Glanzmann Thrombasthenia Patients Research Articles

Glanzmann Thrombasthenia – A Rare Case Report

Glanzmann thrombasthenia is a rare inherited bleeding disorder resulting from mutation in platelet membrane glycoprotein (GP) IIb or IIIa leading to impaired platelet function which is characterized by defective platelet aggregation and diminished clot retraction. Glanzmann thrombasthenia patients commonly visit to physician with features of bleeding. Here we discuss about the case of a 32-years-old young female presented with menorrhagia, ecchymosis and occasional gum bleeding. Her coagulation profile was in favor of Glanzmann thrombasthenia. This rare disease has a good outcome if early diagnosis and proper management can be ensured. Delta Med Col J. Jan 2021;9(2): 101-103

PB1374 Anti-ɑIIbβ3 Antibodies in Glanzmann Thrombasthenia Patients may Generate Procoagulant Platelets by Complement Activation

PB1374 Anti-ɑIIbβ3 Antibodies in Glanzmann Thrombasthenia Patients may Generate Procoagulant Platelets by Complement Activation

Molecular dynamics simulations corroborate recombinant expression studies carried out on three αIIb β-propeller mutations reported in Indian Glanzmann thrombasthenia patients.

Mutations in the αIIb β-propeller domain have long been known to disrupt heterodimerization and intracellular trafficking of αIIbβ3 complexes leading to diminished surface expression and/or function, resulting in Glanzmann thrombasthenia. Our previous study on three β-propeller mutations, namely G128S, S287L, and G357S, showed variable defects in protein transport correlated with the patient's clinical phenotypes. Pulse-chase experiments revealed differences in αIIbβ3 complex maturation among the three mutations. Hence, the current study aims to correlate conformational changes caused by each one of them. Evolutionary conservation analysis, stability analysis, and molecular dynamics simulations of the three mutant structures were carried out. Stability analysis revealed that, while G128S and G357S mutations destabilized the β-propeller structure, S287L retained the stability. Wild-type and mutant β-propeller structures, when subjected to molecular dynamics simulations, confirmed that G128S and G357S were both destabilizing in nature when compared with the wild-type and S287L based on several parameters studied, like RMSD, RMSF, Rg, FEL, PCA, secondary structure, and hydrogen bonds. In our previous study, we demonstrated that mutant S287L αIIbβ3 complexes were more stable than the wild-type αIIbβ3 complexes, as evidenced in pulse-chase experiments. These findings corroborate variable intracellular fates of mutant αIIbβ3 complexes as a result of these β-propeller mutations.

Glanzmann thrombasthenia—A not so rare platelet function disorder in Pakistan

ObjectivesTo determine the prevalence, clinical manifestations and laboratory findings of Pakistani Glanzmann Thrombasthenia patients and to assess the treatment received by these patients. Materials and MethodThis cross-sectional study examined 2542 patients over a period of 10 years who were referred with a suspicion of a platelet function disorder. Clinical details, laboratory parameters and treatment received were noted. ResultsGlanzmann thrombasthenia constituted 80.15% of the platelet function disorders. Of these, males were 112 (34.25%) while females were 215 (65.75%). Median age at diagnosis was 9 yrs 2 months (range of 8 months to 26 years) and at first presentation was 4 years 3 months (range of 3 months to 12 years). Consanguinity was observed in 85.32% of the patients. Common clinical features were easy bruisability (85%), epistaxis (78.6%) and gum bleed (56.3%). Mean haemoglobin was 8.6+2.46 g/dl and mean bleeding time was > 15 min. All patients showed no aggregation with collagen, ADP and epinephrine and a normal response to ristocetin on light transmission aggregometry. ConclusionWe have reported a large number of patients of Glanzmann thrombasthenia, a disorder which is not so rare in our population, emphasizing the importance of early diagnosis and appropriate management.

Activated Platelets Upregulate β2 Integrin Mac-1 (CD11b/CD18) on Dendritic Cells, Which Mediates Heterotypic Cell-Cell Interaction.

