©2007 Schattauer GmbH,Stuttgart

AnniversaryIssueContribution

Ashort history of plateletglycoproteinIbcomplex

Kenneth J. Clemetson 1957–2007)

Theodor Kocher Institute,University of Berne,Berne,Switzerland y(

“Mineisalong and sad tale” said the Mouse,turning to Alice tein bands were detected giving rise to the GPI,GPIIand GPIII and sighing.“It IS along tail,certainly,”said Alice,looking of earlyplateletreceptor nomenclature. In the mid-1970s these down with wonder at the Mouse’s tail; “but why do you call it techniqueswere appliedtoplatelets from the bleeding disorders Anniversar sad?” And she kept on puzzling about it…(1). Glanzmann’sthrombasthenia (3, 13) and Bernard-Soulier syn- th drome (3, 14). These clearly showedthat amajor partofthe GPI 50 1940s band wasabsent in Bernard-Soulier syndrome. The mid-1970s addedtwo-dimensionalnon-reduced/reduced electrophoresis, The historyofplateletGPIbisgenerally recognisedasstarting whichshowedthat manyifnot all membrane glycoproteins con- with the description of Bernard-Souliersyndrome (2),anin- tained disulphide bonds and some consisted of separate chains herited bleeding disorder, nowknown to be caused by the ab- linked by disulphide bridges(15). Thus, GPIb wasshown to con- senceordeficiencyinGPIbcomplexcomponents (3, 4). tain α and β disulphide-linked subunits.Surfacelabelling tech- niques were extensively appliedduring this period to enableeasy 1960s recognition of membrane glycoproteins.Lactoperoxidase-cata- lysediodination (14) and periodate/tritiated borohydride (16) At that time it wasnot clear that aplateletreceptor wasdeficient, gave different labelling patterns related to glycosylation levels and the first clues in thatdirection camefrom studiestowardsthe and tyrosinecontent of the individual glycoproteins. end of 1960s showing first thatBernard-Soulierplatelets had Another technique applied to at that stage wascross- less negative charge thannormal platelets and this wasdue to de- ed immunoelectrophoresis usingnon-ionic detergents, which creased amounts of sialic acid (5).Studiesonthe major ligand has the advantage that the arenot denatured. This was for GPIb, vonWillebrand factor (vWF), and its interactionswith able to distinguishbetween several of the proteins classedasGPI platelets were alsostarting aroundthat time. At first this wasnot and also establish some of their characteristics, including the recognised as such butcameasaside effect of the observation presence of functionalcomplexes(17, 18). This technique, as thatbovine “” agglutinatedhuman platelets. Of well as 125 I-labelled2Dpeptide mapping(19), clearly showed course, it wasnot the fibrinogen that wasresponsible for this but thatglycocalicin is the hydrophilic, extracellulardomain of the contaminating vWF, describedthen as “factor VIII related GPIbα and thatGPIb α contains additionalpeptidesthat arepre- ” (6).In1969 came the first biochemical isolation of (part sumablytransmembrane and cytoplasmic domains. It wasalso of) aplateletreceptor,the extracellulardomain of GPIb, proteo- during this period thatGPIbwas shown to bind to wheat germ lytic fragmentslater calledmacroglycopeptide derivedfrom agglutinin (20)and also to (21).The latter observation “glycocalicin”(7). raised considerable interest butlater also controversy. GPVwas nowshown to be cleaved by thrombin (22). Since Bernard-Sou- 1970s lierplatelets have reduced thrombin binding, and thrombin in- duced responses,this wasascribedtotheir lackofGPIband GPV

The next stage wastomakethe connection betweenplatelets and (21, 23). This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. vWF.This came in the early1970s when Bithelletal. (8) and Weiss et al.(9) showedthat Bernard-Soulier platelets did not ag- 1980s glutinate with bovine fibrinogen preparations. The discovery of the effect of ristocetin allowedinteractions betweenhuman pla- By thisstage most investigatorswere convinced of the roleof telets and human vWF to be studied (10).This period wasalso GPIb as vWF receptorand its absence in Bernard-Soulier syn- notedfor the first applications of acrylamide gelelectrophoresis drome.Attention nowturned to additional components of the methodstoplateletmembranes (11, 12). Three major glycopro- GPIb “complex” and to defining function more clearly.Com-

Correspondence to: Received May4,2007 Dr.K.J.Clemetson Accepted May23, 2007 Theodor KocherInstitute University of Berne Prepublished onlineJune 12, 2007 Freiestrasse 1 doi:10.1160/TH07–05–0327 CH-3012 Berne, Switzerland Te l.: +4131631 41 48, Fax: +41 31 631 37 99 ThrombHaemost 2007; 98: 63–68 E-mail: [email protected]

63 Clemetson: Ashort historyofplatelet glycoproteinIbcomplex

parison of the surfaceproteinsonBernard-Soulierplatelets with structure –the details would be revealed later–followedbytwo thoseonnormal platelets nowshowedthat twoadditional glyco- largedisulphide-linked loopsand then along mucin-likedomain proteins were missing (4). These were glycoprotein Vand glyco- containing manyO-glycosylation sites. Then follows the trans- protein IX.The first monoclonal antibodiesagainst plateletgly- membrane domain, with twocysteines nearthe outer surface, coproteins were alsodeveloped. Oneagainst GPIb α could block and the cytoplasmic domain. Thecloningand sequencing of vWF-induced plateletaggregation, thus definitively establishing GPIbβ (38)and GPIX (39) followedrapidlyand showedthat all

1957–2007) GPIbα as the vWFreceptor on platelets(24). The same antibody threemolecules had overallverysimilardomain compositions co-precipitated a22kDa band corresponding to GPIX from de- butthe latter twoare of course, muchsmaller. y( tergent lysates of platelets providing additionalevidencethat The end of the 1980s also revealed anew protein capable of GPIX wasanintegral partofthe GPIb complex. The availability inducing human plateletaggregation to humanvWF viaGPIb. of antibodiespermittedfunctional studies in newsystems in par- This wasthe snakevenomprotein botrocetin, whichsoon be- ticularthe first approximations to in-vivo systems (25, 26). came avaluable tool for studying this interaction (40). Triton X-114 phase separation wasapplied to platelets and Thepresence in platelets of mRNAcoding for GPIbα was Anniversar most of the known plateletreceptors, including GPV,were iso- also detected during this period (41),and first investigations of th latedinthe detergent phaseinline with their hydrophobic prop- the structureofthe genomic region encoding GPIbα including 50 erties. Surprisingly, however, GPIb-IX separated in thewater chromosomal location were published(42). In addition, acy- phase(27). This is assumed to be due, on the one hand,tothe fact lations of GPIb complexcomponents GPIX, by myristoylation thatthis molecularcomplexishighlyglycosylated and,onthe and GPIb,bypalmitoylation were described (43). other hand,itisalso strongly associatedwith cytoskeletal mol- ecules,such as filamin, of the submembranous cytoskeleton.Ef- 1990s forts at establishingthe stoichiometryofthe GPIb complexusing antibodiestoGPIb α ,GPIXand GPV provided evidencefor a The decade opened withthe molecularcharacterisation of the 2:2:1 relationship(28, 29), whichremained so until recently mutation(s) causing -type vonWillebrand’s disease (30). During this period considerable effort wasalso expendedin showing aGly233Val (44) or aMet239Val (45) change within establishingthe domain structureofGPIb α using variousprotei- one of the disulphide-linked loopsofGPIb α .Thus, mutations in nases and analysing the fragment patterns(31). Receptorswere GPIbα could not onlyreduce vWF binding but, dependingonlo- nowseen as signalling moleculesand GPIbα wasshown to be cation, also increase it. GPIb wasalso shown to be removed phosphorylated (32),with the phosphorylation on Ser166 in- rapidlyfrom the plateletsurfacefollowing activation (46). This creasing in response to PGE1 or PGI2 (33). Theglycosylation of could possibly be explained by aredistribution of membrane to GPIb α wasextensively investigated by several groups and the the surface-connected canalicularsystem. structures of both N- and O-linked oligosaccharideswere re- Newsnakevenomproteinssuch as the alboaggregins, which ported (34–36).Togetherthese resultsgaveapictureofGPIb α as activate platelets via GPIb, were described (47,48).The question an extendedmolecule containing binding sites butwith amucin- whether GPIb is asignalling receptor wasaddressed more seri- likestem. ouslyand binding of vWF wasreported to result in signal trans- Thenextmajor breakthrough and onethat clarified this issue duction (49).Inthis period we were alsoabletoshowhow the wasthe cloning of GPIbα ,which showedthat it wasamemberof distribution of disulphidebonds in GPIbα leads to an unusual the leucine-rich repeat protein family(37). The outer 45 kDa do- “knot”structureatthe coreofthe double-loop domain (50).Al- main consists of several leucine-rich repeatsforming acurved though it hasnever been formally demonstrated,ithas been This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Figure1:Acontemporaryversion of a model of the GPIb-V-IXcomplex. GPV reaches furtherout than in earliermodelsbe- cause the leucine-rich domainsare now thought to fold likethose in GPIbα giving a more open arc. Tw omoleculesofGPIb α linked covalentlyper GPIbβ via disulphidebonds are shown,inline withrecentfindings. The fine de- tail of thebindingsites forligands other than theA1domain of vonWillebrand factor, thrombinand FXIare notyet known. Further connectionstothe cytoskeleton and to signal- ing pathways areknown but areomitted for thesakeofclarity.

