[A mutation of platelet glycoprotein I balpha results in defects in its interaction with immobilized vWF].
OBJECTIVE
To study the importance of glycoprotein (GP) I balpha mutation (A156V) in interaction between mutant expressing cell and immobilized vWF under fluid shear stress.


METHODS
Mutant GP I balpha cDNA was cloned into the EcoR I site of the mammalian expression vector pDX, mutant cDNA was then transfected into CHO betaIX cells. Human vWF was purified from blood cryoprecipitate by glycine and NaCl precipitation and subsequent separation on sepharose 4B column. The purified vWF was immobilized onto a coverslip. Cell rolling was induced in a parallel-plate flow chamber and observed by phase-contrast video microscope.


RESULTS
CHO cells expressing GP I b-IX-V complex could adhere to and roll on immobilized vWF. The A156V mutant cells retained the ability to adhere and roll on vWF matrix, but the rolling speed was significantly faster than that of wild type cells, indicating that the off-rate of the ligand-receptor bond between the mutant and vWF was impaired. Binding of monoclonal antibody AN51 to mutant GP I balpha decreased significantly, indicating that the A156V mutation altered the conformation of the N-terminal ligand-binding region of GP I balpha.


CONCLUSIONS
The mutant GP I balpha has a faster off-rate for its interaction with immobilized vWF. The mutant polypeptide adopts an altered conformation in N-terminal ligand-binding region of GP I balpha. The parallel-plate flow chamber is an extremely useful system in evaluating interaction between GP I balpha and vWF.
Dong
J
J
Division of Hematology/Oncology, Department of Internal Medicine, Baylor College of Medicine and Veterans Affairs Medical Center, Houston, TX 77030, USA.
Li
C
C
Schade
A J
AJ
Sun
L
L
McIntire
L V
LV
Lopez
J A
JA
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics; D006224:Cricetinae; D006801:Humans; D020125:Mutation, Missense; D010957:Plasmids / Q000235:genetics; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism*
D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor