A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia.
We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.
Department of Haematology, University of Cambridge, UK. cg348@cam.ac.uk
Cedric
C
Ghevaert
Alexandre
A
Salsmann
Nicholas A
NA
Watkins
Elisabeth
E
Schaffner-Reckinger
Angela
A
Rankin
Stephen F
SF
Garner
Jonathan
J
Stephens
Graham A
GA
Smith
Najet
N
Debili
William
W
Vainchenker
Philip G
PG
de Groot
James A
JA
Huntington
Mike
M
Laffan
Nelly
N
Kieffer
Willem H
WH
Ouwehand
D000328:Adult; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism* / Q000473:pathology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005340:Fibrinogen / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008533:Megakaryocytes / Q000378:metabolism / Q000473:pathology; D020125:Mutation, Missense; D010375:Pedigree; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding / Q000235:genetics; D013921:Thrombocytopenia / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism
C061248:ITGB3 protein, human; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D005340:Fibrinogen