A 1063G -> A mutation in exon 12 of glycoprotein (GP)IIb associated with athrombasthenic phenotype: mutation analysis of [324E]GPIIb

We report the molecular, genetic and functional analysis of a case of throm basthenic phenotype. The proband showed absence of platelet glycoprotein (G P)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformatio nal polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct s equencing of PCR-amplified GPIIb exon-12 revealed the presence of a G -->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-e xpression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chin ese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPII b-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPI Ib heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, sug gesting that this mutation is the underlying molecular basis for the thromb asthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 3 24E of GPIIb for a positively charged AA other than K prevented the express ion of GPIIb-IIIa complexes. These observations suggest that a domain encom passing 324E of GPIIb is essential for heterodimerization with GPIIIa and i ts substitution for a positively charged residue precludes normal subunit a ssociation.