An extensional strain sensing mechanosome drives adhesion-independent platelet activation at supraphysiological hemodynamic gradients.

BACKGROUND: Supraphysiological hemodynamics are a recognized driver of platelet activation and thrombosis at high-grade stenosis and in blood contacting circulatory support devices. However, whether platelets mechano-sense hemodynamic parameters directly in free flow (in the absence of adhesion receptor engagement), the specific hemodynamic parameters at play, the precise timing of activation, and the signaling mechanism(s) involved remain poorly elucidated. RESULTS: Using a generalized Newtonian computational model in combination with microfluidic models of flow acceleration and quasi-homogenous extensional strain, we demonstrate that platelets directly mechano-sense acute changes in free-flow extensional strain independent of shear strain, platelet amplification loops, von Willebrand factor, and canonical adhesion receptor engagement. We define an extensional strain sensing "mechanosome" in platelets involving cooperative Ca2+ signaling driven by the mechanosensitive channel Piezo1 (as the primary strain sensor) and the fast ATP gated channel P2X1 (as the secondary signal amplifier). We demonstrate that type II PI3 kinase C2α activity (acting as a "clutch") couples extensional strain to the mechanosome. CONCLUSIONS: Our findings suggest that platelets are adapted to rapidly respond to supraphysiological extensional strain dynamics, rather than the peak magnitude of imposed wall shear stress. In the context of overall platelet activation and thrombosis, we posit that "extensional strain sensing" acts as a priming mechanism in response to threshold levels of extensional strain allowing platelets to form downstream adhesive interactions more rapidly under the limiting effects of supraphysiological hemodynamics.

A Review of Design Considerations for Hemocompatibility within Microfluidic Systems.

The manipulation of blood within in vitro environments presents a persistent challenge, due to the highly reactive nature of blood, and its multifaceted response to material contact, changes in environmental conditions, and stimulation during handling. Microfluidic Lab-on-Chip systems offer the promise of robust point-of-care diagnostic tools and sophisticated research platforms. The capacity for precise control of environmental and experimental conditions afforded by microfluidic technologies presents unique opportunities that are particularly relevant to research and clinical applications requiring the controlled manipulation of blood. A critical bottleneck impeding the translation of existing Lab-on-Chip technology from laboratory bench to the clinic is the ability to reliably handle relatively small blood samples without negatively impacting blood composition or function. This review explores design considerations critical to the development of microfluidic systems intended for use with whole blood from an engineering perspective. Material hemocompatibility is briefly explored, encompassing common microfluidic device materials, as well as surface modification strategies intended to improve hemocompatibility. Operational hemocompatibility, including shear-induced effects, temperature dependence, and gas interactions are explored, microfluidic sample preparation methodologies are introduced, as well as current techniques for on-chip manipulation of the whole blood. Finally, methods of assessing hemocompatibility are briefly introduced, with an emphasis on primary hemostasis and platelet function.

Active Micropump-Mixer for Rapid Antiplatelet Drug Screening in Whole Blood.

There is a need for scalable automated lab-on-chip systems incorporating precise hemodynamic control that can be applied to high-content screening of new more efficacious antiplatelet therapies. This paper reports on the development and characterization of a novel active micropump-mixer microfluidic to address this need. Using a novel reciprocating elastomeric micropump design, we take advantage of the flexible structural and actuation properties of this framework to manage the hemodynamics for on-chip platelet thrombosis assay on type 1 fibrillar collagen, using whole blood. By characterizing and harnessing the complex three-dimensional hemodynamics of the micropump operation in conjunction with a microvalve controlled reagent injection system we demonstrate that this prototype can act as a real-time assay of antiplatelet drug pharmacokinetics. In a proof-of-concept preclinical application, we utilize this system to investigate the way in which rapid dosing of human whole blood with isoform selective inhibitors of phosphatidylinositol 3-kinase dose dependently modulate platelet thrombus dynamics. This modular system exhibits utility as an automated multiplexable assay system with applications to high-content chemical library screening of new antiplatelet therapies.

Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin αIIbß3 in mice.

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.

Characterisation of hydrodynamic trapping in microfluidic cross-slot devices for high strain rate applications.

