Sona Nair, Kanjaksha Ghosh, Bipin Kulkarni, Shrimati Shetty, DipikaMohanty, Institute of Immunohaematology (I.C.M.R), 13th Floor,K.E.M. Hospital Campus, Parel, Mumbai – 400 012, India.

Correspondence to: Dr. K. Ghosh, Institute of Immuno-haematology (I.C.M.R), 13th Floor, K.E.M. Hospital Campus, Parel,Mumbai – 400 012, India. Fax: +91-22-4138521; E-mail:mohanty@bom5.vsnl.net.in

Review

Glanzmann’s thrombasthenia: updated

Sona Nair, Kanjaksha Ghosh, Bipin Kulkarni, Shrimati Shetty,Dipika Mohanty

Glanzmann’s thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively morecommon platelet function defect in communities where consanguineous marriages are more frequent. Onclinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gumbleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma orhemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count andmorphology, a greatly prolonged bleeding time, absence of platelet aggregation in response to ADP, collagen,epinephrine, thrombin and to all aggregating agents which ultimately depend on fibrinogen binding to plateletsfor this effect, flow cytometry, studies of GPIIb–IIIa receptors on the platelet membrane surface usingmonoclonal antibodies. The present review describes some of the uncommon features of the disorders and thecurrently available options which the treating physicians should be aware of during the management of thesepatients. Although by definition all patients with Glanzmann’s thrombasthenia have a virtually complete failureof platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins arepresent in normal or near normal amounts but are functionally defective. Understanding the pathophysiologyof the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptorsresulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GTmanagement. It has now been established that different genetic mutations of either GPIIb or IIIa genes resultsin such a heterogeneity of thrombasthenia phenotype. Glanzmann’s thrombasthenia is a paradigm for treatingcoronary artery disease patients with GPIIb–IIIa antibody and inhibitors. By using these medicines we createa temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicingcardiologist. As mutations for different variant forms of GT become known, our understanding of how GPIIb–IIIa molecules can be activated to act as a receptor for fibrinogen molecules will be increased. Suchunderstanding undoubtedly will help us to devise better drugs with GPIIb–IIIa inhibitors. Molecular biologytechniques have enabled us to equivocally detect heterozygote carriers who are clinically asymptomatic.However, there may be several laboratories in the developing world, which have no access to molecular biologytechniques. Development of more robust techniques of quantitation of platelet receptors has enabled anaccurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of theGPIIb–IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the lessdirect method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as16 weeks of intrauterine life can be used for prenatal diagnosis of GT.

Introduction

Glanzmann’s thrombasthenia is an inherited hemorrhagicdisorder of platelets caused by the deficiency or

abnormality of the platelet glycoprotein (GP) IIb and/orIIIa. In 1918 Glanzmann,1 a Swiss pediatrician, descri-bed a group of patients with normal platelet count,impaired clot retraction and prolonged bleeding time. Henamed it hereditary thrombasthenia.

About 50 years later, the development of methods forstudying platelets demonstrated that thrombasthenicpatients failed to aggregate in response to physiologicalagonists such as ADP, epinephrine, collagen and throm-bin,2–5 had markedly reduced2–6 levels of platelet

ISSN 0953-7104 print/ISSN 1369-1635 online/02/070387-07 © 2002 Taylor & Francis Ltd

DOI: 10.1080/0953710021000024394

Platelets (2002) 13, 387– 393

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fibrinogen and had reduced or absent clot retraction. In1966, Caen et al.2 described 15 patients with Glanz-mann’s thrombasthenia in which platelet aggregationwas either nil or drastically decreased but the clotretraction was sometimes only slightly diminished. In1965, Castaldi and Caen7 showed that the plateletfibrinogen was either strongly diminished (in parallelwith the impaired clot retraction) or borderline to thenormal range.

In the mid 1970’s Nurden and Caen8 and Phillips etal.9 discovered that thrombasthenic platelets were defi-cient in both GPIIb and GPIIIa.

