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Platelet receptors in hemostasis
Platelet
receptors
R.K. Andrews
in hemostasis and J.E.B. Fox
Gladstone Foundation Laboratories for Cardiovascular Institute, University of California San Francisco, Current
Opinion
in Cell
Biology
Platelets contain many adhesive receptors on their surface; Fig. 1 shows several families of these proteins. Among these receptors, the glycoprotein (GP) Ib-IX complex appears to be a unique glycoprotein complex that is critical for the initial adhesion of platelets. Many of the other major glycoproteins on the platelet surface are members of families of cell adhesion proteins. Glycoprotein IIb-IIIa is the most abundant member of the integrin family; GP Ia-IIa (a2pl), GP Ic-IIa (aSpl), very late antigen (~~06 (aG@), and the vitronectin receptor (avp3) are other integrins present on platelets (Table 1). Granule membrane protein (GMP)-140, a member of the recently described selectin family, is expressed on the surface of the activated platelet. PECAM-1, a member of the imrnunoglobulin family, has also recently been identified on platelets. This article reviews the role of the GP Ib-IX complex and the integrins in mediating the adhesion, activation, and aggregation of platelets, with emphasis on the most recently published data. In addition, potential involvement of the members of the other families of adhesion proteins in mediating the interactions of platelets at a site of injury is discussed. adhesion
Our understanding of the interactions Involved in the adhesion of platelets to the exposed subendothelium has been derived mainly from the use of the in vifro perfusion chamber in which a blood vessel with the endothelium removed is placed in a chamber through which anticoagulated blood is passed under defined flow conditions. The morphology of the blood vessel is then examined, allowing quantitation of platelets that have adhered to the denuded vessel wall and enabling the distinction to be made between those thar made initial contact, those that spread, and those that formed an aggregate. Use of this system has revealed that a number of different receptors are involved in platelet adhesion and thrombus formation and that the receptors and ligands involved are critically dependent upon the shear rate. The most important receptor in mediating the initial contact of platelets with the subendothelium at high shear rates (> 500s’) is the GP Ib-IX complex which consists of equimolar amounts of three polypeptides: GP Ib,, GP Iba, and GP IX (Fig. 1). Glycoprotein Ib, and GP
CMP-granule LFA-lymphocyte
894
1990,
Current
Biology
2:894-901
Interestingly, leucine-rich domains are found in numerous, apparently unrelated proteins of mammalian, invertebrate and microbial origin [3,4]. Glycoprotein V, an 82kD platelet-membrane protein hydrolyzed during platelet activation by a-thrombin, has also recently been shown to contain multiple homologous leucine-rich domains [ 51. Based on the known adhesion function of GP Ib,, it has been postulated that these proteins represent a class of leucine-rich cell-adhesive proteins (Krantz and Zipursky, EMBO Jl990,9:196+1977). However, it should be noted that there is currently no firm evidence that a leucine-rich sequence of any of these proteins constitutes a site of adhesion. Even though the von Wiiebrand factor (vWf)-binding and leucine-rich regions on GP Ib, are both contained within the same structural domain (see below), the direct involvement of the leucine-rich sequences in vWf binding has not been demonstrated.
Abbreviations membrane protein; CP-glycoprotein; function-associated antigen; VIA-very
@
Research USA
Ibp are disullide-linked, and GP IX is non-covalently associated with GP Ib (Du et al, Blood 1987, 9:1524-1527). Rotary shadowed images reveal that the complex is an elongated structure with a globular domain at the extracytoplasmic amino-terminal end of GP Ib, (Fox et al, J Bio/ Chem 1988, 263:4882+890). Glycoprotein Ib,, GP Ibp, and GP IX have been cloned from human erythrocyte leukemia (HEL) cell CDNA libraries, and each has been shown to be a separate gene product (Lopez et al, Proc Nat1 Acud Sci USA 1987, 84:5615-5619; Lopez et al., Proc Nat1 Acud Sci USA 1988, 85:2135-2139) [ll. Each glycoprotein contains a cytoplasmic domain. Both GP Ibp and GP IX contain cytoplasmic cysteine residues that appear co be palmitylated [ 21. The cytoplasmic domain of the complex is associated with the membrane skeleton (Fox,J Clin Invest 1985, 76:167>1683), a structure that may regulate the functional activity of the receptor (see below). The amino acid sequence of GP Ib,, but not GP Ibp or GP IX, contains a highly glycosylated serine/threonine-rich macroglycopeptide region of about 80 residues located close to the extracytoplasmic side of the membrane (Fig. 2). Glycoprotein Ib, contains seven leucine-rich internal repeat domains toward the aminoterminal, extracytoplasmic end of the protein (residues kuj6-Uel87; Fig. 2). A single homologous leucine-rich domain occurs in the extracytoplasmic portion of both GP Ibl-, and GP IX, and the flanking sequences of these domains in GP Ib,, GP Ibp, and GP IX are also highly conserved.
Introduction
Platelet
Disease, Cardiovascular San Francisco, California,
Ltd
HEL-human late antigen;
ISSN
0955674
erythrocyte vWf-von
leukemia; Willebrand
factor.
Platelet
Table
1. Vascular
cell adhesron
receptors
in hemostasis
Andrews
and Fox
receptors.
Cell types
Soluble or matrw
associated
kgands
Cell-assocrated
kgands
Receptor
Leu-rich family CP lb-IX lntegnn family VLA-1 la-lla
0
l
(alp11
l
0
VLA-3 VLA-4
fa2pl) fa3f31) fa4pl)
Ic-lla VLA-6
fa5pl) (abpll
l
l
l
l
LFA-1 Mac-l
faLp2)
l
wq32) (aXj.32) fallbp3)
l
l
l
l
l
(avp3)
l
p150/90 Ilbllla VNR
0
0
l
l
(a+3
l
.
l
0
0
l
0
0
l
l
l
l
l
l
l
l
l
l
l
l
l l l l
l
l l
.
.
l
l
l
l
l
l
l
l
l l
l
l
l
l l
LAM-l Ig-like family VCAM-1 PECAM-1
l
l
l
l
l
l
l l
l
ICAMICAM-
CP, glycoprotein; receptor;
l l
l
l
Selectrn family CMP-140 ELAM-1
l
l
l
l
l .
l
l
CP IV CP VI
wtronectin
.
LAM,
lymphocyte
vWf.
von
l
l
l
l
adhesron
Wrllebrand
molecule:
LFA. lymphocyte
functton-assocrated
antrgen.
VLA.
very
late antigen;
VNR,
factor
The component of the subendothelial matrix to which GP Ib-IX binds is vWf. Considerable progress has recently been made in delining the vWf-binding domain on GP Ib-IX. In vivo, GP Ib-IX does not bind plasma vWf; the vWf must first bind to unidentified components of the subendothelial matrix. Presumably, this induces a conformational change in the molecule that then allows it to bind GP Ib-IX. However, the binding of soluble vWf can be induced by non-physiological modulators such as the microbial antibiotic glycopeptide known as ristocetin or the snake (Bothr-o~~jururucu) venom protein botrocetin. Botrocetin functions by binding to a regulatory binding site on vWf, but the mechanism of ristocetin activity is obscure (Read ef a,!, Blood 1989, 74:1031-1035) [6,7]. Other assays have used asialo-vWf (neuraminidasetreated native vWf) or bovine vWf, both of which bind
directly to GP Ib-IX in the absence of either ristocetin or botrocetin. These in vitro assay systems have enabled investigators to localize the vWf-binding site on GP Ib-IX to the globular amino-terminal domain of GP Ib, (His1-Arg293>. First, it was discovered that antibodies against this, but not other regions of the complex, inhibit binding of vWf to intact platelets or to purikd GP Ib-Ix (Wicki and Clemetson, Eur J Biocbem 1985,153:1-11; Handa et cd, J Biol Clam 1986,261:1257+12585; Bemdt et al, Biocbem&?y 1988, 27:633&O). Second, the amino-terminal domain isolated from tryptic digests of the purikd receptor completely inhibits binding of native vWf to put&d GP Ib-Ix in the presence of botrocetin [6,8]. Third, this fragment binds directly to immobilized vWf in the presence of risto-
895
8%
Cell-to-cell
contact
and extracellular
matrix
.
(b)
l-l
UI II”
(heavy chain) (116 kD)
(d) N Aminoterminal peptide domain
Ixtracellular
(e)
#
.2.. . L
~-,-J
u
C
CP IX (22 kD)
u C
C
C
GP lb0 (25 kD)
(105 kD)
GP llla
CP Ilb (light chain) (23 kD)
GP Iba
CP IV (88 kD)
CMP-140 (140 kD)
PECAM-1 (130 kD)
(135 kD)
Fig. 1. Platelet adhesion receptors: (a) the glycoprotein (CP) I&X complex; 0, Leu-rich domain; 18, macroglycopeptide. (b) GP Ilb-llla region. Other integrins on platelets are the (allbe3), the major integrin receptor on platelets; q , Cys-rich domain; UN, Ca 2+-binding collagen receptor, GP la-lla (a2pl); the fibronectin receptor, GP Ic-lla (a5Pl); the laminin receptor (abpl); and the vitronectin receptor (avp3). (cl CMP-140, a member of the selectin family; n , lectin-like domain; lZl, ECF-like domain; W, CRB-like domain. (d) PECAM-1, a member of the immunoglobulin (Ig) superfamily; q , Ig-like domain. (e) CPIV; @I, Cys-containing region. C, carboxy terminus; CRB, complement regulatory binding protein; ECF, epidermal growth factor; N, amino terminus; W, transmembrane domain.
cetin (Vicente et al, JSiolCbem 1988,263:18473-18479). That only this domain of GP Ib, binds vWf has been directly confirmed on intact platelets by covalent crosslinking analysis using a monomeric 39/34 kD VWf fragment (Le~~al~~+y,~~) that retains the GP Ib-K-binding site [7]. Further definition of the amino acid sequence in the amino-terminal domain of GP Ib, that binds vWf has been obtained by determining the effect of synthetic peptides based on sequences within this domain. Using this approach, one group observed that a peptide, Ser251-m279, parMy inhibited the vWf-GP Ib-IX interaction [8]. Another group identified A.s~~~~L~s~~~as be-
ing inhibitory [9]. While this approach may provide insight into important regions of primary sequence, several lines of evidence suggest that the three-dimensional organization of the amino-terminal domain is necessary for a fully functional vWf-binding site. First, the active peptides just referred to were much less inhibitory on a molar basis than the native amino-terminal domain was. Second, other peptides from within the amino-terminal sequence were only marginally less inhibitory than the Ser251-Tyr279 peptide. Third, reduction of the disulfide bridges within the purikd amino-terminal domain dras tically reduced its inhibitory activity toward botrocetindependent binding of native VWf and toward binding of asialo-VWf to platelets [8].
