Crucial role for the VWF A1 domain in binding to type IV collagen

Abstract

Von Willebrand factor (VWF) contains binding sites for platelets and for vascular collagens to facilitate clot formation at sites of injury. Although previous work has shown that VWF can bind type IV collagen (collagen 4), little characterization of this interaction has been performed. We examined the binding of VWF to collagen 4 in vitro and extended this characterization to a murine model of defective VWF-collagen 4 interactions. The interactions of VWF and collagen 4 were further studied using plasma samples from a large study of both healthy controls and subjects with different types of von Willebrand disease (VWD). Our results show that collagen 4 appears to bind VWF exclusively via the VWF A1 domain, and that specific sequence variations identified through VWF patient samples and through site-directed mutagenesis in the VWF A1 domain can decrease or abrogate this interaction. In addition, VWF-dependent platelet binding to collagen 4 under flow conditions requires an intact VWF A1 domain. We observed that decreased binding to collagen 4 was associated with select VWF A1 domain sequence variations in type 1 and type 2M VWD. This suggests an additional mechanism through which VWF variants may alter hemostasis.

Figure 1

Location of VWF A1 domain sequence variations and their effect on collagen 4 binding. The VWF A1 domain crystal structure is shown here with orange spheres to indicate Zimmerman Program A1 domain sequence variations found in type 1 subjects, and the blue spheres indicate sequence variations found in type 2M subjects that were associated with reduced or absent VWF–collagen 4 binding.

Figure 2

Collagen 4 binding to VWF depends on multimeric structure and intact VWF A1 domain. Binding of VWF samples to collagen 4 was measured by ELISA and results are expressed as the ratio of collagen 4 binding to VWF:Ag. (A) VWF:CB4/VWF:Ag ratios for human plasma samples from healthy controls (n = 246) and type 3 VWD subjects (n = 24) as well as type 2A (n = 43) and type 2B subjects (n = 19) lacking high-molecular-weight multimers. (B) VWF:CB4/VWF:Ag ratios for recombinant VWF constructs expressed in HEK293T cells with either full-length human VWF sequence or VWF containing the indicated sequence variation (n = 3 separate transfections for each construct). Error bars show mean ± 1 SD.

Figure 3

VWF A1 domain contains key residues for collagen 4 binding. Scanning alanine mutagenesis was performed for VWF A1 domain residues 1387 to 1412. (A) VWF:CB4/VWF:Ag ratios for each alanine mutant as listed on the x-axis. Error bars show mean ± 1 SD. (B) Location of key residues with absent binding (blue spheres) or reduced binding (orange spheres) to collagen 4 in a drawing of the VWF A1 domain crystal structure. Residues that were changed in the scanning alanine mutagenesis are shown in purple. An 11-amino-acid deletion that also affected collagen 4 binding is not shown for clarity.

Figure 4

Murine 1399H VWF demonstrates defect in shear-induced collagen 4 binding. VWF-deficient mice were injected with either WT or p.1399H murine VWF DNA to achieve temporary correction of their VWF levels. Blood samples were obtained 24 hours postinjection. Binding to collagen 4 under flow conditions was assessed using the Venaflux system at 37°C using a shear rate of 1111 s⁻¹. Platelets were labeled with mepacrine for visualization. (A) Platelet adhesion after 180 seconds for a representative sample from a mouse injected with WT VWF (panel 1) and a mouse injected with 1399H VWF (panel 2). Images were obtained using a Zeiss Axio Observer.A1 microscope at ×10 original magnification and a Hamamatsu Orca R2 camera. (B) VWF:Ag levels obtained 24 hours postinjection for WT (n = 4) and 1399H (n = 4) mice. (C) VWF:CB4 levels obtained 24 hours postinjection for WT (n = 4) and 1399H (n = 4) mice. (D) Platelet adhesion as measured by number of platelet aggregates for 1399H-injected mice (n = 3) compared with WT-injected mice (n = 4).