64 Clemetson: Ashort historyofplatelet glycoproteinIbcomplex tacitly(and reasonably)assumedthat the other complexcompo- toskeleton reorganisation (69).Expression of GPIb complex nents, GPIbβ ,GPIXand GPV contain the same type of structural genesintransfected cellswas able to assemble the functionalre- element basedonsimilarsequences.Indeed, it is likelythat this ceptor.Itwas nowshown that Ser609 on GPIbα is phosphory- is acommon featureofmanyrelated leucine-rich repeat proteins. lated, whichhad notbeen observed earlier, using 32Plabelling, Therole of GPIb in plateletresponses to thrombin were em- due to the lowrates of turnoveratthis site(70). phasised by several studiesincluding those with newblocking So farvWF and thrombin hadbeen the onlyknown GPIb li-

monoclonal antibodies. This period alsosaw newinterest in the gands. This wasnow extendedwhen P- wasshown to 1957–2007) cytoskeletal associations of the GPIb complex withdemonstra- bind via the sulphated tyrosines and an O-glycosylation siteof tions of actin-binding protein (nowcalledfilamin) binding (51, the anionic peptide domain (71). Possible signalling mech- y( 52). The possibilityofmonitoring levels of the solubleextracel- anisms were alsoextendedbyexperiments suggesting that FcRγ lular domain, glycocalicin, in plasma raised interest in what this playedanimportant roleinGPIbactivation of the srckinasesFyn might provide as information about the state of the circulation and Lyn(72). and possible pathological conditions (53). Much effortwent into identifying structure-function re- During this period the question also arose about expression lations within GPIbeitherbymutating singleamino acids or Anniversar of GPIb on endothelial cells and what physiological significance swapping specificdomains such as leucine-richrepeatswith th this might have,anarea notyet satisfactorilyexplored(54). An thosefrom other species. 50 interesting and still poorlyunderstood Bernard-Soulier syn- drome variant Leu57Phe in GPIb α wasdescribed (55) that 2000s showedautosomal dominant inheritance unlikethe recessive in- heritancenormallyobserved. Amajor step forward camewith Several papers appeared emphasising the important roleofGPIb the cloning and sequencing of the last componentofthe GPIb in plateletfunction and responses to thrombin complex, GPV (56).Itwas againfound to be amember of the (73–75).Sincethe discovery of the protease activatedreceptors leucine-rich repeat family, this time with 16 repeats, and asimi- (PAR)inthe 1990s, doubt had beencastonthe role of GPIb as a lardomain structuretothe other members. The thrombin cleav- thrombin receptor.For afew years this wasavery controversial age site wasidentified just belowthe disulphide loop knot. The area. Therangeofligandsusing GPIb as receptor wasextended function of the thrombin cleavage remained obscure. Following furtherwith the discoverythat (Mac-1, CD11b/CD18) is a snake venom proteins that agglutinated platelets by enhancing counter-receptor and alittle laterthat factorsXII and XI also VWF binding or directly by cross-linking GPIb,the first to block bind to GPIb (76).The preparation of amouse with GPIbα VWFbinding, echicetin, wasdescribed (57).Manyothersnake “knocked out” providedamodel for BSSand showedthat it venom C-type lectins with similar sequencesand activity were could be rescuedbygene therapy(77). subsequently described(58). The anchorage of GPIb complexinthe cytoskeleton viaits In the mid-1990s came the first description of mutants in interaction with filamin wasshown to be essential for platelet GPIXleading to Bernard-Souliersyndrome (59, 60) and the de- rolling on vWF(78). The question of the role of the membrane- scription of anew cytoplasmic protein associatedwith the GPIb associated cytoskeleton in regulation of GPIbbindingtovWF complex,designated14–3–3 ζ (61).This period alsosaw the first wasalso addressed(79). This is probablyrelated to the changes description of the tandemrepeat polymorphisms of a13amino in GPIbsurfaceexpression following plateletactivation. acidpeptide within the highly glycosylated stem region of Anotherimportant observation in the late 1990s and early GPIbα thatwere subsequentlymuch investigated as apossible 2000s wasbased on the preparation of micewith GPV “knocked sourceofvariation in cardiovasculardiseasesusceptibility(62). out” by twogroups in an effort to establish the function of this The thrombin-binding siteonGPIb α waslocalised to the anionic componentofthe complex(80–83). Although the resultsremain peptide region betweenthe outer domains and the highlyglyco- controversial, evidencewas obtainedsuggesting that the absence sylated stem (63).Anewsnakevenomprotein affecting GPIb of GPV rendered these mice platelets more susceptible to throm- function wasdescribed,this timeametalloproteasethat cleaves bin and theywere shown to form larger thrombi in vivo in re-