Recent evidence suggests interaction of platelets with dendritic cells (DCs), while the molecular mechanisms mediating this heterotypic cell cross-talk are largely unknown. We evaluated the role of integrin Mac-1 (αMβ2, CD11b/CD18) on DCs as a counterreceptor for platelet glycoprotein (GP) Ibα. In a dynamic coincubation model, we observed interaction of human platelets with monocyte-derived DCs, but also that platelet activation induced a sharp increase in heterotypic cell binding. Inhibition of CD11b or GPIbα led to significant reduction of DC adhesion to platelets in vitro independent of GPIIbIIIa, which we confirmed using platelets from Glanzmann thrombasthenia patients and transgenic mouse lines on C57BL/6 background (GPIbα-/-, IL4R-GPIbα-tg, and muMac1 mice). In vivo, inhibition or genetic deletion of CD11b and GPIbα induced a significant reduction of platelet-mediated DC adhesion to the injured arterial wall. Interestingly, only intravascular antiCD11b inhibited DC recruitment, suggesting a dynamic DC-platelet interaction. Indeed, we could show that activated platelets induced CD11b upregulation on Mg2+-preactivated DCs, which was related to protein kinase B (Akt) and dependent on P-selectin and P-selectin glycoprotein ligand 1. Importantly, specific pharmacological targeting of the GPIbα-Mac-1 interaction site blocked DC-platelet interaction in vitro and in vivo. These results demonstrate that cross-talk of platelets with DCs is mediated by GPIbα and Mac-1, which is upregulated on DCs by activated platelets in a P-selectin glycoprotein ligand 1-dependent manner.

Utility of the ISTH bleeding assessment tool (BAT) in diagnosis of Glanzmann Thrombasthenia patients.

Objectives:To assess the utility of ISTH-BAT (International Society on Thrombosis and Hemostasis- Bleeding Assessment Tool) in the diagnosis of Glanzmann Thrombasthenia (GT) in comparison to controls.Methods:It was a case-control study carried out at The Children’s Hospital, Lahore from January 2012 to May 2021. All patients from neonates to 18 years with a final diagnosis of GT were studied retrospectively. The clinical details were collected from hospital records and telephonically on ISTH-BAT questionnaire after taking informed consent. The same proforma was obtained from 75 healthy controls. Data was analyzed on SPSS version 26.Results:Out of 427 patients with suspected platelet function disorders, 133 were diagnosed as GT. The mean age was 7.29±5 years. Male to female ratio was 1.1:1. Among cases, 76.6% were products of consanguineous marriage. Epistaxis was the commonest symptom with highest score (p value<0.001). Cutaneous and oral cavity bleeds were more severe and frequent in patients than controls (p value < 0.004). The median ISTH-BAT score among patients was nine while in control group was one. Sensitivity was 86.4%, specificity was 77.3%, positive predictive value was 0.87 and negative predictive value was 0.76. Area under the receiver operator curve was 0.78 (95% confidence interval 0.82–0.90, p< 0.001*).Conclusion:ISTH-BAT scores were significantly higher in GT patients than controls. So, we recommend the inclusion of ISTH-BAT in diagnostic evaluation of patients with suspected Glanzmann Thrombasthenia.

Weekly low-dose recombinant factor VIIa prophylaxis in Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited disease causing bleeding episodes due to platelet dysfunction. The standard treatment for moderate-severe bleeding is platelet transfusion. Recombinant factor VIIa (rFVIIa) is successfully used in bleeding episodes and invasive procedures. Here, we present a patient with Glanzmann thrombasthenia, whose bleeding episodes could only be controlled by rFVIIa. The patient is a 28 years old male, who has had frequent bleeding episodes unresponsive to local hemostatic agents and tranexamic acid and had an anaphylactoid reaction to platelet transfusion. We started the patient on a low-dose (20 μg/kg) rFVIIa once a week. The patient has no spontaneous bleeding since then. This is the first case report of a Glanzmann thrombasthenia patient on routine prophylaxis with low-dose rFVIIa.