Hydrodynamic trapping of a particle or cluster of particles based on contact and non-contact approaches has brought prominent insights to micro-nano scale applications. Of the non-contact methods, image-based real-time control in cross-slot microfluidic devices is one of the most promising potential platform for single cellular assays. Here, we report results from experiments conducted in two cross-slot microfluidic channels of different widths, with varying real-time delay of the control algorithm and different magnification. Sustained trapping of 5 µm diameter particles was achieved with high strain rates, of order 102 s-1, higher than in any previous studies. Our experiments show that the maximum attainable strain rate is a function of the real-time delay of the control algorithm and the particle resolution (pixel/µm). Therefore, we anticipate that with further reduced time delays and enhanced particle resolution, considerably higher strain rates can be attained, opening the platform to single cellular assay studies where very high strain rates are required.

A microfluidic method to investigate platelet mechanotransduction under extensional strain.

Background: Blood platelets have evolved a complex mechanotransduction machinery to rapidly respond to hemodynamic conditions. A variety of microfluidic flow-based approaches have been developed to explore platelet mechanotransduction; however, these experimental models primarily focus on the effects of increased wall shear stress on platelet adhesion events and do not consider the critical effects of extensional strain on platelet activation in free flow. Objectives: We report the development and application of a hyperbolic microfluidic assay that allows for investigation of platelet mechanotransduction under quasi-homogenous extensional strain rates in the absence of surface adhesions. Methods: Using a combined computational fluid dynamic and experimental microfluidic approach, we explore 5 extensional strain regimes (geometries) and their effect on platelet calcium signal transduction. Results: We demonstrate that in the absence of canonical adhesion, receptor engagement platelets are highly sensitive to both initial increase and subsequent decrease in extensional strain rates within the range of 747 to 3319/s. Furthermore, we demonstrate that platelets rapidly respond to the rate of change in extensional strain and define a threshold of ≥7.33 × 106/s/m, with an optimal range of 9.21 × 107 to 1.32 × 108/s/m. In addition, we demonstrate a key role of both the actin-based cytoskeleton and annular microtubules in the modulation of extensional strain-mediated platelet mechanotransduction. Conclusion: This method opens a window onto a novel platelet signal transduction mechanism and may have potential diagnostic utility in the identification of patients who are prone to thromboembolic complications associated with high-grade arterial stenosis or are on mechanical circulatory support systems, for which the extensional strain rate is a predominant hemodynamic driver.

PI 3-kinase p110beta: a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.

Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces.

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.

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-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.

Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth.

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.

In vitro flow based systems to study platelet function and thrombus formation: Recommendations for standardization: Communication from the SSC on Biorheology of the ISTH.

Experimental videomicroscopic in vitro assays of thrombus formation based on blood perfusion are instrumental in a wide range of basic studies in thrombosis, screening for hereditary or acquired plateletrelated pathologies, and assessing the effectiveness of novel anti-platelet therapies. Here, we discuss application of the broadly used "in vitro thrombosis model": a frequently used assay to study the formation of 3D aggregates under flow, which involves perfusing anticoagulated whole blood over fibrillar collagen in a flow geometry of rectangular cross-section, such as glass microcapillaries or parallel-plate flow chambers. Major advantaged of this assay are simplicity and ability to reproduce the four main stages of platelet thrombus formation, i.e. platelet tethering, adhesion, activation and aggregation under a wide range of hemodynamic conditions. On the other hand, these devices represent, at best, simple reductive models of thrombosis. We also describe how blood flow assays can be used to study various aspects of platelet function on adhesive proteins and discuss the relevance of such flow models. Finally, we propose recommendations for standardization related to the use of this assay that cover collagen source, coating methods, micropatterning, sample composition, anticoagulation, choice of flow device, hemodynamic conditions, quantification challenges, variability, pre-analytical conditions and other issues.

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.

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 alpha IIb beta 3 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.

The PI 3-kinase PI3KC2α regulates mouse platelet membrane structure and function independently of membrane lipid composition.

