Novel Stenotic Microchannels to Study Thrombus Formation in Shear Gradients: Influence of Shear Forces and Human Platelet-Related Factors

Abstract

Thrombus formation in hemostasis or thrombotic disease is initiated by the rapid adhesion, activation, and aggregation of circulating platelets in flowing blood. At arterial or pathological shear rates, for example due to vascular stenosis or circulatory support devices, platelets may be exposed to highly pulsatile blood flow, while even under constant flow platelets are exposed to pulsation due to thrombus growth or changes in vessel geometry. The aim of this study is to investigate platelet thrombus formation dynamics within flow conditions consisting of either constant or variable shear. Human platelets in anticoagulated whole blood were exposed ex vivo to collagen type I-coated microchannels subjected to constant shear in straight channels or variable shear gradients using different stenosis geometries (50%, 70%, and 90% by area). Base wall shears between 1800 and 6600 s^-1, and peak wall shears of 3700 to 29,000 s^-1 within stenoses were investigated, representing arterial-pathological shear conditions. Computational flow-field simulations and stenosis platelet thrombi total volume, average volume, and surface coverage were analysed. Interestingly, shear gradients dramatically changed platelet thrombi formation compared to constant base shear alone. Such shear gradients extended the range of shear at which thrombi were formed, that is, platelets became hyperthrombotic within shear gradients. Furthermore, individual healthy donors displayed quantifiable differences in extent/formation of thrombi within shear gradients, with implications for future development and testing of antiplatelet agents. In conclusion, here, we demonstrate a specific contribution of blood flow shear gradients to thrombus formation, and provide a novel platform for platelet functional testing under shear conditions.

Keywords: platelets; shear gradients; stenosis; thrombosis.

Figure 1

Shear stress in stenoses. The calculated shear fields and maximal wall shear rate (γ_w, s⁻¹) in microchannels with 50% stenosis (A), 70% stenosis (B), and 90% stenosis (C). (D) The Lagrangian variation of wall shear (γ_w, s⁻¹) experienced by a platelet flowing along the stenosis centreline before and after the apex for 50% stenosis (dotted line), 70% stenosis (dashed line), and 90% stenosis (solid line). (E) Three-dimensional view of stenosis channel geometries showing the flow inlet and outlet.

Figure 2

Platelet thrombus formation in straight channels. Images of thrombus formation (demonstrated by contours of thrombus height) all collected under the same conditions from anticoagulated blood from 4 separate donors within collagen-coated channels with constant wall shear rates (γ_w) of 1800 s⁻¹ (A), 3000 s⁻¹ (B), and 6600 s⁻¹ (C) (donors 1–4 are shown from top to bottom panels at each shear rate). Measured thrombi total volume (D), surface coverage (E), and average thrombus volume/area (F) for 4 donors (open circles; red = donor 1, green = donor 2, blue = donor 3, black = donor 4) at 1800 s⁻¹, 3000 s⁻¹, and 6600 s⁻¹, where bars represent mean ± standard deviation. Data in panels (D–F) were evaluated using one-way ANOVA with Tukey’s correction for multiple comparisons. ns = no significance. Larger numbers of smaller thrombi formed at 1800 s⁻¹, while there were lower numbers of larger thrombi at 6600 s⁻¹, and at the intermediate wall shear rate of 3000 s⁻¹, thrombus formation was generally reduced suggesting distinct shear-dependent mechanisms regulating initial adhesion or growth of thrombi. Despite the differences in thrombus geometries, the total volumes of immobilised platelets at 1800 and 6600 s⁻¹ were comparable (A).

Figure 3

Platelet thrombus formation in stenosed channels. Images of thrombus formation (demonstrated by contours of thrombus height) all collected under the same conditions from anticoagulated blood from 4 separate donors within collagen-coated channels with 50% stenosis (A), 70% stenosis (B), and 90% stenosis (C) (donors 1–4 are shown from top to bottom panels at each shear rate, and images represent overlays of four consecutive stenoses for each donor). Calculated wall shear patterns are shown for 50% stenosis (D), 70% stenosis (E), and 90% stenosis (F), and thrombi average volume/area (G) are indicated for each donor (open circles; red = donor 1, green = donor 2, blue = donor 3, black = donor 4) at inlet regions, stenotic regions, and outlet regions for 50% stenosis, 70% stenosis, and 90% stenosis, where bars represent mean ± standard deviation. Data in panel (G) were evaluated using one-way ANOVA with Tukey’s correction for multiple comparisons. ns = no significance; * p ≤ 0.05. Pulsatile shear in stenotic channels compared with straight channels at varying shear (Figure 2) supports a specific role for shear gradients in initiating thrombus formation and regulating thrombus growth.