Glanzmann's thrombasthenia: updated

Glanzmann's thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively more common platelet function defect in communities where consanguineous marriages are more frequent. On clinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gum bleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma or hemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count and morphology, 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 platelets for this effect, flow cytometry, studies of GPIIb-IIIa receptors on the platelet membrane surface using monoclonal antibodies. The present review describes some of the uncommon features of the disorders and the currently available options which the treating physicians should be aware of during the management of these patients. Although by definition all patients with Glanzmann's thrombasthenia have a virtually complete failure of platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins are present in normal or near normal amounts but are functionally defective. Understanding the pathophysiology of the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptors resulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GT management. It has now been established that different genetic mutations of either GPIIb or IIIa genes results in such a heterogeneity of thrombasthenia phenotype. Glanzmann's thrombasthenia is a paradigm for treating coronary artery disease patients with GPIIb-IIIa antibody and inhibitors. By using these medicines we create a temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicing cardiologist. 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. Such understanding undoubtedly will help us to devise better drugs with GPIIb-IIIa inhibitors. Molecular biology techniques 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 biology techniques. Development of more robust techniques of quantitation of platelet receptors has enabled an accurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of the GPIIb-IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the less direct method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as 16 weeks of intrauterine life can be used for prenatal diagnosis of GT.     

Human platelet alloantigen polymorphism in Glanzmann's thrombasthenia and its impact on the severity of the disease

Glanzmann's thrombasthenia (GT) is an autosomal recessive disorder of platelets caused by the deficiency or abnormality of platelet receptors. Several platelet alloantigen systems reside on glycoprotein (GP) IIb and GPIIIa, of which the human platelet antigen 1 (HPA-1) system is important. Studies have shown that, in the normal population, the HPA-1b phenotype results in increased platelet aggregation and increased fibrinogen binding, increasing the risk of myocardial infarction. GT produces severe bleeding, but in a subset of patients has a relatively milder course. Forty-one GT patients and 100 healthy control subjects were genotyped for platelet alloantigens HPA-1 to HPA-6, using PCR-ASA (polymerase chain reaction-allele-specific amplification), and for GPIIb-IIIa expression and fibrinogen binding using flow cytometric techniques. Platelet alloantigen distributions were similar in the patient and control groups. With the exception of the two HPA-1b/1b homozygous patients (> 10%), 25 GT patients had less than 5% aggregation to 6 micro mol/l ADP, and 16 patients showed between 5% and 10% aggregation to 6 micro mol/l ADP. Seven out of 37 patients with HPA-1a/1a phenotype showed 1-5% fibrinogen binding and GPIIb-IIIa receptors. The two HPA-1b/1b patients showed 34.6% and 32% fibrinogen binding and > 10% GPIIb-IIIa receptors. This study determined the platelet alloantigen distribution in a large cohort of unrelated GT patients from western India. GT patients homozygous for HPA-1b/1b had higher levels of platelet aggregation and fibrinogen binding as well as a milder course, as evidenced by infrequent epistaxis and no transfusion requirement to date.     

PMA induces platelet activation of specific antigens (CD62/CD63) in GpIIb-IIIa deficient platelets from Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary platelet disorder resulting from a quantitative or qualitative deficiency of the major platelet membrane complex GPIIb-IIIa (CD41) required for platelet aggregation. We investigated by flow cytometry, the expression of CD41, fibrinogen, and of two platelet activation-related antigens, CD62 and CD63, (i) before and after activation of platelets by PMA, and (ii) on the surface and within the cytoplasm of resting platelets, after permeabilization by saponin. Platelets from a series of normal subjects and from nine members of two GT families, were reacted with FITC-conjugated antibodies and analyzed on a flow cytometer. Fluorescence intensities measured on normal and GT platelets were quantified by using calibrated beads. Results showed lack of both GPIIb-IIIa and fibrinogen, on the platelet surface and also within the cytoplasm in five of these GT patients, whereas GPIIb-IIIa and fibrinogen remained normal in the four other cases. However, CD62 and CD63 antigenic levels were found within normal range for all members of these families, after PMA stimulation and also after platelet permeabilization. This work therefore showed that the lack of CD41 in GT, which causes strong disturbance of platelet aggregation, may not be associated with the deficiency of activation pathways.     

A novel Ser123Pro substitution in the MIDAS domain of integrin 3 associated with variant Glanzmann's thrombasthenia in an Indian patient

We report a novel 465T-->C (S123P) mutation in exon 3 of the GPIIIalpha gene in a patient with type III or variant Glanzmann's thrombasthenia (GT). Though this mutation did not affect fibrinogen binding to GPIIb-IIIalpha in activated platelets, it interfered with the platelet aggregation in a manner similar to GT.     

