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

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.

Methods to Determine the Lagrangian Shear Experienced by Platelets during Thrombus Growth.

Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 µm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time.

Haemodynamic forces on in vitro thrombi: a numerical analysis.

The flow of blood past an axisymmetric thrombus analogue, within an in vitro geometry, is computed via solution of the discrete three-dimensional (3D) Navier-Stokes equations. Particle tracking is used to model the behaviour of thrombocytes (platelets) moving throughout the domain and to investigate behaviour with respect to the platelets. The system is explored using shear rate to quantify the effects an idealised thrombus has with respect to an undisturbed in vitro geometry over 'Poiseuille flow' shear rate conditions applicable to in vivo and in vitro experiments (1,200-10,000 s⁻¹). Local shear rate variations show peaks in shear rate greater than double that of Poiseuille flow conditions. These local shear rate variations are observed to be non-linear, despite the low Reynolds number (5.2-43.4) within the system. Topological transitions of shear rate are observed, limiting the height of peak shear rate within the system, suggesting a thrombus growth limiting behaviour. Temporal gradients of shear rate, measured with respect to individual platelets, were calculated. Multiple regions of peak shear rate gradient were observed throughout the flow, suggesting that platelet-platelet interaction may not be limited to regions near to the surface of the thrombus.

Effect of hemodynamic forces on platelet aggregation geometry.

The shear rate dependence of platelet aggregation geometries is investigated using a combination of in vitro and numerical experiments. Changes in upstream shear rate, γ(Pw), are found to cause systematic changes in mature platelet aggregation geometries. However, γ(Pw) is not the only factor determining the shear rate experienced by a platelet moving over, and adhering to, a platelet aggregation: flow simulations demonstrate that naturally occurring variations in platelet aggregation geometry cause the local shear rate on the surface of a mature platelet aggregation to vary between zero and up to eight times γ(Pw). Additionally, as a platelet aggregation grows, systematic changes in geometry are found, indicating that the local shear field over a growing platelet aggregation will differ from that over mature platelet aggregations.