The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-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 alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb 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 alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb 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 alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.