Mutations in RASGRP2 gene identified in patients misdiagnosed as Glanzmann thrombasthenia patients

IntroductionGlanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. AimWe aimed to study the genetic cause of IPFD mimicking GT. MethodsDuring 2017–2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. ResultsUsing flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbβ3 and αvβ3, as well as normal integrin αIIbβ3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. DiscussionBoth RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. ConclusionPatients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.

Immunization against αIIbβ3 and αvβ3 in Glanzmann thrombasthenia patients carrying the French Gypsy mutation

Unlabelled Box AbstractBackgroundGlanzmann thrombasthenia (GT) is a rare bleeding disorder caused by the absence or the dysfunction of the platelet αIIbβ3 integrin. A founder mutation in the ITGA2B gene was previously identified in French Gypsy patients. Interestingly, this mutation was strongly linked to the human platelet antigen‐1b (HPA‐1b). The HPA‐1bb Gypsy patients are at risk of isoimmunization against αIIbβ3, as this complex is not expressed at their platelet surface. Tentatively, they would, however, not have an increased risk of developing anti‐HPA‐1a alloantibodies by exposure of αIIbβ3 on platelets from random platelet transfusions. However, the β3 chain can also associate with the αv subunit expressed at the platelet surface. Because Gypsy GT patients express normal αvβ3 carrying HPA‐1b epitopes, these patients might develop anti‐HPA‐1a alloantibodies reacting with αvβ3 and/or β3. Objectives/Patients/MethodsTo demonstrate this hypothesis, sera from HPA‐1bb (n = 5) and HPA‐1ab (n = 1) Gypsy GT patients were investigated by modified antigen capture assay using platelets or stable transfected cells. Furthermore, stable transfected cells expressing either αIIbβ3 or αvβ3 together with soluble monomeric chimeric β3 (as absorbent) were used to differentiate anti‐β3 and anti‐αvβ3 reactivity. ResultsOnly HPA‐1bb patients developed alloantibodies reacting with HPA‐1a cells. Further analysis showed that HPA‐1bb patients developed anti‐HPA‐1a alloantibodies reacting with β3 and/or αvβ3. ConclusionIn this study, we found that HPA‐1bb patients who failed to express αIIbβ3 on the platelet surface can develop alloantibodies against HPA‐1a reacting with β3 as well as αvβ3. This is of particular importance as anti‐HPA‐1a alloantibodies might cause fetal neonatal alloimmune thrombocytopenia and/or platelet transfusion refractoriness.

Two homozygous missense mutations in ITGB3 gene as a cause of Glanzmann Thrombasthenia in four consanguineous Pakistani pedigrees.

Glanzmann thrombasthenia (GT) is most common of inherited platelet disorders, resulting from quantitative/qualitative defects in platelet surface integrin αIIbβ3, encoded by ITGA2B and ITGB3 genes. Little is known about clinical and molecular characteristics of GT patients from highly consanguineous Pakistani population. This study analyzed the clinical and molecular spectrum of six GT patients from four unrelated but consanguineous families. Platelet surface expression of αIIbβ3 integrin was determined using flow cytometry analysis. ITGA2B and ITGB3 genes were screened for causative mutations by DNA sequencing. Detected mutations were characterized for their pathogenicity using a variety of in silico tools. Glanzmann thrombasthenia patients in this study generally presented early in life, had a severe course of clinical disease with transfusion dependency for management of bleeding episodes. Molecular analysis revealed 2 homozygous missense mutations in ITGB3 gene, c.422 A˃G (p.Y141C) in three GT patients from a single pedigree with familial segregation and c.1641 C>G (p.C547W) in three unrelated GT patients from three families manifesting type I GT with severe reduction in platelet αIIbβ3 levels. In silico pathogenicity predictions, multiple sequence alignment and 3D protein modeling unanimously suggested deleterious nature of the detected mutations, possibly due to aberrant disulfide bonding. Of note, clinical diversity was observed even among GT patients with same mutation in GT1 family. This study provides an initial yet important account of clinical and genetic characterization of GT in local patients which may spark further studies to help molecular diagnosis, optimal disease management, and genetic counseling based prevention efforts.