GPIIb–GPIIIa complex

The early studies performed with platelets from patientswith thrombasthenia identified GPIIb–IIIa as the plateletfibrinogen receptor.10 A normal single platelet containsapproximately 40 000–80 000 receptors on the surfaceand 20 000–40 000 receptors inside the a-granulemembranes of the platelets.11–13 The genes coding forglycoproteins IIb and IIIa are localized on the long arm ofchromosome 17 (bands q21–23) within a single 260-kbsegment.14 Though human umbilical vein endothelialcells also synthesize GPIIb–IIIa,15 this is evidently underseparate genetic control from that synthesized in themegakaryocyte, since endothelial cells from the umbilicalcord of an infant with Glanzmann’s thrombasthenia werefound to express the complex normally.16

It is normally present as a calcium-dependent hetero-dimer17 at very high density on the platelet surface. TheGPIIb–IIIa complex is composed of one molecule ofGPIIb (mol. wt. 140 000) consisting of a large chain withmolecular weight of 125 000 and a small chain of mol.wt. 25 000 linked by one or more disulfide bonds and onemolecule of GPIIIa (mol. wt. 105 000) which is a singlepolypeptide chain.18 The GPIIb–IIIa receptor (aIIb,b3)is a typical integrin. Its 136-kDa a-subunit, is atransmembrane protein with four calmodulin-likedomains that are able to bind divalent cations. Themature protein has 1008 amino acids, with one trans-membrane domain. The 92-kDa b-subunit consists of asingle polypeptide of 762 amino acids, with a shortcytoplasmic tail, a single transmembrane region and alarge extracellular domain.11 The subunits are non-covalently bound to each other and calcium is required tomaintain the heterodimer structure.19,20 The fibrinogenrecognition specificity of the GPIIb–IIIa receptor isdefined by two peptide sequence. The Arg–Gly–Asp(RGD) sequence was initially identified as the adhesivesequence in fibronectin,21 but is also present in fibrino-gen, von Willebrand factor and vitronectin. All theseligands contain at least one RGD sequence, whereasfibrinogen contains two RGD sequences, one near thecarboxy terminus of each of the two Aa (amino acids572–574) and another at amino acids 95–97. In addition,the carboxy terminal 12 amino acids of each of the

g-chains (amino acids 400–411) contain a sequence thatincludes Lys–Gln–Ala–Gly–Asp–Val and it appears

that the Lys and Gly–Asp form a molecular mimic of theRGD sequence.22 The Lys–Gln–Ala–Gly–Asp–Valsequence is found only in fibrinogen and is probably thepredominant site for the binding of fibrinogen toglycoprotein IIb–IIIa receptors; the RGD sequence mayalso participate.23– 26 Small synthetic peptides containingthe RGD or g-chain sequence inhibit binding offibrinogen to platelets, this observation has been used todevelop therapeutic agents to inhibit platelet thrombusformation and monoclonal antibodies as antiplateletagent.

Polymorphisms of GPIIb–IIIa

Several platelet alloantigen systems reside on GPIIb andGPIIIa molecules,27 of which the HPA-1 system isimportant in various disease processes.28 GPIIb andGPIIIa also contain the antigens responsible for mostcases of post-transfusion purpura and neonatal thrombo-cytopenia.29 HPA-1a and 1b antigen sites are situated onthe membrane glycoprotein IIIa.30,31 The genetic basis of10 alloantigens on the complex is known and nine ofthese involve single nucleotide substitutions. The antigenOe(a) is an exception in that it is the result of an ‘inframe’ deletion of three nucleotides and that it has arisenfrom a mutation in the HPA-1b allele rather than theHPA-1a form or ‘wild type’.32 There is a linkagedisequilibrium between the HPA-1b and 3b alleles, theHPA-3b is associated with a nine-basepair deletion inintron 21 of the GPIIb gene,33 HPA-9bwis linked to theHPA-3b allele34 and a rare leucine 40/arginine 40polymorphism on GPIIIa is linked to HPA-1b.35 Twosilent polymorphisms in the GPIIb gene show completelinkage disequilibrium with HPA-3.36 There are occa-sional studies where Glanzmann’s platelets could not begenotyped because of large deletion in the GPIIIa gene.37

Previous studies have shown difficulties in phenotypingplatelet antigens from Glanzmann’s thrombasthenia pla-telets38 and these platelets were referred to as ‘nullplatelets’.

Vitronectin receptor

The b3 subunit in integrin is also a component of thevitronectin receptor (VnR or avb3). VnR is a rarecomponent in platelets, with about 50 copies being foundat the surface as compared to the 50 000 copies ofGPIIb–IIIa receptor.39 In Glanzmann’s thrombasthenia,VnR is absent if GPIIIa production is affected.40 Thisreceptor can bind many of the same adhesive glycopro-teins on GPIIb–IIIa, although there are differences in theligand preference and binding sequences.41 VnR is foundon endothelial cells, fibroblasts, smooth muscle cells andosteoclasts. VnR complex contains GPIIIa but hasdifferent GPIIb-like peptide. This is homologous toGPIIb. Both bipeptidic structures belong to a family ofadhesion molecules (the cytoadhesins) which share acommon b-chain (GPIIIa or b3 chain) and have differentbut homologus a-chains.42

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Classification of Glanzmann’s thrombasthenia

Although by definition, all patients with Glanzmann’sthrombasthenia have a complete absence of plateletaggregation, the underlying biochemical abnormalityvaries from one kindred to another. Caen,43 in 1972, wasthe first to classify Glanzmann’s thrombasthenia intotype I and type II. According to his classification, type Ipatients were clinically more severely affected, hadabsent clot retraction and greatly reduced plateletfibrinogen. In type II Glanzmann’s thrombastheniapatients, clot retraction and platelet fibrinogen were onlyslightly diminished, although aggregation was equallydefective. Nurden et al., in 1985, found some patientswith type II Glanzmann’s thrombasthenia by the oldcriteria had about 15% of the normal amount of GPIIb–IIIa on their platelets and some type I patients hadresidual amounts of GPIIb and/or GPIIIa. Such differ-ences are genetically strongly suggested by the observa-tions of Coller et al. in 1987 on Glanzmann’s thrombas-thenia patients in Israel.44

The 1990 classification system of George et al.45 isthe most coherent. Patients with severely deficientglycoprotein IIb and IIIa, i.e., less than 5% of normalwere termed type I, patients with moderate IIb–IIIadeficiency, i.e., 10–20% of normal, were termed as typeII, and the patients with half normal to normal IIb–IIIawere termed variants. In addition to this heterogeneityamongst severely GPIIb–IIIa-deficient patients, someGlanzmann’s thrombasthenia patients have been descri-bed with normal or near normal levels of GPIIb–IIIa butwith their platelets lacking fibrinogen and the ability toaggregate.46 In 1986, Ginsberg et al.47 studied a patientwith near normal amounts of GPIIb–IIIa but greatlydefective fibrinogen binding which they presumed wasdue to abnormal surface orientation of GPIIb.

Nurden et al., in 1987, found that the normal level ofGPIIb–IIIa complex in their patient was abnormallysensitive to dissociation by EDTA and failed to bindfibrinogen.48 Fournier et al., in 1989, also found asimilar type of defect. In 1987, Tanoue et al.49 foundnormally dissociable but abnormally glycosylatedGPIIb–IIIa in a patient. Jung et al., in 1988, found apatient with 20% normal GPIIb together with 35% ofabnormal GPIIb of different molecular weight.50 Throm-basthenia Christchurch51 is another thrombasthenic vari-ant in which the patient’s platelets aggregate slightly atlarge doses of ADP or thrombin; the fibrinogen bindingis nil, while the GPIIb–IIIa complex using monoclonalantibodies was quantitatively normal.

Inheritance pattern

Glanzmann’s thrombasthenia has an autosomal recessivepattern of inheritance.52 This disorder has therefore beenobserved in both sexes. Glanzmann’s thrombasthenia,though a very rare disorder, is found very frequently inregions where consanguineous marriages are very com-mon. Heterozygotes appear to be asymptomatic and

generally have normal results in platelet functiontests.53,54 Forty-two cases of Glanzmann’s thrombasthe-nia have been reported from South India55 whereconsanguinous marriage is very frequent.

