(596av) Direct Observation of Von Willebrand Factor Unfolding and Elongation On Collagen During Acute Whole Blood Exposure to Pathological Shear Rates

When blood moves through a severe
arterial stenosis, von Willebrand Factor (vWF) experiences acute
millisecond-scale exposures to pathological wall shear rates (g_w > 5000 s^-1).  Distinct from viscometers or recirculation
systems that shear blood or its components for many seconds, we deployed
microfluidic devices for single-pass perfusion of whole blood or platelet free
plasma (PFP) over fibrillar collagen type 1 surfaces (< 10 msec transit time) at pathological g_w or spatial wall shear rate gradients (grad g_w).   Long vWF fibers (>20 mm) bound to collagen were observed with
fluorescent anti-vWF at g_w
> 30,000 s^-1 during perfusion of PFP (citrate, EDTA, or
recalcified with PPACK to inhibit thrombin), a process occurring at constant g_w and enhanced at zero Ca²⁺
possibly through vWF A2 domain destabilization. 
Rapid deceleration of EDTA-PFP perfusion from 125,000 s^-1 at grad g_w
= -3.7 x 10⁷ s^-1/cm quenched vWF fiber formation.  At 19,400 s^-1, EDTA-whole blood
perfusion resulted in unstable and rolling vWF-platelet nets, while whole blood
perfusion (normal Ca²⁺) generated large vWF/platelet aggregates that
repeatedly grew and embolized, a process blocked by
GR144053 (a_IIbb₃
inhibitor) or anti-GP1b.  PPACK-inhibited
whole blood perfusion at venous shear rate of 200 s^-1
produced a stable, non-growing platelet deposit that was a substrate for
massive but unstable vWF-platelet aggregates when flow was suddenly increased
to 7800 s^-1.  Triggered by
surface collagen and enhanced by platelets via GP1b and a_IIbb₃, vWF unfolding and fiber formation
occurred during acute exposures to pathological shear rates and did not require
spatial shear rate gradients. 

Figure 1, Platelet free citrated-plasma was treated with 1
μg/mL fluorescently labeled anti-vWF  and 5 mM EDTA. The plasma samples were
perfused over a collagen type 1 surface at the indicated wall shear rates. Long
fibers of vWF (>20 μm) appeared at shear rates above ~30,000 s^-1,
with more fibers appearing at higher shear rates. The bar indicates 15 μm.