Multi-step binding of ADAMTS-13 to von Willebrand factor.
BACKGROUND
ADAMTS-13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS-13 distant from the active site.


OBJECTIVE
We hypothesized that not all binding sites for ADAMTS-13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS-13.


METHODS
Immunoprecipiation of VWF-ADAMTS-13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays.


RESULTS
Co-immunoprecipitation demonstrated that ADAMTS-13 binds to native multimeric VWF (K(d) of 79 +/- 11 nmol L(-1)) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50-200). Moreover, results with ADAMTS-13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS-13 spacer, probably thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS-13 to VWF stoichiometry of 0.0040 +/- 0.0004 (mean +/- SEM, n = 10).


CONCLUSIONS
ADAMTS-13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.
Feys
H B
HB
Department of Biochemistry and Molecular Biophysics, Department of Medicine, Washington University, School of Medicine, St Louis, MO, USA.
Anderson
P J
PJ
Vanhoorelbeke
K
K
Majerus
E M
EM
Sadler
J E
JE
D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001665:Binding Sites; D001775:Blood Circulation; D015153:Blotting, Western; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D011489:Protein Denaturation; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human