Clinical manifestations

Glanzmann’s thrombasthenia presents in infancy or earlychildhood with bleeding, multiple bruises followingminimal or unrecognized trauma or with petechiae orecchymoses. Usually the disease is discovered duringchildhood or in very rare cases, during adolescence.56

Epistaxes are very common and gingival bleeding isfrequent due to the shedding of deciduous teeth or eventooth brushing. Epistaxis may be difficult to control withlocal measure and may require abundant blood or platelettransfusions.54,57,59 Cutaneous bleeding is very commonwith easy bruising, numerous petechiae or ecchymoticpatches. Gastrointestinal hemorrhage is frequent in thefirst few years of life. Muscle hematoma and hemar-throses are very rare. Menorrhagia occurs in nearly allfemales at the time of menarche.

Laboratory diagnosis

Patients with Glanzmann’s thrombasthenia have normalplatelet morphology and normal platelet count, pro-longed bleeding time and absent or decreased clotretraction. Platelet aggregation in response to ADP,collagen, epinephrine and thrombin are completelyabsent though some variants of Glanzmann’s thrombas-thenia show aggregation with ADP. The aggregationdefect is seen in response to all other aggregating agentswhich depend upon binding of fibrinogen to the plateletfor their effect. Platelet aggregation in response toristocetin or bovine VWF which act through binding ofVWF to GPIb occur normally. Platelets have a normalinitial slope of ristocetin-induced aggregation, indicat-ing the normal levels of plasma VWF and the normalplatelet GPIb–IX content. The reduced second wave ofaggregation at low doses of ristocetin reflects impairedGPIIb–IIIa function.58 Platelets undergo normal shapechange in response to ADP and thrombin which showstheir ability to undergo metabolic and cytoskeletalchanges in response to agonists. Once the aggregationeffect is seen it can be confirmed by measuring theGPIIb–IIIa complex, GPIIb, GPIIIa separately andfibrinogen binding by using monoclonal antibodies.This is done nowadays by using flow cytometry on fewmicrolitres of blood sample.59 Though most of thepatients appear relatively homogeneous in having noplatelet aggregation in response to ADP and otheragonists and in having little or no detectable GPIIb–IIIacomplex on platelet surface by flow cytometry. Morestudies using sensitive immunoblotting methods for thedetection of GPIIb and GPIIIa in the platelet pool havedemonstrated significant heterogeneity.49,60 The screen-ing for the mutations are done by SSCP and themutations are detected by sequencing. These methods

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are, however, used mainly for research purposes. Clotretraction and PF3 availability are defective in Glanz-mann’s thrombasthenia. Thrombasthenic platelets havetheir normal granule secretion in response to thrombinor high concentrations of collagen, but not in responseto ADP or adrenaline which depend on aggregation fortheir secretory effect.

Detection of heterozygotes (carrier detection)

Heterozygotes for Glanzmann’s thrombasthenia usuallyhave no significant bleeding symptoms and no demon-strable defect of platelet aggregation.61 However, theirplatelets express about half the normal number ofGPIIb–IIIa complexes in the plasma membrane and havea corresponding partial defect of fibrinogen binding.Carrier detection is most accurately performed by DNAanalysis when the defect is known. Carrier detection isdone using a variety of methods for quantitative GPIIb–IIIa determination19,62–64 or fibrinogen binding.59 In1986, Coller et al. showed that a good agreement existedbetween the results of monoclonal antibody bindingassay, polyacrylamide gel electrophoresis and electro-immunoassay methods in a large number of obligatecarriers.

Kunicki et al.19 found that Glanzmann’s thrombasthe-nia heterozygotes expressed 50% of GPIIb–IIIa and 50or 25% of PLA1 antigen suggesting that the genecontrolling the expression of PLA1 and the defect inGlanzmann’s thrombasthenia are not the same. Stor-morken et al.,62 using CIE, found in heterozygotes aconstant level of GPIIb–IIIa (50–60%) with no over-lapping between the heterozygotes and normal. By usingflow cytometry and whole blood, Jennings et al.64

developed a method for distinguishing patients andobligate carriers from normal. The methods for carrierdetection and prenatal diagnosis were largely developedin Israel.65 GPIIIa Taq 1 polymorphism has been used todetermine the carrier status in Glanzmann’s thrombasthe-nia families of Chinese origin.66,67 It is possible torapidly predict the carrier status of family members ofGlanzmann’s thrombasthenia sufferers, both type I andII, by flow cytometric analysis.68

Prenatal diagnosis

The determination of the levels of platelet antigens onplatelets in the human fetus has been studied by Gruel etal.69 PLA1 and Leka antigens are expressed in normalamounts on fetal platelets as early as 16 weeks ofintrauterine life. The GPIIb–IIIa complex quantified bypolyclonal or monoclonal antibodies was in the samerange in fetuses and in adults.

