Blood clots are rapidly assembled hemodynamic sensors: flow arrest triggers intraluminal thrombus contraction

Abstract

Objective: Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops.

Methods and results: The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction.

Conclusions: Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

Figure 1. Microfluidic device to independently control blood flow and transthrombus pressure drop

COMSOL was used to determine the pressure throughout the microfluidic device (A) including the collagen scaffold region (B), where L=250 μm. Human type I polymerized collagen was localized in the scaffold region (C). Following 10 min of CTI-whole blood flow at 1130 s⁻¹, platelets (red), fibrin (green) and their overlap (yellow) form a thrombus on the collagen (D). A confocal image of platelets (red) and fibrin (green) shows the 3D structure the microfluidic device (E).

Figure 2. Flow arrest triggers clot contraction

A thrombus formed in the absence of thrombin and presence of fluorescent 50 nm beads was rinsed with Ca²⁺ buffer for 4.5 min before the cessation of flow caused a rapid contraction. The outline of a pre-retracted thrombus (t’=−1 min) shows the inward retraction of the thrombus following flow stoppage (t’=0 to 2 min) (A). Contraction rate of the upstream and downstream sections of the thrombus were measured before and after flow arrest (n=3 donors) (B). Trajectories of the 50 nm beads represent the contractile response of the thrombus at upstream (red, n=6), middle (blue, n=3), and downstream (green, n=6) locations (C). Stopping the flow caused a significant increase in contraction rate in the Y and Z directions. To quantify these rates, the times before (0-2 min) and after flow cessation (0-1 min, 1-2 min) were monitored for bead velocity in the three sections of the thrombus (D). Downstream contraction rate in Y direction is shown as absolute value for contraction toward the middle region. *, P<0.01; error bars indicate mean ± SD.

Figure 3. Clot permeability

Thrombi formed under 1130 s⁻¹ were pulsed with fluorescent dye at controlled pressure drops. The normalized input and output fluorescent intensities, along with a numerically predicted output from COMSOL were measured over time for clots formed in the absence (A) and presence of TF (B). Thrombus formed without thrombin reduced the residence time of the permeated dye by 30 s compared to platelet/fibrin thrombus. COMSOL simulations were used to numerically calculate permeability over collagen (n=9), platelet (n=6, 5 donors), platelet/fibrin (n=4, 4 donors), and platelet/flow interrupted (n=2, 2 donors) geometries at constant pressure drops (C). †, P<0.01; error bars indicate mean ± SD.

Figure 4. Contraction after flow arrest requires myosin and released ADP/TXA₂

A parallel channel microfluidic device was used to develop thrombus in PPACK, at 1160 s⁻¹. Following the formation of stable thrombi, Ca²⁺ or antagonist buffer was used to rinse the surface for 7 min before stopping flow. Upstream contractions were observed in five sections (blue dashed line) over ten min. Stopping flow without a buffer rinse (A) and with a 10 μM blebbistatin rinse (B) show clot retraction compared to a trace (pink line) before flow cessation. Total clot contraction measured over time compares the effects of blebbistatin and intermediate flow stopping with stopping flow with and without a buffer rinse (C). TXA₂ and ADP antagonist significantly reduced total contraction after flow cessation as compared to buffer (D). n=3 events at 5 discrete points for each time-point indicated, using 5 separate donors; error bars indicate mean ± SD.

Figure 5. Flow cessation triggers platelet intracellular calcium mobilization via ADP/TXA₂ autocrine signaling

Platelets in PPACK whole blood were loaded with Ca²⁺ dye and perfused over collagen at 1160 s⁻¹. Following thrombus formation, whole blood perfusion was switched to Ca²⁺ or antagonist buffer for six minutes prior to stopping flow. Intracellular Ca²⁺ was measured via fluorescent intensity over time for either a Ca²⁺ buffer (A) or ADP and TXA₂ antagonist rinse (B). n=3 events using 2 donors, error bars indicate mean ± SD. A schematic illustrates the flow sensing ability of platelets via convective removal of ADP/TXA₂ (C). Shear (τ) acting on the exterior of aggregated platelets accompanied by the strain placed on interconnected α_IIbβ₃ may signal the inhibition of contraction or dense granule (δ) release.