Josie Carberry

A shear gradient–dependent platelet aggregation mechanism drives thrombus formation

Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

A-Disintegrin-And-Metalloproteinase (ADAM) 10 Activity on Resting and Activated Platelets.

The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectodomain of human GPVI (sGPVI) is proteolytically shed from human platelets by a-disintegrin-and-metalloproteinase 10 (ADAM10). In this study, we used a novel ADAM10-sensitive fluorescence resonance energy transfer sensor to analyze ADAM10-mediated shedding of GPVI from human platelets in response to the exposure of GPVI ligands collagen-related peptide (10 μg/mL), collagen (10 μg/mL), and convulxin (0.1 μg/mL) to shear stress (1000-10000 s(-1), 5 min), or a generic activator of metalloproteinases, N-ethylmaleimide (NEM, 5 mM). Elevated shear, NEM, or ligand engagement of GPVI all induced shedding of GPVI, as detected by release of sGPVI; however, only shear or NEM significantly increased ADAM10 enzyme activity. ADAM10 activity was also detectable on the surface of thrombi formed on a collagen-coated surface under flow conditions. Our findings indicate different mechanisms regulate shear- and ligand-induced shedding and shear forces found within the vasculature can regulate ADAM10 activity.

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, γPw, are found to cause systematic changes in mature platelet aggregation geometries. However, γPw 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 γPw. 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.

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.

Functional Imaging to Understand Biomechanics: A Critical Tool for the Study of Biology, Pathology and the Development of Pharmacological Solutions

We present four case studies of the literature discussing the effects of physical forces on biological function. While the field of biomechanics has existed for many decades, it may be considered by some a poor cousin to biochemistry and other traditional fields of medical research. In these case studies, including cardiovascular and respiratory systems, we demonstrate that, in fact, many systems historically believed to be controlled by biochemistry are dominated by biomechanics. We discuss both the previous paradigms that have advanced research in these fields and the changing paradigms that will define the progressions of these fields for decades to come. In the case of biomechanical effects of flowing blood on the endothelium, this has been well understood for decades. In the cases of platelet activation and liquid clearance from the lungs during birth, these discoveries are far more recent and perhaps not as universally accepted. While only a few specific examples are examined here, it is clear that not enough attention is paid to the possible mechanical links to biological function. The continued development of these research areas, with the inclusion of physical effects, will hopefully provide new insight into disease development, progression, diagnosis and effective therapies.

Polynomial element velocimetry (PEV): a technique for continuous in-plane velocity and velocity gradient measurements for low Reynolds number flows

Particle image velocimetry (PIV) selects the maximum of the cross-correlation map to represent the modal displacement, and a wealth of information stored in the cross-correlation is discarded. We introduce a novel method, termed polynomial element velocimetry (PEV), which results in continuous velocity and velocity gradient measurements. PEV utilizes the extra information stored in the cross-correlation to determine continuous velocity measurements with low levels of measurement noise. In contrast to PIV, the continuous nature of velocity measurements facilitates the direct determination of the velocity gradient. The PEV method is applied to two laboratory flows: flow in a channel and flow behind a circular cylinder at Reynolds number, Re?= 30, and is shown to greatly reduce the noise in the measurements. In addition, the accuracy of PEV is validated using two computer-generated synthetic flows: parabolic flow in a channel and flow past a square cylinder at Re?= 30. In these cases, PEV is shown to reduce the velocity measurement error by up to 45% and the vorticity estimation error by up to 77% when compared to PIV. A key benefit of the PEV method is that it is capable of calculating continuous measures for flow gradient with greatly reduced bias errors. In particular, PEV provides a more accurate measurement of the vorticity near interfaces such as a cylinder wall or channel wall where PIV methods only provide measurement data at half the sampling window size from the wall. Since PEV utilizes the entire shape of the cross-correlation map to determine a local map for the underlying velocity, minimal random error is transmitted to the estimated flow gradient. This feature of the PEV method makes it optimal for flows where flow gradients are well defined and there are insufficient pixels to fully resolve structures in the flow using PIV.

Effect of Shear Rate on Thrombus Growth

In this study micro and nano scale measurement techniques are applied to platelet studies and determination of factors in platelet aggregation and thrombus formation. Conventionally it has been assumed that platelets are stimulated by blood clotting factors and platelet activators to aggregate and form a thrombus at sites of vascular injury. We have recently shown that a primary factor in initiating platelet aggregation is hemodynamic shear. This paper presents the effect of shear rate on the time evolution of thrombus formation and the final geometry of a mature thrombus. A relationship between maximum mature thrombus height and local shear rate is formulated. We have shown that the shear rate is not only an important factor in initiating platelet aggregation but is also one of the main inhibitors of platelet aggregation and thrombus formation. We propose that when the platelets reach a critical height, they encounter a specific local hemodynamic range, which prevents further thrombus growth.© 2010 ASME

A Novel Scanning Method to Measure Shear Rate Around a Thrombus Using Micro Particle Image Velocimetry

The ability of platelets to adhere and aggregate to form a stable thrombus plays a key role in the normal haemostatic process at sites of vascular injury. However, thrombus formation plays a sinister role in arterial diseases leading to heart attacks or strokes.© 2008 ASME