TY - JOUR
T1 - N-terminal flanking region of A1 domain in von Willebrand factor stabilizes structure of A1A2A3 complex and modulates platelet activation under shear stress
AU - Auton, Matthew
AU - Sowa, Katie E.
AU - Behymer, Molly
AU - Cruz, Miguel A.
PY - 2012/4/27
Y1 - 2012/4/27
N2 - von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ibβ. We have analyzed two A1A2A3 tri-domain proteins to demonstrate that the amino acid sequence, Gln1238-Glu1260, in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIbβ binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln1238- Glu1260 stabilizes the structural conformation of the A1A2A3 tri-domain complex. The structural stabilization imparted by this particular region inhibits the binding capacity of the tri-domain protein for GPIbα. Deletion of this region causes a conformational change in the A1 domain that increases binding to GPIbα. Only the truncated protein was capable of effectively blocking ristocetin-induced platelet agglutination. To determine the capacity of activating platelets via the interaction with GPIbα, whole blood was incubated with the N-terminal region truncated or intact tri-A domain protein prior to perfusion over a fibrin- (ogen)-coated surface. At a high shear rate of 1,500 s-1, platelets from blood containing the truncated protein rapidly bound, covering >90% of the fibrin(ogen) surface area, whereas the intact tri-A domain protein induced platelets to bind <10%. The results obtained in this study ascertain the relevant role of the structural association between the N-terminal flanking region of the A1 domain (amino acids Gln1238- Glu1260) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIbα in solution under high shear forces.
AB - von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ibβ. We have analyzed two A1A2A3 tri-domain proteins to demonstrate that the amino acid sequence, Gln1238-Glu1260, in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIbβ binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln1238- Glu1260 stabilizes the structural conformation of the A1A2A3 tri-domain complex. The structural stabilization imparted by this particular region inhibits the binding capacity of the tri-domain protein for GPIbα. Deletion of this region causes a conformational change in the A1 domain that increases binding to GPIbα. Only the truncated protein was capable of effectively blocking ristocetin-induced platelet agglutination. To determine the capacity of activating platelets via the interaction with GPIbα, whole blood was incubated with the N-terminal region truncated or intact tri-A domain protein prior to perfusion over a fibrin- (ogen)-coated surface. At a high shear rate of 1,500 s-1, platelets from blood containing the truncated protein rapidly bound, covering >90% of the fibrin(ogen) surface area, whereas the intact tri-A domain protein induced platelets to bind <10%. The results obtained in this study ascertain the relevant role of the structural association between the N-terminal flanking region of the A1 domain (amino acids Gln1238- Glu1260) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIbα in solution under high shear forces.
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U2 - 10.1074/jbc.M112.348573
DO - 10.1074/jbc.M112.348573
M3 - Article
C2 - 22431729
AN - SCOPUS:84860376548
SN - 0021-9258
VL - 287
SP - 14579
EP - 14585
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 18
ER -