GLANZMANN THROMBASTHENIA RESULTING FROM A SINGLE AMINO-ACID SUBSTITUTION BETWEEN THE 2ND AND 3RD CALCIUM-BINDING DOMAINS OF GPIIB - ROLE OFTHE GPIIB AMINO-TERMINUS IN INTEGRIN SUBUNIT ASSOCIATION

To gain insight into regions of the platelet GPIIb-IIIa complex involv ed in receptor biogenesis and function, we examined the biochemical pr operties of a defective GPIIb-IIIa complex from patient suffering from type II Glanzmann thrombasthenia. Flow cytometric as well as immunobl ot analysis of patient platelets showed significantly reduced levels o f GPIIb and GPIIIa compared with a normal control, Patient platelets, however, retained the ability to retract a fibrin clot, Sequence analy sis of PCR-amplified platelet GPIIb mRNA revealed an Arg(327)-->His am ino acid substitution between the second and third calcium-binding dom ains of the GPIIb heavy chain, a residue that is highly conserved amon g integrin alpha-subunits. The recombinant His(327) form of GPIIb was found to be fully capable of associating with GPIIIa, therefore the ro le of the calcium-binding domains in intersubunit association was furt her examined by constructing amino-terminal segments of GPIIb, that en ded before the first, second, and third calcium-binding domains, All t hree fragments were found to associate with GPIIIa, demonstrating that the calcium-binding domains of GPIIb are not necessary for initial co mplex formation, Regions amino-terminal to the calcium-binding domains of GPIIb may play a heretofore unappreciated role in integrin subunit association.