Platelet Biorheology and Mechanobiology in Thrombosis and Hemostasis: Perspectives from Multiscale Computation.

Thrombosis is the pathological clot formation under abnormal hemodynamic conditions, which can result in vascular obstruction, causing ischemic strokes and myocardial infarction. Thrombus growth under moderate to low shear (<1000 s-1) relies on platelet activation and coagulation. Thrombosis at elevated high shear rates (>10,000 s-1) is predominantly driven by unactivated platelet binding and aggregating mediated by von Willebrand factor (VWF), while platelet activation and coagulation are secondary in supporting and reinforcing the thrombus. Given the molecular and cellular level information it can access, multiscale computational modeling informed by biology can provide new pathophysiological mechanisms that are otherwise not accessible experimentally, holding promise for novel first-principle-based therapeutics. In this review, we summarize the key aspects of platelet biorheology and mechanobiology, focusing on the molecular and cellular scale events and how they build up to thrombosis through platelet adhesion and aggregation in the presence or absence of platelet activation. In particular, we highlight recent advancements in multiscale modeling of platelet biorheology and mechanobiology and how they can lead to the better prediction and quantification of thrombus formation, exemplifying the exciting paradigm of digital medicine.

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 Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function.

Implantable Cardiovascular Therapeutic Devices (CTD), while lifesaving, impart supraphysiologic shear stress to platelets, resulting in thrombotic and bleeding coagulopathy. We previously demonstrated that shear-mediated platelet dysfunction is associated with downregulation of platelet GPIb-IX-V and αIIbß3 receptors via generation of Platelet-Derived MicroParticles (PDMPs). Here, we test the hypothesis that sheared PDMPs manifest phenotypical heterogeneity of morphology and receptor surface expression and modulate platelet hemostatic function. Human gel-filtered platelets were exposed to continuous shear stress. Alterations of platelet morphology were visualized using transmission electron microscopy. Surface expression of platelet receptors and PDMP generation were quantified by flow cytometry. Thrombin generation was quantified spectrophotometrically, and platelet aggregation was measured by optical aggregometry. Shear stress promotes notable alterations in platelet morphology and ejection of distinctive types of PDMPs. Shear-mediated microvesiculation is associated with the remodeling of platelet receptors, with PDMPs expressing significantly higher levels of adhesion receptors (αIIbß3, GPIX, PECAM-1, P-selectin, and PSGL-1) and agonist receptors (P2Y12 and PAR1). Sheared PDMPs promote thrombin generation and inhibit platelet aggregation induced by collagen and ADP. Sheared PDMPs demonstrate phenotypic heterogeneity as to morphology and defined patterns of surface receptors and impose a bidirectional effect on platelet hemostatic function. PDMP heterogeneity suggests that a range of mechanisms are operative in the microvesiculation process, contributing to CTD coagulopathy and posing opportunities for therapeutic manipulation.

A Multiscale Model for Shear-Mediated Platelet Adhesion Dynamics: Correlating In Silico with In Vitro Results.

Platelet adhesion to blood vessel walls is a key initial event in thrombus formation in both vascular disease processes and prosthetic cardiovascular devices. We extended a deformable multiscale model (MSM) of flowing platelets, incorporating Dissipative Particle Dynamics (DPD) and Coarse-Grained Molecular Dynamics (CGMD) describing molecular-scale intraplatelet constituents and their interaction with surrounding flow, to predict platelet adhesion dynamics under physiological flow shear stresses. Binding of platelet glycoprotein receptor Ibα (GPIbα) to von Willebrand factor (vWF) on the blood vessel wall was modeled by a molecular-level hybrid force field and validated with in vitro microchannel experiments of flowing platelets at 30 dyne/cm2. High frame rate videos of flipping platelets were analyzed with a Semi-Unsupervised Learning System (SULS) machine learning-guided imaging approach to segment platelet geometries and quantify adhesion dynamics parameters. In silico flipping dynamics followed in vitro measurements at 15 and 45 dyne/cm2 with high fidelity, predicting GPIbα-vWF bonding and debonding processes, distribution of bonds strength, and providing a biomechanical insight into initiation of the complex platelet adhesion process. The adhesion model and simulation framework can be further integrated with our established MSMs of platelet activation and aggregation to simulate initial mural thrombus formation on blood vessel walls.