Platelet
receptors
in hemostasis
Andrews
and
Fox
Cytoplasmic Amino-terminal
peptide
domain
Macroglycopeptide
domain
domain
I 610
C I
’
’ ’ Leucine-rich
’ ’ domain
’
I
Putative vWf-binding peptides
Fig. 2. Structural and functional domains peptide sequences, Ser2s,-Tyrz,g [Sl and glycosylation; 0, N-linked glycosylation.
I
III
1 I
[II III
Transmembrane domain
II[I f k
Serzsl
I
,
I
’
!A
- Tv279
sP235 - LYs,62
of CP lb,. The location within the amino-terminal domain of leucine-rich repeats Asp 2.s-Lys262 [91, reported to inhibit the vWf-CP lb-IX interaction, are indicated. (The number of O-linked glycosylation sites is under-represented in this diagram.)
and
0,
of two O-linked
Together, the studies described above have localized a distinct binding domain on the GP Ib-IX complex that mediates its interaction with vWf, even though the precise contribution of smaller discrete amino acid sequences involved is yet to be confirmed. Some indication that binding can be induced at different sites depending on the modulator used comes from the Iinding that GP Ib, peptides had different inhibitory activities towards ristocetin-dependent compared with botrocetindependent binding of vWf to platelets [8], and from the report that an anti-GP Ib, monoclonal antibody (OP-Fl) inhibited binding of vWf to platelets in the presence of ristocetin but not in the presence of botrocetin (Nishio et al, 7h-omb Hamost 1989, 62:2I9). Therefore, the exact relationship between these in vitro lindings and the binding of platelets to subendothelial matrix-associated vWf in vivo remains to be determined.
mains to be determined, there is now considerable evidence that the GP Ia-IIa complex (the integrin VIA-2, a2j31) is a functional collagen receptor. First, platelets of a patient lacking GP Ia showed decreased adhesion to collagen (Nieuwenhuis et al., Nature 1985, 318:470-472; Nieuwenhuis et al, Blood 1986, 68:692495). Second, monoclonal antibodies against the complex inhibited the adhesion of platelets to a collagen-coated surface (Kunicki et al, J Biol C&m 1988,263:4516-4519) [ 131. Most importantly, pm-&d GPI-IIa complex has now been shown to bind specifically to collagen [ 141, and the binding of collagen to these proteins has the same divalent cation dependence as that of intact platelets to collagencoated surfaces (Santoro, Cell 1986,46:913-920).
Although GP Ib-IX is the most important receptor in mediating the initial adhesion of platelets to the subendothelial matrix, other receptors may be involved. Antibodies against vWf markedly inhibited adhesion of platelets to rabbit aorta subendothelium at high shear rates ( > 1000 s-t), but they had no effect at low shear rates (< 500~~l; Meyer et al., Br J Haemutol 1984, 57:60+620; Stel et al, Blood 1985, 65:85-90). At the low shear rates, which are comparable to those in the aorta or large blood vessels, the interaction of platelets with collagen or Iibronectin may be important. Recently, a number of platelet receptors for collagen have been proposed, including GP N (also known as GP IIIb or CD36)--a recently cloned 88 kD glycoprotein expressed on platelets, endothelial cells, and monocytes [ lO,ll]and GP VI [ 121. While the physiological importance of these glycoproteins in mediating adhesion to collagen re-
As platelets adhere to the subendothelial matrix, they be come activated. This transition is essential for allowing the subsequent stages of the hemostatic response. Activation is induced either as a consequence of interaction with collagen or by thrombin that is generated as the coagulation cascade is accelerated at a site of injury. The observations that the binding of soluble vWf to GP Ib-IX induced under conditions of high shear (Ikeda et al, Clin Res 1990, 38:426A) and the binding of soluble asialovWf to GP Ib-IX (De Marco et al, J Clin Invest 1985, 75:1198-1203) induced platelet activation raise the possibility that the interaction of GP Ib-IX with vWf in the extracellular matrix may provide an additional mechanism for generating intracellular messages that lead to the activation response. The Iinding that the cyclic AMP-dependent phosphorylation of GP Ibp may inhibit agonistinduced actin polymerization [ 151 also provides some
Platelet
spreading
and aggregation
897
898
Cell-to-cell
contact
and extracellular
matrix
support for the idea that GP Ib-IX may regulate signal transduction. . As platelets become activated, they spread over the subendothelium, increasing the area of contact, and adcltional platelets then aggregate to the initial layer of adherent platelets. The GP IIb-IIla complex is the receptor that mediates this aggregation. As shown in Fig. 1, GP IIb-IIIa is a member of the integrin family of adhesion receptors, consisting of a heterodimeric non-covalent complex of an cr-subunit and a P-subunit. Both subunits of GP IIbIIIa have been cloned and sequenced (Poncz et al., J Biol cbem 1987, 262~8476-8482; Fitzgerald et al, J Biol C&em 1987, 262:3936-39391, and this year the gene structures for platelet GP IIb [ 161 and GP IIIa [ 171 have been described. Recent work has demonstrated that both posttranslational processing and surface expression of the GP IIb-IIIa complex require the presence of both a- and psubunits [ 18,191. On the unstimulated platelet, GP IIb-IIIa is present on the surface but is not active. When platelets are activated, GP IIb-IIIa becomes competent to bind adhesive ligands. Considerable progress has been made in identifying the binding sites for these ligands on the complex [ 201. One of the adhesive ligands that binds to activated GP IIb-IIIa is fibrinogen, and it has long been assumed that platelets aggregate as a consequence of the binding of this multivalent ligand to GP IIb-IIIa However, it is now known that GP IIb-IIIa can also bind the adhesive ligands fibronectin, vWf, and vitronectin (Parise, Curr Opin Cell Biol 1989, 1:947-952). Recently, Weiss et al [21] have investigated the importance of these ligands in mediating the spreading of platelets on the subendothelium and the formation of thrombi. It was observed that at high shear rates, platelets adhered equally well in the presence or absence of fibrinogen. Further, antibodies that prevented the binding of fibrinogen had no effect on the number of spread platelets, nor did they inhibit the size of the thrombus that formed. In contrast, spreading and thrombus formation under these conditions were inhibited by an antibody that prevented the binding of vWf and vitronectin. Other studies have provided evidence that under conditions of high shear, platelets aggregate as a consequence of the binding of vWf to the GP IIb-IIIa complex (Moake et al, Blood 1988,71:1366-1374) [21]. Thus, while fibrinogen may be the ligand that mediates aggregation under conditions of low shear, it is becoming apparent that vWf may be an important ligand medi ating adhesion of spreading platelets and the formation of thrombi at high shear. The question of how the GP IIb-IIIa complex becomes competent to bind adhesive ligands on the activated platelet remains unanswered. It is particularly intriguing to speculate on mechanisms by which activated GP IIbIIIa may bind fibrinogen at low shear rates and vWf at high shear rates. Is this a function of a shear-induced change in vWf, or does it reflect a difference in intracellular messengers generated when platelets are activated under conditions of high shear? Although it is possible that increased access of adhesive ligands to a pre-existing binding site results from an activation-induced change in components of the membrane other than the GP IIb-IIIa
complex (Caller, J Cell Biol 1986, IO3:451-456), the recent demonstration that a monoclonal antibody (D33C) against a discrete sequence of GP IIb induced fibrinogendependent platelet aggregation [22] provides some support for the concept that expression of a functional receptor results from a modification to the GP IIb-IIIa complex itself. The only activation-induced covalent modification that has been described in the complex is phosphorylation of GP IIIa, which appears to be induced on threonine residues by protein kinase C [23]. Future studies will be needed to determine whether this modification al ters the ability of the GP IIb-IIIa complex to bind ligand. Interestingly, as platelets become activated and GP IIbIIIa becomes competent to bind to adhesive ligands in the subendothelium, the GP Ib-IX complex appears to lose its ability to bind vWf. The GP Ib-IX complex is as sociated (via actin-binding protein) with the membrane skeleton (Fox, J Biol Cbem 1985, 260:1197@11977). It has been suggested that this association is responsible for maintaining the GP Ib-IX complex uniformly distributed over the surface of the membrane, thus allowing the maximum number of GP Ib-IX molecules to interact with the multivalent vWf molecule (Fox and Boyles, Blood 1985,66:304a). It is possible that the decreased functional activity of GP Ib-IX observed at early stages of platelet activation (George and Torres, Blood 1988, 71:1253-1259) may result from internalization of the GP Ib-IX complex (Nurden et al, Blood 1989, 74:129a), an event that could be directed by the membrane skeleton. Since activation of calpain has been shown to result in a clustering of the complex on the platelet surface, an additional mechanism may involve activation of this intracellular protease at the later, irreversible stages of platelet activation. In recent studies, amino acid sequences in the GP Ib-IX complex that may mediate the interaction of the complex with the skeleton have been identified (Andrews and Fox, Blood1990, abstract in press). Further, the GP Ib-IX complex has been expressed in a functional form on the surface of HeIa cells, a cell type that contains actin-binding protein (Lopez et al, Blood 1990, abstract in press). It is anticipated that future studies in which GP Ib-IX is expressed in a native form or in a form in which it can no longer associate with the cytoskeleton will allow the importance of the cytoskeleton in regulating its distribution and function to be evaluated further. Stabilization
and contraction
of the thrombus
A developing platelet thrombus is initially fragile and can be readily washed away by the flowing blood. However, activated platelets express procoagulant activity on their surface, thereby accelerating the formation of fibrin. Thus, the fragile aggregate becomes stabilized in a network of polymerizing fibrin. The aggregate may also be stabilized in part by the interaction of thrombospondin with GP N on the platelet surface. It has been suggested that the GP N-thrombospondin complex associates with the GP IIb-IIIa fibrinogen complex (Nachman et al. In i%rombosis and Haemcstus& 1987, edited by Verstraete M et al. International Society on Thrombosis and Haemostasis and Leuven University Press, 1987,
Platelet