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.

Molecular yield of targeted sequencing for Glanzmann thrombasthenia patients

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder. Around 490 mutations in ITGA2B and ITGB3 genes were reported. We aimed to use targeted next-generation sequencing (NGS) to identify variants in patients with GT. We screened 72 individuals (including unaffected family members) using a panel of 393 genes (SHGP heme panel). Validation was done by Sanger sequencing and pathogenicity was predicted using multiple tools. In 83.5% of our cohort, 17 mutations were identified in ITGA2B and ITGB3 (including 6 that were not previously reported). In addition to variants in the two known genes, we found variants in ITGA2, VWF and F8. The SHGP heme panel can be used as a high-throughput molecular diagnostic assay to screen for mutations and variants in GT cases and carriers. Our findings expand the molecular landscape of GT and emphasize the robustness and usefulness of this panel.

Management of Glanzmann's Thrombasthenia – Guidelines based on an expert panel consensus from gulf cooperation council countries

Glanzmann thrombasthenia is a rare disorder, due to quantitative and/or qualitative abnormalities of the platelet integrin αIIbβ3 and/or αIIbβ3. Although it is considered a rare disorder with a global incidence of 1/1,000,000 population, the case is different at the Gulf Cooperation Council countries, where prevalence rate as high as 1/40,000 in Madinah, Saudi Arabia. This makes it necessary to develop patient's management guidelines. Due to limited resources in the literature, experts' consensus was important to develop such guidelines. Experts panel elected to use recombinant activated factor VII (rFVIIa) as the first line of treatment of acute bleeds and reserve platelets transfusion for nonresponding patients or severe bleeds, rFVIIa at high dose (270 μg/kg body weight) may tried upfront. rFVIIa may be tried as prophylactic treatment in patients with frequents bleeds. Experts panel elected to allow girls with Glanzmann thrombasthenia to have menstruation and to adapt a special protocol for this purpose (Madinah protocol). Pregnancy should be managed carefully, where normal delivery encouraged under coverage of rFVIIa. Risk of bleeding should be expected in neonates. Minor surgeries and dental workup can be managed under coverage of rFVIIa. Glanzmann thrombasthenia patients with severe disease should be encouraged for hematopoietic stem cell transplant.

Molecular genetic diagnosis of Tunisian Glanzmann thrombasthenia patients reveals a common nonsense mutation in the ITGA2B gene that seems to be specific for the studied population.

: Glanzmann thrombasthenia is an inherited severe bleeding disease. Mutations associated with Glanzmann thrombasthenia are highly heterogeneous and occur across the two genes coding for the platelet αIIbβ3 integrin. This study was aimed at identifying Glanzmann thrombasthenia-associated novel mutations in Tunisian patients. Seven unrelated Glanzmann thrombasthenia patients issued from high consanguineous families (86%; 6/7 of the patients) were studied. Glanzmann thrombasthenia diagnoses were based on patients' bleeding histories and platelet aggregation tests. Screening of ITGA2B and ITGB3 genes was performed by denaturing high-performance liquid chromatography (DHPLC) analysis. Amplicons with abnormal elution profiles were subjected to direct sequencing. DHPLC/sequencing analysis identified a pathogenic homozygous mutation in exon 26 at position c.2702C>A, inducing a substitution of a serine to a stop codon (p.S901*) in the ITGA2B gene, in three patients. This mutation was only previously reported in a Glanzmann thrombasthenia patient of a Tunisian origin and not in other populations. We diagnosed a pathogenic Glanzmann thrombasthenia mutation in ITGA2B screened by DHPLC that appears to be specific to individuals of Tunisian heritage and that deserves to be investigated in first intention. As a result, we determined that performing prenatal diagnosis and setting a prevention strategy via counselling for affected heterozygote individuals will be helpful for Tunisian Glanzmann thrombasthenia families where there is still a high rate of consanguinity.

