Dataset Information

A conformational transition of the D'D3 domain primes von Willebrand factor for multimerization.

ABSTRACT: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D'D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D'D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

SUBMITTER: Gruber S

PROVIDER: S-EPMC9631632 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A conformational transition of the D'D3 domain primes von Willebrand factor for multimerization.

Gruber Sophia S Löf Achim A Hausch Adina A Kutzki Fabian F Jöhr Res R Obser Tobias T König Gesa G Schneppenheim Reinhard R Aponte-Santamaría Camilo C Gräter Frauke F Brehm Maria A MA Benoit Martin M Lipfert Jan J

Blood advances 20220901 17

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can ...[more]

PMID: 36069828

Similar Datasets

Project description:Essentials The role of von Willebrand factor (VWF) domains in regulating platelet adhesion was studied in vivo. Multimeric VWF with spacers at the N- and C-terminus of VWF-A1 were systematically tested. N-terminal modified VWF avidly bound platelet GpIbα, causing VWD Type2B like phenotype in mice. Novel anti-D'D3 mAbs suggest that changes at the D'D3-A1 interface may be biologically relevant.SummaryBackground Previous ex vivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF in vivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6 nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested ex vivo and in vivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF-/- mice caused profound thrombocytopenia, reduced plasma VWF concentrations, lower multimer distribution, and incessant tail bleeding that is reminiscent of von Willebrand disease type 2B. Platelet plugs were noted in the portal veins and hepatic arteries. An anti-D'D3 mAb DD3.3 that displays enhanced binding to VWF containing the N-terminal mucin insert also exhibited increased binding to wild-type VWF under shear and upon ristocetin addition. Conclusion Conformation changes at the VWF D'D3-A1 interface may be a key regulator of thrombosis in vivo. Structural features at the A1-A2 interface are likely of less significance.

Dataset Information

A conformational transition of the D'D3 domain primes von Willebrand factor for multimerization.

Publications

A conformational transition of the D'D3 domain primes von Willebrand factor for multimerization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets