Abstract
von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ibα. We have analyzed two A1A2A3 tri-domain proteins to demonstrate that the amino acid sequence, Gln(1238)-Glu(1260), in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIbα binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln(1238)-Glu(1260) 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 Gln(1238)-Glu(1260)) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIbα in solution under high shear forces.
Highlights
A conformational change in A1 domain of von Willebrand factor (vWF) is required for GPIb␣ binding
Amino Acid Sequence 1238 –1260 in N-terminal Flanking Region of A1 Domain Regulates Binding to GPIb␣—Previously, we noticed that an N-terminally truncated A1A2A3 (1261– 1874) protein from our laboratory resulted in increased binding activity for GPIb␣ [25]
Tial regulatory role for the amino acid sequence 1238 –1260 in vWF, and we performed comparative studies between the 1261-A1A2A3 and 1238-A1A2A3 tri-domain fragments of vWF to ascertain how this sequence is involved in vWF-platelet interactions
Summary
A conformational change in A1 domain of vWF is required for GPIb␣ binding. Results: N-terminal flanking region of the A1 domain inhibits binding to GPIb␣, stabilizes the A1A2A3 structure, and prevents platelet activation. 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 Gln1238Glu1260) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIb␣ in solution under high shear forces. Because we noticed that an N-terminally truncated tri-domain (amino acids 1261–1874) resulted in apparent binding activity for GPIb␣ higher than that of the longer construct [25], this study has used a number of biophysical and molecular biological methods to demonstrate that the sequence 1238 –1260 stabilizes the tri-domain, inhibits binding to GPIb␣, and prevents activation of platelets under shear flow These results indicate that potential coupling between the N terminus-flanking region of the A1 domain and the A domain complex forms part of the regulatory mechanism of vWF activation
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