Introduction: Obstructive sleep apnea (OSA) and loud snoring are conditions with increased cardiovascular risk and notably an association with stroke. Central in stroke are thrombosis and thromboembolism, all related to and initiaing with platelet activation. Platelet activation in OSA has been felt to be driven by biochemical and inflammatory means, including intermittent catecholamine exposure and transient hypoxia. We hypothesized that snore-associated acoustic vibration (SAAV) is an activator of platelets that synergizes with catecholamines and hypoxia to further amplify platelet activation. Methods: Gel-filtered human platelets were exposed to snoring utilizing a designed vibro-acoustic exposure device, varying the time and intensity of exposure and frequency content. Platelet activation was assessed via thrombin generation using the Platelet Activity State assay and scanning electron microscopy. Comparative activation induced by epinephrine and hypoxia were assessed individually as well as additively with SAAV, as well as the inhibitory effect of aspirin. Results: We demonstrate that snore-associated acoustic vibration is an independent activator of platelets, which is dependent upon the dose of exposure, i.e., intensity x time. In snoring, acoustic vibrations associated with low-frequency sound content (200 Hz) are more activating than those associated with high frequencies (900 Hz) (53.05% vs. 22.08%, p = 0.001). Furthermore, SAAV is additive to both catecholamines and hypoxia-mediated activation, inducing synergistic activation. Finally, aspirin, a known inhibitor of platelet activation, has no significant effect in limiting SAAV platelet activation. Conclusion: Snore-associated acoustic vibration is a mechanical means of platelet activation, which may drive prothrombosis and thrombotic risk clinically observed in loud snoring and OSA.

Background: Obstructive sleep apnea (OSA) is a common disease state with prevalence in the U.S. of near 25%. of the population, characterized by snoring and apnea. Beyond intermittent hypoxemia, OSA imparts increased risk of myocardial infarction (MI) and stroke (CVA), both driven by thrombosis and thromboembolism. Central in thrombosis is platelet activation, traditionally felt to driven by biochemical agonists. We explored the possibility that mechanical vibration generated by OSA snore is also a driver of platelet activation. We hypothesized that OSA snore frequency components induce vibration capable of platelet activation. Here we characterized 1. the frequency content of OSA snore and 2. the effect of specific frequencies ranges on platelet activation, and 3. the optoacoustic vibration pattern generated by snore frequency components. Methods: Snore from OSA patients was recorded in a sleep lab. A snore emulator device (speaker platform) was constructed allowing exposure of human gel filtered platelets to snore via placement on a stage transmitting sound and associated vibration, at clinical sound intensity. Snore was analyzed as to sound frequency content. Gel filtered platelet were exposed to snore sound bandwidths (100 – 1000 Hz.) for 40 min (identical intensities) with activation assessed by thrombin generation (PAS assay). Snore vibration was characterized via assessment of optoacoustic fluid wave patterns generated. Results: OSA snore vibration led to definitive platelet activation. Notably, low frequency snore bandwidths induced greater degrees of activation than high frequency exposure - 53.26% activation at 300 hz vs. 13.44% at 900 hz. (Fig. 1a). Low frequency bandwidths induced noticeably greater fluid oscillation than high frequency bandwidth exposure. (Fig. 1b). Conclusion: OSA snore is capable of inducing vibrations which impart adequate mechanical energy to fluid to induce platelet activation. Within snore, specific frequency bandwidths, i.e. low frequencies, appear to be more activating than others. Correlating with platelet activation is detectable visual fluid disturbance which may be characterized via opto-acoustic patterns. Modulation of sound and vibration associated with OSA snore may offer opportunity as a means of limiting thrombotic consequences and adverse events associated with OSA.

Background: Obstructive sleep apnea (OSA) is an independent risk factor for thrombotic stroke. OSA stroke is felt to be driven by hypoxia and post apneacatecholamine surge, leading to platelet activation and thrombosis. OSA is also associated with significant snoring and related neck vibration. In our work related to thrombogenicity of mechanical circulatory support (MCS) devices we demonstrated that device vibration and related shear are biomechanical activators of platelets. Recently we showed that snore-associated vibration is platelet activating. As OSA snore, vibration is imparted to major neck blood vessels, i.e. carotid and jugular, and activated platelets interact with vessel endothelial cells, the effect of vibration on endothelial thrombogenicity warrants further definition. Methods: OSA snore recordings were obtained from the human sleep lab. A speaker-platform device, validated to generate snore neck vibrations, was fabricated (snore emulator). Human umbilical vein endothelial cells (HUVECs) grown on petri dishes, placed on the emulator and exposed to snore vibrations for 4,8,12, 24,or 36 hours at 37oC., non-vibration cultures serving as controls. HUVEC viability (LDH assay) and platelet adhesion (inactivated platelets bound, measured via scanning EM) were serially assessed. Results: HUVEC exposed to snore vibration remained viable, though with a progressive decline in viability vs control, i.e. a 28.9% and 46.5% reduction at 24h and 36h, respectively (p<0.05). Interestingly, vibration led to a disproportionally greater increase in thrombogenicity, with net platelet adhesion increasing by 64% and 86% at 24h and 36h respectively (p<0.05) Conclusion: Vibration, as a general biomechanical stimulus, previously demonstrated to increase platelet activation, also leads to endothelial dysfunction, including loss of viability, with an even greater increase in endothelial thrombogenicity. Further definition of vibration-mediated endothelial dysfunction has implication for developing translational therapeutics for OSA, as well as providing insight for endothelial-contacting therapeutic device design.

