A Ser752-->Pro substitution in the cytoplasmic domain of beta3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the alphaIIbbeta3 integrin and the platelet granule pool of fibrinogen

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.     

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.     

Activation of integrin alphaIIbbeta3 expressed in Chinese hamster ovary cells is required for interaction with solid-phase von Willebrand factor

Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-alphaIIbbeta3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-alphaIIbbeta3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-alphaIIbbeta3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-alphaIIbbeta3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated alphaIIbbeta3 is absolutely required for spread cells to resist detachment and strengthens the importance of the alphaIIbbeta3 activation step for adhesion and spreading to VWF.     

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.     

Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αIIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3, αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.     

Dissociation between fibrinogen and fibrin interaction with platelets in patients with different subtypes of Glanzmann's thrombasthenia: studies in an ex vivo perfusion chamber model

To explore the possible role of a residual or variant alphaIIbbeta3 integrin (alphaIIbbeta3) in thrombogenesis, we used a new ex vivo perfusion chamber model to examine blood from patients with different subtypes of Glanzmann's thrombasthenia (GT). Non-anticoagulated blood was perfused through capillaries coated with type III collagen for 4.5 min (shear rate: 1600/s). Platelet deposition was quantified as platelet adhesion and mean thrombus size volume; fibrin and von Willebrand Factor (VWF) were specifically revealed by immunohistochemistry. In two patients with variant and in one patient with type II GT, platelet adhesion was maximal and we observed an unexpected formation of thrombi that were smaller than normal in size. These thrombi were surrounded by a thick meshwork that displayed a strong staining for fibrin and VWF. In two patients with heterozygous GT, platelet adhesion and thrombogenesis were normal. In two patients with type I GT, there was no thrombus formation, although platelet adhesion was also maximal. These data suggest the existence of a substitute pathway for thrombogenesis mediated by fibrin and possibly alphaIIbbeta3 (alphaIIbbeta3 at a reduced level, as in type II, and/or abnormal) as this fibrin network was not observed in type I GT with no alphaIIbbeta3. These interactions might facilitate haemostasis and even lead to thrombosis under certain favourable conditions. Furthermore, these data might have pharmacological relevance to the development of anti-alphaIIbbeta3 antithrombotic agents.     

Phosphatidylserine surface expression and integrin αIIbβ3 activity on thrombin/convulxin stimulated platelets/particles of different sizes

Platelets stimulated by a combination of thrombin/convulxin have been shown to develop two to three populations characterized by different phosphatidylserine (PS) surface expression and integrin αIIbβ3 activity. To determine how these markers are distributed on the surface of platelets/particles, we studied Annexin V and PAC-1 binding to platelets/particles of different sizes by flow cytometry analysis and evaluated influences of calpain and caspase inhibitors on thrombin/convulxin-activated platelets. Analysed platelets/particles were divided by their sizes, according to the standard size beads, into seven populations from 0·37 to 4·8 μm. PAC-1 binding/μm² was almost equal in platelets/particles ranging from 1·2 to 4·8 μm and was significantly lower on smaller-sized particles sizes (0·37–0·7 μm). PS surface exposure/μm² was high in the particles of 0·37–1·2 μm and very low in platelets (2·6–4·8 μm). Upon thrombin/convulxin stimulation caspase inhibitors prevented microparticle (MP) formation, while a calpain inhibitor stimulated MP formation. It was also shown that stimulated platelets are heterogeneous not only in their ability to activate αIIbβ3 integrin complex and expose PS on their surface, but also in the distribution of activation markers, which strongly depends on platelet/particle size and that platelets/particles of different sizes provide different responses to the same stimulus.     

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p -chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y₁₂ ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y₁₂ receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y₁₂ thiol-dependent reaction near the final αIIbβ3-dependent events of aggregation. We then used 3-( N -maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in αIIbβ3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the αIIb and β3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in αIIbβ3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of αIIbβ3 by Mn²⁺. Our results suggest that the thiols in αIIbβ3 that are blocked by pCMBS are important in the activation of this integrin.     

