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

Authors: 
Colace, T., University of Pennsylvania
Diamond, S. L., University of Pennsylvania


When blood moves through a severe
arterial stenosis, von Willebrand Factor (vWF) experiences acute
millisecond-scale exposures to pathological wall shear rates (gw > 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 gw or spatial wall shear rate gradients (grad gw).   Long vWF fibers (>20 mm) bound to collagen were observed with
fluorescent anti-vWF at gw
> 30,000 s-1 during perfusion of PFP (citrate, EDTA, or
recalcified with PPACK to inhibit thrombin), a process occurring at constant gw and enhanced at zero Ca2+
possibly through vWF A2 domain destabilization. 
Rapid deceleration of EDTA-PFP perfusion from 125,000 s-1 at grad gw
= -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 (aIIbb3
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 aIIbb3, 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.