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Prepublished online as a Blood First Edition Paper on November 7, 2002; DOI 10.1182/blood-2002-07-2266.
Submitted July 30, 2002
Accepted October 22, 2002
Two novel mutations in the  IIb calcium-binding domains identify hydrophobic regions essential for  IIb 3 biogenesis
W Beau Mitchell, JiHong Li, Fiza Singh, Alan D Michelson, James Bussel, Barry S Coller, and Deborah L French*
Department of Pediatrics, Mount Sinai School of Medicine, New York, NY, USA
Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA
Center for Platelet Function Studies, University of Massachusetts, Worcester, MA, USA
Department of Medicine, Cornell Medical Center, New York, NY, USA
Laboratory for Blood and Vascular Biology, Rockefeller University, New York, NY, USA
* Corresponding author; email: debbie.french{at}mssm.edu.
The recently published crystal structure of the external domains of V3 confirms the prediction that the amino-terminal portion of V, which shares 40% homology with IIb, folds into a -propeller structure, and that the four 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 two patients with Glanzmann thrombasthenia. One patient inherited a V298F mutation in the second domain, and the other patient inherited an I374T mutation in the third domain. Mammalian cells expression studies were performed with normal and mutant IIb and 3 cDNA constructs. By flow cytometry, expression of IIbV298F/3 in transfected cells was 28% of control, and expression of IIbI374T/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 V298 and I374 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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