Shear-Mediated Platelet Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function.

Objective: Implantable cardiovascular therapeutic devices (CTD) including stents, percutaneous heart valves and ventricular assist devices, while lifesaving, impart supraphysiologic shear stress to platelets resulting in thrombotic and bleeding device-related coagulopathy. We previously demonstrated that shear-mediated platelet dysfunction is associated with downregulation of platelet GPIb-IX-V and αIIbß3 receptors via generation of platelet-derived microparticles (PDMPs). Here, we test the hypothesis that shear-generated PDMPs manifest phenotypical heterogeneity of their morphology and surface expression of platelet receptors, and modulate platelet hemostatic function. Approach and Results: Human gel-filtered platelets were exposed to continuous shear stress and sonication. Alterations of platelet morphology were visualized using transmission electron microscopy. Surface expression of platelet receptors and PDMP generation were quantified by flow cytometry. Thrombin generation was quantified spectrophotometrically, and platelet aggregation in plasma was measured by optical aggregometry. We demonstrate that platelet exposure to shear stress promotes notable alterations in platelet morphology and ejection of several distinctive types of PDMPs. Shear-mediated microvesiculation is associated with the differential remodeling of platelet receptors with PDMPs expressing significantly higher levels of both adhesion (α IIb ß 3 , GPIX, PECAM-1, P-selectin, and PSGL-1) and agonist-evoked receptors (P 2 Y 12 & PAR1). Shear-mediated PDMPs have a bidirectional effect on platelet hemostatic function, promoting thrombin generation and inhibiting platelet aggregation induced by collagen and ADP. Conclusions: Shear-generated PDMPs demonstrate phenotypic heterogeneity as to morphologic features and defined patterns of surface receptor alteration, and impose a bidirectional effect on platelet hemostatic function. PDMP heterogeneity suggests that a range of mechanisms are operative in the microvesiculation process, contributing to CTD coagulopathy and posing opportunities for therapeutic manipulation.

Age-restricted functional and developmental differences of neonatal platelets.

BACKGROUND: Developmental ontogeny of neonatal thrombopoiesis retains characteristics that are distinct from adults although molecular mechanisms remain unestablished. METHODS: We applied multiparameter quantitative platelet responses with integrated ribosome profiling/transcriptomic studies to better define gene/pathway perturbations regulating the neonatal-to-adult transition. A bioinformatics pipeline was developed to identify stable, neonatal-restricted platelet biomarkers for clinical application. RESULTS: Cord blood (CB) platelets retained the capacity for linear agonist-receptor coupling linked to phosphatidylserine (PS) exposure and α-granule release, although a restricted block in cross-agonist activation pathways was evident. Functional immaturity of synergistic signaling pathways was due to younger ontogenetic age and singular underdevelopment of the protein secretory gene network, with reciprocal expansion of developmental pathways (E2F, G2M checkpoint, c-Myc) important for megakaryocytopoiesis. Genetic perturbations regulating vesicle transport and fusion (TOM1L1, VAMP3, SNAP23, and DNM1L) and PS exposure and procoagulant activity (CLCN3) were the most significant, providing a molecular explanation for globally attenuated responses. Integrated transcriptomic and ribosomal footprints identified highly abundant (ribosome-protected) DEFA3 (encoding human defensin neutrophil peptide 3) and HBG1 as stable biomarkers of neonatal thrombopoiesis. Studies comparing CB- or adult-derived megakaryocytopoiesis confirmed inducible and abundant DEFA3 antigenic expression in CB megakaryocytes, ~3.5-fold greater than in leukocytes (the most abundant source in humans). An initial feasibility cohort of at-risk pregnancies manifested by maternal/fetal hemorrhage (chimerism) were applied for detection and validation of platelet HBG1 and DEFA3 as neonatal thrombopoiesis markers, most consistent for HBG1, which displayed gestational age-dependent expression. CONCLUSIONS: These studies establish an ontogenetically divergent stage of neonatal thrombopoiesis, and provide initial feasibility studies to track disordered fetal-to-adult megakaryocytopoiesis in vivo.

Shear-Mediated Platelet Activation is Accompanied by Unique Alterations in Platelet Release of Lipids.

