Shear-mediated Platelet Activation Research Articles

Impact of rise and fall phases of shear on platelet activation and aggregation using microfluidics.

Blood flow disorders are often the result of the non-physiological narrowing of blood arteries caused by atherosclerosis and thrombus. The blood then proceeds through rising-peak-decreasing phases as it passes through the narrow area. Although abnormally high shear is known to activate platelets, the shear process that platelets undergo in small arteries is complex. Thus, understanding how each shear phase affects platelet activation can be used to improve antiplatelet therapy and decrease the risk of side effects like bleeding. Blood samples were sheared (68.8 ms,5200s-1) in vitro by the microfluidic technique, and platelet activation levels (P-selectin and integrin αIIbβ3) and von Willebrand factor (vWF) binding to platelets were analyzed by flow cytometry. Post-stenosis platelet aggregation was dynamically detected using microfluidic technology. We studied TXA2, P2Y12-ADP, and integrin αIIbβ3-fibrinogen receptor pathways by adding antiplatelet drugs, such as acetylsalicylic acid (ASA, an active ingredient of aspirin that inhibits platelet metabolism), ticagrelor (hinders platelet activation), and tirofiban (blocks integrin αIIbβ3 receptor) in vitro, respectively, to determine platelet activation function mediated by transient non-physiological high shear rates. We demonstrated that platelets can be activated under transient pathological high shear rates. The shear rise and fall phases influenced shear-induced platelet activation by regulating the binding of vWF to platelets. The degree of platelet activation and aggregation increased with multiple shear rise and fall phases. ASA did not inhibit shear-mediated platelet activation, but ticagrelor and tirofiban effectively inhibited shear-mediated platelet activation. Our data demonstrated that the shear rise and fall phases play an important role in shear-mediated platelet activation and promote platelet activation and aggregation in a vWF-dependent manner. Blocking integrin αIIbβ3 receptor and hindering P2Y12-ADP were beneficial to reducing shear-mediated platelet activation.

On-Chip Platelet Activation Assessment: Microfluidic Emulation of Shear Stress Profiles Induced by Mechanical Circulatory Support Devices.

Mechanical circulatory support devices (MCSDs), although proved to be a pillar in the clinical setting of advanced heart failure, are afflicted by thromboembolic complications. Shear-mediated platelet activation has been recognized to drive thromboembolic events in patients implanted with MCSDs. Despite this, to date, a clinically reliable diagnostic test for assessing platelet response to stress stimuli is still missing. Here, we describe and apply the previously developed device thrombogenicity emulation methodology to the design of a microfluidic platform able to replicate shear stress profiles representative of MCSDs. The device-specific shear-mediated platelet activation is finally assessed by the platelet activity state assay, which measures real-time thrombin production, as a marker of platelet activation level. This technique can be employed to emulate the shear stress patterns of different MCSDs, such as mechanical heart valves, ventricular assist devices, and stents.

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

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. 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. 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. 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. The online version contains supplementary material available at 10.1007/s12195-021-00692-x.

Multi-constituent simulation of thrombus formation at LVAD inlet cannula connection: Importance of Virchow's triad.

As pump thrombosis is reduced in current-generation ventricular assist devices (VAD), adverse events such as bleeding or stroke remain at unacceptable rates. Thrombosis around the VAD inlet cannula (IC) has been highlighted as a possible source of stroke events. Recent computational fluid dynamics (CFD) studies have attempted to characterize the thrombosis risk of different IC-ventricle configurations. However, purely CFD simulations relate thrombosis risk to ad hoc criteria based on flow characteristics, with little consideration of biochemical factors. This study investigates the genesis of IC thrombosis including two elements of the Virchow's triad: endothelial injury and hypercoagulability. To this end a multi-scale thrombosis simulation that includes platelet activity and coagulation reactions was performed. Our results show significant thrombin formation in stagnation regions (|u| < 0.005m/s) close to the IC wall. In addition, high shear-mediated platelet activation was observed over the leading-edge tip of the cannula. The current study reveals the importance of biochemical factors to the genesis of thrombosis at the ventricular-cannula junction in a perioperative state. This study is a first step toward the long-term objective of including clinically relevant pharmacological kinetics such as heparin or aspirin in simulations of inflow cannula thrombosis.

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

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. 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. 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. 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

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. 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 αIIbβ3 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. Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbβ3 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 αIIbβ3 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. 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.

