Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbß3 in Fibrin-Mediated Microthrombus Formation.

OBJECTIVE: Fibrin is considered to strengthen thrombus formation via integrin αIIbß3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s-1) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbß3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca2+ signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers. CONCLUSIONS: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbß3 through signaling via Syk and low-level Ca2+ rises.

Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

Heterogeneity of Integrin αIIbß3 Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis.

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin αIIbß3 functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.

Integrin ß3 Deficiency Results in Hypertriglyceridemia via Disrupting LPL (Lipoprotein Lipase) Secretion.

OBJECTIVE: Integrin ß3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether ß3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with ß3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The ß3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that ß3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine)720-722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/ß3/LPL complex that is required for ß3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in ß3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH720-722-mutated ß3 genes. CONCLUSIONS: This study reveals a hypertriglyceridemia in both ß3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of ß3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with ß3-deficient Glanzmann thrombasthenia.

Clinico-hematological and thromboelastographic profiles in glanzmann's thrombasthenia.

: Glanzmann's thrombasthenia is a rare inherited bleeding disorder characterized by the quantitative or qualitative defect of glycoprotein IIb/IIIa receptor on platelets which leads to ineffective aggregation. Light transmittance aggregometry is considered as the gold standard for diagnosis of Glanzmann's thrombasthenia. Thromboelastography (TEG) is a global hemostatic assay which measures clot formation, clot strengthening and fibrinolysis. This study evaluates the clinical, laboratory and TEG profiles in patients with Glanzmann's thrombasthenia. Bleeding score by (International Society on Thrombosis and Haemostasis) ISTH-bleeding assessment tool (bleeding score), laboratory tests to diagnose Glanzmann's thrombasthenia, and TEG parameters were correlated in 11 Glanzmann's thrombasthenia patients. Seventeen participants with normal bleeding score were included as controls. Bleeding score was increased in all patients. The highest bleeding score was in an adult female (26), whereas the lowest score (4) was in two children of less than 1 year. Majority of TEG parameters (except R-time) showed a statistically significant difference between Glanzmann's thrombasthenia patients and controls (K-time: P < 0.001, angle: P < 0.001, maximum amplitude: P < 0.001). The average time required to record the maximum amplitude was 23 min. Maximum amplitude was markedly reduced in all Glanzmann's thrombasthenia patients with an average of 20.9 mm (reference range 44-68 mm) having 100% sensitivity. The thromboelastographic profile of Glanzmann's thrombasthenia showed a consistently reduced maximum amplitude. Hence reduced maximum amplitude with a normal platelet count, significant bleeding score and prolonged bleeding time could potentially be used as a preliminary algorithm for the diagnosis of Glanzmann's thrombasthenia.

A Glanzmann thrombasthenia family associated with a TUBB1-related macrothrombocytopenia.

BACKGROUND: Macrothrombocytopenia (MTP) is a rare but enigmatic complication of Glanzmann thrombasthenia (GT), an inherited bleeding disorder caused by the absence of platelet aggregation due to deficiencies of the αIIbß3 integrin. OBJECTIVES: We report a family with type I GT and a prolonged bleeding time but unusually associated with congenital mild thrombocytopenia and platelet size heterogeneity with giant forms. METHODS AND RESULTS: Sanger sequencing of DNA from the propositus identified 2 heterozygous ITGB3 gene mutations: p.P189S and p.C210S both of which prevent αIIbß3 expression and are causative of GT but without explaining the presence of enlarged platelets. High-throughput screening led to the detection of a predicted disease-causing heterozygous mutation in the TUBB1 gene: p.G146R, encoding ß1-tubulin, a component of the platelet cytoskeleton and a gene where mutations are a known cause of MTP. CONCLUSIONS: Family screening confirmed that this rare phenotype results from oligogenic inheritance while suggesting that the GT phenotype dominates clinically.

A Successful Renal Transplant in a Pediatric Patient With Glanzmann Thrombasthenia and Hyperimmunization.

We report the case of a patient with type 2 Glanzmann thrombasthenia who underwent successful kidney transplant with his mother's kidney. He started dialysis at 13 months. The patient had been diagnosed with Glanzmann thrombasthenia at 9 years old, after hemorrhagic shock, during which multiple transfusions were required and hyperimmunization had developed. At 12 years old, he received a kidney transplant. Before transplant, ABO- and HLA-compatible platelet donors were identified and convened to donate forthe surgery and in case of emergency. Bleeding was prevented withprophylacticHLA-matched platelet transfusion and tranexamic acid. After transplant, diuresis started immediately with excellent graft function and no severe bleeding. However, after week 5, several episodes of macroscopic hematuria occurred, with obstruction and anuria. The double J ureteric stent was replaced 4 times in 2 months. Finally, the ureteric stent was removed 9 months later. At 22 months after kidney transplant, the patient has a normal graft function and no further bleeding has occurred, underlying the importance of multidisciplinary management.