PI3KC2α is a phosphoinositide 3-kinase with a recently reported function in platelets; PI3KC2α-deficient mouse platelets have altered membrane structure and impaired function. Yet, how these membrane changes cause platelet dysfunction remains unknown. Here, focused ion beam-scanning electron microscopy of PI3KC2α-deficient platelet ultrastructure reveals a specific effect on the internal membrane structure, while liquid chromatography-tandem mass spectrometry profiling of 294 lipid species shows unaltered lipid composition. Functionally, PI3KC2α-deficient platelets exhibit impaired thrombosis specifically under conditions involving membrane tethering. These studies indicate that the structural changes in PI3KC2α-deficient platelets are limited to the membrane, occur without major changes in lipid composition, and selectively impair cell function during thrombus formation. These findings illustrate a unique mechanism that may be targetable for anti-thrombotic benefit.

The mode of anesthesia influences outcome in mouse models of arterial thrombosis.

BACKGROUND: Arterial thrombosis models are important for preclinical evaluation of antithrombotics but how anesthetic protocol can influence experimental results is not studied. OBJECTIVES: We studied how three most commonly used rodent anesthetics affect the induction of thrombosis and thrombus resolution with antiplatelet agent integrilin (Eptifibatide). METHODS: The Folts, electrolytic, and FeCl3 models of carotid artery thrombosis were evaluated. The extent of blood flow reduction required to elicit cyclic flow reductions (CFR) was examined in the Folts model. The occlusion time and stability following electrolytic or FeCl3 injury was assessed. The efficacy of Eptifibatide was studied in each cohort and clot composition following FeCl3 application was assessed histologically. RESULTS: Isoflurane and ketamine-xylazine (ket-x) elicited higher basal blood flow velocities. For reliable CFR in the Folts model, a higher degree of blood flow reduction was required under ket-x and isoflurane. For the FeCl3 and electrolytic models, injury severity had to be increased in mice under ket-x anesthesia to achieve rapid occlusion. FeCl3-injured artery sections from ket-x and isoflurane-treated mice showed vessel dilatation and clots that were more fibrin/red-cell rich compared to pentobarbitone. Integrilin led to cycle abolishment for all three Folts-injury cohorts but for the electrolytic model a 2.5-fold higher dose was required to restore blood flow under pentobarbitone. Integrilin after FeCl3 arterial injury was partially ineffective in isoflurane-treated mice. CONCLUSIONS: Anesthesia impacts rodent carotid artery occlusion experiments and alters integrilin efficacy. It is important to consider anesthetic protocols in animal experiments involving pharmacological agents for treatment of atherothrombosis.

Elastomeric microvalve geometry affects haemocompatibility.

This paper reports on the parameters that determine the haemocompatibility of elastomeric microvalves for blood handling in microfluidic systems. Using a comprehensive investigation of blood function, we describe a hierarchy of haemocompatibility as a function of microvalve geometry and identify a "normally-closed" v-gate pneumatic microvalve design that minimally affects blood plasma fibrinogen and von Willebrand factor composition, minimises effects on erythrocyte structure and function, and limits effects on platelet activation and aggregation, while facilitating rapid switching control for blood sample delivery. We propose that the haemodynamic profile of valve gate geometries is a significant determinant of platelet-dependent biofouling and haemocompatibility. Overall our findings suggest that modification of microvalve gate geometry and consequently haemodynamic profile can improve haemocompatibility, while minimising the requirement for chemical or protein modification of microfluidic surfaces. This biological insight and approach may be harnessed to inform future haemocompatible microfluidic valve and component design, and is an advance towards lab-on-chip automation for blood based diagnostic systems.

The impact of blood rheology on the molecular and cellular events underlying arterial thrombosis.

There is an increasing appreciation of the importance of disturbed blood flow, especially turbulent flow, in the pathogenesis of vascular disease. However, the precise mechanism(s) by which rheological changes accelerate the atherothrombotic process remains incompletely understood. Atherosclerotic lesions typically develop in vascular regions exhibiting bifurcated or curved architectures. Such regions exhibit complex blood flow profiles with considerable divergence from uniform laminar flow. These altered flow behaviours can promote deposition of pro-atherogenic lipids and proteins to the vessel wall and modulate the adhesive function of endothelial, platelets and leukocytes. Once developed, atherosclerotic lesions can further exacerbate flow disturbances, establishing a potential hazardous cycle of accelerated atherogenesis. At the cellular level, alterations in fluid flow can lead to significant changes in signal transduction, leading to a variety of functional and morphological changes. In particular, disturbed rheology has a significant impact on the adhesion and activation mechanisms utilised by platelets and leukocytes with high shear, playing an important role in accelerating platelet activation and thrombus growth. This review focuses on the impact of blood rheology on the cellular and molecular events underlying thrombosis, with particular emphasis on the role of platelets in this process.

Application of a strain rate gradient microfluidic device to von Willebrand's disease screening.

Von Willebrand's disease (VWD) is the most common inherited bleeding disorder caused by either quantitative or qualitative defects of von Willebrand factor (VWF). Current tests for VWD require relatively large blood volumes, have low throughput, are time-consuming, and do not incorporate the physiologically relevant effects of haemodynamic forces. We developed a microfluidic device incorporating micro-contractions that harnesses well-defined haemodynamic strain gradients to initiate platelet aggregation in citrated whole blood. The microchannel architecture has been specifically designed to allow for continuous real-time imaging of platelet aggregation dynamics. Subjects aged ≥18 years with previously diagnosed VWD or who presented for evaluation of a bleeding disorder, where the possible diagnosis included VWD, were tested. Samples were obtained for device characterization as well as for pathology-based testing. Platelet aggregation in the microfluidic device is independent of platelet amplification loops but dependent on low-level platelet activation, GPIb/IX/V and integrin αIIbß3 engagement. Microfluidic output directly correlates with VWF antigen levels and is able to sensitively detect aggregation defects associated with VWD subtypes. Testing demonstrated a strong correlation with standard clinical laboratory-based tests. Head-to-head comparison with PFA100® demonstrated equivalent, if not improved, sensitivity for screening aggregation defects associated with VWD. This strain rate gradient microfluidic prototype has the potential to be a clinically useful, rapid and high throughput-screening tool for VWD as well as other strain-dependent platelet disorders. In addition, the microfluidic device represents a novel approach to examine the effects of high magnitude/short duration (ms) strain rate gradients on platelet function.

Nonlinear Dynamic Modelling of Platelet Aggregation via Microfluidic Devices.

The recent application of new microfluidic technologies and methods has facilitated significant progress in the understanding of the fundamental mechanisms governing blood platelet function and how these parameters affect pathological thrombus formation. In-line with these new bioengineering approaches, the application of nonlinear dynamic systems analysis holds particular potential to extend our understanding of the complex interplay between mechanical and biochemical factors that underlie this complex biological phenomenon. In this paper we propose a simple mathematical model of the main dynamics of platelet aggregation/disaggregation observed experimentally in a novel microfluidic device that approximates a severe arterial stenosis. We apply dynamic systems theory (system identification) to explore the dynamics of the biomechanical platelet aggregation response to a range of shear stress rates, inhibiting blood-born chemical pathways of platelet activation (ADP, TXA2, and thrombin). We demonstrate that the proposed model is able to replicate experimental results with low variation, and suggest that the reduced set of model parameters has the potential to be used as a simplified way to evaluate the biomechanical dynamics of platelet aggregation. The proposed model has application to the development of automatic controllers within the context of microfluidic systems that may show great utility in the clinical assessment of platelet hyperfunction.

A multimode-TIRFM and microfluidic technique to examine platelet adhesion dynamics.

Fluorescence microscopy techniques have provided important insights into the structural and signalling events occurring during platelet adhesion under both static and blood flow conditions. However, due to limitations in sectioning ability and sensitivity these techniques are restricted in their capacity to precisely image the adhesion footprint of spreading platelets. In particular, investigation of platelet adhesion under hemodynamic shear stress requires an imaging platform with high spatial discrimination and sensitivity and rapid temporal resolution. This chapter describes in detail a multimode imaging approach combining total internal reflection fluorescence microscopy (TIRFM) with high speed epifluorescence and differential interference contrast (DIC) microscopy along with a novel microfluidic perfusion system developed in our laboratory to examine platelet membrane adhesion dynamics under static and flow conditions.