pp 81-91), although the derailed mechanisms of these secondary interactions are not understood. Similarly, evidence has been provided that binding of vitronectin to the vitronectin receptor has a role in mediating thrombus formation, although the relative importance of this receptor compared with GP IIb-IIIa is not known [ 241. Contraction of the platelet thrombus is an important secondary stage in wound healing. It is thought that fibrin binds externally to the GP IIb-IIIa complex, which in turn associates intracellularly with the cytoskeleton (Phillips et al, J Cell Biol1980, 86:77-86). As the cytoskeleton contracts, the fibrin clot is retracted. An important question in understanding the molecular mechanisms involved in this response is how does the GP IIb-IIIa complex associate with the cytoskeleton? Although talin redistributes to the periphery of the ceU upon platelet activation (Beckerle et al, J Cell Bioll989,109:3333-3346), future studies will be needed to determine the physiological role of this protein in mediating actin-membrane interactions in platelets. An interesting observation related to the mechanisms regulating clot retraction is that when GP IIb-IIIa binds an adhesive l&and, it undergoes a conformational change that exposes new epitopes. Several antibodies against these l&and-induced binding sites have been raised; one of them (PMI-2) inhibits clot retraction [25]. One mechanism that has been proposed to explain this observation is that occupancy of the fibrinogen-binding site on GP IIb-IIIa induces expression of a distinct site on GP IIb-IIIa to which fibrin (and PMI-2) binds [25]. Altematively, Isenberg et al. (I Cell Biol 1987, 104:16551663) have shown that occupancy of the fibrinogen-binding site results in clustering of the GP IIb-IIIa molecules, an other event that could conceivably be required for clot retraction to occur (for example, by inducing association of the cytoplasmic domain of the complex with the cy toskeleton). Finally, ligand binding to GP IIb-IIIa appears to regulate the intracellular activation of tyrosine kinase, yet another event that could conceivably be involved in regulating post-aggregation events such as clot retraction [26]. Future studies will be needed to elucidate the way in which occupancy of GP IIb-IIIa regulates intracellular events in platelets that have already aggregated and are thus a part of a platelet thrombus, and to elucidate how these events regulate the contraction of the thrombus. Other
adhesion
receptors
The interaction of platelets with white cells (neutrophils, monocytes, and/or lymphocytes) at the site of vascular injury may affect platelet aggregate formation and other platelet responses @ngi et al, Blood 1986, 67:629-636; Maclouf et al, Blood 1989, 74:703707). Therefore, while there is as yet no clear understanding of mechanisms, it appears possible that receptors of the selectin and immunoglobulin families will prove to have Important functions in regulating hemostasis. Selectins (GMP-140, EL&-l, and the lymphocyte homing receptor IAM-1) and members of the immunoglobulin superfamily (PECAM-1, VCAM-1, and ICAM- and -2) are present on platelets, endothelial cells, and/or vascular cells (Table 1). GMP-140 (Fig. lc) is a granule mem-
receptors
in hemostasis
Andrews
and Fox
brane component of platelets and endothelial cells that is expressed on the cell surface within seconds of ceU activation [27]. Cytokine-induced expression of ELAM1 on endothellal cells requires protein synthesis, and maximum surface expression occurs after several hours (Bevilacqua et al, Science 1989, 243:116&1165). GMP140 and ELAM-1 regulate the adhesion of neutrophils and perhaps monocytes to activated platelets and endothelial cells, and target white cell recruitment to the site of vascular damage [28,29] (Hamburger and McEver, Blood 1990, 75:550-554). PECAM-1 (Fig. Id) is the only member of the immunoglobulin superfamily identified so far on platelets, and is probably involved in cell-cell recognition and adhesion [30]. It appears likely that further studies on these recently described receptors will greatly increase our understanding of the way in which the interactions of a variety of cells act together to form a functional thrombus and thus to mediate hemostasis efficiently. Conclusions
and future
directions
Many of the receptors on platelets have now been characterized, and in several cases the molecular details of their Interactions with adhesive ligands have been elucidated. There are still many important questions yet to be answered, and new techniques and lines of research will be required to answer them. First, we need to use more physiological models to assess whether the domains on GP Ib-IX to which soluble vWf binds are the same as those that mediate the interaction of vWf with the subendothelial matrix in vivo. Second, investigation of the contribution of features other than the amino acid sequence of GP Ib, in regulating the binding of the GP Ib-IX complex to vWf is required. For example, what is the role, if any, of GP Ibb and GP IX in vWf binding to the GP Ib-IX complex; what is the role of the leucine-rich repeats; and what is the role of N and Olinked carbohydrate on the GP Ib-IX complex in regulating vWf binding? What is the role of GP V, another leucine-rich glycoprotein that, like the GP Ib-IX complex, is missing from Bernard-Soulier platelets? The GP Ib-IX complex has recently been expressed in mammalian cells in a functional form (Lopez et al, Blood 1990, abstract in press). It appears probable that the use of cells expressing this complex will allow some of these questions to be addressed. Third, we must consider how the GP Ib-IX complex associates with the membrane skeleton, and how this regulates the function of the complex. Fourth, what are the intracellular mediators that induce GP IIb-IIIa to express l&and-binding activity, and how does GP IIb-IIIa associate with the cytoskeleton? A fifth question is how are platelet adhesion receptors involved in signal transduction? For example, how does the shear-induced binding of vWf to GP Ib-IX initiate platelet activation? How does l&and binding to GP IIb-IIIa regulate intracellular events? Sixth, what is the importance of shear in vim2 It is becoming increasingly clear that the shear rate can have profound Influences on the binding of adhesive ligands to receptors. Seventh, which are the important ligands and receptors in the circulation? This is particularly important in terms of the development of specific inhibitors to prevent the formation of platelet thrombi at inappropriate sites. For example,
899
900
Cell-to-cell
contact
and
extracellular
matrix
which ligands and receptors are involved in the formation of thrombi at sites of rupturing atherosclerotic plaques? Finally, what is the role of the recently described selectins and members of the immunoglobulin superfamily of adhesive receptors, both on platelets and on the other cell types that are recruited into a developing thrombus, in mediating and stabilizing the platelet thrombus? It is to be hoped that the next few years will provide the answers to some of these questions. Annotated reading l
me
references
Of interest Of outstanding
and recommended
interest
1.
HIclcz~ MJ, WLLUAMS SA. ROTH GJ: Human platelet glycoproteIn IX: an adhesive prototype of Ieucine-rich glycoproteins with Bank-center-Bank strucmres. froc Nafl Acad Sci Cl.%4 1989, t366773-6rn. The predicted amino acid sequence for platelet GP IX cloned from a HEL cell cDNA library shows it to consist of 160 residues contain. ing a single leucine-rich sequence in the exuacytoplasmk domain and a putative transmembrane domain. The leucine-rich domain and its amino- and carboxyterminal llanking sequences are highly homologous to those found in the u- and P-subunits of GP Ib. l
0
2. a
MUSZBEK 1 LWXATA M: Glycoprotein lb and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages. J Biol c%em 1989, 26497169719. This work shows that a cysteine residue on the cytoplasmic domain of GP lbg and of GP IX may be covalently linked to palmItIc acid in platelets, although the stoichiometry and function of this modification await further study. P, COUMBATTI A: The carboxyl teminuS of the chicken a3 chain of collagen VI is a unique mosaic structure with glycoprotein lb-like, libronectin type III, and Kunits modules. J Btil Gem 1969, 264:2023%20239. Strucmral domains identified in the predicted primary sequence of the chicken collagen VI a3 chain include the KUII~Q domain common to the KUI-I~Q protease inhibitors and dendrotoxins; the type A domain found in other adhesive glycoproteins such as vWf and the a-subunits of platelet GP la-IIa (a2pl) and of the 82 integrin fm the fibronectin type III domain; and leucine-rich and serine/threonine-rich domains that are also found in GP lb-IX. Among the IeucIne-rich protein family, only collagen Vl a3 chain, E&ericbia co/i tra-E protein, GP lbg, and GP IX are known to contain a single leucine-rich repeat.
3. a
BONAIDO
4. 0
MIKOL DD, GULCHER JR, STEFANSSON K: The oligodendrocytemyeIin glycoprotein belongs to a distinct family of proteins and contains the HNK-1 carbohydrate. J Cell Biol1990, 110:471479. The 433.amino-acid glycoprotein that is glycosylphosphatidylinositol. linked to ollgodendrocytes and central nervous system my&n is structurally related fo platelet GP lb,, in terms of the structural organization of leucine-rich and sedne/threonine-rich domains, although the two proteins have no known functional relationship.
T, FUJ~~~URA K, MAEW S, TAKEMOTO M, ODA K, FUJIMOTO T, OYAMA R. SUZLJKI M, ICHIHARA-TANAKA K, TITANI K, KUR~MOTO A: Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein lb. Blood 1990, 75:234%2356. Partial amino acid sequence analysis of purified platelet GP V showed that this glycoprotein contaIned 10 or more leucine.rich repeat domains homologous to those found in GP lb-IX. Glycoprotein V, which is miss ing together with GP lb-IX on the surface of Bernard-SouIier syndrome platelets, currently has no identied function but is the only known platelet surface substrate for a-thrombin. 5. aa
6. l
SHIMOMUM
AND~%X Ry BOOTH WJ, GORMAN JJ, CA~TAIDI PA BERNDT MC: Purification of botrocetin from Bothrops jururucu venom. Analysii of the botrocetin-mediated Interaction between von WIebrand factor and the human datelet membrane glycoprotein lb-IX complex. Bicdemist& $3$ 28:83174326.
The isolated 45 kD amino-terminal peptide fragment of GP lb,. but not the serine/threonine-rich macroglycopeptide fragment, was shown to inhibit binding of purified vWf to purified GP lb-IX complex competitively in the presence of botrocetin. Also, monoclonal antibodies against the amino-terminal peptide domain inhibited the botrocetin.dependent vWfCP lb-IX interaction. 7. 0
ANDRRX’S RK, GORMAN JJ, BOOTH W’J, COWNO GL CASTAIDI PA, BEKNDT MC: Cross-linking of a monomeric 39/34kDa dispase fragment of von Wiiebrand factor (Leu48O/Val481-Gly-718) to the N-terminal region of the achain of membrane glycoprotein lb on intact platelets with bis(sulfosuccinimidyI)subcrate. Eiocbemisftv 1989, 28:8326-8336. A monomeric dispase fragment of vWf that retained the abiliry to bind fo GP Ib-IX was covalently crosslinked exclusively to the amino-terminal peptide region of GP Ib, on intact platelets. providing direct evidence that this region contains the vWf-binding site. 8. a
VICENI-IZ V, HOUGH’IFN Q RUGGEIU ZM: Identification of a site in the a-chain of platelet glycoprotein Ib that participates in von Wiiebrand factor binding. - I Biol C&m 1990, i65:274-280. A comprehensive analysis of 15.residue synthetic peprides from overlapping sequences within the amino-terminal region of GP lb, identified a number of sequences that blocked the binding of vWf fo platelets in the presence of ristocetin or bouocetin. While .some of these sequences participate in binding vWf, a fully functional binding site probabiy requires higher structure of the domain. In related experimems, reduction of disuffide bonds in the isolated amino-terminal fragment abolished botrocetin-dependent, but not ristocetin-dependent, inhibitory activity of the fragment. 9.
KATAGW Y, HAYA~HI Y. YA~~OTO K, TANOUE K, KOSAKI G, YA&%ZAKI H: Localization of von Wiiebrand factor and thrombin-interactive domains on human platelet glycoproteIn lb. lh-omb Huemast 1990, 631122-126. Partialiy overlapping synthetic peptides A.sp235-Lys,,~ and Phez,eThrzdo from within the amino-terminal peptide region of GP lb, were identified as inhibitors of the interaction of GP lb-IX on platelets with vWf in the presence of ristocerin and with a-thrombin, respectively, l
10.