Seligsohn et al.70 measured GPIIb–IIIa in fetal bloodsamples by monoclonal antibody binding assay andKaplan et al.71 used the less direct method of PLA1typing. These methods can be used accurately in familieswith type I Glanzmann’s thrombasthenia and can evendetect the heterozygous state in utero72 but cannot be

applied for the diagnosis of molecular variants ofGlanzmann’s thrombasthenia with normal or subnormallevels of platelet GPIIb–IIIa complexes.73,74 In suchcases, prenatal diagnosis must rest on the demonstrationof a platelet aggregation defect75 or fibrinogen bindingusing micromethods.59 The experience of Seligsohn etal.65 shows that fetal sampling for prenatal diagnosis ofGlanzmann’s thrombasthenia carries a high risk of deathfrom continuing hemorrhage to the affected fetus. Thisunderlines the importance of detailed counseling of theparents before the procedure is undertaken. In each case,parents must have informed counseling based on thecomplete genetic, clinical and laboratory knowledge ofthe disease and it’s familial severity.

Treatment

At present, there is no curative treatment short ofallogenic BMT. The management of GT consists mainlyin avoiding the risk of hemorrhagic trauma. Thetreatment of bleeding in these patients is very difficult,especially in cases where repeated platelet transfusionsresulted in antiglycoprotein (GP) IIb–IIIa or anti HLAalloimmunisation. Recombinant factor VIIa (rVIIa) hasbeen used as first line therapy in three patients with GT.This treatment resulted in excellent clinical efficacy andtolerance in two cases.75 Use of recombinant factor VIIahas been reported in three patients with inherited type IGT undergoing invasive procedures.76 In 2001, Poon77

reviewed the proposed mechanisms of action, clinicaleffectiveness and safety of rVIIa for the treatment ofbleeding disorders. The use of this drug may be limitedbecause of the cost factor.77 Drugs such as aspirin, whichmay further interfere with platelet function, should not beused. Good dental hygiene and care is essential tominimize gum bleeding and tooth extraction. Minorbleeding can be controlled by local measures. Folic acidand oral iron may be needed in patients with continuoushemorrhage to avoid anaemia and iron deficiency.Antifibrinolytic agents, such as tranexamic acid, mayhelp to control bleeding after tooth extractions. Menor-rhagia is often most severe from the time of menarche.Severe menorrhagia due to GT has been treated withhydrothermal ablation.78 Hormonal therapy beforemenarche can help control menorrhagia. Prepubertalcounselling is very important in these cases. The maintherapeutic approach is supportive therapy by platelettransfusions. The main limiting factor in this case is therisk of development of platelet antibodies. These may bedirected against platelet-specific alloantigens expressedon GPIIb–IIIa complex (PLA1, Baka, Pena) and morecommonly against HLA determinants. For this reason,platelets from a single HLA-compatible donor should beused whenever possible.

The only available curative treatment for GT is bonemarrow transplantation and that only when the hemor-rhagic manifestations are severe and refractory to plateletinfusions. It has been successfully performed by Bellucciet al.79 in a GT patient.

