ADAMTS-13 Binds Platelets through Gpib.

Abstract 3044

Poster Board II-1020

ADAMTS-13 cleaves high molecular weight von Willebrand factor (VWF) realeased by the endothelium in order to prevent massive intravascular platelets adhesion and aggregation as pathologically observed in thrombotic thrombocytopenic purpura. ADAMTS-13 is present at low levels in plasma. We therefore surmised that platelets are able to specifically bind the metalloprotease on their surface, hereby concentrating the enzyme where it is most required. Immunofluorescent studies were then performed to see whether or not ADAMTS-13 bound to the platelet plasma membrane using as primary antibody a murine anti-human ADAMTS-13 monoclonal antibody (13E2). A positive membrane fluorescent signal was detected using as a source of platelets either a normal donor or a patient with type III Von Willebrand disease, demonstrating that ADAMTS-13 is located on the platelet surface independently from VWF. This results were confirmed by flow cytometry analysis. In all 10 normal individuals tested 13E2 binding to not-permeabilized platelets was detected with a FITC antimurine secondary antibody. With this as background, 96 wells polystyrene NUNC plates were coated either with albumin or fibrinogen or VWF or recombinant ADAMTS-13 (each at 10 μg/ml) and then blocked with 5% albumin overnight. Washed platelets (100,000/μl), incubated with divalent cations, were then let adhere to the wells for 1 hr at 37°C. After extensive washing adherent platelets were lysed, p-nitrophenilphosphate was added and the reaction was stopped with NaOH 2M. Detection was done by assessing optical density at 405 nm. Binding of washed platelets preincubated with 2mM CaCl2 and/or 2mM MgCl2 to wells covered with immobilized recombinant ADAMTS-13 was significantly higher than binding to wells coated with albumin (p<0.001), was at the same levels of the binding to wells covered with recombinant VWF and approximately half of binding to the wells coated with fibrinogen. When washed platelets were preincubated with EDTA 2mM binding to the wells coated with fibrinogen or recombinant ADAMTS-13 decreased at the same degree of the wells covered with albumin Activation of platelets, obtained by preincubating platelets with ADP (5 μM) or collagen (10 μg/ml), significantly increased their binding to recombinant ADAMTS-13 (p<0.001) as compared to the binding of non-activated platelets. In a set of experiments platelets were preincubated with blocking antibodies against αaIIbβ3 or GpIb (10 μg/ml) or murine IgG. did not effect the levels of binding to recombinant ADAMTS-13 while the binding to fibrinogen and VWF was totally abolished. To confirm the data washed platelets obtained from a patient with Bernard-Soulier syndrome (BSS) in which GpIb/IX/V expression was below 1% were let adhere to wells covered with albumin, fibrinogen, VWF or recombinant ADAMTS-13 and platelet adhesion was detected as above: while adhesion to fibrinogen was normal, platelets not expressing Gpib/IX/V did not adhere to VWF or ADAMTS-13. In conclusion we demonstrated that platelets bind ADAMTS-13 on their surface through GpIb/IX/V; the binding is specific, activation and divalent cations dependent, inhibited by EDTA, and not mediated by VWF on the membrane of platelets.