First description of an IgM monoclonal antibody causing αIIb ß3 integrin activation and acquired Glanzmann thrombasthenia associated with macrothrombocytopenia.

Essentials Acquired Glanzmann thrombasthenia (GT) is generally caused by anti-αIIb ß3 autoantibodies. We report the case of a man with an acquired GT phenotype associated with macrothrombocytopenia. Perturbed platelet function were associated with an activating anti-αIIb ß3 IgM autoantibody. This novel clinical entity raises interesting questions about the αIIb ß3 integrin signaling. SUMMARY: Background Acquired Glanzmann thrombasthenia (GT) is a bleeding disorder generally caused by anti-αIIb ß3 autoantibodies. Objectives We aimed to characterize the molecular mechanism leading to a progressive GT-like phenotype in a patient with chronic immune thrombocytopenia. Patient, Methods, and Results The patient suffered from repeated episodes of gastrointestinal bleeding; further studies indicated a moderate platelet aggregation defect. A few months later, platelet function showed abolished aggregation using all agonists, but normal agglutination with ristocetin. No platelet-bound antibodies were detected, but the presence of large amounts of an IgM type antibody detected together with αIIb ß3 in the patient permeabilized platelets suggested that this IgM was an autoantibody causing the internalization of the complex. This was confirmed by the fact that the patient IgM bound to normal platelets but not to platelets from GT type I patients. Moreover, patient's plasma activated αIIb ß3 on controls' platelets as evidenced by increased PAC-1 binding. We also demonstrated that the patient plasma triggered αIIb ß3 outside-in signaling, as ß3 Tyr773 and FAK were phosphorylated, and increased the rate of actin polymerization in resting platelets reflecting an impairment of cytoskeletal reorganization. Because different signs of dysmegakaryopoiesis were also observed in our patient, we evaluated the ability of its serum to impair proplatelets formation and showed that it significantly decreased the number of proplatelet-bearing megakaryocytes in controls' bone marrow stem cells culture compared with normal serum. Conclusions We present the case of a patient with a progressive and severely perturbed platelet function associated with the presence of an IgM activating autoantibody directed against αIIb ß3 .

Low Concentrations of Recombinant Factor VIIa May Improve the Impaired Thrombin Generation of Glanzmann Thrombasthenia Patients.

INTRODUCTION: Glanzmann thrombasthenia (GT) is a rare bleeding disorder. The disease is caused by the lack or dysfunction of platelet membrane glycoprotein IIb/IIIa (integrin αIIbß3) which is essential for platelet aggregation. Bleeding episodes are usually managed by platelet transfusions. Recombinant activated factor VII (rFVIIa) is a common adjunct or alternative treatment option. OBJECTIVE: This article evaluates GT patients' response to increasing concentrations of rFVIIa using an ex vivo thrombin generation assay to elaborate the knowledge in which rFVIIa treats a platelet dysfunction for bleeding episodes and preoperative management. MATERIALS AND METHODS: Twenty-four GT patients in a non-bleeding state were enrolled into the study. Thrombin generation was measured in platelet-rich plasma (PRP) in the presence of 0.7 to 7.0 µg/mL rFVIIa. Clinical data of rFVIIa used to treat GT patients' bleeding episodes was collected, and patients' follow-up course was documented. RESULTS: Thrombin generation was significantly decreased in GT patients compared with controls. An individual response to rFVIIa spiking was noted in GT patients' PRP. In the majority of patients, a significant improvement in thrombin generation was already demonstrated with low concentrations (0.7 µg/mL) of rFVIIa. CONCLUSION: Thrombin generation is improved in the majority of GT patients following ex vivo spiking with rFVIIa. The magnitude of this improvement is individual and was noted at low concentrations of rFVIIa. There is a need for a prospective clinical trial to find the optimal doses or rFVIIa for treatment of GT patients.

A unique phenotype of acquired Glanzmann thrombasthenia due to non-function-blocking anti-αIIbß3 autoantibodies.

