A 13-bp deletion in αIIb gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of alpha(IIb)beta3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13-bp deletion in the alpha(IIb) gene that causes in-frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13-bp deletion, and determined the mechanism by which the 13-bp deletion abolishes alpha(IIb)beta3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13-bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300-600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective alpha(IIb)beta3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in alpha(IIb) gene, TT1616-7 deletion in beta3 gene, and IVS14: -3C --> G in beta3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of beta3 and was expressed as dysfunctional alpha(IIb)beta(3). None of 15 unrelated Jordanian patients carried any of the described mutations.     

Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion.

BACKGROUND: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. OBJECTIVE AND METHODS: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin-dependent platelet adhesion utilizing both human and murine platelets. RESULTS: Platelets can adhere to fibronectin via the integrin alpha(IIb)beta(3), leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes alpha(IIb)beta(3)-mediated Ca(2+) mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C gamma2. In contrast, studies with blocking antibodies and mice demonstrate that alpha(5)beta(1) and alpha(v)beta(3) support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither alpha(5)beta(1) nor alpha(v)beta(3) is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP). CONCLUSIONS: These observations demonstrate that integrins alpha(5)beta(1) and alpha(v)beta(3) support platelet adhesion and the generation of filopodia but that, in contrast to the integrin alpha(IIb)beta(3), are unable to promote formation of lamellipodia.     

Novel Mutations in the GPIIb and GPIIIa Genes in Glanzmann Thrombasthenia

BACKGROUND: Glanzmann thrombasthenia (GT) is an inherited autosomal recessive platelet disorder characterized by a complete or partial lack, or mutation, of the GPIIb/IIIa complex (integrin α(IIb)β(3)) on the thrombocytes' surface, leading to a severe bleeding syndrome. MATERIAL AND METHODS: Molecular genetic analysis was performed in patients with suspected GT. The aim of the present study was the identification of new natural variants, their impact on platelet function, and their relation to the risk of bleeding. RESULTS: Expression of the platelet integrin α(IIb)β(3) was determined by flow cytometry. Mutations were identified through sequencing of cDNA and genomic DNA. In addition, platelet function studies (PAC-binding, aggregations) were implemented. The study included 25 patients revealing 13 mutations (GPIIb: n = 9; GPIIIa: n = 4). Two of the 13 mutations were previously described (T207I; L214P). The remaining mutations have not been published yet, whereas 1 mutation in 2 unrelated families was identical (3062 T→C). CONCLUSION: All patients with less than 25% of present α(IIb)β(3) have a medical history of bleeding.     

Three novel β-propeller mutations causing Glanzmann thrombasthenia result in production of normally stable pro-αIIb, but variably impaired progression of pro-αIIbβ3 from endoplasmic reticulum to Golgi

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa). OBJECTIVES: To determine the molecular basis of GT and characterize the mutations by in vitro expression studies. PATIENTS: We studied three unrelated patients from southern India whose diagnosis was consistent with GT. RESULTS: Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature alpha(IIb) in the G128S mutant, in contrast to 6% and 33% of the normal amount of mature alpha(IIb) in the S287L and G357S mutants, respectively. Pulse-chase analysis demonstrated pro-alpha(IIb) in the mutants comparable with the normal pro-alpha(IIb), but no conversion to mature alpha(IIb) in the G128S mutant, and only trace conversion to mature alpha(IIb) in the S287L and G357S mutants. The disappearance of pro-alpha(IIb) in the three mutants was similar to that in cells expressing normal alpha(IIb)beta3 or alpha(IIb) only. All three mutants demonstrated pro-alpha(IIb)beta3 complexes and co-localized with an ER marker by immunofluorescence. The G128S mutant showed no co-localization with a Golgi marker, and the other two mutants showed minimal and moderate co-localization with the Golgi marker. CONCLUSIONS: These three beta-propeller mutations do not affect the production of pro-alpha(IIb), its ability to complex with beta3, or its stability, but do cause variable defects in transport of pro-alpha(IIb)beta3 complexes from the endoplasmic reticulum to the Golgi.     

Double heterozygosity for a novel missense mutation of Ile304 to Asn in addition to the missense mutation His280 to Pro in the integrin beta3 gene as a cause of the absence of platelet alphaIIbbeta3 in Glanzmann's thrombasthenia.

