(596ar) Interstitial Permeation of Human Blood Clots Formed Under Flow Using Controlled Pressure Gradients in a Microfluidic Model of Bleeding

Traumatic
injuries and genetic bleeding disorders provide a sustained clinical need to
understand the complex transport properties of intraluminal thrombi. We
developed a microfluidic device to measure intraluminal thrombi permeabilities
under controlled pressure gradients. Unlike many previous in vitro
permeability methods, this device incorporates the critical mechanical and
biological thrombi properties that can only be obtained under hemodynamic
conditions. The PDMS microfluidic device maintains blood pressure and flow rate
by utilizing two constant volume syringe pumps and a LabVIEW interface. The
main channel is perfused with anti-coagulated human whole blood at an initial
shear rate of 1100 s^-1. After flowing blood contacts the localized collagen
or collagen/lipidated tissue factor scaffold; the controlled pressure gradient
forces blood to permeate through the structure. The pressure gradient is
maintained by a downstream infusion of Ca²⁺ buffer controlled by
LabVIEW and measured using three pressure transducers. Permeate velocity was
measured using a pulse of texas red dye and a numerical model was employed to
determine the permeabilities of the collagen scaffold, platelet layer, and
platelet/fibrin layer. The device was validated by calculating a
permeability of collagen (1.98x10^-11 ± 6.01x10^-12 cm²) over varying
pressures (12-23.4 mm Hg) and comparing to previous literature values. The
resulting permeabilities for a platelet (5.45x10^-14± 9.66x10^-15 cm²)
and platelet/fibrin layer (2.71x10^-14± 3.56x10^-15 cm²) formed over
10 minutes of flow provide insight into the relative resistance that each supply
to the cessation of bleeding. Noticeably, the formation of fibrin and presence
of thrombin provided a 50% reduction in permeability under hemodynamic
conditions. In addition to permeability measurements, we observed decreased platelet
accumulation with increased pressure drop (13.8-23.4 mm Hg) across the
intraluminal thrombi in PPACK whole blood. This response can be explained by
the increased permeation of ADP and TXA₂ through the forming
thrombi. The design and validation of our microfluidic device allowed for
permeability measurements of intraluminal thrombi with and without thrombin. In
the absence of thrombin, PPACK whole blood formed a platelet mass which was >350
fold less permeable than collagen alone. An additional 50% reduction of
permeability was observed when thrombin was present and platelet/fibrin clots
were formed.