GPIbα in the middle of the sulphatedtyrosine/anionic peptide sponse to thrombin.Onthe other hand,some experiments sug- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. domain, and wastobecomeavaluable tool in studying GPIb gestedthat GPV might have arole in collagenbinding(83). function (64). Mutations in GPIb β leading to Bernard-Soulier During this period the first results appeared suggesting that syndrome were nowalso described (65), leaving onlyGPV GPIb might interact withGPVI, the recently characterisedcol- where arole in Bernard-Soulier syndrome had not beenfound. lagensignalling receptor,inits responses to its specificligands During this decade considerableprogresswas also madein (84). Snake venom C-type lectins interacting with both receptors understanding the regulation of GPIb complexgene expression to produceamaximalactivation of platelets hadalso been char- that would be later applied to studies of expression in mega- acterised(85). karyocytes(66). Towards the end of the 1990s the demonstration Binding of thrombin to GPIb caused conformational changes of tyrosinephosphorylation in response to GPIb specific platelet in the active site leading to enhanced cleavage of PAR1, suggest- activation (67)and aproposal for arole of the Fcγ RIIA receptor ing this might be the physiological role (86). in this processwere major advances (68).The first studiesap- In 2002 there wasaburst of structural activity with several pearedthat implicated GPIb as areceptor in bacterial binding to groups reporting crystallographic data on the outerdomain of platelets. Studiesshowedthat the interactions betweenvWF and GPIbα alone(87, 88) or in complexwith the A1 domain of vWF GPIb during platelet rolling induced actin polarisation and cy- (88,89). These clarified the natureofthe final complexformed

65 Clemetson: Ashort historyofplatelet glycoproteinIbcomplex

Ta ble1:Platelet GPIb ligandsand theirfunction. question that has arisen is what role lipid rafts play in GPIbfunc- tion. It seems that onlyafraction of GPIb molecules are present Ligand Function in lipid rafts and these maywellbethe acylated ones whichare A1 domain of vonWillebrand Critical in platelet bindingtoexposed therefore morehydrophobic (96).Calmodulin wasshown to be a factor sub-endothelium or activated endothe- cytoplasmic componentinteracting with the GPIb complexand, lium perhaps, regulating proteolytic cleavage of GPIbα and GPV

1957–2007) Thrombin Binding to GPIb has amajorrole in pla- (97). teletresponses to lowconcentrations of Evidencehas been foundthat soluble GPVI and glycocalicin y( thrombin (presentation to PAR-1?) canbind togethersuggesting functional interactions between Mac-1 Platelet bindingtomacrophages.Removal these molecules (98).Thrombospondin-1 wasshown to be yet of “chilled”platelets from the circulation anotherligand forGPIb, particularlyinsmallervessels where P-selectin (CD62P)Critical in platelet bindingtoactivated vWF is less expressed (99).Otherrecently demonstrated ligands endothelium or activated leukocytes include factor XI (100), factor XII (101), and (102, Anniversar FXI, FXII Critical roles in amplification of pro- 103). th coagulant cascade Recentyearshaveseen manystudies using in-vivo models of 50 High-molecular-weightkininogen Supports GPIb interaction with Mac-1 haemostasis and thrombosis to examine the roleofthe individual components of plasma, platelets andendothelial cellsinphysio- Thrombospondin-1 Can replace vonWillebrand factor in pla- teletbinding to small vesselsubendothe- logical andpathological processes. Depending on the type of in- lium at highshear juryinflictedand which ligands are exposed,receptors will be im- Collagen Indirectlyvia A1 and A3 domainsofvon plicatedtodifferent degrees.Itisneverthelessimpressive to see Willebrand factor the role that GPIb plays in many of these models (104). Following the failureof α IIbβ 3inhibitorstoreducecardiovasculardisease SolubleGPVIMay affect platelet responsestocollagen in apreventative context, there hasbeen alot of interestinalter- native strategies. GPIb has beensuggested as atargetinmany studiesindicating thatitmay be possibletouse GPIb antagonists, betweenthese twomolecules and howthe binding is regulated, such as small antibodiesorsimilar molecules,toprevent throm- butstill leaveanumber of questions unanswered. These struc- bosis without having an undue effect on haemostasis.Why this tures were quicklyfollowedbythose of complexesofthe outer should be is not completelyclear, andthe increasingnumberof domain of GPIbα withthrombin whichrevealed alot of newin- GPIb ligands would suggest that this is counterintuitive.How- formation butalso raised alot of newquestions (90, 91). These ever,perhaps targeting the specific ligands ratherthanGPIbitself structures supported earlier suggestions that thrombin bound to mightbeanalternative strategy andallowmore flexibility. GPIbα might be able to interact with otherGPIb α molecules on Eachyear morepublications on GPIb appear,but that does the same or otherplatelets, or with PAR1 or GPV. not change the fact thatthereisstill alot about this fascinating Morelight wasshed on howvWF/GPIbinteractions cause molecularcomplexthat remains unknown. Despiteintensive re- plateletrolling by the discoverythat this occurs via the formation search and many interesting publications over the past fewyears of “tethers”, long, pseudopod-like, membrane structures that are we still understand onlyverypoorlyhow clustering GPIb leads drawn out from the plateletsurfacefollowing contact betweena to plateletactivation. Improving this is amajor target forthe plateletand avWF-coated surface (92, 93). Evidently, this in- coming decade.Some recent excellent reviewshaveaddressed volves apatch of GPIb molecules linked to the cytoskeleton. this area (105–107).Onthe structural side, recent studies (30) “Tether”formation ends with the slowing of the platelet, with the demonstrate that thereare twoGPIb β molecules linked to each breaking of the “tether”, or with the tether detaching from the GPIbα and notone, showing howmuch progressweneed to surface. This process mayalso be involved in microparticle make in studying the complex as awhole. There arestill major formation. technical hurdlestocrossinorder to solvethese problems, but

Recent years have shown an increased importancebeing newmethodologiesmay allowusthis possibility. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. giventoGPIbinplateletfunction. It waswell-known thatpla- The question whether the GPIbcomplexisexpressed in other telets thathad been chilledbefore transfusion were rapidlyelim- cells,such as endothelial cells and tumour cells,and what its role inated, and this is whyplateletconcentrates have to be kept there is, whichwas once of intense interest,seemstohave warm.However,this naturallygives problems with bacterial slippedinto the background. Newinformation maywellbring it growth and restricts the length of time thatplateletconcentrates to the foreground again. There areundoubtedlymore GPIb li- canbestored to five days. Thus, studies showing that GPIb is gands than have beendescribed so far, and the list will continue clustered during chilling and that this has acritical role in platelet to lengthen. elimination because α M β 2-integrin on macrophages recognises This short review,describing howknowledge of one particu- exposed β -N-acetylglucosamine on GPIb(94).This has led to ef- lar receptor complexhas developedovernearly60years,has forts to increase glycosylation of platelets in ordertocover this necessarilyconcentrated on onlysome aspects. Iapologise to sugar residue as amethodtoallowplateletconcentrates storage those colleagues whose work has notbeen fullyaddressed,due to at lowertemperatures (95). lackofspace, whichalso limitedreference to their work. Several Recent years have shown an intensifiedinterest in activation earlier reviewsshould be consulted for more detailed references mechanisms and signalling pathwaysinvolving GPIb, and one and to fill these gaps.

66 Clemetson: Ashort historyofplatelet glycoproteinIbcomplex

Acknowledgements Work performed at the Theodor Kocher Institute wassupported by grant #31–107754.04fromthe SwissNational Science Foundation.

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