267629 Direct Observation of Von Willebrand Factor Unfolding and Elongation On Collagen During Acute Whole Blood Exposure to Pathological Shear Rates

Wednesday, October 31, 2012
Hall B (Convention Center )
Thomas Colace and Scott L. Diamond, Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA

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.

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