Essentials Acquired Glanzmann thrombasthenia (aGT) is generally caused by function-blocking antibodies (Abs). We demonstrated a unique aGT case due to marked reduction of αIIbß3 with anti-αIIbß3 Abs. The anti-αIIbß3 Abs of the patient did not inhibit platelet function but reduced surface αIIbß3. Internalization of αIIbß3 induced by the Abs binding may be responsible for the phenotype. SUMMARY: Background Acquired Glanzmann thrombasthenia (aGT) is a bleeding disorder generally caused by function-blocking anti-αIIbß3 autoantibodies. Aim We characterize an unusual case of aGT caused by marked reduction of surface αIIbß3 with non-function-blocking anti-αIIbß3 antibodies (Abs). Methods A 72-year-old male suffering from immune thrombocytopenia since his 50s showed exacerbation of bleeding symptom despite mild thrombocytopenia. Platelet aggregation was absent with all agonists but ristocetin. Analysis of αIIbß3 expression and genetic analysis were performed. We also analyzed effects of anti-αIIbß3 Abs of the patient on platelet function and αIIbß3 expression. Results Surface αIIbß3 expression was markedly reduced to around 5% of normal, whereas his platelets contained αIIbß3 to the amount of 40-50% of normal. A substantial amount of fibrinogen was also detected in his platelets. There were no abnormalities in ITGA2B and ITGB3 cDNA. These results indicated that reduced surface αIIbß3 expression caused a GT phenotype, and active internalization of αIIbß3 was suggested. Anti-αIIbß3 IgG Abs were detected in platelet eluate and plasma. These Abs did not inhibit PAC-1 binding, indicating that the Abs were non-function-blocking. Surface αIIbß3 expression of a megakaryocytic cell line and cultured megakaryocytes tended to be impaired by incubation with the patient's Abs. After 2 years of aGT diagnosis, his bleeding symptom improved and surface αIIbß3 expression was recovered to 20% of normal with reduction of anti-αIIbß3 Abs. Conclusion We demonstrated a unique aGT phenotype due to marked reduction of surface αIIbß3. Internalization induced by anti-αIIbß3 Abs may be responsible in part for the phenotype.

Morphometric analysis of spread platelets identifies integrin αIIbß3-specific contractile phenotype.

Haemostatic platelet function is intimately linked to cellular mechanics and cytoskeletal morphology. How cytoskeletal reorganizations give rise to a highly contractile phenotype that is necessary for clot contraction remains poorly understood. To elucidate this process in vitro, we developed a morphometric screen to quantify the spatial organization of actin fibres and vinculin adhesion sites in single spread platelets. Platelets from healthy donors predominantly adopted a bipolar morphology on fibrinogen and fibronectin, whereas distinguishable, more isotropic phenotypes on collagen type I or laminin. Specific integrin αIIbß3 inhibitors induced an isotropic cytoskeletal organization in a dose-dependent manner. The same trend was observed with decreasing matrix stiffness. Circular F-actin arrangements in platelets from a patient with type II Glanzmann thrombasthenia (GT) were consistent with the residual activity of a small number of αIIbß3 integrins. Cytoskeletal morphologies in vitro thus inform about platelet adhesion receptor identity and functionality, and integrin αIIbß3 mechanotransduction fundamentally determines the adoption of a bipolar phenotype associated with contraction. Super-resolution microscopy and electron microscopies further confirmed the stress fibre-like contractile actin architecture. For the first time, our assay allows the unbiased and quantitative assessment of platelet morphologies and could help to identify defective platelet behaviour contributing to elusive bleeding phenotypes.

Clinical and molecular insights into Glanzmann's thrombasthenia in China.

Glanzmann's thrombasthenia (GT) is a rare bleeding disorder characterized by spontaneous mucocutaneous bleeding. The disorder is caused by quantitative or qualitative defects in integrin αIIbß3 (encoded by ITGA2B and ITGB3) on the platelet and is more common in consanguineous populations. However, the prevalence rate and clinical characteristics of GT in non-consanguineous populations have been unclear. We analyzed 97 patients from 93 families with GT in the Han population in China. This analysis showed lower consanguinity (18.3%) in Han patients than other ethnic populations in GT-prone countries. Compared with other ethnic populations, there was no significant difference in the distribution of GT types. Han females suffered more severe bleeding and had a poorer prognosis. We identified a total of 43 different ITGA2B and ITGB3 variants, including 25 previously unidentified, in 45 patients. These variants included 14 missense, 4 nonsense, 4 frameshift, and 3 splicing site variants. Patients with the same genotype generally manifested the same GT type but presented with different bleeding severities. This suggests that GT clinical phenotype does not solely depend on genotype. Our study provides an initial, yet important, clinical and molecular characterization of GT heterogeneity in China.

