Study On The Clinical Features And Molecular Mechanisms Of Bernard-Soulier Syndrome And Glanzmann Thrombasthenia

With the great advances in the human genome sequence map completion and molecular biology, many hereditary bleeding disorders have been found, and genetic diagnosis has also become the main direction for the future development of hereditary hemorrhagic disease research. Bernard-Soulier Syndrome is a rare hereditary bleeding disorder characterized by moderate to severe thrombocytopenia, enlarged platelets, and a tendency to have profuse and often spontaneous bleeding. Glanzmann is an autosomal recessive bleeding syndrome affecting the megakaryocyte lineage and characterized by a quantitative or qualitative abnormality of GPIIb IIIa,absent or severely reduced platelet aggregation in response to adenosine 5’-diphosphate(ADP).However, the phenotype of BSS and GT are highly variable and they could not be easily recognized. As a result, the patients are often misdiagnosed with immune thrombocytopenia, and they are at risk of receiving inappropriate treatments. Therefore, a correct definition of the clinical and laboratory spectrum, along with accurate genetic diagnosis is important for the diagnosis and treatments of BSS and GT.In our study, the genetic diagnosis and molecular mechanisms is exoploed in thirteen patients.The maximum aggregation of platelet in response to the physiological agonists are tested; Flow cytometry tested the platelet GPIb, GPIX, GPIIb and GPIIIa positive percentage of patient and control; The full-length coding of the five genes GPIbα, GPIbβ, GPIX, GPIIb and GPIIIa are amplified by PCR; PCR products are directly subjected to DNA sequencing analysis; Mutant plasmids are constructed(GPIbα987G>A, MT1; GPIbα1480 del A, MT2 and GPIIIa 719G>A, MT3); The plasmids are transfected into Chinese hamster ovary(CHO) cells; GPIb-GPIX and GPIIb IIIa cytoplasmic expressions in the transfected CHO cells are analyzed using Western blot;All exons and exon-intron boundaries of FLNA gene are amplified by PCR followed by direct sequencing. Spreading of platelets is performed to explore the FLNA distribution in the platelet. The FLNA, Syk and syk-P of cytoplasmic expressions in the case II patient is analyzed using Western blot.For 3 BSS patients, the maximum aggregation of platelet induced ristocetin was significantly lower(range, 0%-3.5%;mean, 1.2%)in vitro, and case II ’s aggregation to collagen is reduced(range,10%-20%; mean,12%).Platelet surface expression of GPIbα was markedly decreased(range,0.5%-7.1%;mean,3.2%). For 10 GT patients, the maximum aggregation of platelet induced ADP was lessen even absent(range, 0%-20.9%; mean,6.1%) in vitro. Platelet surface expression of GPIIb IIIa was greatly decreased: GPIIb(range, 0.1%-98.7%;mean,19.6%); GPIIIa(range, 0.3%-100%; mean, 56.1%). The GPIIb IIIa expression of case 7 was >50% and the binding of fibrinogen to activated platelets of case 7 is significantly reduced,the rate of which was14% to normal.In our study 15 candidate pathogenic mutations were confirmed in 13 patients,including 8 missense mutations,2 nonsense mutations,5 splicing site mutations. 1 mutation was confirmed in FLNA gene, 2 mutations in GPIBA gene and GPIIIa, respectively, and 10 mutations were found in GPαIIb gene. Meanwhile,Expression of GPIbα and GPIX in CHO cells transfected MT1 were remarkably reduced,but they were detected in the cytoplasma.The surface expression of GPIX was found on the CHO cells transfected MT2, the rate of which was 18% to normal. However, GPIbα was not detected both on and in the transfeted CHO cells.The mutant GPIIIa MT3 can be synthesized in CHO cells and the surface expression of mutant GPIIIa was nearly normal. We next examined the Flamin A, and no significant difference between control and case II was observed, moreover, GPIIIa and FLNA were colocalized in cytosol of spreading platelets. Furthermore, we tested the tyrosine kinase Syk phosphorylation in the signaling pathway of GPVI induced by Cvx in unstirred platelets, since the expression of GPVI chain was normal on case 2 platelets. Quantification of Syk-P showed it was low in her platelets. These results was confirmed the effect of FLNA mutations on the platelet aggregation induced by collagen.