Identification of a rheology dependent platelet aggregation mechanism

There is a growing body of evidence suggesting that the dynamics of platelet aggregation are regulated by shear (rheology) dependent platelet aggregation mechanisms that operate alongside traditional soluble agonist dependent aggregation mechanisms. Rheology-dependent platelet aggregation requires the biomechanical adhesive and signalling function of GPIb and integrin αIIbβ3, with the contribution of each receptor to the initiation of aggregation dependent on local hemodynamic conditions. In contrast, soluble agonists amplify and potentiate platelet activation and play a major role in stabilising formed aggregates. Unravelling the dynamics of platelet aggregation and thrombus formation in vivo therefore requires consideration of the co-operative interplay between soluble agonist and rheology-dependent platelet aggregation mechanisms. This thesis focuses on the role of hemodynamics (rheology) in platelet activation and aggregation. Chapter 3 describes the characterisation of the dynamic behaviour of platelet aggregation in vivo. In Chapter 4 it is established that platelet aggregation is greatly enhanced by acute changes in blood flow conditions. A novel rheology dependent platelet aggregation mechanism is identified that operates in the presence of shear microgradients. In chapter 5 I further characterise rheology dependent platelet aggregation and establish the relative role of soluble agonists in this process. Chapter 6 describes a novel and highly localised platelet morphological shape change that forms the basis of a mechano-sensory mechanism that allows platelets to respond to local hemodynamic conditions. This mechanism, involving membrane tether restructuring, increases the ability of discoid platelets to aggregate within low or decelerating shear zones in the blood flow

Identification of a rheology dependent platelet aggregation mechanism

There is a growing body of evidence suggesting that the dynamics of platelet aggregation are regulated by shear (rheology) dependent platelet aggregation mechanisms that operate alongside traditional soluble agonist dependent aggregation mechanisms. Rheology-dependent platelet aggregation requires the biomechanical adhesive and signalling function of GPIb and integrin αIIbβ3, with the contribution of each receptor to the initiation of aggregation dependent on local hemodynamic conditions. In contrast, soluble agonists amplify and potentiate platelet activation and play a major role in stabilising formed aggregates. Unravelling the dynamics of platelet aggregation and thrombus formation in vivo therefore requires consideration of the co-operative interplay between soluble agonist and rheology-dependent platelet aggregation mechanisms. This thesis focuses on the role of hemodynamics (rheology) in platelet activation and aggregation. Chapter 3 describes the characterisation of the dynamic behaviour of platelet aggregation in vivo. In Chapter 4 it is established that platelet aggregation is greatly enhanced by acute changes in blood flow conditions. A novel rheology dependent platelet aggregation mechanism is identified that operates in the presence of shear microgradients. In chapter 5 I further characterise rheology dependent platelet aggregation and establish the relative role of soluble agonists in this process. Chapter 6 describes a novel and highly localised platelet morphological shape change that forms the basis of a mechano-sensory mechanism that allows platelets to respond to local hemodynamic conditions. This mechanism, involving membrane tether restructuring, increases the ability of discoid platelets to aggregate within low or decelerating shear zones in the blood flow

Monitoring in vitro thrombus formation with novel microfluidic devices

Cardiovascular disease is a major cause of mortality globally and is subject to ongoing research to improve clinical treatment. It is established that activation of platelets and coagulation are central to thrombosis, yet at different extents in the arterial and venous system. In vitro perfusion chamber technology has contributed significant knowledge on the function of platelets in the thrombotic process under shear conditions. Recent efforts to downscale this technique with a variety of microfluidic devices has opened new possibilities to study this process under precisely controlled flow conditions. Such microfluidic devices possess the capability to execute platelet function tests more quickly than current assays, while using small blood samples. Gradually becoming available to the clinic now, they may provide a new means to manage the treatment of cardiovascular diseases, although accurate validation studies still are missing. This review highlights the progress that has been made in monitoring aspects of thrombus formation using microfluidic devices

Monitoring in vitro thrombus formation with novel microfluidic devices

Cardiovascular disease is a major cause of mortality globally and is subject to ongoing research to improve clinical treatment. It is established that activation of platelets and coagulation are central to thrombosis, yet at different extents in the arterial and venous system. In vitro perfusion chamber technology has contributed significant knowledge on the function of platelets in the thrombotic process under shear conditions. Recent efforts to downscale this technique with a variety of microfluidic devices has opened new possibilities to study this process under precisely controlled flow conditions. Such microfluidic devices possess the capability to execute platelet function tests more quickly than current assays, while using small blood samples. Gradually becoming available to the clinic now, they may provide a new means to manage the treatment of cardiovascular diseases, although accurate validation studies still are missing. This review highlights the progress that has been made in monitoring aspects of thrombus formation using microfluidic devices