INTRODUCTION: Platelet activation by mechanical means such as shear stress exposure, is a vital driver of thrombotic risk in implantable blood-contacting devices used in the treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical shear-mediated platelet activation. METHODS: Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. RESULTS: We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. CONCLUSIONS: We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have known procoagulant properties. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00692-x.

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.

In Vitro Measurements of Shear-Mediated Platelet Adhesion Kinematics as Analyzed through Machine Learning.

Platelet adhesion to blood vessel walls in shear flow is essential to initiating the blood coagulation cascade and prompting clot formation in vascular disease processes and prosthetic cardiovascular devices. Validation of predictive adhesion kinematics models at the single platelet level is difficult due to gaps in high resolution, dynamic morphological data or a mismatch between simulation and experimental parameters. Gel-filtered platelets were perfused at 30 dyne/cm2 in von Willebrand Factor (vWF)-coated microchannels, with flipping platelets imaged at high spatial and temporal resolution. A semi-unsupervised learning system (SULS), consisting of a series of convolutional neural networks, was used to segment platelet geometry, which was compared with expert-analyzed images. Resulting time-dependent rotational angles were smoothed with wavelet-denoising and shifting techniques to characterize the rotational period and quantify flipping kinematics. We observed that flipping platelets do not follow the previously-modeled modified Jefferey orbit, but are characterized by a longer lift-off and shorter reattachment period. At the juncture of the two periods, rotational velocity approached 257.48 ± 13.31 rad/s. Our SULS approach accurately segmented large numbers of moving platelet images to identify distinct adhesive kinematic characteristics which may further validate the physical accuracy of individual platelet motion in multiscale models of shear-mediated thrombosis.

Rapid analysis of streaming platelet images by semi-unsupervised learning.

We developed a fast and accurate deep learning approach employing a semi-unsupervised learning system (SULS) for capturing the real-time noisy, sparse, and ambiguous images of platelet activation. Outperforming several leading supervised learning methods when applied to segment various platelet morphologies, the SULS detects their complex boundaries at submicron resolutions and it massively decreases to only a few hours for segmenting streaming images of 45 million platelets that would have taken 40 years to annotate manually. For the first time, the fast dynamics of pseudopod formation and platelet morphological changes including membrane tethers and transient tethering to vessels are accurately captured.

A multiscale model for multiple platelet aggregation in shear flow.

We developed a multiscale model for simulating aggregation of multiple, free-flowing platelets in low-intermediate shear viscous flow, in which aggregation is mediated by the interaction of αIIbß3 receptors on the platelet membrane and fibrinogen (Fg). This multiscale model uses coarse grained molecular dynamics (CGMD) for platelets at the microscales and dissipative particle dynamics (DPD) for the shear flow at the macroscales, employing our hybrid aggregation force field for modeling molecular level receptor ligand bonds. We define an aggregation tensor and use it to quantify the molecular level contact characteristics between platelets in an aggregate. We perform numerical studies under different flow conditions for platelet doublets and triplets and evaluate the contact area, detaching force and minimum distance between different pairs of platelets in an aggregate. We also present the dynamics of applied stress and velocity magnitude distributions on the platelet membrane during aggregation and quantify the increase in stress in the contact region under different flow conditions. Integrating the knowledge from our previously validated models, together with new aggregation scenarios, our model can dynamically quantify aggregation characteristics and map stress and velocity distribution on the platelet membrane which are difficult to measure in vitro, thus providing an insight into mechanotransduction bond formation response of platelets to flow-induced shear stresses. This modeling framework, together with the tensor method for quantifying inter-platelet contact, can be extended to simulate and analyze larger aggregates and their adhesive properties.

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

[Figure: see text].

A predictive multiscale model for simulating flow-induced platelet activation: Correlating in silico results with in vitro results.

Flow-induced platelet activation prompts complex filopodial formation. Continuum methods fail to capture such molecular-scale mechanisms. A multiscale numerical model was developed to simulate this activation process, where a Dissipative Particle Dynamics (DPD) model of viscous blood flow is interfaced with a Coarse Grained Molecular Dynamics (CGMD) platelet model. Embedded in DPD blood flow, the macroscopic dynamic stresses are interactively transferred to the CGMD model, inducing intra-platelet associated events. The platelets activate by a biomechanical transductive linkage chain and dynamically change their shape in response. The models are fully coupled via a hybrid-potential interface and multiple time-stepping (MTS) schemes for handling the disparity between the spatiotemporal scales. Cumulative hemodynamic stresses that may lead to platelet activation are mapped on the surface membrane and simultaneously transmitted to the cytoplasm and cytoskeleton. Upon activation, the flowing platelets lose their quiescent discoid shape and evolve by forming filopodia. The model predictions were validated by a set of in vitro experiments, Platelets were exposed to various combinations of shear stresses and durations in our programmable hemodynamic shearing device (HSD). Their shape change was measured at multiple time points using scanning electron microscopy (SEM). The CGMD model parameters were fine-tuned by interrogating a parameter space established in these experiments. Segmentation of the SEM imaging streams was conducted by a deep machine learning system. This model can be further employed to simulate shear mediated platelet activation thrombosis initiation and to study the effects of modulating platelet properties to enhance their shear resistance via mechanotransduction pathways.

Shear-Induced Platelet Activation is Sensitive to Age and Calcium Availability: A Comparison of Adult and Cord Blood.

INTRODUCTION: Antiplatelet therapy for neonates and infants is often extrapolated from the adult experience, based on limited observation of agonist-induced neonatal platelet hypoactivity and poor understanding of flow shear-mediated platelet activation. Therefore, thrombotic events due to device-associated disturbed flow are inadequately mitigated in critically ill neonates with indwelling umbilical catheters and infants receiving cardiovascular implants. METHODS: Whole blood (WB), platelet-rich plasma (PRP), and gel-filtered platelets (GFP) were prepared from umbilical cord and adult blood, and exposed to biochemical agonists or pathological shear stress of 70 dyne/cm2. We evaluated α-granule release, phosphatidylserine (PS) scrambling, and procoagulant response using P-selectin expression, Annexin V binding, and thrombin generation (PAS), respectively. Activation modulation due to depletion of intracellular and extracellular calcium, requisite second messengers, was also examined. RESULTS: Similar P-selectin expression was observed for sheared adult and cord platelets, with concordant inhibition due to intracellular and extracellular calcium depletion. Sheared cord platelet Annexin V binding and PAS activity was similar to adult values in GFP, but lower in PRP and WB. Annexin V on sheared cord platelets was calcium-independent, with PAS slightly reduced by intracellular calcium depletion. CONCLUSIONS: Increased PS activity on purified sheared cord platelets suggest that their intrinsic function under pathological flow conditions is suppressed by cell-cell or plasmatic components. Although secretory functions of adult and cord platelets retain comparable calcium-dependence, PS exposure in sheared cord platelets is uniquely calcium-independent and distinct from adults. Identification of calcium-regulated developmental disparities in shear-mediated platelet function may provide novel targets for age-specific antiplatelet therapy.

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.

Assessment of neonatal, cord, and adult platelet granule trafficking and secretion.

Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.

Circulatory loop design and components introduce artifacts impacting in vitro evaluation of ventricular assist device thrombogenicity: A call for caution.

