(309b) 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.