N-terminal flanking region of A1 domain in von Willebrand factor stabilizes structure of A1A2A3 complex and modulates platelet activation under shear stress

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.

FIGURE 1.

Binding of recombinant A1A2A3 proteins to platelet GPIbα. Increasing concentrations of recombinant 1261-A1A2A3 (circles) or 1238-A1A2A3 (squares) tri-domain proteins were incubated with immobilized fixed platelets in the presence (filled symbols) or absence (open symbols) of ristocetin. Bound protein was determined by ELISA as described under “Experimental Procedures.” The graphs are representative of two separate experiments. Each point represents the mean ± S.D. (error bars) of values obtained from a triplicate assay.

FIGURE 2.

Effect of A1A2A3 proteins in RIPA. 1261-A1A2A3 protein (0.5 μm) or 1238-A1A2A3 protein (1.0 μm) was incubated with platelet-rich plasma for 2 min at 25 °C. Platelet agglutination was then initiated with the addition of ristocetin (1.0 mg/ml). This figure represents two separate experiments using different two blood donors.

FIGURE 3.

Effect of 1261-A1A2A3 protein in flow-dependent platelet adhesion to immobilized fibrinogen. Whole blood mixed with 1238- or 1261-A1A2A3 (250 nm) was perfused over a surface coated with fibrinogen at 1,500 s⁻¹ shear rates. After a 2-min perfusion, the plates were washed with buffer, and several frames of attached platelets were recorded. The photomicrographs represent two separate assays using two different blood donors.

FIGURE 4.

Circular dichroism spectra. CD of 1238-A1A2A3 (black) and the N-terminally truncated 1261-A1A2A3 (gray) tri-domains were acquired at 5 °C. Mean residue molar ellipticity (deg cm²/dmol per residue) data are an average of three spectral scans of 1 μm protein taken in 0.1-nm intervals from 200 to 260 nm. Inset, spectral scan of the N-terminal peptide at 20 μm. Solid black lines are a 20-data point (2-nm window) smoothing of the data.

FIGURE 5.

Thermal unfolding of A1A2A3 proteins. Thermal unfolding of 1238-A1A2A3 (black) and the N-terminally truncated 1261-A1A2A3 (gray) tri-domains was monitored by CD at 222 nm (A) and by DSC (B). Mean residue molar ellipticity (deg cm²/dmol per residue) on left was obtained by continuous scanning at 1 ºC/min taking data at 3-s intervals. CD results are an average of two scans of 1.1 μm 1238-A1A2A3 and six scans of 0.6 μm 1261-A1A2A3 to minimize data scatter and maximize signal/noise ratio. Solid black lines are a 20-data point (1 ºC window) smoothing of the data. Excess heat capacity (cal/mol per K) on right was obtained by continuous scanning of ∼3 μm protein at 1 ºC/min. Low temperature transitions observed by CD were not detectable by calorimetry. Insets, second derivatives of the data identify the midpoint temperatures that comprise these overlapping transitions (see “Results”).

FIGURE 6.

Reactivity of the 1238-A1A2A3 and 1261-A1A2A3 tri-domains and vWF to antibody A108. A, structure of the A1 domain (1AUQ) with sequences recognized by A108 is indicated. The N-terminal sequence is not present in the crystal structure. B, tri-domains (250 nm) and purified plasma vWF (0.5 μg/ml) were incubated with immobilized antibody A108 or rabbit IgG, and the captured protein was detected by ELISA (see “Experimental Procedures”). Data (mean ± S.D. (error bars)) are representative of two separate triplicate experiments.