Vibro-Acoustic Platelet Activation: An Additive Mechanism of Prothrombosis with Applicability to Snoring and Obstructive Sleep Apnea.

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.

Shear-mediated platelet activation in the free flow II: Evolving mechanobiological mechanisms reveal an identifiable signature of activation and a bi-directional platelet dyscrasia with thrombotic and bleeding features.

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 αIIbß3 activation or P-selectin exposure due to platelet degranulation, as is commonly observed in biochemical activation. Rather, downregulation of GPIb, αIIbß3, 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 αIIbß3. 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.

Platelet Dysfunction During Mechanical Circulatory Support: Elevated Shear Stress Promotes Downregulation of αIIbß3 and GPIb via Microparticle Shedding Decreasing Platelet Aggregability.

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Patterned Electrospinning: A Method of Generating Defined Fibrous Constructs Influencing Cell Adhesion and Retention.

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.

Platelet Activation via Shear Stress Exposure Induces a Differing Pattern of Biomarkers of Activation versus Biochemical Agonists.

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 αIIbß3 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 αIIbß3 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 αIIbß3 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.

Acellular porcine heart matrices: whole organ decellularization with 3D-bioscaffold & vascular preservation.

Regenerative medicine, particularly decellularization-recellularization methods via whole-organ tissue engineering, has been increasingly studied due to the growing donor organ shortage. Though numerous decellularization protocols exist, the ideal decellularization protocol for optimal recellularization is unclear. This study was performed to optimize existing heart decellularization protocols and compare current methods using the detergents SDS (sodium dodecyl sulfate), Triton X-100, OGP (octyl ß-D-glucopyranoside), and CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) through retrograde aortic perfusion via aortic cannulation of a whole porcine heart. The goal of decellularization is to preserve extracellular matrix integrity and architecture, which was analyzed in this study through histology, microscopy, DNA analysis, hydroxyproline content analysis, materials analysis and angiography. Effective decellularization was determined by analyzing the tissue organization, geometry, and biological properties of the resultant extracellular matrix scaffold. Using these parameters, optimal decellularization was achieved between 90 and 120 mmHg pressure with 3% SDS as a detergent. Relevance for patients: This study provides important information about whole heart decellularization, which will ultimately contribute to heart bioengineering.