A point mutation Thr(799)Met on the alpha(2) integrin leads to the formation of new human platelet alloantigen Sit(a) and affects collagen-induced aggregation.
A new platelet-specific alloantigen, termed Sit(a), was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sit(a) alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sit(a) epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sit(a)-positive platelets showed C(2531)-->T(2531) point mutation, resulting in Thr(799)Met dimorphism. Analysis of genomic DNA from 22 Sit(a)-negative normal individuals showed that the Thr(799) is encoded by ACG(2532) (90.9%) or ACA(2532) (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme Mae III showed that the Met(799) form of GPIa is restricted to Sit(a) (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sit(a) exclusively reacted with the Glu(505)Met(799), but not with the Glu(505)Thr(799) and the Lys(505)Thr(799) isoforms. In contrast, anti-Br(a) (HPA-5b) only recognized the Lys(505)Thr(799) form, whereas anti-Br(b) (HPA-5a) reacted with both Glu(505)Thr(799) and Glu(505)Met(799) isoforms. These results demonstrated that the Met(799) is responsible for formation of the Sit(a) alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Br(a) and Br(b) epitopes, respectively. Platelet aggregation responses of Sit(a) (+) individuals were diminished in response to collagen, indicating that the Thr(799)Met mutation affects the function of the GPIa/IIa complex.
Santoso
S
S
Institute for Clinical Immunology, Justus Liebig University Giessen, Giessen, Germany. Santoso@immunologie.med.uni-giessen.de
Amrhein
J
J
Hofmann
H A
HA
Sachs
U J
UJ
Walka
M M
MM
Kroll
H
H
Kiefel
V
V
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D016824:Antigens, Human Platelet / Q000235:genetics*; D001792:Blood Platelets / Q000502:physiology*; D006224:Cricetinae; D006801:Humans; D039421:Integrin alpha2; D008715:Methionine / Q000235:genetics; D010974:Platelet Aggregation / Q000235:genetics*; D017354:Point Mutation; D013912:Threonine / Q000235:genetics
D015703:Antigens, CD; D016824:Antigens, Human Platelet; D039421:Integrin alpha2; D013912:Threonine; D008715:Methionine