OQUENIXI P, HUNDT E, LUXUR J, SEED B: CD36 directly mediates cytoadherence of Plusmodium fuhpunrm parasitized erythrocytes. Cd 1989, 58:95-101. The cDNA clone for CD36 from a human placental library predicted a protein of 471 residues, including a putative transmembrane domain, and nine potential N-glycosylation sites. CD36 is identical fo platelet membrane GP IV (proposed receptor for thrombospondin and col. lagen) and is expressed on the surface of Plusmodium fukiparuminfected erythrocytes. l
11. 0
TANNIN NN, KRAusz U, JA~UEWN GA: ldentilication of gly coprotein IV (CD36) as a primary receptor for plateletcollagen adhesion. J Biol C5em 1989, 264~75767583. A role for platelet GP IV in adhesion fo collagen-coated surfaces and in collagen-induced aggregation is suggested by studies with anti-GP IV antibodies and puriIied GP IV. 12. 0
MOROI M, JUNG SM, OKLIMA M, SHINMY~ZU K: A patient with platelets deficient in glycoproteln VI that lack both collagen-induced aggregation and adhesion. J Clin Irwest 1989, B4:144@1445. The studies described in this report of a platelet GP VI-deficient patient provide the first indication of a possible physiological role for GP Vl as a collagen receptor. COUIR BS, BEER JH, SCUDDER LE, STEINBERG MH: Collagenplatelet interactions: Evidence for a direct interaction of collagen with platelet GPla/lla and an indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins. Blood 1989, 74:182-192. Studies using an anti-GP la-IIa monoclonal antibody (6Fl) suggested a functional role for the platelet GP la-lla integrin complex (a2pl) as a receptor involved in adhesion of platelets to collagen. This antibody blocked the adhesion of platelets to collagen-coated plastic but did not inhibit collagen-induced platelet aggregation. 13.
0
14.
STAAIZ WD, RAJPARA SM. WAYNER FA, CARTER WG, SNORO SA: The membrane glycoprotein la-IIa (VLA-2) complex medIafes the Mg+ +dependent adhesion of platelets to colIagen. J Cell Biol 1989, lOJ3:1917-1924. When purified platelet membrane GP la-IIa (a2pl) was incorporated into phosphatidylcholine Iiposomes. they behaved like platelets in that l .
Platelet they bound to collagen in the presence of Mg*+, but did not bind if Mg*+ was replaced by Ca*+. Glycoprotein la-IIa apparentty did not support adhesion of the Uposomes to other adhesive glycoproteins. 15.
FOX JEB, BERN~?’ MC: Cyclic AMP-dependent phosphory lation of zlvcooroteln lb inhibits collaeen-induced wlvmerization‘of a actIn in platelets. J “Biol Cbem i98$, 264:952&9526. Analysis of cAMF-dependent phosphorylation of platelet proteins and response to activation in normal platelets compared with BemardSouller platelets (which lack GP Ib-IX) indicated a role for phosphorylatlon of GP Ibg in regulating collagen-induced actin polymerization. This supports the concept that the GP lb-IX complex may regulate sig nal transduction in platelets.
l
16. 0
HE~DENREICH R, EISMAN R, SURREY S, DELGROSSO K, BEJS, SCHWA E, PONCZ M: Organization of the gene for platelet glycoprotein IIb. Bbclwn&Q 1990, 29:1232-1244. The analysis of the genomic organization of both subunits of the GP IIb-IIla complex (see [17]) is important for future studies on the reg ulation of GP IIb-IIIa expression and function, and on the evolutionary relationships between GP IIb-UIa and the other related members of the integrin superfamily of adhesive glycoproteins. 17. 0
Zthmm AB, GIDWTIZ S, LORD S, SCHWA E, BENNETT JS, WHITE GC II, PONCZ M: The genomic organization of platelet gly coprotein IIIa. J Biol Gem 1990, 265:85m595. see [16]. 18. 0
O’TCXX TE, Lonvs JC, PLOW EF, GIASS AA, HARPER JR, GINSBERG MH: Efficient surface expression of platelet GP Ilb-IIIa requires both subunits. Biocd 1989, 74314-18. When either GP IIb cDNA or GP IIIa cDNA was uansfected into COS cells, each protein was synthesized, but there was no surface expression of either giycoprotein, and no proteolytic processing of GP IIb into light and heavy chains. However, normal GP IIb-IIIa complex was expressed by COS cells uansfected with both GP Ub and GP IIIa cDNAs. ROSA J-P, MCEVER RP: Processing and assembly of the integrin, glycoprotein IIb-III& in HEL cells. J Bzbl @em 1989, 264:1259&12603. Studies on expression of GP IIb-IIla in a human etythroleukemic cell line demonstrated that after translation, heterodimers of GP Ub and GP IIIa that formed in the endopla.smic reticulum, but not uncomplexed GP IIb, were transported to the Golgi apparatus where GP IIb was cleaved to heavy and light chains and its carbohydrate was modified. This demonstrated the importance of GP IIIa in surface expression of hlly processed GP IIb. 19. 0
20. 00
D’Souz~ SE, GINSBERG MH, BURKE TA, P~ovi EF: The Ugand binding site of the platelet integrin receptor GP I&-IIIa Is proximal to the second calcium binding domain of its a subunit. J Biol Gem 1990, 265:344&3446. In this paper, a synthetic peptide analogous to the carboxy-terminal peptide of the fibrinogen y-chain was covalently crosslinked to a dlsCrete sequence of GP Ub (residues 294-314) located at the second calcium-binding domain. An RGD-containing pepride had prevlousiy been crosslinked to a discrete region of GP UIa (residues lwl71). The combined studies deline distinct regions of both GP Ub and GP IIIa that are proximal to the l&and-binding site on the complex. 21. 00
WEISS HJ, HAWIGER J, RUGGERI ZM, TUR~~TO VT. THL%ARAJAN P, HOFFMANN T: Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invesf 1989, 83~288-297. At high shear, human platelet adhesion and thrombus formation in catheterized, perfused rabbit aorta was blocked by antibodies and synthetic peptides that inhibit intemction of adhesive ligands with platelet GP IIb-IIIa. The GP IIb-IIIa-mediated platelet adhesion and thrombus formation at high shear rate proceeded normally without fibrinogen and were also inhibited by an an&GP IIb-ma monoclonal antlbrxly that allows normal fibrinogen binding but blocks binding of vWf and libronectin. This is the first indication that, at high shear rates, adhesion of platelets occurs independently of Ebrinogen. 22. l
GUUNO D, RYCKEWAERT G: IdentIficatIon of a GP Ilb that interacts duces aggregation. J
J-J, ANDRIEUX A, RABIJ.X M-J, MARGUERIE monoclonal antibody against platelet with a calcium-binding site and inBiol &em 1990, 265:957%9581.
receptors
in hemostasis
Andrews
and
Fox
Binding of a monoclonal antibody, D33C, to a discrete peptide sequence on the GP IIb heavy chain (residues 426-437) induced Ebrinogen-dependent platelet aggregation. This tinding suggests that the functional actit-ion of GP IIb-ma may result from a specilic conformational change in the complex. 23.
PARISE LV, Cruss AB, NANNUA L, WARDEU MR: Glycoprotein IIIa is has horylated in intact human platelets. Blood 1990, 75:236$-23&. The P-subunit (GP ma) of the GP IIb-ma complex, but not GP IIb, was shown to be phosphorylated, predominately on threonine residues, on its cytoplasmlc domain. The degree of phosphotylation increased fourfold on thrombin-activated platelets, and this increase was blocked by inhibitors of protein kinase C. This phosphoryhtion potentially regulates some aspects of GP IIb-IIIa function. l o
24. 0
ASCH E, PODACK E: Vitronectin binds to activated human platelets and lays a role in platelet aggregation. J Clin Invest 1990, 8 P :1372-1378. Studies with anti-vitronectin and anti-GP IIb-IIIa antibodies suggested that vltronectin released by activated platelets is involved in PI&et aggregation in vitro through its RGD-dependent binding to GP nb-ma. 25.
FREL~NGER AL III, COHEN I. Ptow EF, Shimr MA, ROBERTS J, IAM SC-T, GINSBERG MH: Selective inhibition of integrin function by antibodies specilic for li and-occupied receptor conformers. BIOI &em 1990, 2 %5:634ti352. Contraction of p Q teletilibrin clots, but not Ebrinogen binding to activated platelets, was inhibited by monoclonal antibodies that only recognize epitopes on GP IIb-ma which has bound an RGD-containing peptide. This suggests the formation of functionally important sites on GP IIb-ma induced by &and binding. l e
26. 0
FERREU JE JR, m GS: ‘Qrosine-specific protein phosphorylation is regulated by Proc Nat1 Acud Sci USA 1&c%%$2?&~a in ‘latelets’ Selective phosphorylation of tyrosine’residues of platelet cytoplasmic proteins in response to thrombin was inhibited by synthetic peptides that blocked the binding of Ugands to GP IIb-IIIa. These phosphorylation events were absent in Glarrzmann’s thrombasthenia platelets, which lack GP IIb-ma. These results suggest a role for GP IIb-ma in regulating intracellular tyrosine phosphotylation, although the physiological significance of this function is unknown. 27.
JOHN.STON GI, Coon RG, McEVER RP: Clonin a granule membrane protein of platelets an 3 se uence similarity to proteins involved in l e an2 inRammation. Cell 1989 56:103%1044. The primary sequence of GMP-140 bredkted from the from a human endothelial cell Library contains multiple mains comparable with other adhesive receptors of the ELAM-1 and IAM-1.
of GMP-140, endotheliw ceil adhesion cDNA cloned stn~ctutal doselectin family,
28. 0
MEN E. CEU A, GUBEKT GE, FURIE BC, ERBAN JK, BONFAE~ R, WAGNER DD, FURIE B: PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 1989, 59305312. The rapid, actlvdtiomdependent surface expression of GMP-140 on platelets allows their calcium-dependent specific adhesion to neutrophils. NeutrophiIs also bound to purified GMP-140. These studies provide a functional role for GMF-140 and identify a receptor involved in intercellular adhesion of platelets and Inllammatoty cells. 29.