Mechanical circulatory support (MCS) devices continue to be hampered by thrombotic adverse events (AEs), a consequence of device-imparted supraphysiologic shear stresses, leading to shear-mediated platelet activation (SMPA). In advancing MCS devices from design to clinical use, in vitro circulatory loops containing the device under development and testing are utilized as a means of assessing device thrombogenicity. Physical characteristics of these test circulatory loops may also contribute to inadvertent platelet activation through imparted shear stress, adding inadvertent error in evaluating MCS device thrombogenicity. While investigators normally control for the effect of a loop, inadvertent addition of what are considered innocuous connectors may impact test results. Here, we tested the effect of common, additive components of in vitro circulatory test loops, that is, connectors and loop geometry, as to their additive contribution to shear stress via both in silico and in vitro models. A series of test circulatory loops containing a ventricular assist device (VAD) with differing constituent components, were established in silico including: loops with 0~5 Luer connectors, a loop with a T-connector creating 90° angulation, and a loop with 90° angulation. Computational fluid dynamics (CFD) simulations were performed using a k - ω shear stress transport turbulence model to platelet activation index (PAI) based on a power law model. VAD-operated loops replicating in silico designs were assembled in vitro and gel-filtered human platelets were recirculated within (1 hour) and SMPA was determined. CFD simulations demonstrated high shear being introduced at non-smooth regions such as edge-connector boundaries, tubing, and at Luer holes. Noticeable peaks' shifts of scalar shear stress (sss) distributions toward high shear-region existed with increasing loop complexity. Platelet activation also increased with increasing shear exposure time, being statistically higher when platelets were exposed to connector-employed loop designs. The extent of platelet activation in vitro could be successfully predicted by CFD simulations. Loops employing additional components (non-physiological flow pattern connectors) resulted in higher PAI. Loops with more components (5-connector loop and 90° T-connector) showed 63% and 128% higher platelet activation levels, respectively, versus those with fewer (0-connector (P = .023) and a 90° heat-bend loop (P = .0041). Our results underscore the importance of careful consideration of all component elements, and suggest the need for standardization in designing in vitro circulatory loops for MCS device evaluation to avoid inadvertent additive SMPA during device evaluation, confounding overall results. Specifically, we caution on the use and inadvertent introduction of additional connectors, ports, and other shear-generating elements which introduce artifact, clouding primary device evaluation via introduction of additive SMPA.

A Multiscale Model for Recruitment Aggregation of Platelets by Correlating with In Vitro Results.

INTRODUCTION: We developed a multiscale model to simulate the dynamics of platelet aggregation by recruitment of unactivated platelets flowing in viscous shear flows by an activated platelet deposited onto a blood vessel wall. This model uses coarse grained molecular dynamics (CGMD) for platelets at the microscale and dissipative particle dynamics (DPD) for the shear flow at the macroscale. Under conditions of relatively low shear, aggregation is mediated by fibrinogen via αIIbß3 receptors. METHODS: The binding of αIIbß3 and fibrinogen is modeled by a molecular-level hybrid force field consisting of Morse potential and Hooke law for the nonbonded and bonded interactions, respectively. The force field, parametrized in two different interaction scales, is calculated by correlating with the platelet contact area measured in vitro and the detaching force between αIIbß3 and fibrinogen. RESULTS: Using our model, we derived, the relationship between recruitment force and distance between the centers of mass of two platelets, by integrating the molecular-scale inter-platelet interactions during recruitment aggregation in shear flows. Our model indicates that assuming a rigid-platelet model, underestimates the contact area by 89% and the detaching force by 93% as compared to a model that takes into account the platelet deformability leading to a prediction of a significantly lower attachment during recruitment. CONCLUSIONS: The molecular-level predictive capability of our model sheds a light on differences observed between transient and permanent platelet aggregation patterns. The model and simulation framework can be further adapted to simulate initial thrombus formation involving multiple flowing platelets as well as deposition and adhesion onto blood vessels.

Prothrombotic activity of cytokine-activated endothelial cells and shear-activated platelets in the setting of ventricular assist device support.

BACKGROUND: We systematically analyzed the synergistic effect of: (i) cytokine-mediated inflammatory activation of endothelial cells (ECs) with and (ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of left ventricular assist device (LVAD) support. METHODS: Intact and shear-activated human platelets were exposed to non-activated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, and 70 dynes/cm2, 10 minutes) via a hemodynamic shearing device. ECs were activated via exposure to inflammatory tumor necrosis factor-α (TNF-α 10 and 100 ng/ml, 24 hours), consistent with inflammatory activation recorded in patients on LVAD circulatory support. RESULTS: Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm2 shear-activated platelets vs intact platelets: +80%, p < 0.001). Importantly, inflammatory activation of ECs amplified platelet prothrombinase activity progressively with increasing shear stress magnitude and TNF-α concentration: thrombin generation of 70 dynes/cm2 shear-activated platelets was 2.6-fold higher after exposure and adhesion to 100 ng/ml TNF-α‒activated ECs (p < 0.0001). CONCLUSIONS: We demonstrated synergistic effect of SMPA and cytokine-mediated EC inflammatory activation to enhance EC‒platelet adhesion and platelet prothrombotic function. These mechanisms may contribute to intraventricular thrombosis in the setting of mechanical circulatory support.