ADAMTS13 binds to CD36: a potential mechanism for platelet and endothelial localization of ADAMTS13.
BACKGROUND
ADAMTS13 cleaves ultralarge von Willebrand factor (VWF) and plays a significant role in vascular biology and thrombotic thrombocytopenic purpura. CD36, a transmembrane protein present on endothelial cells and platelets (PLTs), binds to thrombospondin via three thrombospondin type 1 repeats. ADAMTS13 contains eight thrombospondin type 1 repeats.


METHODS
An enzyme-linked immunoassay was used to explore the binding of recombinant human CD36 (rHuCD36) to recombinant human ADAMTS13 (rHuADAMTS13). A competition assay between rHuADAMTS13 and recombinant human (rHu)-thrombospondin-2 for binding to CD36 was then performed. Subsequently, binding of rHuADAMTS13 to PLT membrane fragments expressing CD36 (PLT glycoprotein IV) and glycoprotein Ib/IX was assessed. To examine the functional significance of an ADAMTS13-CD36 interaction, ADAMTS13 activity measured by a fluorescence resonance energy transfer assay was investigated in the presence of either rHuCD36 or concentrated PLTs.


RESULTS
rHuCD36 bound to rHuADAMTS13 in a dose-dependent fashion. rHu-thrombospondin-2 competed with ADAMTS13 for CD36 occupancy, but even high concentrations of rHu-thrombospondin-2 failed to completely block binding of rHuADAMTS13 to rHuCD36. rHuADAMTS13 bound to PLT membrane fragments expressing CD36 (PLT glycoprotein IV) in preference to PLT membrane fragments expressing glycoprotein Ib/IX. ADAMTS13 activity was not inhibited by the presence of either rHuCD36 or concentrated PLTs.


CONCLUSIONS
rHuADAMTS13 binds to both rHuCD36 and PLT membrane CD36 in vitro. The binding of CD36 to rHuADAMTS13 with retention of its enzymatic activity is consistent with a proposed role for CD36 in localizing ADAMTS13 on the endothelial cell surface where it regulates the cleavage of VWF.
Davis
Amanda K
AK
Department of Pathology, Massachusetts General Hospital, Boston, USA. am.davis@alfred.org.au
Makar
Robert S
RS
Stowell
Christopher P
CP
Kuter
David J
DJ
Dzik
Walter H
WH
D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019699:Thrombospondins / Q000235:genetics / Q000378:metabolism; D000099262:Thrombospondin 2
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D001798:Blood Proteins; D018955:CD36 Antigens; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins; D000099262:Thrombospondin 2; C099604:ADAMTS13 protein, human