Intercellular calcium communication regulates platelet aggregation and thrombus growth

The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet–platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin αIIbβ3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth

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.

Generation of asymmetric (85/15) blood streams at micro-scale stenosis to investigate the role of early aggregate development.

<p><i>Top stream (85): labelled whole blood. Bottom stream (15): Autologus platelet-poor-plasma (PPP). a) Representative DIC images of blood perfusion experiments over 10 minutes of monitoring in a device that produces two non-symmetrically focused streams (</i><i> and </i><i>). It can be observed that contrary to previous experiments, no aggregate was formed downstream of the contraction. b) Representative (n = 3) epi-fluorescence images of the same experiment as (a)). No aggregate was formed downstream of the contraction. This data demonstrates that initial aggregate formation was driven by platelets skimming within </i><i> of the stenosis apex.</i></p

A microfluidics flow focusing device was used to investigate the role of mass transport in micro stenosis.

<p><i>The device was designed to operate with negative pressure (on-chip sampling), using a single syringe pump. a) Schematic of the variables used to determine the thickness of the blood streams. Different widths of the focused stream () was achieved by changing the hydraulic resistance of the inlet feeder channels. See (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074123#pone.0074123.s001" target="_blank">Text S1</a>). b) A 3D representation of a device fabricated in PDMS-glass (cover-slip of </i><i>). c) Detail of the sampling section. Pc, Pf are the reservoirs from the focused and core stream, Lc and Lf the variables to modulate the thickness of the stream. See (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074123#pone.0074123.s001" target="_blank">Text S1</a>). d) Detail of the microcontraction (stenosis). e) Assembly of the device, showing that Bovine Serum Albumin was used to prevent unspecific adhesion of platelets to the glass surface. f) Schematic of the implemented valve to assist the purging of the device.</i></p

Generation of symmetric (50/50) blood streams at micro-scale stenosis.

<p><i>Region 50/50, n = 3. a) DIC images of blood perfusion experiments over 10 minutes of monitoring in a device that produces two symmetrically focused streams (</i><i> and </i><i>), where the fluorescent stream is at the bottom. It can be observed that an aggregate formed downstream of the contraction using two streams. b) Representative (n = 3) epi-fluorescence image of the same experiment as (a)), a strong fluorescent platelet incorporation into the developing aggregate was observed which corresponds to the DIC visible aggregate. c) Same experiment as a) but the fluorescent stream was located at the bottom. d) Representative fluorescence image of the same experiment as (c)), showing that no fluorescently label aggregation occurred downstream of the contraction using two stream. (White bar: Scale bar </i><i>.)</i></p

Fluidic performance using two streams of fluorescent and non-fluorescent blood, compared with CFD simulations using two species, homogeneous and Newtonian fluid, a.1) Two symmetric streams are generated of .

<p><i>Re = 0.78 and Pe = 770 a.2) Two streams are generated of </i><i> and </i><i>, at the same Re and Pe number. a.3) Two streams of </i><i> and </i><i> at the same Re and Pe. b)Concentration profile across the channel from CFD and experiments with blood at </i><i> upstream the contraction (xx). c)Concentration profile across the channel from CFD and experiments with blood at the contraction (yy).</i></p