GENG J-G, BEXLKQUA MP, MOORE I& MclMVRE TM, Prt~~orr SM, I(IM JM, Buss GA Zwuhw GA, McEVER RP: Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nahtre 1990, 343:757-760. NeutrophUs and related ceU lines specifically adhered to immobilized put&d GMP-140 or to COS cells transfected with GMP-140 cDNA The interaction of neutrophils and perhaps monocytes, with GMP-140 on endothelial cells and platelets [see 28) is likely to be an important mechanism for rapid neutrophil adhesion at sites of vascular injury. l
NM PJ, BERNDT MC, GOSKI J, \xTHITE GC II, LYMAN S, PADDOCK C, MUUER WA: PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamil Science 1990, 247:121+1222. PECAM-1 cloned r rom a human endothelial ceU cDNA library consists of 711 amino acid residues and contains a putative transmembrane domain, a 117.residue cytoplasmic domain, and a 574.residue extracytoplasmlc region including six immunoglobulin-like repeat domains. PECAM-1 may function in ceU recognition and adhesion and is the only member of the immunoglobulin superfamily found thus far on platelets. 30. a*
901
receptors
R.K. Andrews
in hemostasis and J.E.B. Fox
Gladstone Foundation Laboratories for Cardiovascular Institute, University of California San Francisco, Current
Opinion
in Cell
Biology
Platelets contain many adhesive receptors on their surface; Fig. 1 shows several families of these proteins. Among these receptors, the glycoprotein (GP) Ib-IX complex appears to be a unique glycoprotein complex that is critical for the initial adhesion of platelets. Many of the other major glycoproteins on the platelet surface are members of families of cell adhesion proteins. Glycoprotein IIb-IIIa is the most abundant member of the integrin family; GP Ia-IIa (a2pl), GP Ic-IIa (aSpl), very late antigen (~~06 (aG@), and the vitronectin receptor (avp3) are other integrins present on platelets (Table 1). Granule membrane protein (GMP)-140, a member of the recently described selectin family, is expressed on the surface of the activated platelet. PECAM-1, a member of the imrnunoglobulin family, has also recently been identified on platelets. This article reviews the role of the GP Ib-IX complex and the integrins in mediating the adhesion, activation, and aggregation of platelets, with emphasis on the most recently published data. In addition, potential involvement of the members of the other families of adhesion proteins in mediating the interactions of platelets at a site of injury is discussed. adhesion
Our understanding of the interactions Involved in the adhesion of platelets to the exposed subendothelium has been derived mainly from the use of the in vifro perfusion chamber in which a blood vessel with the endothelium removed is placed in a chamber through which anticoagulated blood is passed under defined flow conditions. The morphology of the blood vessel is then examined, allowing quantitation of platelets that have adhered to the denuded vessel wall and enabling the distinction to be made between those thar made initial contact, those that spread, and those that formed an aggregate. Use of this system has revealed that a number of different receptors are involved in platelet adhesion and thrombus formation and that the receptors and ligands involved are critically dependent upon the shear rate. The most important receptor in mediating the initial contact of platelets with the subendothelium at high shear rates (> 500s’) is the GP Ib-IX complex which consists of equimolar amounts of three polypeptides: GP Ib,, GP Iba, and GP IX (Fig. 1). Glycoprotein Ib, and GP
CMP-granule LFA-lymphocyte
894
1990,
Current
Biology
2:894-901
Interestingly, leucine-rich domains are found in numerous, apparently unrelated proteins of mammalian, invertebrate and microbial origin [3,4]. Glycoprotein V, an 82kD platelet-membrane protein hydrolyzed during platelet activation by a-thrombin, has also recently been shown to contain multiple homologous leucine-rich domains [ 51. Based on the known adhesion function of GP Ib,, it has been postulated that these proteins represent a class of leucine-rich cell-adhesive proteins (Krantz and Zipursky, EMBO Jl990,9:196+1977). However, it should be noted that there is currently no firm evidence that a leucine-rich sequence of any of these proteins constitutes a site of adhesion. Even though the von Wiiebrand factor (vWf)-binding and leucine-rich regions on GP Ib, are both contained within the same structural domain (see below), the direct involvement of the leucine-rich sequences in vWf binding has not been demonstrated.
Abbreviations membrane protein; CP-glycoprotein; function-associated antigen; VIA-very
@
Research USA
Ibp are disullide-linked, and GP IX is non-covalently associated with GP Ib (Du et al, Blood 1987, 9:1524-1527). Rotary shadowed images reveal that the complex is an elongated structure with a globular domain at the extracytoplasmic amino-terminal end of GP Ib, (Fox et al, J Bio/ Chem 1988, 263:4882+890). Glycoprotein Ib,, GP Ibp, and GP IX have been cloned from human erythrocyte leukemia (HEL) cell CDNA libraries, and each has been shown to be a separate gene product (Lopez et al, Proc Nat1 Acud Sci USA 1987, 84:5615-5619; Lopez et al., Proc Nat1 Acud Sci USA 1988, 85:2135-2139) [ll. Each glycoprotein contains a cytoplasmic domain. Both GP Ibp and GP IX contain cytoplasmic cysteine residues that appear co be palmitylated [ 21. The cytoplasmic domain of the complex is associated with the membrane skeleton (Fox,J Clin Invest 1985, 76:167>1683), a structure that may regulate the functional activity of the receptor (see below). The amino acid sequence of GP Ib,, but not GP Ibp or GP IX, contains a highly glycosylated serine/threonine-rich macroglycopeptide region of about 80 residues located close to the extracytoplasmic side of the membrane (Fig. 2). Glycoprotein Ib, contains seven leucine-rich internal repeat domains toward the aminoterminal, extracytoplasmic end of the protein (residues kuj6-Uel87; Fig. 2). A single homologous leucine-rich domain occurs in the extracytoplasmic portion of both GP Ibl-, and GP IX, and the flanking sequences of these domains in GP Ib,, GP Ibp, and GP IX are also highly conserved.
Introduction
Platelet
Disease, Cardiovascular San Francisco, California,
Ltd
HEL-human late antigen;
ISSN
0955674
erythrocyte vWf-von
leukemia; Willebrand
factor.
Platelet
Table
1. Vascular
cell adhesron
receptors
in hemostasis
Andrews
and Fox
receptors.
Cell types
Soluble or matrw
associated
kgands
Cell-assocrated
kgands
Receptor
Leu-rich family CP lb-IX lntegnn family VLA-1 la-lla
0
l
(alp11
l
0
VLA-3 VLA-4
fa2pl) fa3f31) fa4pl)
Ic-lla VLA-6
fa5pl) (abpll
l
l
l
l
LFA-1 Mac-l
faLp2)
l
wq32) (aXj.32) fallbp3)
l
l
l
l
l
(avp3)
l
p150/90 Ilbllla VNR
0
0
l
l
(a+3
l
.
l
0
0
l
0
0
l
l
l
l
l
l
l
l
l
l
l
l
l l l l
l
l l
.
.
l
l
l
l
l
l
l
l
l l
l
l
l
l l
LAM-l Ig-like family VCAM-1 PECAM-1
l
l
l
l
l
l
l l
l
ICAMICAM-
CP, glycoprotein; receptor;
l l
l
l
Selectrn family CMP-140 ELAM-1
l
l
l
l
l .
l
l
CP IV CP VI
wtronectin
.
LAM,
lymphocyte
vWf.
von
l
l
l
l
adhesron
Wrllebrand
molecule:
LFA. lymphocyte
functton-assocrated
antrgen.
VLA.
very
late antigen;
VNR,
factor
The component of the subendothelial matrix to which GP Ib-IX binds is vWf. Considerable progress has recently been made in delining the vWf-binding domain on GP Ib-IX. In vivo, GP Ib-IX does not bind plasma vWf; the vWf must first bind to unidentified components of the subendothelial matrix. Presumably, this induces a conformational change in the molecule that then allows it to bind GP Ib-IX. However, the binding of soluble vWf can be induced by non-physiological modulators such as the microbial antibiotic glycopeptide known as ristocetin or the snake (Bothr-o~~jururucu) venom protein botrocetin. Botrocetin functions by binding to a regulatory binding site on vWf, but the mechanism of ristocetin activity is obscure (Read ef a,!, Blood 1989, 74:1031-1035) [6,7]. Other assays have used asialo-vWf (neuraminidasetreated native vWf) or bovine vWf, both of which bind
directly to GP Ib-IX in the absence of either ristocetin or botrocetin. These in vitro assay systems have enabled investigators to localize the vWf-binding site on GP Ib-IX to the globular amino-terminal domain of GP Ib, (His1-Arg293>. First, it was discovered that antibodies against this, but not other regions of the complex, inhibit binding of vWf to intact platelets or to purikd GP Ib-Ix (Wicki and Clemetson, Eur J Biocbem 1985,153:1-11; Handa et cd, J Biol Clam 1986,261:1257+12585; Bemdt et al, Biocbem&?y 1988, 27:633&O). Second, the amino-terminal domain isolated from tryptic digests of the purikd receptor completely inhibits binding of native vWf to put&d GP Ib-Ix in the presence of botrocetin [6,8]. Third, this fragment binds directly to immobilized vWf in the presence of risto-
895
8%
Cell-to-cell
contact
and extracellular
matrix
.
(b)
l-l
UI II”
(heavy chain) (116 kD)
(d) N Aminoterminal peptide domain
Ixtracellular
(e)
#
.2.. . L
~-,-J
u
C
CP IX (22 kD)
u C
C
C
GP lb0 (25 kD)
(105 kD)
GP llla
CP Ilb (light chain) (23 kD)
GP Iba
CP IV (88 kD)
CMP-140 (140 kD)
PECAM-1 (130 kD)
(135 kD)
Fig. 1. Platelet adhesion receptors: (a) the glycoprotein (CP) I&X complex; 0, Leu-rich domain; 18, macroglycopeptide. (b) GP Ilb-llla region. Other integrins on platelets are the (allbe3), the major integrin receptor on platelets; q , Cys-rich domain; UN, Ca 2+-binding collagen receptor, GP la-lla (a2pl); the fibronectin receptor, GP Ic-lla (a5Pl); the laminin receptor (abpl); and the vitronectin receptor (avp3). (cl CMP-140, a member of the selectin family; n , lectin-like domain; lZl, ECF-like domain; W, CRB-like domain. (d) PECAM-1, a member of the immunoglobulin (Ig) superfamily; q , Ig-like domain. (e) CPIV; @I, Cys-containing region. C, carboxy terminus; CRB, complement regulatory binding protein; ECF, epidermal growth factor; N, amino terminus; W, transmembrane domain.
cetin (Vicente et al, JSiolCbem 1988,263:18473-18479). That only this domain of GP Ib, binds vWf has been directly confirmed on intact platelets by covalent crosslinking analysis using a monomeric 39/34 kD VWf fragment (Le~~al~~+y,~~) that retains the GP Ib-K-binding site [7]. Further definition of the amino acid sequence in the amino-terminal domain of GP Ib, that binds vWf has been obtained by determining the effect of synthetic peptides based on sequences within this domain. Using this approach, one group observed that a peptide, Ser251-m279, parMy inhibited the vWf-GP Ib-IX interaction [8]. Another group identified A.s~~~~L~s~~~as be-
ing inhibitory [9]. While this approach may provide insight into important regions of primary sequence, several lines of evidence suggest that the three-dimensional organization of the amino-terminal domain is necessary for a fully functional vWf-binding site. First, the active peptides just referred to were much less inhibitory on a molar basis than the native amino-terminal domain was. Second, other peptides from within the amino-terminal sequence were only marginally less inhibitory than the Ser251-Tyr279 peptide. Third, reduction of the disulfide bridges within the purikd amino-terminal domain dras tically reduced its inhibitory activity toward botrocetindependent binding of native VWf and toward binding of asialo-VWf to platelets [8].