A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood

This paper reports the development of a platform technology for measuring platelet function and aggregation based on localized strain rate micro-gradients. Recent experimental findings within our laboratories have identified a key role for strain rate micro-gradients in focally triggering initial recruitment and subsequent aggregation of discoid platelets at sites of blood vessel injury. We present the design justification, hydrodynamic characterization and experimental validation of a microfluidic device incorporating contraction&ndash;expansion geometries that generate strain rate conditions mimicking the effects of pathological changes in blood vessel geometry. Blood perfusion through this device supports our published findings of both in vivo and in vitro platelet aggregation and confirms a critical requirement for the coupling of blood flow acceleration to downstream deceleration for the initiation and stabilization of platelet aggregation, in the absence of soluble platelet agonists. The microfluidics platform presented will facilitate the detailed analysis of the effects of hemodynamic parameters on the rate and extent of platelet aggregation and will be a useful tool to elucidate the hemodynamic and platelet mechano-transduction mechanisms, underlying this shear-dependent process.<br /

Structural and hydrodynamic simulation of an acute stenosis-dependent thrombosis model in mice

Platelet activation under blood flow is thought to be critically dependent on the autologous secretion of soluble platelet agonists (chemical activators) such as ADP and thromboxane. However, recent evidence challenging this model suggests that platelet activation can occur independent of soluble agonist signalling, in response to the mechanical effects of micro-scale shear gradients. A key experimental tool utilized to define the effect of shear gradients on platelet aggregation is the murine intravital microscopy model of platelet thrombosis under conditions of acute controlled arteriolar stenosis. This paper presents a computational structural and hydrodynamic simulation of acute stenotic blood flow in the small bowel mesenteric vessels of mice. Using a homogeneous fluid at low Reynolds number (0.45) we investigated the relationship between the local hydrodynamic strain-rates and the severity of arteriolar stensosis. We conclude that the critical rates of blood flow acceleration and deceleration at sites of artificially induced stenosis (vessel side-wall compression or ligation) are a function of tissue elasticity. By implementing a structural simulation of arteriolar side wall compression, we present a mechanistic model that provides accurate simulations of stenosis in vivo and allows for predictions of the effects on local haemodynamics in the murine small bowel mesenteric thrombosis model.

LDL-receptor-related protein regulates beta2-integrin-mediated leukocyte adhesion

Beta2-integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. In the search for molecular mechanisms leading to this clustering, we have identified low-density lipoprotein (LDL) receptor-related protein (LRP) as a new partner for beta2-integrins at the leukocyte surface. Immobilized recombinant LRP fragments served as an adhesive surface for blood-derived leukocytes and the U937 cell line. This adhesion was decreased up to 95% in the presence of antibodies against beta2-integrins, pointing to these integrins as potential partners for LRP. Using purified proteins, LRP indeed associated with the alphaMbeta2 complex and the alphaM and alphaL I-domains (K(d, app) approximately 0.5 microM). Immunoprecipitation experiments and confocal microscopy revealed that endogenously expressed LRP and alphaLbeta2 colocalized in monocytes and U937 cells. Furthermore, activation of U937 cells resulted in clustering of alphaLbeta2 and LRP to similar regions at the cell surface, indicating potential cooperation between both proteins. This was confirmed by the lack of alphaLbeta2 clustering in U937 cells treated by antisense oligonucleotides to down-regulate LRP. In addition, the absence of LRP resulted in complete abrogation of beta2-integrin-dependent adhesion to endothelial cells in a perfusion system, demonstrating the presence of a previously unrecognized link between LRP and leukocyte functio

Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner

Rupture of a vulnerable atherosclerotic plaque causes thrombus formation and precipitates cardiovascular diseases. In addition to the thrombogenic content of a plaque, also the hemodynamic microenvironment plays a major role in thrombus formation. How the altered hemodynamics around a plaque promote pathological thrombus formation is not well understood. In this study, we provide evidence that plaque geometries result in fluid mechanical conditions that promote platelet aggregation and thrombus formation by increased accumulation and activity of von Willebrand factor (vWF) at poststenotic sites. Resonant-scanning multiphoton microscopy revealed that in vivo arterial stenosis of a damaged carotid artery markedly increased platelet aggregate formation in the stenotic outlet region. Complementary in vitro studies using microfluidic stenotic chambers, designed to mimic the flow conditions in a stenotic artery, showed enhanced platelet aggregation in the stenotic outlet region at 60-80% channel occlusion over a range of input wall shear rates. The poststenotic thrombus formation was critically dependent on bloodborne vWF and autocrine platelet stimulation. In stenotic chambers containing endothelial cells, flow provoked increased endothelial vWF secretion in the stenotic outlet region, contributing to exacerbated platelet aggregation. Taken together, this study identifies a role for the shear-sensitive protein vWF in transducing hemodynamic forces that are present around a stenosis to a prothrombogenic microenvironment resulting in spatially confined and exacerbated platelet aggregation in the stenosis outlet region. The developed stenotic microfluidic chamber offers a realistic platform for in vitro evaluation of shear-dependent thrombus formation in the setting of atherosclerosis