To develop a new human blood microfluidic assay to interrogate core/shell mechanics.
METHODS: A 2-stage assay perfused whole blood over
collagen/±
tissue factor (TF) for 180 seconds at 100 s-1 wall shear rate, followed by
buffer perfusion at either 100 s-1 (venous) or 1000 s-1 (arterial). This microfluidic assay used an extended channel height (120 µm), allowing
buffer perfusion well before occlusion.
RESULTS: Clot growth on
collagen stopped immediately with
buffer exchange, revealing ~10% reduction in platelet fluorescence intensity (at 100 s-1) and ~30% (at 1000 s-1) by 1200 seconds.
Thrombin generation (on
collagen/TF) reduced erosion at either
buffer flow rate.
P-selectin-positive platelets were stable (no erosion) against 1000 s-1, in contrast to
P-selectin negative platelets.
Thrombin inhibition (with
D-Phe-Pro-Arg-CMK) reduced the number of
P-selectin-positive platelets and lowered
thrombus stability through the reduction of
P-selectin-positive platelets. Interestingly,
fibrin inhibition (with H-
Gly-Pro-Arg-Pro-
OH acetate salt) increased the number of
P-selectin-positive platelets but did not lower stability, suggesting that
fibrin was only in the core region.
Thromboxane inhibition reduced
P-selectin-positive platelets and caused a nearly 60% reduction of the clot at arterial
buffer flow. P2Y1 antagonism reduced clot size and the number of
P-selectin-positive platelets and reduced the stability of
P-selectin-negative platelets.
CONCLUSION: The 2-stage assay (extended channel height plus
buffer exchange) interrogated platelet stability using human blood. Under all conditions,
P-selectin-positive platelets never left the clot.