BACKGROUND: Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder characterized by a defect in the expression or the function of alphaIIbbeta3. Objectives: The purpose of the present study was to identify genetic defects in a GT patient. METHODS: The expression of alphaIIbbeta3 was determined by flow cytometric analysis and Western blotting. We analyzed the cDNA sequences of both alphaIIb and beta3, and performed transfection experiments using COS7 cells to confirm that a specific mutation was responsible for the GT case. RESULTS: Flow cytometric analysis and Western blotting showed remarkably reduced expression of alphaIIbbeta3. Sequence analysis of the patient's cDNA indicated a new missense mutation that led to the amino acid substitution of Ile304 (ATC) with Asn (AAC) in exon 6 of the beta3 gene. This was in addition to the missense mutation of His280 (CAT) to Pro (CCT) in exon 5, which had been previously reported. The missense mutation of Ile304 (ATC) to Asn (AAC) in beta3 was found to be responsible for this GT case. This was because transfection experiments using COS7 cells indicated that alphaIIbbeta3 possessing Asn304 in beta3 was not expressed on the surface of the transfected cells. In addition, immunoprecipitation analysis demonstrated that alphaIIbbeta3 was absent inside the transfected COS7 cells possessing Asn304 in beta(3). CONCLUSION: In this study, we describe a new missense mutation (ATC to AAC) at position 1009 in exon 6 that leads to an amino acid substitution (Ile304 to Asn) in beta3, which is responsible for this GT case.     

三个遗传性血小板无力症家系临床特性和基因分析

目的 对三个遗传性血小板无力症(GT)家系进行整合素αⅡbβ3临床特性分析和基因突变检测.方法 经BPC、血涂片、出血时间(BT)、凝血常规检查、血小板聚集试验和流式细胞术进行实验检测;用PCR法对先证者αⅡbβ3基因所有外显子及其侧翼序列进行扩增;PCR产物纯化后直接测序,检测其基因突变.突变位点经直接测序排除基因多态性,103名健康人作对照.结果 三个家系的先证者BPC正常,血小板不聚集,BT延长,凝血常规指标检查正常,对ADP、凝血酶、肾上腺素、胶原、花生四烯酸等多种诱聚剂反应低下,而对瑞斯托霉素反应基本正常;家系1和家系3先证者的血小板膜表面αⅡbβ3的含量极度降低,家系2先证者的血小板膜表面αⅡb阳性血小板为63%,β3阳性血小板为76%.家系1先证者αⅡb基因存在G10A纯合突变,β3基因存在G1412T纯合突变.家系2先证者β3基因存在G1199A和1525delC复合杂合突变.家系3先证者在αⅡbβ3基因未检测到突变.结论 G10A和G1412T复合纯合突变是导致家系1先证者发生GT的原因,G1199A和1525delC复合杂合突变是导致家系2先证者发生GT的原因.G10A、G1412T和1525delC为国际首次报道的突变.     

Insertion of a C in the exon 28 of integrin alphaIIb gene leading to a frameshift mutation is responsible for Glanzmann thrombasthenia in a Japanese case.

BACKGROUND: Glanzmann thrombasthenia (GT) is a hereditary bleeding disorder caused by a qualitative or quantitative defect in the integrin alphaIIbbeta3. OBJECTIVE: Our objective is to identify the gene mutation that resulted in GT. PATIENTS AND METHODS: The patient was a 66-year-old male with a history of frequent bleeding. The expression levels of the integrin proteins in the platelets were determined by flow cytometry and Western blot analysis. The sequences of genomic DNA and mRNA encoding for alphaIIb and beta3 were analyzed by the dye-terminator cycle sequencing method. For transfection experiments, expression vectors encoding for wild-type alphaIIb, mutated alphaIIb, beta3, green fluorescent protein (GFP) fusion wild-type alphaIIb, GFP fusion mutated alphaIIb and DsRed fusion beta3 were constructed. These vectors were transfected to COS-7 cells, and the expression levels were determined. RESULTS: The alphaIIb protein was remarkably reduced in the patient's platelets, and gene analysis showed that the patient possessed compound heterozygous mutations in the alphaIIb gene. One was a C --> G substitution at the splice acceptor site (- 3) of exon 26 (CAG -->GAG) and the other was the insertion of an additional C at the region including six C bases between 2911 and 2916 in exon 28 (InsC). Transfection experiments using COS-7 cells showed that alphaIIb containing InsC had expressed and formed a complex with beta3, but had not been transported to the Golgi apparatus. CONCLUSIONS: In the present study the novel mutation InsC, leading to a frameshift that affects the transmembrane domain and the cytoplasmic tail, was found to be responsible for GT.     

一例GPⅡb基因突变导致血小板无力症的诊断

目的对1例血小板无力症（GT）患者进行实验诊断,分析引起血小板膜糖蛋白（GP）缺陷的突变基因。方法通过对先证者凝血常规、血小板计数、血小板聚集功能、出血时间、GPⅡb/Ⅲa（CD41/CD61）含量等项目的检测和血涂片中血小板形态及分布的观察,进行临床表型诊断。通过聚合酶链反应（PCR）扩增结合测序的方法,分析先证者GPⅡb/Ⅲa基因的所有外显子区及其侧翼序列,家系成员仅在相应突变区域进行PCR扩增和测序。同时以100名健康人作为对照进行分析,以排除基因多态性。结果先证者凝血常规检测和血小板计数正常,但出血时间延长,血小板聚集功能异常;表现为对多种生理性诱聚剂反应低下,但对瑞斯托霉素反应正常,血块退缩不良;流式细胞术检测CD41、CD61含量显著降低;血涂片中血小板散在,无聚集现象。经测序,发现先证者GPⅡb基因23号外显子存在18183A〉C杂合变异,导致GPⅡb蛋白Q778P替换。与100名健康对照者测序比较,排除基因多态性,证实其为基因突变。结论 GPⅡb基因的Q778P杂合突变是导致该先证者患GT的原因之一。     

三个遗传性血小板无力症家系临床表型和基因表型诊断的研究

目的对3个遗传性血小板无力症（glanzmann thrombasthenia，GT）家系进行血小板膜糖蛋白Ⅱb、Ⅲa（GPⅡb／GPⅢa）基因突变的检测。方法应用PCR对先证者GPⅡb／GPⅢa基因所有外显子及其侧翼序列进行扩增；PCR产物纯化后直接测序，检测其突变基因。突变位点经直接测序证实排除基因多态性。结果3个家系的先证者PLT均正常，血小板形态分散，出血时间（BT）延长，凝血象正常，对二磷酸腺苷（ADP）、凝血酶、肾上腺素、胶原、花生四烯酸等多种诱聚剂反应低下，而对瑞斯托霉素反应基本正常；家系1和家系2先证者的血小板膜表面CD41，（GPⅡb）／CD61（GPⅢa）的含量极度降低，分别为0．16％／1．8％、0．9％／3．7％，家系3先证者的血小板膜表面GPⅡb阳性血小板为10．1％，GPⅢa阳性血小板为12．8％。免疫印迹法几乎检测不到家系1和家系3先证者的α／Ⅱb。蛋白，家系2先证者的α／Ⅱb蛋白含量明显降低。家系1先证者在GPⅡb基因存在T2255G和C2671T复合杂合突变，家系2先证者在GPⅡb基因存在A2334C纯合突变，家系3先证者在GPⅡb基因存在C1750T和69-79del复合杂合突变。结论T2255G和C2671T复合杂合突变是导致家系1先证者发生GT的原因，A2334C纯合突变是导致家系2先证者发生GT的原因，C1750T和69-79del复合杂合突变是导致家系3先证者发生GT的原因。     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

三个遗传性血小板无力症家系的表型和基因型诊断分析

目的对3个遗传性血小板无力症(GT)家系进行临床表型和基因型诊断,探讨其分子发病机制。方法通过凝血指标检测、血小板(PLT)聚集试验、出血时间测定和流式细胞仪检测,明确3个遗传性GT家系的诊断,用PCR扩增先证者的αⅡb及β3基因的所有外显子及其侧翼序列,利用直接测序法进行基因序列分析。针对先证者的基因突变位点,对其家系成员进行相应外显子基因检测,同时选择100名健康人对照以排除基因多态性。结果 3个家系的先证者血小板计数及凝血四项检测均正常,而出血时间(BT)延长;血小板对多种诱聚剂反应低下,而对瑞斯托霉素反应基本正常;流式细胞术检测结果显示,家系2及家系3先证者为Ⅰ型GT,家系1先证者为Ⅱ型GT。基因检测结果显示3个家系的基因突变均位于αⅡb基因,分别是17号外显子14502CT导致R(Arg)553X(stop)纯合无义突变;26号外显子18859CT导致Q(Gln)860X(stop)纯合无义突变;15号外显子14103-14104insT、14105AC、14106GC、14107AT导致氨基酸移码突变;其家系部分成员检测到相应位点的杂合突变。结论纯合无义突变18859CT、纯合插入突变14103-14104insT及3个纯合性错义突变14105AC、14106GC、14107AT是导致先证者2及先证者3发生Ⅰ型GT的原因,而14502CT纯合无义突变是先证者1发生Ⅱ型GT的原因。其中,纯合性插入突变14103-14104insT及纯合性点突变14105AC、14106GC、14107AT为国际首次报道的新突变。     

Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.     

von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of botrocetin and ristocetin, respectively

BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.     

Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function

BACKGROUND: The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits alpha(IIb) and beta(3), is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of alpha(IIb)beta(3), thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to alpha(IIb)beta(3) triggers cytoskeletal changes and granule release (outside-in signaling). AIM: Genetic approaches to characterize the molecular pathways involved in alpha(IIb)beta(3) signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). RESULTS: alpha(IIb)beta(3) activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIbalpha(+) cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of alpha(IIb)beta(3) outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of alpha(IIb)beta(3) activation. CONCLUSION: Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and alpha(IIb)beta(3) signaling in the native cellular environment.     

The evidence for the use of recombinant human activated factor VII in the treatment of bleeding patients with quantitative and qualitative platelet disorders

There are increasing reports suggesting that high-dose recombinant human activated factor VII (rFVIIa) is effective in the treatment and prevention of bleeding in patients with quantitative and qualitative platelet disorders. These clinical observations are supported by evidence that FVIIa binds weakly to activated platelet surface and at high concentration improves thrombin generation. In experimental models, this improved thrombin generation enhances platelet adhesion in thrombocytopenic conditions and enhances adhesion and aggregation of platelets lacking glycoprotein IIbIIIa (integrin alpha(IIb)beta(3)), characteristic of the qualitative platelet disorder Glanzmann thrombasthenia (GT). There is a need for clinical trials to confirm the safety and efficacy of rFVIIa in patients with various quantitative and qualitative platelet defects, either by itself or in combination with other hemostatic agents such as platelet transfusion. Pending the availability of such data, rFVIIa may be considered in severe bleeding in thrombocytopenia and GT patients with platelet antibodies and refractory to platelet transfusions and other standard treatments. An international survey suggests that rFVIIa at about 90 microg/kg every 2 hours for 3 or more doses could be used for GT patients with severe bleeding, but confirmation by larger studies is needed. For GT patients undergoing surgery and for treatment and prevention of bleeding in thrombocytopenic patients, the optimal rFVIIa regimen remains to be defined.     

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia

Summary.  This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (≤ 10% of control) platelet content of GPIIb–IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3'-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both.     

Critical role of ADP interaction with P2Y12 receptor in the maintenance of αIIbβ3 activation: association with Rap1B activation

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.     

Bleeding tendency and impaired platelet function in a patient carrying a heterozygous mutation in the thromboxane A2 receptor

Summary. Background: Thromboxane A₂ receptor (TXA₂R) abnormality appears to dominantly disturb platelet function. Objectives: To reveal a molecular genetic defect in a patient with TXA₂R abnormality and investigate the mechanism for the impaired response to TXA₂. Patient: The proband (OSP‐2, PT) was a 7‐year‐old Japanese girl, suffering from repeated mucocutaneous bleeding. Methods and results: U46619 (2.5 and 10 μm )‐induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA₂R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA₂R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA₂R. We then examined α_IIbβ₃ activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained α_IIbβ₃ and Rap1B activation in the proband. In addition, platelet secretion as monitored by P‐selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y₁₂ has been shown to play a critical role in the sustained α_IIbβ₃ activation (Kamae et al. J Thromb Haemost 2006; 4 : 1379). As expected, small amounts of exogenous ADP (0.5 μm ) partially restored the sustained α_IIbβ₃ activation induced by U46619. Conclusion: Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA₂R mutation is, at least in part, as a result of the decrease in ADP secretion.     

A tripeptide mimetic of von Willebrand factor residues 981–983 enhances platelet adhesion to fibrinogen by signaling through integrin βIIbβ3

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.