The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction

Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the ‘RGD pocket’ formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, but not unactivated, Ps adhere well to immobilized 'D98;' 2. cells expressing normal αIIbβ3 could bind to: a) fbg in the absence of EDTA, but not in the presence of EDTA, b) D100 in the absence or presence of EDTA, c) 'D98' in the presence, but not the absence, of EDTA; 3. cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3, adhere well to immobilized 'D98.' These data support there being two separate mechanisms of binding, one involving the interaction of γ-404-411 (in fbg and D100) with the αIIbβ3 RGD binding pocket, and another involving a site on D100 and 'D98' (but cryptic in fbg) that interacts with a site on αIIbβ3 exposed by receptor activation or induced by EDTA.We have now studied adhesion of αIIbβ3 to the cross-linked fibrin fragment D-dimer (D-d). We predicted it to be similar to the interaction of ‘D98‘ with αIIbβ3 because γ-K406, which is important in the binding of fbg to αIIb, participates in the antiparallel reciprocal factor XIIIa-mediated transglutaminase crosslinks between K406 and Q398 on two different γ-chains, and so is unlikely to be able to interact with the RGD pocket.Methods: D-d, purified from cross-linked fibrin after plasmin digestion, was obtained from Haematologic Technologies and found to contain a dominant band with the expected Mr and immunoblot pattern using antibodies to the individual fbg chains. Fibrinogen, 'D98,' and HEK293 cells expressing normal and mutated αIIbβ3 were prepared as we previously described (Zafar et al., Blood Advances 2017), and the adhesion assays were conducted as described in that paper. Clot retraction was performed by adding BSA, 'D98,' or D-d (200 µg/ml) to washed Ps (2 X 108/ml) + thrombin (0.2 U/ml) in a glass aggregometer cuvette.Results: αIIbβ3-mediated adhesion to immobilized D-d (10 µg/ml coating concentration) was similar to adhesion to 'D98' in that 1. Unactivated P adhered well to fbg, but poorly to 'D98' and D-d (n=4); 2. Activated Ps (10 µM thrombin receptor activating peptide) adhere ~2-fold better than unactivated Ps to fbg, but ~10-fold better to 'D98' and D-d (p<0.001 for unactivated vs activated Ps with both 'D98' and D-d; n=4); 3. HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) adhere well to fibrinogen, but not 'D98' and D-d; 4. HEK293 cells expressing the constitutively active aIIbF992A/F993Aβ3 receptor [αIIb(FF)β3-HEK] bound well to fbg, 'D98, and D-d (p<0.001 for both 'D98' and D-d compared to normal αIIbβ3; n=5); 5. EDTA treatment of Ps results in loss of adhesion to fbg, but significantly enhanced adhesion to 'D98' and D-d (p<0.05 for each ligand with and without EDTA; n=4); 6. EDTA treatment of αIIbβ3-HEK results in loss of adhesion to fbg, but significantly enhanced adhesion to both 'D98' and D-d (p<0.01 for each ligand with and without EDTA; n=5); 7. Adhesion of αIIb(FF)β3-HEK to D-d is like adhesion to 'D98' in being inhibitable by mAbs 7E3 and 10E5, eptifibatide, tirofiban, and RUC-4 (p<0.01 for all); n=4); 8. Adhesion of αIIb(FF)β3-HEK to D-d is similar to adhesion to 'D98' in being inhibitable by mutating β3 D119 to A in the MIDAS (p<0.01; n=6); 9. EDTA-induced adhesion of HEK-αIIbβ3 to both 'D98' and D-d is not inhibitable by mutating β3 D119 to A. 10. 'D98' and D-d inhibited clot retraction, but BSA did not.Conclusion: We conclude that cross-linked fibrin fragment D-dimer, like fibrinogen fragment ‘D98‘, interacts with activated αIIbβ3 via sites other than the γ-404-411 peptide, and that this interaction contributes to clot retraction. DisclosuresColler:Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.