Platelet
receptors
in hemostasis
Andrews
and
Fox
Cytoplasmic Amino-terminal
peptide
domain
Macroglycopeptide
domain
domain
I 610
C I
’
’ ’ Leucine-rich
’ ’ domain
’
I
Putative vWf-binding peptides
Fig. 2. Structural and functional domains peptide sequences, Ser2s,-Tyrz,g [Sl and glycosylation; 0, N-linked glycosylation.
I
III
1 I
[II III
Transmembrane domain
II[I f k
Serzsl
I
,
I
’
!A
- Tv279
sP235 - LYs,62
of CP lb,. The location within the amino-terminal domain of leucine-rich repeats Asp 2.s-Lys262 [91, reported to inhibit the vWf-CP lb-IX interaction, are indicated. (The number of O-linked glycosylation sites is under-represented in this diagram.)
and
0,
of two O-linked
Together, the studies described above have localized a distinct binding domain on the GP Ib-IX complex that mediates its interaction with vWf, even though the precise contribution of smaller discrete amino acid sequences involved is yet to be confirmed. Some indication that binding can be induced at different sites depending on the modulator used comes from the Iinding that GP Ib, peptides had different inhibitory activities towards ristocetin-dependent compared with botrocetindependent binding of vWf to platelets [8], and from the report that an anti-GP Ib, monoclonal antibody (OP-Fl) inhibited binding of vWf to platelets in the presence of ristocetin but not in the presence of botrocetin (Nishio et al, 7h-omb Hamost 1989, 62:2I9). Therefore, the exact relationship between these in vitro lindings and the binding of platelets to subendothelial matrix-associated vWf in vivo remains to be determined.
mains to be determined, there is now considerable evidence that the GP Ia-IIa complex (the integrin VIA-2, a2j31) is a functional collagen receptor. First, platelets of a patient lacking GP Ia showed decreased adhesion to collagen (Nieuwenhuis et al., Nature 1985, 318:470-472; Nieuwenhuis et al, Blood 1986, 68:692495). Second, monoclonal antibodies against the complex inhibited the adhesion of platelets to a collagen-coated surface (Kunicki et al, J Biol C&m 1988,263:4516-4519) [ 131. Most importantly, pm-&d GPI-IIa complex has now been shown to bind specifically to collagen [ 141, and the binding of collagen to these proteins has the same divalent cation dependence as that of intact platelets to collagencoated surfaces (Santoro, Cell 1986,46:913-920).
Although GP Ib-IX is the most important receptor in mediating the initial adhesion of platelets to the subendothelial matrix, other receptors may be involved. Antibodies against vWf markedly inhibited adhesion of platelets to rabbit aorta subendothelium at high shear rates ( > 1000 s-t), but they had no effect at low shear rates (< 500~~l; Meyer et al., Br J Haemutol 1984, 57:60+620; Stel et al, Blood 1985, 65:85-90). At the low shear rates, which are comparable to those in the aorta or large blood vessels, the interaction of platelets with collagen or Iibronectin may be important. Recently, a number of platelet receptors for collagen have been proposed, including GP N (also known as GP IIIb or CD36)--a recently cloned 88 kD glycoprotein expressed on platelets, endothelial cells, and monocytes [ lO,ll]and GP VI [ 121. While the physiological importance of these glycoproteins in mediating adhesion to collagen re-
As platelets adhere to the subendothelial matrix, they be come activated. This transition is essential for allowing the subsequent stages of the hemostatic response. Activation is induced either as a consequence of interaction with collagen or by thrombin that is generated as the coagulation cascade is accelerated at a site of injury. The observations that the binding of soluble vWf to GP Ib-IX induced under conditions of high shear (Ikeda et al, Clin Res 1990, 38:426A) and the binding of soluble asialovWf to GP Ib-IX (De Marco et al, J Clin Invest 1985, 75:1198-1203) induced platelet activation raise the possibility that the interaction of GP Ib-IX with vWf in the extracellular matrix may provide an additional mechanism for generating intracellular messages that lead to the activation response. The Iinding that the cyclic AMP-dependent phosphorylation of GP Ibp may inhibit agonistinduced actin polymerization [ 151 also provides some
Platelet
spreading
and aggregation
897
898
Cell-to-cell
contact
and extracellular
matrix
support for the idea that GP Ib-IX may regulate signal transduction. . As platelets become activated, they spread over the subendothelium, increasing the area of contact, and adcltional platelets then aggregate to the initial layer of adherent platelets. The GP IIb-IIla complex is the receptor that mediates this aggregation. As shown in Fig. 1, GP IIb-IIIa is a member of the integrin family of adhesion receptors, consisting of a heterodimeric non-covalent complex of an cr-subunit and a P-subunit. Both subunits of GP IIbIIIa have been cloned and sequenced (Poncz et al., J Biol cbem 1987, 262~8476-8482; Fitzgerald et al, J Biol C&em 1987, 262:3936-39391, and this year the gene structures for platelet GP IIb [ 161 and GP IIIa [ 171 have been described. Recent work has demonstrated that both posttranslational processing and surface expression of the GP IIb-IIIa complex require the presence of both a- and psubunits [ 18,191. On the unstimulated platelet, GP IIb-IIIa is present on the surface but is not active. When platelets are activated, GP IIb-IIIa becomes competent to bind adhesive ligands. Considerable progress has been made in identifying the binding sites for these ligands on the complex [ 201. One of the adhesive ligands that binds to activated GP IIb-IIIa is fibrinogen, and it has long been assumed that platelets aggregate as a consequence of the binding of this multivalent ligand to GP IIb-IIIa However, it is now known that GP IIb-IIIa can also bind the adhesive ligands fibronectin, vWf, and vitronectin (Parise, Curr Opin Cell Biol 1989, 1:947-952). Recently, Weiss et al [21] have investigated the importance of these ligands in mediating the spreading of platelets on the subendothelium and the formation of thrombi. It was observed that at high shear rates, platelets adhered equally well in the presence or absence of fibrinogen. Further, antibodies that prevented the binding of fibrinogen had no effect on the number of spread platelets, nor did they inhibit the size of the thrombus that formed. In contrast, spreading and thrombus formation under these conditions were inhibited by an antibody that prevented the binding of vWf and vitronectin. Other studies have provided evidence that under conditions of high shear, platelets aggregate as a consequence of the binding of vWf to the GP IIb-IIIa complex (Moake et al, Blood 1988,71:1366-1374) [21]. Thus, while fibrinogen may be the ligand that mediates aggregation under conditions of low shear, it is becoming apparent that vWf may be an important ligand medi ating adhesion of spreading platelets and the formation of thrombi at high shear. The question of how the GP IIb-IIIa complex becomes competent to bind adhesive ligands on the activated platelet remains unanswered. It is particularly intriguing to speculate on mechanisms by which activated GP IIbIIIa may bind fibrinogen at low shear rates and vWf at high shear rates. Is this a function of a shear-induced change in vWf, or does it reflect a difference in intracellular messengers generated when platelets are activated under conditions of high shear? Although it is possible that increased access of adhesive ligands to a pre-existing binding site results from an activation-induced change in components of the membrane other than the GP IIb-IIIa
complex (Caller, J Cell Biol 1986, IO3:451-456), the recent demonstration that a monoclonal antibody (D33C) against a discrete sequence of GP IIb induced fibrinogendependent platelet aggregation [22] provides some support for the concept that expression of a functional receptor results from a modification to the GP IIb-IIIa complex itself. The only activation-induced covalent modification that has been described in the complex is phosphorylation of GP IIIa, which appears to be induced on threonine residues by protein kinase C [23]. Future studies will be needed to determine whether this modification al ters the ability of the GP IIb-IIIa complex to bind ligand. Interestingly, as platelets become activated and GP IIbIIIa becomes competent to bind to adhesive ligands in the subendothelium, the GP Ib-IX complex appears to lose its ability to bind vWf. The GP Ib-IX complex is as sociated (via actin-binding protein) with the membrane skeleton (Fox, J Biol Cbem 1985, 260:1197@11977). It has been suggested that this association is responsible for maintaining the GP Ib-IX complex uniformly distributed over the surface of the membrane, thus allowing the maximum number of GP Ib-IX molecules to interact with the multivalent vWf molecule (Fox and Boyles, Blood 1985,66:304a). It is possible that the decreased functional activity of GP Ib-IX observed at early stages of platelet activation (George and Torres, Blood 1988, 71:1253-1259) may result from internalization of the GP Ib-IX complex (Nurden et al, Blood 1989, 74:129a), an event that could be directed by the membrane skeleton. Since activation of calpain has been shown to result in a clustering of the complex on the platelet surface, an additional mechanism may involve activation of this intracellular protease at the later, irreversible stages of platelet activation. In recent studies, amino acid sequences in the GP Ib-IX complex that may mediate the interaction of the complex with the skeleton have been identified (Andrews and Fox, Blood1990, abstract in press). Further, the GP Ib-IX complex has been expressed in a functional form on the surface of HeIa cells, a cell type that contains actin-binding protein (Lopez et al, Blood 1990, abstract in press). It is anticipated that future studies in which GP Ib-IX is expressed in a native form or in a form in which it can no longer associate with the cytoskeleton will allow the importance of the cytoskeleton in regulating its distribution and function to be evaluated further. Stabilization
and contraction
of the thrombus
A developing platelet thrombus is initially fragile and can be readily washed away by the flowing blood. However, activated platelets express procoagulant activity on their surface, thereby accelerating the formation of fibrin. Thus, the fragile aggregate becomes stabilized in a network of polymerizing fibrin. The aggregate may also be stabilized in part by the interaction of thrombospondin with GP N on the platelet surface. It has been suggested that the GP N-thrombospondin complex associates with the GP IIb-IIIa fibrinogen complex (Nachman et al. In i%rombosis and Haemcstus& 1987, edited by Verstraete M et al. International Society on Thrombosis and Haemostasis and Leuven University Press, 1987,
Platelet