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Pitfalls and fallacies in the diagnosis ofGlanzmann’s thrombasthenia

Glanzmann’s thrombasthenia is an extremely rare dis-order and its clinical manifestations are similar to manyother platelet function disorders. Therefore, one has to beextremely careful while diagnosing such patients. Adetailed clinical history of the patient which includes thefamily history of bleeding, the type of bleeding, theseverity of bleeding, and history of the transfusionsgiven to the patient is very important. The diagnosis forsuch patients can be done at a three-stage level. Stage Iincludes all the routine tests, including the bleeding time,clotting time, clot retraction, platelet count and morphol-ogy and screening coagulation. Abnormalities in thebleeding time and clot retraction with normal plateletcount morphology in a consanguineous family setting arehighly suggestive. This basic facility is available in mosthospitals, even at the district level. Patients showingsome abnormality in stage I screening should then besent for more specific tests carried out at Stage II andStage III. Stage II includes the platelet aggregation tests.This testing facility is now available at quite a fewcentres in India. The platelet aggregation tests along withclinical and other diagnostic tests are confirmatory andcan be used to distinguish between the various plateletfunction disorders like GT, BSS and vWD. Stage III testsinvolve tests in which defective GPIIb–IIIa receptors ofGlanzmann’s thrombasthenia in platelets are studied bytechniques like flow cytometry. The receptor analysisusing respective monoclonal antibodies give a confirma-tory and distinctive diagnosis between the differentplatelet receptor function disorders. It is very importantfor the clinicians to take a detailed bleeding history of thepatients and to do an initial screening test beforeproceeding with any of the specialized tests. There areseveral conditions other than the platelet function defectthat may cause bleeding in patients. In such cases, theabove-mentioned tests may not be useful. Patients whoare on different types of anti-epileptic drugs like valproicacid, can occasionally produce antibody to GPIIb–IIIaand give an abnormal platelet aggregation and receptorstudy. In cases of coronary artery disease patients whoare already on b-blockers and ADP blockers, ticlopidineand clopidogrel may cause bleeding. The platelet func-tion studies done in such patients will be abnormal. Incases of ITP, platelet studies may also give abnormalresults due to the antibody developed against GPIIb–IIIain a subset of such patients.

Therefore a practicing clinician should be very carefulin his approach towards such cases while giving adiagnosis. The field of GT is evolving and a largenumber of investigations are available to explore thedefects in such a patient.

Hence, if the tests are not selected properly the patientwill unnecessarily have to bear heavy expenditurewithout any benefit from these tests. Increasinglynowadays GPIIb–IIIa inhibitors like eptifibatide is beingused for patients with coronary artery disease. These

drugs produce GT-like abnormalities in the platelets, andcertain studies80 showed increased incidence of anti-bodies to GPIIb–IIIa receptors. Patients who are onGPIIb–IIIa receptor-blocking drugs will continue toshow GT-like defects even when the offending drug isstopped, as long as the antibody-blocking GPIIb–IIIareceptors are present in the circulation.

Recently a gain in function in GPIIb–IIIa receptor hasbeen reported.81 In this condition, GPIIb–IIIa receptorsremain in a perpetually activated state without anystimulus to the platelet, and this perpetual activationleads to platelet exhaustion and GT-like defects in theplatelet. Existence of this kind of mutation or polymor-phism in GPIIb–IIIa receptors suggests that eventuallywe may find a class of mutations or polymorphism(HPA-1b is an example), in platelet GPIIb–IIIa whichwill lead to spontaneous aggregation or reduced thresh-old to aggregation towards different agonists predispos-ing these patients to atherothrombotic hazards.

This angle of gain in function mutation in GT patientsopens a totally new area of investigating and prognosti-cating patients with atherothrombotic disease.

Many GT patients who have received platelet infu-sions eventually produce anti-GPIIb–IIIa antibodies.Antibodies from some of these patients could be ofimmense therapeutic value. Antibodies engineered fromthese patients by experiments in nature may be rearedand nurtured by proper expansion of lymphocytes fromsuch patients using techniques such as Epstein–Barrvirus (EBV)-driven expansion and immortalisation ofsuch cell clones; combining the powerful tools ofinformation technology (loss and gain in function ofGPIIb–IIIa receptors correlating with three-dimensionalchanges in the receptor function using modern concept ofproteomics). Studies in association with advances incombinational chemistry will eventually allow us todevelop many orally active molecules capable of alteringGPIIb–IIIa receptor function in future both in positiveand negative directions.

Thus GT, once thought of a simple disease due toplatelet function defect, has resulted in the discovery ofmany variants and a model for elucidating the complexinteraction of the genes, receptors of the platelets.

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