Purpose: Blood-contacting implantable devices, e.g. ventricular assist devices, valves, stents, while effective therapeutically in augmenting or replacing lost native function, subject circulating blood cells, notably platelets, to unnatural excessive shear stress. This “hypershear” has been shown to lead to platelet activation and microparticle generation, paradoxically driving both thrombosis and bleeding. We previously demonstrated that the biomechanical parameter of cell stiffness, which varies with patient conditions, is a determinant of shear-mediated platelet activation (SMPA). Measuring platelet stiffness to date has been a cumbersome lab-based technique. Here we report on the development of a simple, portable chip system using dielectrophoresis (DEP) to measure stiffness. Methods: A microfluidic chip with contained electrodes was designed for exposure of platelets to oscillating electromagnetic force (EMF), inducing DEP, to deform (stretch/relax) platelets. An optical system allowed for process visualization, with images captured, analyzed, and stiffness (Young’s modulus) calculated via computational algorithm. A graphical user interface (GUI) allowed user control of the DEP protocol and data/results depiction. Human gelfiltered platelets from healthy volunteers were used in all studies. Results: The assembled chip system and controlling electronics were effective in generating EMF oscillations, adequate for platelet deformation. Optical image visualization, recording and analysis allowed calculation of individual platelet stiffness = 2.1 +/- 0.7 kdyn/cm2 (n=6) (Figure 1). Conclusion: A microfluidic construct effectively generates EMF which can induce DEP. This system can generate functional alterations in platelet dimensions, a factor of underlying stiffness, allowing quantitation of platelet stiffness. Further incorporation of this chip system into increasingly portable devices will advance this useful biomarker from the lab to the bedside. Figure 1. Young’s Modulus of platelet following exposure to differing electromagnetic force (Dielectrophoresis)

Shear-mediated platelet activation (SMPA) in the “free flow” is the net result of a range of cell mechanobiological mechanisms. Previously, we outlined three main groups of mechanisms including: 1) mechano-destruction - i.e. additive platelet (membrane) damage; 2) mechano-activation - i.e. activation of shear-sensitive ion channels and pores; and 3) mechano-transduction - i.e. “outside-in” signaling via a range of transducers. Here, we report on recent advances since our original report which describes additional features of SMPA. A clear “signature” of SMPA has been defined, allowing differentiation from biochemically-mediated activation. Notably, SMPA is characterized by mitochondrial dysfunction, platelet membrane eversion, externalization of anionic phospholipids, and increased thrombin generation on the platelet surface. However, SMPA does not lead to integrin αIIbβ3 activation or P-selectin exposure due to platelet degranulation, as is commonly observed in biochemical activation. Rather, downregulation of GPIb, αIIbβ3, and P-selectin surface expression is evident. Furthermore, SMPA is accompanied by a decrease in overall platelet size coupled with a concomitant, progressive increase in microparticle generation. Shear-ejected microparticles are highly enriched in GPIb and αIIbβ3. These observations indicate the enhanced diffusion, migration, or otherwise dispersion of platelet adhesion receptors to membrane zones, which are ultimately shed as receptor-rich PDMPs. The pathophysiological consequence of this progressive shear accumulation phenomenon is an associated dyscrasia of remaining platelets – being both reduced in size and less activatable via biochemical means – a tendency to favor bleeding, while concomitantly shed microparticles are highly prothrombotic and increase the tendency for thrombosis in both local and systemic milieu. These mechanisms and observations offer direct clinical utility in allowing measurement and guidance of the net balance of platelet driven events in patients with implanted cardiovascular therapeutic devices.

Supplemental Digital Content is available in the text. Objective: Mechanical circulatory support has emerged as lifesaving therapy for patients with advanced heart failure. However, mechanical circulatory support remains limited by a paradoxical coagulopathy accompanied by both thrombosis and bleeding. While mechanisms of mechanical circulatory support thrombosis are increasingly defined, mechanical circulatory support-related bleeding, as related to shear-mediated alteration of platelet function, remains poorly understood. We tested the hypothesis that platelet exposure to elevated shear stress, while a defined prothrombotic activator of platelets, coordinately induces downregulation of key platelet adhesion receptors GPIb-IX-V, αIIbβ3, and P-selectin, thus decreasing platelet functional responsiveness to physiological stimuli. Approach and Results: Human gel-filtered platelets were exposed to continuous or pulsatile shear stress in vitro. Surface expression of platelet receptors and platelet-derived microparticle generation were quantified by flow cytometry. Shedding of receptor soluble forms were assessed via ELISA, and platelet aggregation was measured by optical aggregometry. We demonstrate that platelet exposure to elevated shear stress led to a downregulation of GPIb and αIIbβ3 receptors on platelets with a progressive increase in the generation of platelet-derived microparticles expressing elevated levels of αIIbβ3 and GPIb on their surface. No shear-mediated shedding of GPIb and β3 subunit soluble fragments was detected. Soluble P-selectin was extensively shed from platelets, while surface expression of P-selectin on platelets was not significantly altered by shear. Shear-mediated downregulation of GPIb and αIIbβ3 on platelets was associated with an evident decrease of platelet aggregatory response induced by ADP and TRAP 6 (thrombin receptor activating peptide 6). Conclusions: Our data clearly indicate that accumulation of shear stress, consistent with supraphysiologic conditions characterizing device-supported circulation (1) induces adequate platelet degranulation, yet (2) causes downregulation of primary platelet adhesion receptors via ejection of receptor-enriched platelet-derived microparticles, thus mechanistically limiting platelet activation and the aggregatory response.

