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Prepublished online as a Blood First Edition Paper on November 7, 2002; DOI 10.1182/blood-2002-07-2266.
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Blood, 15 March 2003, Vol. 101, No. 6, pp. 2268-2276
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Two novel mutations in the  IIb calcium-binding domains
identify hydrophobic regions essential for IIb 3
biogenesis
W. Beau Mitchell,
Ji Hong Li,
Fiza Singh,
Alan D. Michelson,
James Bussel,
Barry S. Coller, and
Deborah L. French
From the Departments of Pediatrics and Medicine, Mount
Sinai School of Medicine, Department of Medicine, Cornell Medical
Center, New York, NY, Center for Platelet Function Studies, University
of Massachusetts Medical School, Worcester, and Laboratory of Blood and
Vascular Biology, Rockefeller University, New York, NY.
The recently published crystal structure of the external domains of
 V 3 confirms the prediction that the aminoterminal portion of V, which shares 40% homology with IIb, folds into a
-propeller structure and that the 4 calcium-binding domains are
positioned on the bottom of the propeller. To gain insight into the
role of the calcium-binding domains in IIb biogenesis, we
characterized mutations in the second and third calcium-binding domains
of IIb in 2 patients with Glanzmann thrombasthenia. One patient
inherited a Val298Phe mutation in the second domain, and the
other patient inherited an Ile374Thr mutation in the third domain.
Mammalian cell expression studies were performed with normal and mutant IIb and 3 cDNA constructs. By flow cytometry, expression of IIb Val298Phe/3 in transfected cells was 28% of control, and expression of IIbIle374Thr/3 was 11% of control. Pulse-chase analyses showed that both mutant pro-IIb subunits are retained in
the endoplasmic reticulum and degraded. Mutagenesis studies of the
Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of IIb3 is dramatically
affected by structural variations in these regions of the
calcium-binding domains. Energy calculations derived from a new model
of the IIb -propeller indicate that these mutations
interfere with calcium binding. These data suggest that the IIb
calcium-binding domains play a key structural role in the
-propeller, and that the structural integrity of the
calcium-binding domains is critical for integrin biogenesis.
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