Delimiting the autoinhibitory module of von Willebrand factor

Abstract

Essentials The self-inhibitory mechanism of von Willebrand factor (VWF) remains unclear. Residues flanking the A1 domain of VWF form a discontinuous autoinhibitory module (AIM). rVWF_1238-1493 exhibited greater thermostability and inactivity than its shorter counterparts. The cooperative coupling between the N- and C- AIM regions are required for inhibiting A1.

Summary: Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF_1238-1493 ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF_1238-1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF_1238-1461 , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF_1271-1493 , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

Keywords: blood platelets; hemostasis; platelet GPIb-IX complex; tandem mass spectrometry; von Willebrand factor.

Figure 1

Binding and thermostability analysis of A1 fragments. (A) An AIM model from the previous study [12] and an illustration of A1 fragments. The N- and C-terminal sequences flanking the von Willebrand factor (VWF) A1 domain were designated as NAIM (orange) and CAIM (celestial blue). The starting and ending residue numbers of each fragment are labeled. O-glycosylation sites (filled ovals) and the 1272-1458 disulfide bond are marked. (B) A Coomassie blue-stained polyacrylamide gel showing the purified proteins under non-reducing (N.R.) and reducing (R.) conditions. (C) Superimposed gel filtration chromatograms of A1 fragment from Superdex 200 10/300 GL SEC column. (D) Sedimentation velocity results for rVWF1238- 1493 showing the fitted interference scans and residuals (upper) and sedimentation coefficient distributions (lower). Every second scan and every third data point are shown for clarity. (E, G) ELISA measurements (n = 3) of the isotherms of A1 fragments and human plasma-derived VWF binding to immobilized GPIb-IX in the absence (E) and presence (G) of 1.5 mg/mL ristocetin. (F) Representative histogram of flow cytometry measurement to determine the binding of A1 fragments to platelets in platelet rich plasma. (H) SYPRO Orange fluorescence intensity changes over the temperature of each A1 fragment (n=4). Temperature increment is 1°C per minute. (a.u., arbitrary unit) (I) Whisker plot of the melting temperature measurements from (H). Mean value ± standard deviations are marked under each plot. A paired t-test was conducted using GraphPad Prism 5 (GraphPad Software, Inc. CA the USA) and the p-value was marked.

Figure 2

Hydrogen-deuterium exchange (HDX) characterization of A1 fragments. (A) Residual HDX heat map of each A1 fragments from 10 s to 10 000 s. The line for 0 s denoted the results obtained without exchange. Relative fractional deuterium uptake was plotted using the rainbow color scale. Secondary structures of each region were labeled on top. Denaturation of rVWF1238-1493 (rVWF1238-1493 +GdnHCl) was performed as described [9]. Three representative peptides around Gly1479 were selected to show their raw mass spectra and deuterium uptake in the native (red) and denatured (blue) forms. (B) 3D projection of HDX data at the indicated exchange time. HDX data were mapped to the structure of the A1 domain (Protein Data Bank, 1SQ0, only residues 1269-1466 were shown in the structure) using PyMOL. (C) 3D projection of the difference in relative fractional uptake at 10 000 s of exchange between rVWF1238-1493 and rVWF1238-1472, as defined by the color code in the figure.

Figure 3

Cooperation of the N- and C- AIM is required for its inhibitory effect. (A) Illustration of rVWF1238- 1461 that misses the C-AIM. (B) Comparing the rVWF1271-1493 and rVWF1238-1461 by a coomassie blue- stained polyacrylamide gel under nonreducing conditions. (C) Comparing the binding to immobilized GPIb- IX among rVWF1261-1472, rVWF1271-1493, rVWF1238-1461, and rVWF1238-1493 by ELISA (n = 3). (D) Representative histogram of flow cytometry measurement to determine the binding of rVWF1261-1472, rVWF1271-1493, and rVWF1238-1461 to platelets in platelet rich plasma. (E) Whisker plot of the melting temperatures of A1 fragments. Mean value ± standard deviation (n=4) was marked under each plot. A paired t-test was conducted using GraphPad Prism 5 (GraphPad Software, Inc. CA the USA) and the pvalue was marked.