Regulatory Role of D'D3 Domain in VWF-A1 Mediated Platelet Thrombus Formation: Application towards Understanding Von Willebrand Disease

Introduction: von Willebrand factor (VWF) is a multimeric blood glycoprotein that plays a crucial role during blood coagulation by recruiting platelets to vascular injury site [1]. Improper regulation of VWF-platelet interaction results in various cardiovascular disorders like stroke, heart attack and other bleeding disorders called von Willebrand disease. The 3-D structural organization of VWF is believed to regulate the platelet binding function of VWF-A1 domain [2]. However, the precise mechanism by which domain level organization controls VWF function is not known. Here, we tried to address this by applying a “systems approach”.

Materials and Methods: Dimeric VWF (ΔPro-VWF) was constructed by deleting the propeptide section of full-length VWF cDNA. ΔD'D3-VWF was identical to ΔPro-VWF only it lacked the VWF-D'D3 domain. Recombinant GpIbα was synthesized as a fusion protein with human IgG tail. All proteins were purified from mammalian expression systems. ΔPro-VWF was crosslinked using BS³, a reagent that links proximal amines. This was proteolytically cleaved and subjected to high resolution tandem mass spectrometry (LTQ Orbitrap). C++ programs were written to identify cross-linked peptide pairs from the MS data. Experiments that validated the MS studies examined the effect of D'D3 domain deletion on the binding of VWF-A1 to platelet receptor GpIbα in static ELISA studies and flow chamber based shear assays. Additionally, anti-D'D3 mAbs were generated in mice and the effect of these reagents on shear induced platelet aggregation (SIPA) was studied.

Results and Discussion: Analysis of the cross-linked VWF tandem mass spectrometry data revealed three cross-linked peptide pairs linking VWF D'D3 and A1 domain. One of these A1 peptides formed a major interaction interface with platelet GpIbα. Based on this, studies were designed to assay the role of D'D3 in regulating VWF-A1 function. To this end, the deletion of the D'D3 domain (i.e. ΔD'D3-VWF) resulted in a dimeric protein that efficiently bound GpIbα even under static conditions and in the absence of ristocetin. ΔPro-VWF, on the other hand, only bound GpIbα when ristocetin was added.  Under low shear conditions (1dyn/cm²) robust platelet translocation was observed on ΔD'D3-VWF, but not ΔPro-VWF, coated surfaces. At higher shear conditions (10-20 dyn/cm²), while platelet accumulation was comparable on the two surfaces, platelet rolling velocity on ΔD'D3-VWF was ~ 50% lower compared to ΔPro-VWF. Finally, one of the anti-D'D3 mAb generated (clone DD3.1) blocked SIPA by ~50% in the absence but not presence of ristocetin.

Model for VWF- GpIbα interaction under shear.  A. D'D3 domain shields VWF-A1 binding to GpIbα. B. Upon shear, D'D3 releases A1. C. This enables VWF-A1 binding to GpIbα.

Conclusions: The data demonstrate that, in the native state, VWF-D'D3 domain sterically inhibits the VWF-A1 domain and this reduces the rate of VWF binding to platelets. Shear stress or agonist (e.g. ristocetin) releases this steric inhibition. This then allows VWF to bind platelet-Gp1bα efficiently. Such regulation of D'D3-A1 interaction may be a critical feature that controls platelet adhesion rates at sites of vascular injury. While von Willebrand disease (VWD) mutations have been identified, the molecular mechanism of mutation manifesting into the disease state is not well understood. Based on these results, it is likely that some of the VWD 2B or 2M mutations affecting VWF-GpIbα interaction affect the domain level regulation of VWF instead of directly affecting A1-GpIbα affinity.

References: [1] Madabhushi, S.R., et al., Blood, 2012, March 27 (Epub ahead of print).

                     [2] Singh, I et al., Biophys J., 2009;96(6):2313-20.