pp 81-91), although the derailed mechanisms of these secondary interactions are not understood. Similarly, evidence has been provided that binding of vitronectin to the vitronectin receptor has a role in mediating thrombus formation, although the relative importance of this receptor compared with GP IIb-IIIa is not known [ 241. Contraction of the platelet thrombus is an important secondary stage in wound healing. It is thought that fibrin binds externally to the GP IIb-IIIa complex, which in turn associates intracellularly with the cytoskeleton (Phillips et al, J Cell Biol1980, 86:77-86). As the cytoskeleton contracts, the fibrin clot is retracted. An important question in understanding the molecular mechanisms involved in this response is how does the GP IIb-IIIa complex associate with the cytoskeleton? Although talin redistributes to the periphery of the ceU upon platelet activation (Beckerle et al, J Cell Bioll989,109:3333-3346), future studies will be needed to determine the physiological role of this protein in mediating actin-membrane interactions in platelets. An interesting observation related to the mechanisms regulating clot retraction is that when GP IIb-IIIa binds an adhesive l&and, it undergoes a conformational change that exposes new epitopes. Several antibodies against these l&and-induced binding sites have been raised; one of them (PMI-2) inhibits clot retraction [25]. One mechanism that has been proposed to explain this observation is that occupancy of the fibrinogen-binding site on GP IIb-IIIa induces expression of a distinct site on GP IIb-IIIa to which fibrin (and PMI-2) binds [25]. Altematively, Isenberg et al. (I Cell Biol 1987, 104:16551663) have shown that occupancy of the fibrinogen-binding site results in clustering of the GP IIb-IIIa molecules, an other event that could conceivably be required for clot retraction to occur (for example, by inducing association of the cytoplasmic domain of the complex with the cy toskeleton). Finally, ligand binding to GP IIb-IIIa appears to regulate the intracellular activation of tyrosine kinase, yet another event that could conceivably be involved in regulating post-aggregation events such as clot retraction [26]. Future studies will be needed to elucidate the way in which occupancy of GP IIb-IIIa regulates intracellular events in platelets that have already aggregated and are thus a part of a platelet thrombus, and to elucidate how these events regulate the contraction of the thrombus. Other
adhesion
receptors
The interaction of platelets with white cells (neutrophils, monocytes, and/or lymphocytes) at the site of vascular injury may affect platelet aggregate formation and other platelet responses @ngi et al, Blood 1986, 67:629-636; Maclouf et al, Blood 1989, 74:703707). Therefore, while there is as yet no clear understanding of mechanisms, it appears possible that receptors of the selectin and immunoglobulin families will prove to have Important functions in regulating hemostasis. Selectins (GMP-140, EL&-l, and the lymphocyte homing receptor IAM-1) and members of the immunoglobulin superfamily (PECAM-1, VCAM-1, and ICAM- and -2) are present on platelets, endothelial cells, and/or vascular cells (Table 1). GMP-140 (Fig. lc) is a granule mem-
receptors
in hemostasis
Andrews
and Fox
brane component of platelets and endothelial cells that is expressed on the cell surface within seconds of ceU activation [27]. Cytokine-induced expression of ELAM1 on endothellal cells requires protein synthesis, and maximum surface expression occurs after several hours (Bevilacqua et al, Science 1989, 243:116&1165). GMP140 and ELAM-1 regulate the adhesion of neutrophils and perhaps monocytes to activated platelets and endothelial cells, and target white cell recruitment to the site of vascular damage [28,29] (Hamburger and McEver, Blood 1990, 75:550-554). PECAM-1 (Fig. Id) is the only member of the immunoglobulin superfamily identified so far on platelets, and is probably involved in cell-cell recognition and adhesion [30]. It appears likely that further studies on these recently described receptors will greatly increase our understanding of the way in which the interactions of a variety of cells act together to form a functional thrombus and thus to mediate hemostasis efficiently. Conclusions
and future
directions
Many of the receptors on platelets have now been characterized, and in several cases the molecular details of their Interactions with adhesive ligands have been elucidated. There are still many important questions yet to be answered, and new techniques and lines of research will be required to answer them. First, we need to use more physiological models to assess whether the domains on GP Ib-IX to which soluble vWf binds are the same as those that mediate the interaction of vWf with the subendothelial matrix in vivo. Second, investigation of the contribution of features other than the amino acid sequence of GP Ib, in regulating the binding of the GP Ib-IX complex to vWf is required. For example, what is the role, if any, of GP Ibb and GP IX in vWf binding to the GP Ib-IX complex; what is the role of the leucine-rich repeats; and what is the role of N and Olinked carbohydrate on the GP Ib-IX complex in regulating vWf binding? What is the role of GP V, another leucine-rich glycoprotein that, like the GP Ib-IX complex, is missing from Bernard-Soulier platelets? The GP Ib-IX complex has recently been expressed in mammalian cells in a functional form (Lopez et al, Blood 1990, abstract in press). It appears probable that the use of cells expressing this complex will allow some of these questions to be addressed. Third, we must consider how the GP Ib-IX complex associates with the membrane skeleton, and how this regulates the function of the complex. Fourth, what are the intracellular mediators that induce GP IIb-IIIa to express l&and-binding activity, and how does GP IIb-IIIa associate with the cytoskeleton? A fifth question is how are platelet adhesion receptors involved in signal transduction? For example, how does the shear-induced binding of vWf to GP Ib-IX initiate platelet activation? How does l&and binding to GP IIb-IIIa regulate intracellular events? Sixth, what is the importance of shear in vim2 It is becoming increasingly clear that the shear rate can have profound Influences on the binding of adhesive ligands to receptors. Seventh, which are the important ligands and receptors in the circulation? This is particularly important in terms of the development of specific inhibitors to prevent the formation of platelet thrombi at inappropriate sites. For example,
899
900
Cell-to-cell
contact
and
extracellular
matrix
which ligands and receptors are involved in the formation of thrombi at sites of rupturing atherosclerotic plaques? Finally, what is the role of the recently described selectins and members of the immunoglobulin superfamily of adhesive receptors, both on platelets and on the other cell types that are recruited into a developing thrombus, in mediating and stabilizing the platelet thrombus? It is to be hoped that the next few years will provide the answers to some of these questions. Annotated reading l
me
references
Of interest Of outstanding
and recommended
interest
1.
HIclcz~ MJ, WLLUAMS SA. ROTH GJ: Human platelet glycoproteIn IX: an adhesive prototype of Ieucine-rich glycoproteins with Bank-center-Bank strucmres. froc Nafl Acad Sci Cl.%4 1989, t366773-6rn. The predicted amino acid sequence for platelet GP IX cloned from a HEL cell cDNA library shows it to consist of 160 residues contain. ing a single leucine-rich sequence in the exuacytoplasmk domain and a putative transmembrane domain. The leucine-rich domain and its amino- and carboxyterminal llanking sequences are highly homologous to those found in the u- and P-subunits of GP Ib. l
0
2. a
MUSZBEK 1 LWXATA M: Glycoprotein lb and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages. J Biol c%em 1989, 26497169719. This work shows that a cysteine residue on the cytoplasmic domain of GP lbg and of GP IX may be covalently linked to palmItIc acid in platelets, although the stoichiometry and function of this modification await further study. P, COUMBATTI A: The carboxyl teminuS of the chicken a3 chain of collagen VI is a unique mosaic structure with glycoprotein lb-like, libronectin type III, and Kunits modules. J Btil Gem 1969, 264:2023%20239. Strucmral domains identified in the predicted primary sequence of the chicken collagen VI a3 chain include the KUII~Q domain common to the KUI-I~Q protease inhibitors and dendrotoxins; the type A domain found in other adhesive glycoproteins such as vWf and the a-subunits of platelet GP la-IIa (a2pl) and of the 82 integrin fm the fibronectin type III domain; and leucine-rich and serine/threonine-rich domains that are also found in GP lb-IX. Among the IeucIne-rich protein family, only collagen Vl a3 chain, E&ericbia co/i tra-E protein, GP lbg, and GP IX are known to contain a single leucine-rich repeat.
3. a
BONAIDO
4. 0
MIKOL DD, GULCHER JR, STEFANSSON K: The oligodendrocytemyeIin glycoprotein belongs to a distinct family of proteins and contains the HNK-1 carbohydrate. J Cell Biol1990, 110:471479. The 433.amino-acid glycoprotein that is glycosylphosphatidylinositol. linked to ollgodendrocytes and central nervous system my&n is structurally related fo platelet GP lb,, in terms of the structural organization of leucine-rich and sedne/threonine-rich domains, although the two proteins have no known functional relationship.
T, FUJ~~~URA K, MAEW S, TAKEMOTO M, ODA K, FUJIMOTO T, OYAMA R. SUZLJKI M, ICHIHARA-TANAKA K, TITANI K, KUR~MOTO A: Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein lb. Blood 1990, 75:234%2356. Partial amino acid sequence analysis of purified platelet GP V showed that this glycoprotein contaIned 10 or more leucine.rich repeat domains homologous to those found in GP lb-IX. Glycoprotein V, which is miss ing together with GP lb-IX on the surface of Bernard-SouIier syndrome platelets, currently has no identied function but is the only known platelet surface substrate for a-thrombin. 5. aa
6. l
SHIMOMUM
AND~%X Ry BOOTH WJ, GORMAN JJ, CA~TAIDI PA BERNDT MC: Purification of botrocetin from Bothrops jururucu venom. Analysii of the botrocetin-mediated Interaction between von WIebrand factor and the human datelet membrane glycoprotein lb-IX complex. Bicdemist& $3$ 28:83174326.
The isolated 45 kD amino-terminal peptide fragment of GP lb,. but not the serine/threonine-rich macroglycopeptide fragment, was shown to inhibit binding of purified vWf to purified GP lb-IX complex competitively in the presence of botrocetin. Also, monoclonal antibodies against the amino-terminal peptide domain inhibited the botrocetin.dependent vWfCP lb-IX interaction. 7. 0
ANDRRX’S RK, GORMAN JJ, BOOTH W’J, COWNO GL CASTAIDI PA, BEKNDT MC: Cross-linking of a monomeric 39/34kDa dispase fragment of von Wiiebrand factor (Leu48O/Val481-Gly-718) to the N-terminal region of the achain of membrane glycoprotein lb on intact platelets with bis(sulfosuccinimidyI)subcrate. Eiocbemisftv 1989, 28:8326-8336. A monomeric dispase fragment of vWf that retained the abiliry to bind fo GP Ib-IX was covalently crosslinked exclusively to the amino-terminal peptide region of GP Ib, on intact platelets. providing direct evidence that this region contains the vWf-binding site. 8. a
VICENI-IZ V, HOUGH’IFN Q RUGGEIU ZM: Identification of a site in the a-chain of platelet glycoprotein Ib that participates in von Wiiebrand factor binding. - I Biol C&m 1990, i65:274-280. A comprehensive analysis of 15.residue synthetic peprides from overlapping sequences within the amino-terminal region of GP lb, identified a number of sequences that blocked the binding of vWf fo platelets in the presence of ristocetin or bouocetin. While .some of these sequences participate in binding vWf, a fully functional binding site probabiy requires higher structure of the domain. In related experimems, reduction of disuffide bonds in the isolated amino-terminal fragment abolished botrocetin-dependent, but not ristocetin-dependent, inhibitory activity of the fragment. 9.
KATAGW Y, HAYA~HI Y. YA~~OTO K, TANOUE K, KOSAKI G, YA&%ZAKI H: Localization of von Wiiebrand factor and thrombin-interactive domains on human platelet glycoproteIn lb. lh-omb Huemast 1990, 631122-126. Partialiy overlapping synthetic peptides A.sp235-Lys,,~ and Phez,eThrzdo from within the amino-terminal peptide region of GP lb, were identified as inhibitors of the interaction of GP lb-IX on platelets with vWf in the presence of ristocerin and with a-thrombin, respectively, l
10.
OQUENIXI P, HUNDT E, LUXUR J, SEED B: CD36 directly mediates cytoadherence of Plusmodium fuhpunrm parasitized erythrocytes. Cd 1989, 58:95-101. The cDNA clone for CD36 from a human placental library predicted a protein of 471 residues, including a putative transmembrane domain, and nine potential N-glycosylation sites. CD36 is identical fo platelet membrane GP IV (proposed receptor for thrombospondin and col. lagen) and is expressed on the surface of Plusmodium fukiparuminfected erythrocytes. l
11. 0
TANNIN NN, KRAusz U, JA~UEWN GA: ldentilication of gly coprotein IV (CD36) as a primary receptor for plateletcollagen adhesion. J Biol C5em 1989, 264~75767583. A role for platelet GP IV in adhesion fo collagen-coated surfaces and in collagen-induced aggregation is suggested by studies with anti-GP IV antibodies and puriIied GP IV. 12. 0
MOROI M, JUNG SM, OKLIMA M, SHINMY~ZU K: A patient with platelets deficient in glycoproteln VI that lack both collagen-induced aggregation and adhesion. J Clin Irwest 1989, B4:144@1445. The studies described in this report of a platelet GP VI-deficient patient provide the first indication of a possible physiological role for GP Vl as a collagen receptor. COUIR BS, BEER JH, SCUDDER LE, STEINBERG MH: Collagenplatelet interactions: Evidence for a direct interaction of collagen with platelet GPla/lla and an indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins. Blood 1989, 74:182-192. Studies using an anti-GP la-IIa monoclonal antibody (6Fl) suggested a functional role for the platelet GP la-lla integrin complex (a2pl) as a receptor involved in adhesion of platelets to collagen. This antibody blocked the adhesion of platelets to collagen-coated plastic but did not inhibit collagen-induced platelet aggregation. 13.
0
14.
STAAIZ WD, RAJPARA SM. WAYNER FA, CARTER WG, SNORO SA: The membrane glycoprotein la-IIa (VLA-2) complex medIafes the Mg+ +dependent adhesion of platelets to colIagen. J Cell Biol 1989, lOJ3:1917-1924. When purified platelet membrane GP la-IIa (a2pl) was incorporated into phosphatidylcholine Iiposomes. they behaved like platelets in that l .
Platelet they bound to collagen in the presence of Mg*+, but did not bind if Mg*+ was replaced by Ca*+. Glycoprotein la-IIa apparentty did not support adhesion of the Uposomes to other adhesive glycoproteins. 15.
FOX JEB, BERN~?’ MC: Cyclic AMP-dependent phosphory lation of zlvcooroteln lb inhibits collaeen-induced wlvmerization‘of a actIn in platelets. J “Biol Cbem i98$, 264:952&9526. Analysis of cAMF-dependent phosphorylation of platelet proteins and response to activation in normal platelets compared with BemardSouller platelets (which lack GP Ib-IX) indicated a role for phosphorylatlon of GP Ibg in regulating collagen-induced actin polymerization. This supports the concept that the GP lb-IX complex may regulate sig nal transduction in platelets.
l
16. 0
HE~DENREICH R, EISMAN R, SURREY S, DELGROSSO K, BEJS, SCHWA E, PONCZ M: Organization of the gene for platelet glycoprotein IIb. Bbclwn&Q 1990, 29:1232-1244. The analysis of the genomic organization of both subunits of the GP IIb-IIla complex (see [17]) is important for future studies on the reg ulation of GP IIb-IIIa expression and function, and on the evolutionary relationships between GP IIb-UIa and the other related members of the integrin superfamily of adhesive glycoproteins. 17. 0
Zthmm AB, GIDWTIZ S, LORD S, SCHWA E, BENNETT JS, WHITE GC II, PONCZ M: The genomic organization of platelet gly coprotein IIIa. J Biol Gem 1990, 265:85m595. see [16]. 18. 0
O’TCXX TE, Lonvs JC, PLOW EF, GIASS AA, HARPER JR, GINSBERG MH: Efficient surface expression of platelet GP Ilb-IIIa requires both subunits. Biocd 1989, 74314-18. When either GP IIb cDNA or GP IIIa cDNA was uansfected into COS cells, each protein was synthesized, but there was no surface expression of either giycoprotein, and no proteolytic processing of GP IIb into light and heavy chains. However, normal GP IIb-IIIa complex was expressed by COS cells uansfected with both GP Ub and GP IIIa cDNAs. ROSA J-P, MCEVER RP: Processing and assembly of the integrin, glycoprotein IIb-III& in HEL cells. J Bzbl @em 1989, 264:1259&12603. Studies on expression of GP IIb-IIla in a human etythroleukemic cell line demonstrated that after translation, heterodimers of GP Ub and GP IIIa that formed in the endopla.smic reticulum, but not uncomplexed GP IIb, were transported to the Golgi apparatus where GP IIb was cleaved to heavy and light chains and its carbohydrate was modified. This demonstrated the importance of GP IIIa in surface expression of hlly processed GP IIb. 19. 0
20. 00
D’Souz~ SE, GINSBERG MH, BURKE TA, P~ovi EF: The Ugand binding site of the platelet integrin receptor GP I&-IIIa Is proximal to the second calcium binding domain of its a subunit. J Biol Gem 1990, 265:344&3446. In this paper, a synthetic peptide analogous to the carboxy-terminal peptide of the fibrinogen y-chain was covalently crosslinked to a dlsCrete sequence of GP Ub (residues 294-314) located at the second calcium-binding domain. An RGD-containing pepride had prevlousiy been crosslinked to a discrete region of GP UIa (residues lwl71). The combined studies deline distinct regions of both GP Ub and GP IIIa that are proximal to the l&and-binding site on the complex. 21. 00
WEISS HJ, HAWIGER J, RUGGERI ZM, TUR~~TO VT. THL%ARAJAN P, HOFFMANN T: Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invesf 1989, 83~288-297. At high shear, human platelet adhesion and thrombus formation in catheterized, perfused rabbit aorta was blocked by antibodies and synthetic peptides that inhibit intemction of adhesive ligands with platelet GP IIb-IIIa. The GP IIb-IIIa-mediated platelet adhesion and thrombus formation at high shear rate proceeded normally without fibrinogen and were also inhibited by an an&GP IIb-ma monoclonal antlbrxly that allows normal fibrinogen binding but blocks binding of vWf and libronectin. This is the first indication that, at high shear rates, adhesion of platelets occurs independently of Ebrinogen. 22. l
GUUNO D, RYCKEWAERT G: IdentIficatIon of a GP Ilb that interacts duces aggregation. J
J-J, ANDRIEUX A, RABIJ.X M-J, MARGUERIE monoclonal antibody against platelet with a calcium-binding site and inBiol &em 1990, 265:957%9581.
receptors
in hemostasis
Andrews
and
Fox
Binding of a monoclonal antibody, D33C, to a discrete peptide sequence on the GP IIb heavy chain (residues 426-437) induced Ebrinogen-dependent platelet aggregation. This tinding suggests that the functional actit-ion of GP IIb-ma may result from a specilic conformational change in the complex. 23.
PARISE LV, Cruss AB, NANNUA L, WARDEU MR: Glycoprotein IIIa is has horylated in intact human platelets. Blood 1990, 75:236$-23&. The P-subunit (GP ma) of the GP IIb-ma complex, but not GP IIb, was shown to be phosphorylated, predominately on threonine residues, on its cytoplasmlc domain. The degree of phosphotylation increased fourfold on thrombin-activated platelets, and this increase was blocked by inhibitors of protein kinase C. This phosphoryhtion potentially regulates some aspects of GP IIb-IIIa function. l o
24. 0
ASCH E, PODACK E: Vitronectin binds to activated human platelets and lays a role in platelet aggregation. J Clin Invest 1990, 8 P :1372-1378. Studies with anti-vitronectin and anti-GP IIb-IIIa antibodies suggested that vltronectin released by activated platelets is involved in PI&et aggregation in vitro through its RGD-dependent binding to GP nb-ma. 25.
FREL~NGER AL III, COHEN I. Ptow EF, Shimr MA, ROBERTS J, IAM SC-T, GINSBERG MH: Selective inhibition of integrin function by antibodies specilic for li and-occupied receptor conformers. BIOI &em 1990, 2 %5:634ti352. Contraction of p Q teletilibrin clots, but not Ebrinogen binding to activated platelets, was inhibited by monoclonal antibodies that only recognize epitopes on GP IIb-ma which has bound an RGD-containing peptide. This suggests the formation of functionally important sites on GP IIb-ma induced by &and binding. l e
26. 0
FERREU JE JR, m GS: ‘Qrosine-specific protein phosphorylation is regulated by Proc Nat1 Acud Sci USA 1&c%%$2?&~a in ‘latelets’ Selective phosphorylation of tyrosine’residues of platelet cytoplasmic proteins in response to thrombin was inhibited by synthetic peptides that blocked the binding of Ugands to GP IIb-IIIa. These phosphorylation events were absent in Glarrzmann’s thrombasthenia platelets, which lack GP IIb-ma. These results suggest a role for GP IIb-ma in regulating intracellular tyrosine phosphotylation, although the physiological significance of this function is unknown. 27.
JOHN.STON GI, Coon RG, McEVER RP: Clonin a granule membrane protein of platelets an 3 se uence similarity to proteins involved in l e an2 inRammation. Cell 1989 56:103%1044. The primary sequence of GMP-140 bredkted from the from a human endothelial cell Library contains multiple mains comparable with other adhesive receptors of the ELAM-1 and IAM-1.
of GMP-140, endotheliw ceil adhesion cDNA cloned stn~ctutal doselectin family,
28. 0
MEN E. CEU A, GUBEKT GE, FURIE BC, ERBAN JK, BONFAE~ R, WAGNER DD, FURIE B: PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 1989, 59305312. The rapid, actlvdtiomdependent surface expression of GMP-140 on platelets allows their calcium-dependent specific adhesion to neutrophils. NeutrophiIs also bound to purified GMP-140. These studies provide a functional role for GMF-140 and identify a receptor involved in intercellular adhesion of platelets and Inllammatoty cells. 29.
GENG J-G, BEXLKQUA MP, MOORE I& MclMVRE TM, Prt~~orr SM, I(IM JM, Buss GA Zwuhw GA, McEVER RP: Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nahtre 1990, 343:757-760. NeutrophUs and related ceU lines specifically adhered to immobilized put&d GMP-140 or to COS cells transfected with GMP-140 cDNA The interaction of neutrophils and perhaps monocytes, with GMP-140 on endothelial cells and platelets [see 28) is likely to be an important mechanism for rapid neutrophil adhesion at sites of vascular injury. l
NM PJ, BERNDT MC, GOSKI J, \xTHITE GC II, LYMAN S, PADDOCK C, MUUER WA: PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamil Science 1990, 247:121+1222. PECAM-1 cloned r rom a human endothelial ceU cDNA library consists of 711 amino acid residues and contains a putative transmembrane domain, a 117.residue cytoplasmic domain, and a 574.residue extracytoplasmlc region including six immunoglobulin-like repeat domains. PECAM-1 may function in ceU recognition and adhesion and is the only member of the immunoglobulin superfamily found thus far on platelets. 30. a*
901