A critical component of tissue engineering is the ability to functionally replace native tissue stroma. Electrospinning is a technique capable of forming fibrous constructs with a high surface area for increased cell-material interaction and enhanced biocompatibility. However, physical and biological properties of electrospun scaffolds are limited by design controllability on a macroscale. We developed a methodology for generating electrospun scaffolds with defined patterns and topographic features to influence physical properties and biological interactions. Five unique design electrospinning target collectors were fabricated to allow for generation of defined polymeric scaffold patterns including lines, sinusoids, squares, zigzags, and solid. Poly(lactic-co-glycolic) acid was electrospun under identical conditions utilizing these varied targets, and constructs generated were examined as to their physical configuration, mechanical and chemical properties, and their ability to foster vascular smooth muscle cell adhesion and retention at 24 h. Modifying collector designs led to significant differences in fiber target coverage ranging from 300 mm2 for solid (100% of the target area) to 217.8 mm2 for lines (72.6% of the target area). Measured fiber excess, residual open area, and contact angle (hydrophobicity) followed the same trend as fiber target coverage with respect to the collector pattern: lines > sinusoids > squares > zigzags > solid. Similarly, the line design allowed for the greatest cell adhesion and retention (258 ± 31 cells), whereas solid exhibited the lowest (150 ± 15 cells); p < 0.05. There was a strong direct correlation of cell adhesion to construct residual open area (R2 = 0.94), normalized fiber excess (R2 = 0.99), and fiber grammage (R2 = 0.72), with an inverse relationship to fiber target coverage (R2 = 0.94). Our results demonstrate the ability to utilize patterned collectors for modifying macroscopic and microscopic electrospun scaffold features, which directly impact cell adhesion and retention, offering translational utility for designing specific tissue constructs.

Objective: Mechanical circulatory support has emerged as lifesaving therapy for patients with advanced heart failure. However, mechanical circulatory support remains limited by a paradoxical coagulopathy accompanied by both thrombosis and bleeding. While mechanisms of mechanical circulatory support thrombosis are increasingly defined, mechanical circulatory support-related bleeding, as related to shear-mediated alteration of platelet function, remains poorly understood. We tested the hypothesis that platelet exposure to elevated shear stress, while a defined prothrombotic activator of platelets, coordinately induces downregulation of key platelet adhesion receptors GPIb-IX-V, alpha(IIb)beta(3), and P-selectin, thus decreasing platelet functional responsiveness to physiological stimuli. Approach and Results: Human gel-filtered platelets were exposed to continuous or pulsatile shear stress in vitro. Surface expression of platelet receptors and platelet-derived microparticle generation were quantified by flow cytometry. Shedding of receptor soluble forms were assessed via ELISA, and platelet aggregation was measured by optical aggregometry. We demonstrate that platelet exposure to elevated shear stress led to a downregulation of GPIb and alpha(IIb)beta(3) receptors on platelets with a progressive increase in the generation of platelet-derived microparticles expressing elevated levels of alpha(IIb)beta(3) and GPIb on their surface. No shear-mediated shedding of GPIb and beta(3) subunit soluble fragments was detected. Soluble P-selectin was extensively shed from platelets, while surface expression of P-selectin on platelets was not significantly altered by shear. Shear-mediated downregulation of GPIb and alpha(IIb)beta(3) on platelets was associated with an evident decrease of platelet aggregatory response induced by ADP and TRAP 6 (thrombin receptor activating peptide 6). Conclusions: Our data clearly indicate that accumulation of shear stress, consistent with supraphysiologic conditions characterizing device-supported circulation (1) induces adequate platelet degranulation, yet (2) causes downregulation of primary platelet adhesion receptors via ejection of receptor-enriched platelet-derived microparticles, thus mechanistically limiting platelet activation and the aggregatory response.

Background Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. Methods Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin alpha IIb beta 3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. Results Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin alpha IIb beta 3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of alpha IIb beta 3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. Conclusion Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.

Introduction: Mechanical circulatory support (MCS) has emerged as a lifesaving therapy for patients with advanced and end-stage heart failure. Sadly, MCS therapy is limited by a paradoxical coagulo...