THE ARG633HIS SUBSTITUTION RESPONSIBLE FOR THE PRIVATE PLATELET ANTIGEN GRO(A) UNRAVELED BY SSCP ANALYSIS AND DIRECT SEQUENCING

We have previously described the private or family platelet antigen, G ro(a), which was identified in a case of neonatal alloimmune thrombocy topenia. The Gro(a) antigen was found to be located on the GP IIIa (be ta(3)) subunit of the GP IIb/IIIa complex, the most prominent fibrinog en receptor of platelets. Initial experiments to characterize the Gro( a) antigen at the molecular genetic level were unsuccessful. We theref ore decided to use a different strategy to unravel the molecular basis of this antigen. Platelet GP IIIa mRNA of a Gro(a(+)) and a Gro(a(-)) donor was amplified with suitable primers in a reverse transcriptase- polymerase chain reaction (RT-PCR) and subjected to single-strand conf ormational polymorphism (SSCP) analysis. Three regions of the amplifie d GP ma cDNA derived from the Gro(a(+)) donor showed a different SSCP pattern when compared to that of the Gro(a(-)) donor. Direct nucleotid e sequence analysis of these three segments revealed that two of them contained silent substitutions, A1163C, A1553G and G1565A. The first a nd the latter changes were described previously. In the third segment a G1996A mutation was found, predicting an arginine --> histidine subs titution at position 633 of the mature glycoprotein. PCR-ASRA (allele- specific restriction enzyme analysis) performed on cDNA as well as on genomic DNA with the restriction enzyme MaeIII showed that the His633 form of GPIIIa is restricted to the Gro(a(+)) phenotype. The observed mutation is three amino acids upstream of the mutation underlying the HPA-8/Sr system (Arg636Cys), suggesting this region of GP IIIa to be s usceptible for mutations. Moreover, the presence of a silent mutation and two low-frequency forms of the silent polymorphisms strongly sugge sts that the G1996A mutation did not occur in a direct ancestral allel e.