1. A Point Mutation Thr799Met on the 2 Integrin Leads to the Formation of New Human Platelet Alloantigen Sita and Affects Collagen-Induced Aggregation
by Sentot Santoso, Julia Amrhein, Heiko A. Hofmann, Ulrich J.H. Sachs, Matthias M. Walka, Hartmut Kroll, and Volker Kiefel
Blood
Volume 94(12):
December 15, 1999
©1999 by American Society of Hematology

2. The reactivities of anti-Sita with paternal platelets in MAIPA assay using MoAbs FMC25 (anti-GPIb/IX), Gi9 (anti-GPIa/IIa), SAM-1 (anti-GPIc/IIa), Gi5 (anti-GPIIb/IIIa), and B1G6 (anti-β2m) as capture antibodies.
The reactivities of anti-Sita with paternal platelets in MAIPA assay using MoAbs FMC25 (anti-GPIb/IX), Gi9 (anti-GPIa/IIa), SAM-1 (anti-GPIc/IIa), Gi5 (anti-GPIIb/IIIa), and B1G6 (anti-β2m) as capture antibodies.
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

3. The pedigrees of the index family Sit (A) and family Dre (B).
The pedigrees of the index family Sit (A) and family Dre (B). Solid symbols represent Sita (+), open symbols represent Sita (−) individuals. The child with NAIT is indicated. The Bra and Brb phenotypes of Sita-phenotyped individuals are shown.
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

4. Immunoprecipitation analysis of biotin surface-labeled platelets derived from a Sita (−) and a Sita(+) individual with anti-Sita (lanes 1 and 4), anti-Bra (lanes 2 and 5), and anti-Brb (lanes 3 and 6).
Immunoprecipitation analysis of biotin surface-labeled platelets derived from a Sita (−) and a Sita(+) individual with anti-Sita (lanes 1 and 4), anti-Bra (lanes 2 and 5), and anti-Brb (lanes 3 and 6). Immunoprecipitates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreduced conditions, transferred onto nitrocellulose membrane, and visualized using streptavidin-horseradish peroxidase and chemiluminescent substrate.
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

5. Nucleotide sequence analyses of amplified GPIa cDNA derived from 2 Sita (+) individuals (A.I.1 and A.II.1; Fig 2).
Nucleotide sequence analyses of amplified GPIa cDNA derived from 2 Sita (+) individuals (A.I.1 and A.II.1; Fig 2). PCR product encompassing nucleotides was subcloned into the plasmid vector pGEM-5Zf and sequenced on both strands using primers corresponding to the SP6 and T7 RNA polymerase promotor sequences. The base exchanges of the wild-type (WT) CA or CG to the mutant TG at positions 2531 and 2532 (arrows) result in a Thr799 (ACA or ACG) → Met799 (ATG) substitution.
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

6. PCR strategy for the elucidation of the GPIa gene surrounding the polymorphic base at position 2531 (arrow).
PCR strategy for the elucidation of the GPIa gene surrounding the polymorphic base at position 2531 (arrow). Genomic DNA was amplified by PCR using primer pairs P61, P42 and P6, P7. PCR products were sequenced for the determination of exon-intron boundaries. The polymorphic exon 20 (bases ) encoding the amino acids Glu782-Pro828 is flanked by 2 introns (421 and 1,200 bp) of phases 2 and 0 (italic). For genotyping analysis of the polymorphic bases 2531 and 2532 (bold), a 630-bp fragment was amplified using primer pair P61, P83.
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

7. Restriction map of the 630-bp PCR products (top).
Restriction map of the 630-bp PCR products (top). The arrow indicates the position of the restriction site for MaeIII endonuclease. The length of the restriction fragments is shown above. RFLP analysis of PCR-amplified genomic DNA derived from peripheral blood cells (PBL) of Sita-phenotyped individuals. DNA fragments were analyzed on 1.6% agarose gel stained with ethidium bromide (bottom). The undigested 630-bp PCR product is shown in lane 1. Lanes 2 and 3 represent the analysis of MaeIII-digested PCR product derived from DNA of a Sita (+) heterozygous individual and a Sita (−) individual, respectively. Lane 4, DNA size standards (pBR328 DNA.BglI + pBR328 DNA.HinfI).
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

8. Immunoprecipitation analysis of allele-specific recombinant GPIa isoforms.
Immunoprecipitation analysis of allele-specific recombinant GPIa isoforms. Recombinant forms of GPIa/IIa complex were produced in CHO cells transfected either with Glu505Thr799 (left panel), Glu50Met799 (middle panel), or Lys505Thr799 (right panel) form of GPIa. After surface labeling with biotin, cell lysates were immunoprecipitated with anti-Brb (lanes 1), anti-Sita (lanes 2), and anti-Bra (lanes 3). Immunoprecipitates were analyzed on 7.5% SDS-PAGE under nonreduced conditions, transferred onto nitrocellulose membrane, and visualized using chemiluminescence substrate.
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology

9. Platelet aggregation of a Sita (+) donor (B. III
Platelet aggregation of a Sita (+) donor (B.III.3; curves 1 and 3) and a Sita (−) individual (B.III.2; curves 2 and 4) after stimulation with 2.5 μg/mL collagen (curves 1 and 2) or 10 μg/mL collagen (curves 3 and 4).
Platelet aggregation of a Sita (+) donor (B.III.3; curves 1 and 3) and a Sita (−) individual (B.III.2; curves 2 and 4) after stimulation with 2.5 μg/mL collagen (curves 1 and 2) or 10 μg/mL collagen (curves 3 and 4).
Sentot Santoso et al. Blood 1999;94:
©1999 by American Society of Hematology