 1988). THE LEUCINE-RICH GLYCOPROTEIN (LRG) GENE FAMILY

The leucine-rich glycoprotein (LRG) gene family defines a new evolving family of proteins that share a common structural motif composed of a leucine-rich, 24-amino acid consensus sequence: P X X L L X X X X X L X X L X L S X N X L X X L (Takahashi e t a l 1985). Members o f the LRG gene family include such widely divergent proteins as yeast adenylate cyclase (Katoaka et aI1985), the Yersinia pestis yopM gene product (Leung

KIEFFER & PHILLIPS


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& Straley 1989), the Drosphila chaoptin (Reinke et al 1988) and Toll gene product (Hashimoto et al 1988), the human e< 2 leucine-rich serum glycoprotein (Takahashi et al 1985), the fibroblast chondroitinjdermatan sulfate proteoglycan (PG40) (Krusius & Ruoslahti 1986), the placental ribonuclease inhibitor (Lee et al 1988), the lutropin-choriogonadotropin receptor (McFarland et al 1989), CD 14 (Setoguchi et al 1989), as well as the oligodendrocyte-myelin glycoprotein (Mikol et al 1990). No common functional role has been identified for proteins of the LRG family. The LRG gene products on platelets include the a and fJ subunits of GP Ib, GP IX, and GP V.

The Glycoprotein

/b-/X Complex

The GP Ib-IX complex is the most prominent sialoglycoprotein of the platelet membrane (25,000 molecules per platelet) and contributes to the net negative charge of the platelet surface (reviewed in Clemetson, 1985). As viewed by rotary shadowing electron microscopy (Figure 2d), the GP Ib-IX complex has an elongated, 60 nm long dumbbell shape with a larger globular domain on one end used for membrane insertion and a smaller globular domain oriented externally to the plasma membrane (Fox et al 1988a). GP Ib consists of two disulfide-linked subunits, GP Iba (Mr 145,000) and GP IbfJ (Mf 24,000) and is tightly complexed with GP IX (Mf 17,000) in a I: I heterodimer (Du et a1 1987; Fox et aI1988a). GP IbfJ and GP IX, which are both transmembrane glycoproteins and form the larger globular domain, are palmitoylated through thioester linkages to cysteine residues (Muszbeck & Lapocata 1989). This pal mytoylation might be responsible for the tight but noncovalent association between GP Ibf3 and GP IX. The elongated, protruding part corresponds to GP Iba, with the smaller globular domain representing the N-terminal 45-kd fragment of GP Iba (Wicki & Clemetson 1987). The complete primary amino acid sequences of the IX and f3 chain of GP Ib (Lopez et al 1987, 1988) and GP IX (Hickey et al 1989) had been established from cDNA clones and were facilitated by studies showing that these glycoproteins were present on HEL cells (Kieffer et al 1986). The mature GP Iba protein is 6 10 amino acids long and is characterized by an extracellular domain of 485 amino acids, a transmembrane segment of 29 amino acids, and a carboxyl-terminal cytoplasmic domain of 100 amino acids long. The extracytoplasmic portion of GP Ibe< contains seven tandem repeats of the 24 amino acid LRG motif. Nine cysteine residues are present in GP Iba, all of which are located in the extracytoplasmic domain. Two cysteine residues are located immediately preceding the putative membrane-spanning segment and are likely to form the disulfide bond(s) with the fJ chain of GP lb. Indeed, trypsin cleavage of the GP Ib=

=

==


345

IX complex releases the large extracellular domain of GP Iba, termed glycocalicin, and leaves a membrane-bound fragment of GP Iba of Mr 25,000 disulfide-linked to GP Ibf) and associated with GP IX (Du et al 1987). The GP Ibf) protein is characterized by an extracytoplasmic domain of 122 residues, a 25-residue transmembrane domain, and a 34residue cytoplasmic tail. Ten cysteines are present, nine of which are in the extracellular segment. The unpaired cysteine in the cytoplasmic seg ment has a reactive thio1 group (Kalomiris & Coller 1985) and is most likely the palmitoylation site. GP IX is composed of an extracellular region of 134 residues, a 19-residue transmembrane domain, and a very short cytoplasmic tail of 6 residues. GP IX contains nine cysteine residues of which eight are found in the extracellular domain. The ninth cysteine is located within the transmembrane domain. A major characteristic of both GP Ibf) and GP IX, which share 60% sequence homology in their extra cellular domain (1. A. Lopez, personal communication), is the presence of a single 24-amino acid LRG motif in their extracellular domain. A further characteristic of GP Iba, GP Ibf), and GP IX is the presence of flanking sequences on either side of the LRG segment that resemble similar flanking segments found in other membranes of the LRG family (Hickey et al 1989).


=

RECEPTOR FUNCTION The major role of the GP Ib-IX complex to platelet function is to bind to immobilized von Willebrand factor on exposed vascular subendothelium and thus initiate adhesion of platelets to the subendothelium at the site of vessel injury. This was established initially by the study of platelets from patients with Bernard Soulier syndrome, an inherited bleeding disorder (reviewed in George et al 1984). Platelets from these individuals are defective in adhesion, do not bind von Willebrand factor, and lack the GP Ib-IX complex. In support of this function for GP Ib, monoclonal antibodies directed against GP Iba also block adhesion of normal platelets to immobilized von Willebrand factor (Weiss et aI1986). Interestingly, GP Ib does not bind soluble von Willebrand factor in plasma; apparently this protein undergoes a conformational change upon binding to the extracellular matrix that then exposes a recognition sequence for GP Ib-IX. The antibiotic ristocetin most likely is able to induce a similar change in the structure of soluble von Willebrand factor. The von Wille brand factor binding domain of the GP Ib-IX complex has been nar rowed to a sequence encompassing amino acids 251 through 279 of GP Iba (Vincente et al 1990). The GP Iba binding domain of von Willebrand factor resides in a tryptic fragment extending from residue 449 to 728 of the constituent subunit that does not contain an RGD sequence (Fujimura et al 1986). This domain is formed by residues contained in two dis-


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continuous sequences linked in the native molecule by disulfide binding (Mohri et al 1989; Andrews et al 1989). The GP Ib-IX complex has also been reported to bind thrombin (Harmon & Jamieson 1986) as quinine/quinidine drug-dependent antibodies (Berndt et al 1985). The cytoplasmic domain of the GP Ib-IX complex has a major function in linking the plasma membrane to the submembranous skeleton, a struc ture in platelets that is composed of short actin filaments linked to actin binding protein (ABP) and that functions to stabilize the plasma membrane and maintain the discoid shape of the platelet (Fox et al 1988b). In resting platelets, the GP Ib-IX complex interacts with the Mr 540,000 dimeric actin-binding protein. Upon platelet stimulation, this association is dis rupted by an endogenous Ca 2+ -dependent protease, which rapidly cleaves the Mr 270,000 ASP subunit into a defined set of three fragments of Mr 190,000, 100,000, and 90,000, the latter being derived from the Mr 100,000 fragment (Ezzell et al 1988). The Mr 100,000/90,000 frag mentes) contains the GP Ib-binding site as well as the actin-binding region, whereas the 190,000 fragment contains the ASP self-association site. This segregated domain structure allows the ASP dimer both to cross-link actin =

=

=

=

=

filaments and to attach to the platelet membrane via the GP Ib-IX complex.

Interestingly, platelets of Bernard Soulier patients, which lack GP Ib-IX, are large, polymorphic cells, whose size and shape may be due to a lack of membrane-cytoskeleton association. When resting platelets are incubated with 32p-phosphate, only the GP IbP subunit of the GP Ib-IX complex becomes phosphorylated predominantly at serine residues and to a lesser extent at threonine residues (Wyler et aI1986). Fox et al (1987) have shown that phosphorylation of the P chain of GP Ib as well as ABP is increased under conditions that increase the concentration of cAMP, which suggests that the GP Ib-IX complex may also be involved in regulating intracellular reactions in platelets by maintaining platelets in a resting state. One conse quence of phosphorylation is to limit actin polymerization, a process that occurs readily during platelet activation. In vitro phosphorylation of the purified GP Ib-IX complex by bovine AMP-dependent protein kinase has revealed serine 166 as the amino acid that is phosphorylated in GP IbP (Wardell et al 1989). The CI. and P chains of GP Ib are coded by two distinct genes; the gene for GP IbCl. is located on chromosome 17 (Wenger et al 1989). The GP IbCl. gene is rather uncommon for an eukaryotic gene because the protein is encoded by a single exceptionally large exon of 2.4 kb (Wenger et al 1988). The single intron in this gene is 233 bp long and is located 6 bp upstream from the ATG translation initiation codon in the 5' nontranslated region. Similar to the GP JIb gene, the 5' untranslated GENE STRUCTURE


347

region of GP TbO( is also devoid of typical TATA or CAAT consensus sequences in at least 2794 bp sequence,

5'

upstream of the putative

initiation transcription site (Wenger et al 1989). Nonetheless, several sequences with homology to established consensus sequences specific for transcription regulation factors are present. Interestingly, similar to the GP lIb gene, a binding site for GF-I is also present in the GP IbO( promoter (Wenger et al 1988, 1989), which provides further evidence for a close regulatory mechanism common to erythroid- and megakaryocytic-specific


gene expression.

