Platelet GpIbα Binding to von Willebrand Factor Under Fluid Shear: Contributions of the D'D3‐Domain, A1‐Domain Flanking Peptide and O‐Linked Glycans

Abstract

BackgroundVon Willebrand Factor (VWF) A1‐domain binding to platelet receptor GpIbα is an important fluid‐shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N‐terminus of the A1‐domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D′D3‐domain, A1‐domain N‐terminal flanking peptide (NFP), and O‐glycans on this peptide.Methods and ResultsFull‐length dimeric VWF (ΔPro‐VWF), dimeric VWF lacking the D′D3 domain (ΔD′D3‐VWF), and ΔD′D3‐VWF variants lacking either the NFP (ΔD′D3NFP─‐VWF) or just O‐glycans on this peptide (ΔD′D3OG─‐VWF) were expressed. Monomeric VWF‐A1 and D′D3‐A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro‐VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50‐ to 100‐fold higher than other proteins lacking the D′D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on‐rate of D′D3‐A1 binding to immobilized GpIbα (kon=1.8±0.4×104 (mol/L)−1·s−1; KD=1.7 μmol/L) was reduced compared with the single VWF‐A1 domain (kon=5.1±0.4×104 (mol/L)−1·s−1; KD=1.2 μmol/L). Thus, VWF‐D′D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1‐GpIbα binding. Here, in ELISA, the number of apparent A1‐domain sites available for binding GpIbα on ΔPro‐VWF was ≈50% that of the ΔD′D3‐VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro‐VWF<ΔD′D3‐VWF<ΔD′D3NFP─‐VWF<ΔD′D3OG─‐VWF.ConclusionsWhereas VWF‐D′D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D′D3‐domain and N‐terminal peptide regulate platelet translocation and thrombus formation.

Application of microfluidic devices in studies of thrombosis and hemostasis

“…To elaborate on this concept, the authors also made anti-D′D3 monoclonal antibodies that sterically inhibit the A1 domain of VWF from binding platelet GpIbα in flow based assays. Additionally, to demonstrate the importance of structural features N-terminal to the A1-domain in regulating VWF-GpIbα binding, various mutants were created [20]. Functional studies performed with these constructs using microfluidics show that the D′D3 domain of VWF is a key regulator that inhibits the binding of platelet GpIbα to the VWF-A1 domain and thrombus formation under physiological shear.…”

“…Depending on the nature of the measurements, these assays can monitor multiple output parameters including: immobilized platelet number and aggregate morphology, platelet activation via cell surface P-selectin, cell surface phosphatidylserine via Annexin-V measurements, fibrin formation and also local thrombin production [20, 43, 44]. Using blood from FVIII −/− mice or human blood supplemented with anti-FIXa aptamer, early studies by Ogawa et al [45] , showed that thrombus formation patterns in haemophilia are regulated by fluid shear.…”

A biophysical view on von Willebrand factor activation

“…An auto-inhibitory role in regulating the A1-GPIbα interaction might further be played by VWF's glycans, as suggested by several studies that showed that (partial) deglycosylation of VWF enhances binding to GPIbα (De Marco et al, 1985;Fallah et al, 2013;Madabhushi et al, 2014). Unfortunately however, the possible influence of glycans has only rarely been taken into consideration in studies investigating VWF's activation, so that insights into this subject are very limited.…”

“…In contrast, the plusD'D3 construct lacked domains D4‐CK and was thus N‐terminally dimerized by intra‐dimer disulfides between the D'D3 domains. It could be argued that this difference partially contributed to the observed shielding effect of D'D3, but later on Madabhushi et al () showed the same effect also with C‐terminally linked dimers lacking D'D3. Additional evidence for a direct inhibition of the A1‐GPIbα interaction through D'D3 was provided by showing that the presence of isolated D'D3 domain peptides directly inhibited ristocetin‐stimulated platelet agglutination mediated by the C‐terminally dimerized ΔD'D3 construct (Ulrichts et al, ).…”

“…However, several further experiments performed under shear flow yielded results that were in line with the abovementioned studies. In particular, A domain constructs lacking the A1 N-terminal flanking peptide exhibited enhanced binding to GPIbα peptides as well as to platelets under flow (Auton et al, 2012;Ju, Dong, Cruz, & Zhu, 2013;Madabhushi, Zhang, Kelkar, Dayananda, & Neelamegham, 2014). Ulrichts et al (2006) further postulated that also the D'D3 domain serves as a masking environment that inhibits binding to the GPIb-IX-V receptor.…”