Redox control of β2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.
BACKGROUND
β(2) -Glycoprotein I (β(2) GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β(2) GPI in thrombus formation is unknown. We have recently shown that β(2) GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of β(2) GPI can take place on the platelet surface.


METHODS
β(2) GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe N(a)-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β(2) GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced β(2) GPI.


RESULTS
We demonstrate that the Cys288-Cys326 disulfide in domain V of β(2) GPI is the predominant disulfide reduced by thioredoxin-1. Reduced β(2) GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β(2) GPI reduced by thioredoxin-1, in comparison with non-reduced β(2) GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF.


CONCLUSIONS
Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of β(2) GPI with VWF may contribute to the redox regulation of platelet adhesion.
Passam
F H
FH
Department of Immunology, Allergy and Infectious Diseases, University of New South Wales, St George Hospital, Sydney, Australia.
Rahgozar
S
S
Qi
M
M
Raftery
M J
MJ
Wong
J W H
JW
Tanaka
K
K
Ioannou
Y
Y
Zhang
J Y
JY
Gemmell
R
R
Qi
J C
JC
Giannakopoulos
B
B
Hughes
W E
WE
Hogg
P J
PJ
Krilis
S A
SA
D000818:Animals; D001777:Blood Coagulation; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction; D010973:Platelet Adhesiveness; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012310:Ristocetin / Q000494:pharmacology; D013438:Sulfhydryl Compounds; D013879:Thioredoxins / Q000378:metabolism*; D053482:beta 2-Glycoprotein I / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D053482:beta 2-Glycoprotein I; D014841:von Willebrand Factor; D012310:Ristocetin; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases; D003545:Cysteine