Anti-αIIb ß3 immunization in Glanzmann thrombasthenia: review of literature and treatment recommendations.

Glanzmann thrombasthenia (GT) is caused by inherited defects of the αIIb ß3 platelet glycoprotein. This bleeding disorder can be treated with platelet transfusion therapy, but some patients will be immunized and begin to form anti-human leucocyte antigen (HLA) and/or anti-αIIb ß3 antibodies. These antibodies can bind and interfere with the function of the transfused platelets, rendering treatment ineffective. However, platelet transfusion refractoriness attributable to HLA antibodies may be managed by the selection of compatible donors, although they are not always readily available, particularly in an emergency. Thus, anti-αIIb ß3 antibodies represent one of the most severe complications in GT. Both genetic and environmental factors may contribute to the risk of anti-αIIb ß3 development, but the underlying pathogenic mechanisms are still unknown. This review will summarize the current knowledge of the risk factors for development of anti-αIIb ß3 antibodies in patients with GT and discuss how these findings may influence the clinical management of patients.

Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation.

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving αIIbß3. SUMMARY: Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-αIIbß3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of αIIbß3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbß3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

Evaluation of platelet surface glycoproteins in patients with Glanzmann thrombasthenia: Association with bleeding symptoms.

BACKGROUND & OBJECTIVES: Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbß3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies. METHODS: Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed. RESULTS: Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002). INTERPRETATION & CONCLUSIONS: Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.

Should studies on Glanzmann thrombasthenia not be telling us more about cardiovascular disease and other major illnesses?

Glanzmann thrombasthenia (GT) is a rare inherited bleeding disorder caused by loss of αIIbß3 integrin function in platelets. Most genetic variants of ß3 also affect the widely expressed αvß3 integrin. With brief mention of mouse models, I now look at the consequences of disease-causing ITGA2B and ITGB3 mutations on the non-hemostatic functions of platelets and other cells. Reports of arterial thrombosis in GT patients are rare, but other aspects of cardiovascular disease do occur including deep vein thrombosis and congenital heart defects. Thrombophilic and other risk factors for thrombosis and lessons from heterozygotes and variant forms of GT are discussed. Assessed for GT patients are reports of leukemia and cancer, loss of fertility, bone pathology, inflammation and wound repair, infections, kidney disease, autism and respiratory disease. This survey shows an urgent need for a concerted international effort to better determine how loss of αIIbß3 and αvß3 influences health and disease.

Alterations of the platelet proteome in type I Glanzmann thrombasthenia caused by different homozygous delG frameshift mutations in ITGA2B.

Glanzmann thrombasthenia (GT) is one of the best characterised inherited platelet function disorders but global platelet proteome has not been determined in these patients. We investigated the proteome and function of platelets from two patients with type I GT, caused by different homozygous ITGA2b mutations, from family members and unrelated controls. The global proteome of highly purified washed platelets was quantified by liquid chromatography-mass spectrometry (LC-MS) and targeted MS-methods. Platelet function was analysed by flow cytometry, light transmission aggregometry and flow-based assays. Platelets from GT patients showed less than 5 % relative levels of the integrin subunit αIIb and 5-9 % fibrinogen compared to controls. These patients demonstrated loss of αIIbß3-dependent platelet function, but normal platelet granule secretion induced by physiological agonists. Platelets from heterozygous family members of a patient expressed 50-60 % of control αIIb levels which were sufficient for normal αIIbß3-dependent platelet function. Studying type I GT as model disease we established quantitative LC-MS to detect and clearly distinguish normal platelets, platelets from GT heterozygotes and platelets from GT patients. Diminished levels of factor XIIIB chain, plasminogen and carboxypeptidase 2B were identified in thrombasthenic platelets. Additionally, GT platelets showed up to 2.5-fold increased levels of FcγRIIA and laminin-α4 chain. Elevated levels of platelet FcγRIIA was associated with increased CD63-surface expression after FcγRIIA-crosslinking in one GT-patient which might present a compensatory mechanism of platelet activation in GT. We demonstrate that quantitative LC-MS based proteomics is suitable to validate known but also to identify previously unknown protein level changes of dysfunctional platelets.