Activation of recombinant alphaIIbbeta3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to alphaIIbbeta3

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin alphaIIbbeta3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce alphaIIbbeta3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of alphaIIbbeta3. By comparing the effects of two potent platelet alphaIIbbeta3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated alphaIIbbeta3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated alphaIIbbeta3-dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT-activated alphaIIbbeta3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.     

Intracellular trafficking of the alphaIIbbeta3 receptor antagonist, abciximab, in normal and Glanzmann's disease megakaryocytes

The alphaIIbbeta3 platelet receptor antagonist abciximab (c7E3Fab, ReoPro(R)) has proved to be effective in preventing arterial thrombosis. However, its binding capacity to the platelet precursors, megakaryocytes (MKs), which also express alphaIIbbeta3, is not known. The purpose of this study was to establish whether abciximab is able to react with alphaIIbbeta3 located on human MKs, and to follow its subsequent intracellular trafficking. MKs were grown from CD34+ progenitors from normal subjects and from a patient with type I Glanzmann's thrombasthenia, and abciximab was added at day 10 of culture (4 microgram/ml). Cells were fixed at day 12, cryosectioned, and immunolabelled for abciximab. Labelling was prominent on the MK plasma membrane; it also lined the demarcation membration system. Interestingly, alpha-granule membranes were labelled showing that the antibody was internalized and further stored into MK secretory granules. Abciximab was also strongly detected on and in newly-formed platelets. Glanzmann's disease MKs (which completely lacked alphaIIbbeta3) were consistently negative, confirming that the antibody fragment was specifically interacting with alphaIIbbeta3. In conclusion, this study demonstrated that abciximab: (i) binds MK plasma membrane and demarcation membranes, (ii) trafficks into alpha-granules, and (iii) is expressed on and in nascent platelets. These findings could be taken in account when monitoring anti-alphaIIbbeta3 receptor therapy.     

Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α-granule membrane

The vitronectin receptor (VnR or α_vβ₃) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β₃ subunit in common: GP IIb–IIIa complexes (or α_IIbβ₃) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the α_v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, α_vβ₃ was mostly detected in internal membrane systems including those of α-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of α_vβ₃ was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb–IIIa, although intracellular staining was present in EM and α-granules were again labelled.     

Prevalence of allo‐immunization anti‐HLA and anti‐integrin αIIbβ3 in Glanzmann Thromboasthenia patients

Summary. Platelet transfusions, main therapy of Glanzmann Thromboasthenia (GT), can induce an allo‐immunization against human leucocyte antigen and integrin αIIbβ3. We have investigated in our GT patients the rate of allo‐immunization and of refractoriness to platelet transfusions. From 1975 until December 2005, we have followed 17 GT patients: 14 type 1, 3 variant type; nine females, eight males; median age at diagnosis 9.8 years (range 1–44.5); median age at the time of the study 35.5 years (range 23.6–68.5). In our patients, 121 bleeding episodes occurred (24 severe, 37 moderate, and 60 mild). Ten major and 22 minor surgical procedures have been performed. Two spontaneous deliveries and three caesarian sections with five live births were performed; moreover, one late foetal loss occurred, and one voluntary abortion was performed. Sixteen of 17 patients have been transfused at least once in life with platelets and/or red blood cells (RBC). All transfused patients have been investigated for the presence of anti‐HLA and anti‐integrin αIIbβ3 allo‐antibodies. The positiveness of allo‐antibodies has been demonstrated in 4/16 transfused patients (25%): isolated for anti‐HLA in two; isolated for anti‐integrin αIIbβ3 in one; and combined in one. In spite of the presence of allo‐antibodies, platelet transfusions have always been effective and the haemostasis was not compromised.     

Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures

Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1β (IL-1β) on the expression of β3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of β3-EEC at concentrations in the range of 0.06–3 nM; however, leptin exhibited a significantly greater effect than IL-1β. We also determined the regulatory effects of IL-1β on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1β upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1β was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of β3-integrin and leptin/leptin receptor expression by IL-1β in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1β action on β3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.     

Variants in the human beta 1-, beta 2-, and beta 3-adrenergic receptor genes are not associated with morbid obesity in children and adolescents

AIM: beta-adrenergic receptors (beta-ARs) are of key importance for the regulation of lipolysis and thermogenesis by catecholamines. Genetic defects in expression or function of beta(1)- beta(2)- and/or beta(3)-AR could affect energy homeostasis and predispose an individual towards the development of obesity. We therefore investigated the possible association of polymorphisms in the beta-adrenergic receptor genes with early onset obesity. METHODS: Frequencies of the following variants were assessed in extremely obese children and healthy underweight controls: Gly/Ser in codon 49 and Arg/Gly in codon 389 of the beta(1)-AR, Arg/Gly in codon 16 and Gln/Glu in codon 27 of the beta(2)-AR, Trp/Arg in codon 64 of the beta(3)-AR. RESULTS: The Ser49 allele in the beta(1)-AR gene was found at a frequency of 0.131 in obese and 0.136 in lean subjects (p = 0.835), while the Gly389 allele in the beta(1)-AR had a frequency of 0.319 in obese and 0.328 in lean subjects (p = 0.802). Gly16 in the beta(2)-AR was found with a frequency of 0.590 in obese and 0.611 in lean subjects (p = 0.591) and the Glu27 allele in the beta(2)-AR had a frequency of 0.380 in obese and 0.420 in lean subjects (p = 0.298). CONCLUSION: We did not detect significant differences for allele and carrier frequencies of individual polymorphisms. Together with previously obtained data on genotype distribution of a beta(3)-AR variant in the same study group, no significant differences were found between obese and lean subjects for the distribution of individuals with variants in none, one, two or all three beta-ARs. Our data make it unlikely that polymorphisms in beta-ARs are involved in the pathogenesis of early onset obesity.     

Energy expenditure, body composition and insulin response to glucose in male twins discordant for the Trp64Arg polymorphism of the beta3-adrenergic receptor gene

AIM: The tryptophan to arginine change in position 64 (Trp64Arg) polymorphism of the beta3-adrenergic receptor (beta3AR) gene has been associated with an increased prevalence of obesity, insulin resistance and type 2 diabetes. In this, decreased rates of energy expenditure and impaired insulin secretion could play a role. METHODS: In 10 male twin pairs discordant for the Trp64Arg polymorphism, we examined insulin response to glucose by an oral glucose tolerance test (OGTT), a frequently sampled intravenous glucose tolerance test (FSIGT), body composition by the bioimpedance method, dual-energy X-ray absorptiometry scanning and energy expenditure by indirect and direct calorimetry. RESULTS: Twins heterozygous for the Trp64Arg polymorphism showed significantly lower fat mass independent of the method used, and significantly lower fasting insulin and glucose concentrations compared with their homozygous wild-type co-twins. Correspondingly, insulin resistance and insulin secretion determined by homeostasis model assessment were significantly lower in twins carrying the Trp64Arg polymorphism. However, there were no significant differences in adiponectin levels, insulinogenic index assessed by OGTT, or insulin sensitivity, acute insulin response to glucose, glucose effectiveness or insulin disposition index assessed by minimal modelling of the FSIGT. Furthermore, there were no differences in sleeping, resting or post-prandial energy expenditure. CONCLUSIONS: In male twins with a high similarity in genetic and environmental background, the Trp64Arg polymorphism of the beta3AR gene is associated with lower fat mass, fasting insulin levels and an appropriate insulin response to glucose. Thus, heterozygosity for the Trp64Arg variant is unlikely to increase the risk of obesity, insulin resistance or type 2 diabetes.