A novel approach using ancillary tests to guide treatment of Glanzmann thrombasthenia patients undergoing surgical procedures

BackgroundGlanzmann thrombasthenia (GT) is a disorder of platelet function. Standard therapy includes platelet transfusions, which may be hampered by antiplatelet antibodies. AimsTo assess potential correlation between bleeding and number of active platelets in GT patients undergoing surgery. Clinical peri- operative patients' hemostasis was compared with flow cytometry analysis (FC), and whole blood clot formation. MethodsGT patients undergoing surgery were included. Blood counts, platelet activation studies, FC and rotational thromboelastography (ROTEM) were performed as ancillary tests to estimate the effectiveness of treatment. ResultsA total of 4 GT patients undergoing 5 surgeries were included. Consecutive FC analysis following platelet transfusions showed gradual decrease of donor platelets with a nadir of 3280 platelets in patients who experienced no post procedural bleeding following minor procedures. After major surgery, bleeding occurred when donor platelets decreased to 2600–4280. Decline in donor platelets was associated with reduced clot firmness as noted by ROTEM. ConclusionResults suggest that very low number of active donor platelets may suffice to achieve proper hemostasis in certain procedures. Our study points to the potential role of consecutive FC examinations to demonstrate the number of donor platelets as an ancillary tool for decision making in GT patients undergoing surgery.

PC161: Titanium platelet rich fibrin membrane in the treatment of Glanzmann's thrombasthenia patient's gingival recession defect

PC161: Titanium platelet rich fibrin membrane in the treatment of Glanzmann's thrombasthenia patient's gingival recession defect

Molecular characterization of Glanzmann's thrombasthenia in Iran: identification of three novel mutations.

: Quantitative and/or qualitative defects of the platelet membrane glycoprotein IIb/IIIa complex lead to the clinical entity of Glanzmann's thrombasthenia. A large variety of mutations and polymorphisms are responsible for the aberrant expression and defective activity of this heterodimeric complex. The current study aimed to determine the pattern of mutations among Iranian population with Glanzmann's thrombasthenia. A total of 20 patients with Glanzmann's thrombasthenia have been evaluated. All exons and splice sites of ITGA2B and ITGB3 genes were amplified using touchdown PCR. Mutation screening was analyzed using conformation sensitive gel electrophoresis heteroduplex PCR, and DNA sequencing. In addition to finding one previously identified mutation and polymorphism, the experimenters explored 3 and 2 novel mutations and polymorphisms, respectively. One substitution mutation, two deletions of a single nucleotide, one insertion of a single nucleotide, two synonymous polymorphisms, and one missense polymorphism were found using Sanger sequencing method. All detected mutations were homozygous which will most likely contribute to the pathogenesis of Glanzmann's thrombasthenia. Furthermore, it suggested ITGB3 as the mainly affected gene impaired in the patients with Glanzmann's thrombasthenia. As expected, the molecular results were consistent with the phenotypic findings so that GPIIb/IIIa complex was disrupted due to mutations in all type-I Glanzmann's thrombasthenia patients. It is concluded that intronic alterations or epigenetic regulations could be responsible for aberrant expression and/or defective activity of GPIIb/IIIa complex among other patients.

Perioperative ROTEM and ROTEMplatelet monitoring in a case of Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia is a rare, inherited disease, which consists in abnormality of the glycoprotein IIa/IIIb genes that causes impaired platelet function. Glanzmann's thrombasthenia patients show prolongation of bleeding time and may experience life-threatening hemorrhage after surgery. We present a case of a young woman having Glanzmann's thrombasthenia, who underwent gynecological operation for ovarian cyst. Perioperative monitoring included thromboelastometry with ROTEM and impedance aggregometry with ROTEMplatelet. In this case, despite acceptable ROTEM results after platelet transfusion, ROTEMplatelet was more indicative of hemostatic impairment and reflected the bleeding occurred in the postoperative period. When treating patients with congenital or acquired platelet dysfunction, some intrinsic limits of thromboelastometry to assess platelet function may be overcome with the combined use of a point-of-care device that measures platelet aggregation.

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction

AbstractIn addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbβ3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbβ3. The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2. CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbβ3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation.