אילמ"ר - האיגוד הישראלי למדעי המעבדה הרפואית

אילמ"ר - האיגוד הישראלי למדעי המעבדה הרפואית

כינוס שנתי ה-46 | 2012

פוסטרים להצגה בכינוס

A MUTATION IN THE FIBRINOGEN RECEPTOR – THE INTEGRIN αIIbβ3, THAT CAUSES GLANZMANN THROMBASTHENIA BLEEDING DISORDER, ABROGATES THE TRANSITION OF THE RECEPTOR TO THE ACTIVE STATE

Hauschner Hagit, Mor-Cohen Ronit, Seligsohn Uri and Rosenberg Nurit
From the Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer 52621 and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

 

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by decrease or absence of functional fibrinogen receptor – the integrin αIIbβ3. αIIbβ3 is a calcium-dependent heterodimer complex that is expressed exclusively in megakaryocytes and platelets. Most of the complex is extracellular and only short tails of αIIb and β3 are inside the cytoplasm. Both cytoplasmic tails play an important role in regulating αIIbβ3 functions. In this study, we examined the effect of the β3 cytoplasmic tail on the activation process of αIIbβ3 by using an IVS14: -3C>G mutation that causes variant Glanzmann thrombasthenia (Rosenberg et al. 2005). This mutation termed 742ins causes alternative splicing and AG insertion into exon 15 predicting a reading frame shift with an extension of 40 residues (743-802) at the C-terminus of β3. We expressed the natural 742ins and 3 artificial mutations in BHK cells along with wild type (WT) αIIb as follows: β3-742stop, a truncated mutant to evaluate the effect of deleted 742-762 residues; β3-749stop, a truncated mutant that preserves the NPLY conserved sequence that is essential for binding talin and other proteins; and β3-749ins, in which the aberrant tail starts after the NPLY conserved sequence. Surface expression of all four mutants was at least 50% of WT expression but almost no soluble fibrinogen binding following activation with activating antibodies (LIBS6 or PT25) was observed. Preservation of the conserved NPLY sequence in 749stop did not improve fibrinogen binding.  The presence of the aberrant tail in 742ins and 749ins increased the deleterious effect on soluble fibrinogen binding apart from the absence of the correct sequence as exhibited by 742stop and 749stop. Activation of the αIIbβ3 mutants by the activating antibodies was achieved only when both PT25 and LIBS6 were used together or following pre-treatment with a disulfide reducing agent (5mM DTT). Activation of αIIbβ3-742ins was also achieved when a second Cys560Ser mutation in the EGF4 domain that creates a constitutively active receptor was introduced. These data suggest that the ectodomain of the 4 αIIbβ3 mutants is tightly locked in an inactive conformation but can be forced to become active only by strong stimuli.

 

 

לתכנית הכנס     חזרה לריכוז הפוסטרים 2012
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