Glycoprotein V Glycoprotein V (GP V) (Mf

=

82,000) is another platelet membrane gly

coprotein that also has the LRG motif (F. Lanza et aI, unpublished observation). This glycoprotein may be associated with the GP Ib-IX complex in platelet membranes, and it is unique in that it is the only major platelet membrane glycoprotein that is hydrolyzed by thrombin during platelet activation (Berndt & Phillips 1981). When GP V is hydrolyzed, a water soluble fragment of GP V, GP Vo (Mf

=

69,000), is released; the

presumptive membrane-bound fragment has not yet been identified. The possible function of GP V hydrolysis in thrombin-induced platelet acti vation remains controversial. Tn support of this function, thrombin acti vates platelets by a proteolytic mechanism and GP V is the only thrombin substrate on the platelet surface. Release of GP Vo, however, does not correlate with platelet stimulation, and GP V antibodies have failed to block platelet stimulation. This action argues against GP V as the thrombin receptor (Bienz et al 1986). GP V is deficient in platelets of patients with Bernard Soulier syndrome, which also display defective thrombin-induced platelet activation (see George et al 1984).

THE SELECTIN GENE FAMILY Selectins are a family of vascular cell surface receptors that are char acterized by a lectin-like domain at their amino terminus, an adjacent epidermal growth factor-like domain, followed by multiple short conSensus repeat units homologous to the complement regulatory proteins. One selectin is ELAM-l , which is induced by cytokines on the surface of endothelial cells and functions as a receptor for neutrophils (Bevilacqua et al 1989). Another has been termed MEL-14 in mice (Lasky et al 1989; Siegelman et al 1989) and LAM-l in human (Tedder et al 1989; Bowen et al 1989) and is found on the cell surface of lymphocytes where it functions in the homing of l ymphocytes to specialized l ymph node endothelium. GMP-140 is the only member of this family present in platelets.

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Granule Membrane Protein 140

Granule membrane protein 140 (GMP- 140), also called PADGEM or CD62, is a 140 kd integral membrane glycoprotein present in the alpha granules of platelets, which becomes expressed on the surface of activated platelets upon granule secretion. GMP- 140 is also a constituent of endo thelial cells where it is present in the membranes of Weibel Palade bodies and becomes expressed on the endothelial cell surface following release of these organelles (for references, see McEver et al 1989; Larsen et al 1989). GMP- 1 40 contains a N-terminal lectin domain, an epidermal growth fac tor domain characteristic of the selectins, and nine repeats of the comp lement regulatory protein motif (Johnston et al 1989). GMP-140 is thus the largest selectin molecule, since ELAM- I has six complement-binding repeats and MEL-14 has only two. Two members of the selectin gene family, Lam-l and GMP- 140 have been mapped to the same region on chromosome I , bands 23-25, thus suggesting that this family of proteins may be encoded by a cluster of loci (Tedder et al 1989). G MP- 140 has been shown to function as a receptor that mediates adherence of neutrophils and monocytes to activated platelets and endo thelial cells (Larsen et al 1989; Hamburger & McEver 1990). This inter action is Ca 2+ -dependent and can be inhibited by anti-GMP-140 anti bodies, purified GMP- 140, or GMP-140 contained in phospholipid vesicles. Neutrophils attach to purified GMP- 140, and GMP-140 phos pholipid vesicles bind specifically to cells expressing the GMP- 140 recog nition site. Also, COS cells transfected with GMP-140 mediate leukocyte adhesion (Geng et al 1990). Thus similar to ELAM- l , endothelial cell GMP- 140 might promote leucocyte adhesion and emigration in inflam matory processes. The functional role of platelet GMP- 140 in platelet leucocyte interactions is less clear. As suggested by Larsen et al ( 1989), platelet GMP- 140 might serve to localize leukocytes to the site of vascular injury. Alternatively, platelet GMP-140 might function as a recognition system for macrophages to remove activated platelets from the circulating blood. The cellular recognition sites for G MP- 140 and other selectins are still unknown. The high sequence homology between the lectin domains of the three glycoproteins, however, suggests that the cellular recognition sites might be carbohydrate-dependent. THE IMMUNOGLOBIN GENE SUPERFAMILY

The immunoglobulin gene superfamily is composed of several subfamilies of glycoproteins that are essentially involved in cellular recognition mech anisms and that all share a common structure called the immunoglobulin homology unit (reviewed in Williams & Barclay 1988). Members of the



349

immunoglobulin gene superfamily include cell surface glycoproteins of the immune system directly involved in antigen interaction such as immu noglobulins, the T cell receptor, and the major histocompatibility complex HLA class I and class II molecules; a family of growth promoting receptors such as the PDGF receptor, or the CSF- I receptor; and the family of cell adhesion molecules (CAM) including the neural cell adhesion molecule (N-CAM), the leukocyte ICAM-I and ICAM-2, and the recently identified platelet and endothelial cell PECAM- l . Two members of the immu noglobulin gene superfamily, PECAM-I and HLA class I molecules, are present on the platelet surface. PECAM- i

The platelet-endothelial cell adhesion molecule-l (PECAM- I ) (Newman et al 1990) is a 130-kd glycoprotein that was initially termed GP IIa on platelets and endothelial cells (van Mourik et al 1985) and the leukocyte differentiation antigen CD31 on blood monocytes, neutrophils, and mitogen-induced lymphoblasts (see Knapp et al 1989). The eDNA de duced primary structure of PECAM-1 predicts an N-terminal extracellular domain of 574 amino acids with nine potential asparagine-linked gly cosylation sites, a 19-residue transmembrane domain, and a 1 18-residue cytoplasmic tail (Newman et al 1990). The extracellular domain contains 12 cysteine residues that are spaced approximately 50 amino acids apart and flanked by six immunoglobulin-like homology units. The immu noglobulin-like domains in PECAM- I are of the C2 subgroup, which are predominantly found in the cell adhesion molecule (CAM) subfamily of the Ig-like proteins (Williams & Barclay 1988). The cytoplasmic domain of PECAM-l contains a tyrosine that could serve as a phosphorylation site for tyrosine kinase. The structural homology of PECAM-l to other CAM family members such as N-CAM and ICAM-l and its localization in endothelial cells at the intercellular junctions supports the hypothesis that PECAM-l may be involved in cellular interactions. OTHER PLATELET PROTEINS

A major platelet glycoprotein that appears unrelated to any known gene family is GP IV. GP IV, also known as GP IIIb or CD36, is highly glyco sylated and is distinguished by its high resistance to proteases in intact platelets (McGregor et al 1989; Tandon et aI1989). GP IV is also present in melanoma cells, monocytes, and endothelial cells (see Knapp et a1 1 989) as well as in nucleated erythroid cells (Kieffer et al 1989). GP IV is a rather late megakaryocytic marker, since it appears at a later stage of mega karyocytic differentiation than GP lIb-lIla or GP Ib (W. Vainchenker,


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personal communication). The deduced amino acid sequence of pla cental GP IV predicts a 438-residue extracellular domain, a 24-residue transmembrane domain, and a short cytoplasmic tail of six residues (Oquendo et al 1989). A second hydrophobic sequence of 23-amino acids, starting at residue seven of the N-terminal extracellular domain, suggests that GP IV might have two transmembrane domains (Tandon et al 1989; Oquendo et aI1989). All the cysteine residues of the extracellular domain are confined to a central domain encompassing residues 241 to 332. GP IV is unique in that it has no sequence homology with any other known protein. A first indication for a functional role of GP IV in cellular adhesion mechanisms came from the observation that a monoclonal antibody to GP TV (OKM5) inhibited thrombospondin binding to thrombin-activated platelets (Asch et al 1987). More recently it has been shown that purified GP IV binds to thrombospondin in a specific and Ca 2+ -dependent manner and that mono specific anti-GP IV antibodies interfere with the expression of endogenous thrombospondin at the surface of thrombin-activated platelets and partially inhibit thrombin-induced platelet aggregation (McGregor et al 1989). A role for GP IV as an alternative collagen receptor has also been reported (Tandon et al 1989). Purified GP IV binds to collagen type I fibrils, and antibodies raised against purified GP IV com pletely inhibit collagen-induced platelet aggregation. Soluble GP IV also competes with membrane-bound GP IV and inhibits platelet activation induced by collagen. The observation that the monoclonal antibody OKM5 inhibited in vitro binding of plasmodium falciparum infected erythrocytes to C32 melanoma cells suggested yet another role for GP IV as a receptor involved in cytoadherence of parasitized red blood cells, possibly by interacting �ith a ligand on the surface of infected erythrocytes (reviewed in Howard & GiJladoga 1989). GP IV purified from platelets mediates specific attach ment of infected red cells and inhibits or reverses binding of infected cells to umbilical vein endothelial cells or melanoma cells. Expression of cDNA encoding placental GP IV in COS7 cells supports specific cytoadherence of infected red cells, and this cytoadherence can be blocked by pretreatment of COS7 transfectants with OKM5 (Oquendo et aI 1989). No specific bind ing of thrombospondin to GP IV-transfected COS cells could be demon strated, however, which suggests that transfected GP IV lacks some essential modification or component necessary for thrombospondin receptor activity. SUMMARY AND CONCLUSION

The understanding of the structure and function of platelet membrane glycoproteins has been facilitated by studies showing that they belong to



35 1

larger gene families of cell surface receptors involved in cellular inter actions. In some instances (e.g. GP lIb-Ilia and GP Ib-IX) the study of the platelet proteins has served as a prototype for relatively newly described gene families (e.g. integrins and LRG proteins, respectively). In other instances, e.g. PECAM-l , the background of information on immu noglobulin domain-containing proteins has served to indicate functions. Receptor-ligand interactions have been characterized at the molecular level, and studies of genetic defects affecting platelet receptors have con tributed significantly to understanding structure-function relationships. Gene transfection studies provide encouraging results that might lead to gene therapy. The knowledge about platelet ligand-receptor processes contributes not only to our understanding of normal platelet function, but also to a more generalized understanding of adhesive mechanisms used by many cells to interact with their environment. Literature Cited Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., Berndt, M . C. 1989. Cross-linking of a monomeric 39/34-kDa fragment of von Willebrand factor (Ieu-480/val-48 1 -gly-78 1 ) to the N terminal region of the ()( chain of mem brane glycoprotein Ib on intact platelets with bis (sulfosuccinimidyl) substrate. Biochernstry 28: 8326-36 Arnaout, M. A. 1 990. Structure and function of the leucocyte adhesion molecules CDI I /CD l 8. Blood 75: 1 037-50 Asch , A. S., Barnwell, J . , Silverstein, R. L . , Nachman, R. L. 1 9 8 7 . Isolation o f the thrombospondin membrane receptor. J. Clin. Invest. 79: 1 054-61 Banga, H. S., Simons, E. R., Brass, L. F., Rittenhouse, S . E . 1986. Activation of phospholipase A and C in human platelets exposed to epinephrine. Role of gly coproteins IIb/IIIa and dual role of epi nephrine. Proc. Natl. A cad. Sci. USA 83: 9 1 9 7-9201 Beer, 1., Coller, B. S. 1989. Evidence that platelet glycoprotein lIla has a large di sulfide bonded loop that is susceptible to proteolytic cleavage. J. Bioi. Chern. 264: 1 7564-73 Berndt, M . c., Phillips, D. R. 1 98 1 . Puri fication and preliminary physico-chemical characterization of human platelet mem brane glycoproteinV. J. Bioi. Chern. 256: 59-65 Berndt, M. c., Chong, B. H . , Bull, H. A., Zola, Z . , Castaldi, P. A. 1 985. Molecu lar characterization of quinine/quinidine drug dependent antibody platelet interac-

tion using monoclonal antibodies. Blood 66: 1 292-301 Bevilacqua, M. P., Stengelin, S., Gimbrone, M. A., Seed, B. 1 989. Endothelial leu cocyte adhesion molecule I: An inducible receptor for neutrophils related to com plement regulatory proteins and lectins. Science 243: 1 1 60-65 Bienz, D., Schnippering, W., Clemetson, K. J. 1 986. GlycoproteinV is not the thrombin activation receptor on human blood plate lets. Blood 68: 720-25 Bodary, S. C, Napier, M. A., McLean, J. W. 1989. Expression of recombinant platelet glycoprotein lIb-IlIa results in a func tional fibrinogen-binding complex. J. Bioi. Chern. 264: 1 8859-62 Bowen, B . R., Nguyen, T, Lasky, L. A. 1989. Characterization of a human homo logue of the murine peripheral lymph node homing receptor. J. Cell Biul. 1 09: 421-27 Bray, P. F., Barsh, G., Rosa, J. P., Luo, X. Y, Magenis, E., Shuman, M. A. 1988. Physical linkage of the genes for platelet membrane glycoproteins JIb and lIla. Proc. Nat!. Acad. Sci. USA 85: 868387 Bray, P. F., Rosa, J.-P., Lingappa, V. R . , Kahn, Y W., McEver, R. P., Shuman, M. A. 1 986. Biogenesis of the platelet recep tor for fibrinogen: Evidence for separate precursors for glycoproteins lIb and IlIa. Proc. Nat!. Acad. Sci. USA 83: 1 480-84 Calvete, ]. J., Henschen, A., Gonzalez Rodriguez, J. 1 989. Complete localization of the intrachain disulphide bonds and the N-glycosylation points in the ()( subunit of

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