Disruption of the long-range GPIIIa Cys(5)-Cys(435) disulfide bond results in the production of constitutively active GPIIb-IIIa (alpha(IIb)beta(3)) integrin complexes.
The major platelet integrin alpha(IIb)beta(3), also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys(5)-Cys(435) disulfide bond of the beta(3) subunit results in the production of isoforms that bind some, but not all, anti-Pl(A1) alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogen-mimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa- and GPIIb-Ala435IIIa-transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125(FAK) on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.
Blood Research Institute, Blood Center of Southeastern Wisconsin, 8727 Watertown Plank Road, PO Box 2178, Milwaukee, WI 53233, USA. qsun@bcsew.edu
Qi-Hong
QH
Sun
Chao-Yan
CY
Liu
Ronggang
R
Wang
Cathy
C
Paddock
Peter J
PJ
Newman
D019943:Amino Acid Substitution; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D003553:Cystine / Q000378:metabolism* / Q000502:physiology; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3; D016297:Mutagenesis, Site-Directed; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction; D014162:Transfection
D015703:Antigens, CD; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D003553:Cystine; D005340:Fibrinogen