Classification of Iranian patients with Glanzmann's Thrombasthenia using a flow cytometric method

Glanzmann's Thrombasthenia (GT) is a rare inherited autosomal recessive platelet disorder caused by a deficiency or dysfunction of the GPIIb-IIIa receptor on platelets, which is characterized by a lack of platelet aggregation in response to multiple physiologic agonists and a life-long bleeding disorder. Flow cytometry is a rapid and highly sensitive method that can detect reduced levels of receptors, as well as absolute deficiency. The aim of this study was to classify Iranian GT patients by a flow cytometric method, and to correlate these findings with the severity of clinical bleeding. The expression of GPIIb-IIIa on the platelet surface was assessed in 123 GT patients using quantitative flow cytometry to determine the most common subtype among these patients. We used a panel of antibodies to detect the expression of glycoproteins GPIb, GPIIb, GPIIIa, as well as Integrin αv. Patients were also interviewed with regard to the severity and frequency of bleeding, according to history and gender, in order to evaluate the nature of their bleeding phenotype, and classify them as mild, moderate or severe bleeders, in accordance with the Glanzmann's Thrombasthenia Italian Team (GLATIT) protocol. In the detailed analysis of the results of our investigation, 95 out of 123 (77.5%) were classified as type I; 20 (16%) as type II with residual GPIIb-IIIa, and eight (6.5%) as GT variants. The variant type was diagnosed by the inability of GPIIb-IIIa to bind fibrinogen, as evidenced by the absence of platelet aggregation in response to physiologic agonists. There was no significant correlation between bleeding severity and different subtypes of GT. This study demonstrates that GT type I is the most common subtype among Iranian patients. There was no correlation between severity of symptoms and cytometric phenotype of the disease. The identification of families at risk may significantly decrease the incidence of the severe form of the disorder if genetic counseling is provided.     

Normal expression of the adhesive structure Mo-1 on monocytes from patients with Glanzmann's thrombasthenia

The glycoprotein complex GPIIb-IIIa of the platelet membrane and CD18 of the monocyte membrane have been established as being the central figure for the adhesion processes of the corresponding cells. The molecular structure of these 2 GPs bears some similarities. It was proposed recently that LFA-1 and Mo-1 (CD18 family) and GPIIb-IIIa might be encoded by the same gene. For this purpose, we studied the expression of Mo-1, Mo-2 (as control) receptors on monocytes and GPIIIa and GPIIb-IIIa on platelets of two GT patients as compared to normal healthy subjects. Monoclonal antibodies anti Mo-1, MO-2, AP-3 and AP-2 were used to measure the expression of the respective antigens by indirect immunofluorescence procedure. The fluorescence of the labelled cells was analysed with an Ortho Cytofluorograph 50-H. The resulting Mean Fluorescence Intensity (MFI) values of AP-2 and AP-3 showed that the patients had a total absence of GPIIb-IIIa antigens. However, Mo-1, as well as those of control Mo-2, in patients (MO-1: 672 and 716; Mo-2: 453 and 637) were similar to that of normal subjects (Mo-1: 735 +/- 74; Mo-2: 585 +/- 35; mean +/- SEM). Therefore, the normal expression of Mo-1 receptors on the monocytes of Glanzmann's thrombasthenia patients suggests different genomic regulations for Mo-1 antigens on the monocyte and GPIIb-IIIa complex on the platelet.     

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (alphaIIbbeta3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia-like phenotype

This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of beta3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa-transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.     

Protein A Sepharose immunoadsorption can restore the efficacy of platelet concentrates in patients with Glanzmann's thrombasthenia and anti-glycoprotein IIb-IIIa antibodies

Type I Glanzmann's thrombasthenia is a rare congenital platelet function disorder, characterized by undetectable platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa). Severe bleeding is controlled by transfusion of normal platelets, leading in some cases to the occurrence of anti-GPIIb-IIIa isoantibodies, which induces a loss of transfused platelet efficacy. We used immunoadsorption on protein A Sepharose (IA-PA), which has been shown to be efficient in decreasing the titre of antibodies in several immune diseases, in three patients with Glanzmann's thrombasthenia and anti-GPIIb-IIIa isoantibodies on five different occasions. IA-PA was well tolerated with no deleterious side-effects reported. It induced a dramatic decrease of total immunoglobulin (Ig)G, including anti-GPIIb-IIIa isoantibody levels, as assessed by the monoclonal antibody-specific immobilization of platelet antigens test and the ex vivo inhibition of normal platelet aggregation induced by the patient's platelet-rich or platelet-poor plasma. Elimination of the antibody was associated with a correction of the bleeding time following platelet transfusion. IA-PA combined with platelet transfusion made it possible to control two life-threatening haemorrhages, and allowed two surgical procedures and one bone marrow transplantation to be performed safely. Our experience suggests that IA-PA, which restores the haemostatic efficacy of platelet transfusion, is a valuable therapeutic strategy in patients with Glanzmann's thrombasthenia and anti-GPIIb-IIIa isoantibodies.     

Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.     

Diagnosis of Glanzmann's thrombasthenia and carrier detection using monoclonal antibodies to platelet glycoprotein IIb and IIIa in immunoblotting.

Glanzmann's thrombasthenia is a rare hemorrhagic syndrome, characterized by a quantitative or functional defect of the platelet glycoprotein GPIIb-IIIa complex. The authors describe a method to diagnose thrombasthenic patients and identify carrier subjects by using monoclonal antibodies specific for GPIIb and GPIIIa in an immunoblotting technique. The immunoreaction patterns of two thrombasthenic patients lacking GPIIb or GPIIIa, respectively, are shown. The described method produces further evidence concerning the biochemical heterogeneity of Glanzmann's thrombasthenia.     

Molecular pathology of inherited Glanzmann's thrombasthenia. Report of 11 cases]

Glycoprotein IIb-IIIa (GPIIb-IIIa) concentration was studied in 11 patients with Glanzmann's thrombasthenia (GT) with sensitive Western blotting technique. 7 patients with severe GPIIb-IIIa deficiency (less than 10% of the normal) were designated as type I (64% of patients), 2 patients with moderate GPIIb-IIIa deficiency (10-25% of the normal) as type II (18%) and 2 patients with GPIIb-IIIa 40-100% of the normal as variants (18%). Southern Blotting was used to analyze the GPIIb and GPIIIa genes in the 11 patients. The results showed that there were no major deletions or insertions in either the GPIIb or GPIIIa genes. However, a small change in GPIIb gene was demonstrated in two sibling patients and the abnormality of GPIIIa gene was found in another two patients. These observations combined with those from literature provide a basis for discussing the molecular pathology of Glanzmann's thrombasthenia.     

Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions

Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.     

Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3' GPIIIa cDNA. However, in one family, a 5' 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5' 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.     

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.     

A novel (288delC) mutation in exon 2 of GPIIb associated with type I Glanzmann's thrombasthenia

This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb-IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site. causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb-IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb-IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb-IIIa requires the integrity of exons 2 and 3 of GPIIb.     

Double heterozygosity of the GPIIb gene in a Swiss patient with Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) results from a qualitative or quantitative defect of GPIIb–IIIa complexes (integrin α_IIbβ₃)_, the fibrinogen receptor on platelets. This integrin plays a critical role in platelet aggregation. In this report we describe the molecular abnormalities of a patient with clinical and laboratory findings typical of type I Glanzmann's thrombasthenia. SDS-PAGE with Western blotting revealed an absence of GPIIb but small amounts of normally migrating GPIIIa in his platelets. A non-radioactive PCR-SSCP procedure and direct sequence analysis of PCR-amplified DNA fragments showed the patient to be a compound heterozygote for mutations in the GPIIb gene. A single point mutation (G to A) at nucleotide 1064 of the cDNA derived from the mother's allele led to a Glu³²⁴ to Lys amino acid substitution in GPIIb. It was responsible for a MscI restriction site in exon 12 of the GPIIb gene. This amino acid substitution changes the electric charge between the second and third Ca⁺⁺-binding domains of GPIIb. The second mutation was inherited from his father and is in exon 18 of the GPIIb gene. It was a T → C base transition at position 1787 of GPIIb cDNA and results in a Ile⁵⁶⁵ to Thr substitution. The two GPIIb mutations identified in this study will provide new information on GPIIb–IIIa structure and biosynthesis.     

Heterogeneity of membrane surface proteins in Glanzmann's thrombasthenia

Studies in several laboratories have suggested that platelets from patients with Glanzmann's thrombasthenia are deficient in two major membrane glycoproteins and that this membrane defect is uniform from patient to patient. We have used an improved electrophoretic technique to study further the surface composition of normal and thrombasthenic platelets. Platelets from three unrelated thrombasthenic patients were labeled by either lactoperoxidase-catalyzed iodination or the neuraminidase-galactose oxidase-[3H]NaBH4 technique and the labeled proteins were separated by two dimensional isoelectric focusing SDS polyacrylamide gel electrophoresis. With both techniques, the major radiolabeled proteins were clearly separated from each other and were present as a horizontal collection of discrete spots that suggest charge heterogeneity. Most of the labeled proteins had an acidic isoelectric point. Compared to normal platelets, platelets from patients with Glanzmann's disease contained no electrophoretically identifiable fibrinogen. In two patients with thrombasthenia, there was total absence of surface glycoproteins GPIIb and GPIII, while a third patient with thrombasthenia, who was clinically indistinguishable from the previous two patients, had decreased, but detectable, amounts of GPIIb and GPIII. These studies suggest that there are at least two phenotypic patterns of membrane abnormalities in Glanzmann's thrombasthenia involving GPIIb and GPIII and may indicate genetic heterogeneity in this disease.     

The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium

Platelet adherence to human artery subendothelium in blood from eight normal subjects, four patients with Glanzmann's thrombasthenia (deficiency of platelet membrane glycoproteins IIb and IIIa: GPIIb-IIIa), two patients with Bernard-Soulier syndrome (deficiency of platelet membrane glycoprotein Ib: GPIb) and one patient with von Willebrand's disease (VWD subtype III. deficient in factor VIII-von Willebrand factor: FVIII-VWF) was compared at various wall shear rates (300, 500, 1000, 1800 and 2500 s-1). Platelet adherence in blood from the patients with Glanzmann's thrombasthenia was within the normal range at shear rates below 1000 s-1. There was some decrease in adhesion at higher shear rates and platelets were less spread out on the subendothelium than normally at all shear rates. Platelet aggregate formation was almost totally absent. Platelet adherence in blood from patients with the Bernard-Soulier syndrome was strongly impaired at all shear rates. Platelet adherence in blood from the patient with VWD subtype III was normal at shear rates of 300 and 500 s-1, but impaired at shear rates above 1000 s-1. Aggregate formation was also decreased at these shear rates. Platelet adhesion was strongly inhibited by a monoclonal antibody against glycoprotein Ib, which had previously been shown to inhibit ristocetin-induced aggregation, at shear rates of 500 and 1800 s-1 but not at 300 s-1. Platelet adhesion at 1800 s-1 was also inhibited, though to a lesser extent, by two antibodies against GPIIb-IIIa. These antibodies also inhibited platelet aggregate formation. The data indicates that GPIb is involved in adhesion at the same shear rates as von Willebrand factor. Absence or inhibition of GPIIb-IIIa primarily causes a defect of aggregate formation but GPIIb-IIIa may also play a role in adhesion, particularly at high shear rates. The defect of adhesion in the Bernard-Soulier syndrome may be dependent on factors other than a deficiency of GPIb alone.     

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid-phase fibrinogen and binding affinity for the RGD-mimetic (3)H-SC52012, a non-peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [(3)H]-SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3' end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [(3)H]-SC52012 with a higher affinity than wild-type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.     

Platelet HDL(3) binding sites are not related to integrin alpha(IIb)beta(3) (GPIIb-IIIa)

Early studies considered that fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha(IIb)beta(3)) is the binding site for low-density lipoprotein (LDL) and high-density lipoprotein type 3 (HDL(3)). Recent data, however, do not support the hypothesis that the binding of LDL to human intact resting platelets is related to integrin alpha(IIb)beta(3). In this study we present evidence that platelet integrin alpha(IIb)beta(3) is also not involved in the interaction of HDL(3) and human intact resting platelets. Firstly, specific ligands for platelet integrin alpha(IIb)beta(3), such as fibrinogen, vitronectin, von Willebrand factor and fibronectin, were unable to inhibit the binding of HDL(3) to intact resting platelets. Secondly, the HDL(3) binding characteristics (K(d) and B(max) values), the activation of protein kinase C (PKC) and the inhibition of thrombin-induced inositoltriphosphate (IP(3)) formation and calcium (Ca(2+)) mobilization mediated by HDL(3) particles were similar in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, which are characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. In contrast, nitrosylation of tyrosine residues of HDL(3) by tetranitromethane fully abolished both the ability of particles to interact with its specific binding sites and the functional effects. Thirdly, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Bak(a/B) and anti-PL(A1/2)), anti-integrin subunits (anti-alpha(V) and anti-beta(3)), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, M8 and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect the binding of HDL(3) particles to human intact resting platelets. Overall results show that neither the GPIIb-IIIa complex nor GPIIb or GPIIIa individually are the membrane binding proteins for HDL(3)on intact resting platelets.