A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope.
We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.
Flug
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F
Department of Pediatrics, New York University Medical School, NY 10016.
Espinola
R
R
Liu
L X
LX
SinQuee
C
C
DaRosso
R
R
Nardi
M
M
Karpatkin
S
S
D000595:Amino Acid Sequence; D016824:Antigens, Human Platelet; D004247:DNA / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D039661:Integrin beta3; D007519:Isoantigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007930:Leucine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011110:Polymorphism, Genetic; D011392:Proline; D011487:Protein Conformation; D013381:Subtilisins
D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007519:Isoantigens; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D004247:DNA; D011392:Proline; C035801:CCSGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D013381:Subtilisins; D007930:Leucine