Yin and Yang of MCS-Related Coagulopathy: Shear Stress Promotes Platelet Prothrombosis and Microparticle Generation While Inducing Integrin Downregulation and Decreased Aggregability

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 coagulopathy accompanied by both thrombosis and bleeding. We hypothesized that shear-mediated alterations of platelet hemostatic function, i.e. integrin expression, procoagulant activity, and aggregation, mechanistically drive MCS-related coagulopathy. Human platelets were exposed to shear stress in a hemodynamic shearing device (30-70 dyn/cm2, 10 min). Integrin αIIbβ3 & GPIb surface expression/activation, phosphatidylserine externalization (PSE) and microparticles (MPs) were quantified by flow cytometry. Platelet procoagulant activity was verified by thrombin generation assay. Platelet aggregation was measured by optical aggregometry. Shear stress exposure induced notable PSE, simultaneously promoting thrombin generation on platelets and MP. Paradoxically, shear-mediated platelet activation (SMPA) did not induce integrin αIIbβ3 activation. Moreover, high shear (70 dyn/cm2) prevented activation and upregulation of αII2bβ3 mediated by biochemical agonists. Aggregometry findings further validate flow cytometry results, as high shear did not promote aggregation, rather decreased ADP- and TRAP-6 induced aggregation. Likewise, SMPA led to the evident reduction of GPIb surface expression, as indicated by significant drop of GPIb+ platelet number and their fluorescence intensity (73.5 ± 2.9% & 953.1 ± 101.0 AU vs. 94.3 ± 0.7% & 1620 ± 72.4 AU for intact platelets; M ± SEM, ANOVA, p ≤ 0.05). Shear-mediated decrease of GPIb expression along with evident augmentation of GPIb- & αIIbβ3-positive MPs (3.3 ± 0.6% & 12.5 ± 2.8%, respectively) supports integrin shedding as an operative mechanism. SMPA promotes platelet PSE and procoagulant MP generation, thus facilitating thrombin formation and driving prothrombosis. Yet, shear stress did not promote platelet aggregation, rather impaired agonist-induced platelet aggregation via integrin αIIbβ3 and GPIb shedding from the platelets into MPs. This dichotomic effect of shear on platelets mechanistically defines the paradoxical prothrombotic state with a concomitant propensity of bleeding in MCS patients.

MCS Hypershear Modulates Platelet Membrane Fluidity, Lipid Species, and is Gender Specific

Shear-mediated platelet activation (SMPA) is a main driver of MCS thrombosis. A target of MCS shear, driving activation, is the platelet membrane - leading to potential alteration of biophysical properties. Here, we examined the effect of shear stress-accumulation on platelet membrane fluidity and on platelet lipid species profile - both retained (membrane) and free (ejected) post-activation. Human gel-filtered platelets (20,000 plts/uL) were exposed to shear in a hemodynamic shearing device (70 dynes/cm2 x 10'); or to thrombin (1U/mL x 10') as a chemical agonist comparator. Membrane fluidity was determined by ໿fluorescence anisotropy of the probe TMA-DPH (1uM, 5' @ 37C). Platelet lipids - both retained (cellular) and free (ejected) following activation - were extracted and analyzed via high resolution liquid-chromatography tandem mass spectrometry (LC-MS/MS). Shear stress accumulation increases platelet membrane fluidity - with a 6% increase noted even for the brief shear exposure examined. Shear stress exposure results in significant alteration of overall platelet lipid composition, with increase in supernatant lipid species relative to the non-activated samples such that 72% of identified lipid species have >2-fold increase. Lipid species altered in the supernatant of sheared platelets were unaltered under thrombin treatment. Interestingly, within the limits of our analysis, a gender-based difference in retained lipid species was seen with shear but not with thrombin activation. Male donors showed at least a 2-fold decrease in 53% of lipids identified in the pellet of the sheared platelets using negative mode MS. In contrast, female donors had only 6% of lipids decrease >2-fold relative to non-activated platelets, while 77% of lipids showed a less than 2-fold change in either direction. Shear stress has a specific impact on platelet membranes - leading to an increase in fluidity, and alteration of platelet lipidomic profile, which differs from biochemical activation, with evident gender-based differences. Specifically, shear leads to an increase in the number of lipids ejected across all donors, and a male-preferred decrease in retained lipid species. This sex-based difference in platelet composition resulting from shear exposure raises the issue of altered responsiveness that may impart gender differences in in MCS outcomes.

Circulatory loop design and components introduce artifacts impacting invitro 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, invitro 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 insilico 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 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 (1hour) 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.

P2Y12 Receptor Antagonists: Do They Really Inhibit Shear-Mediated Platelet Activation within MCS?

Purpose Shear-mediated platelet activation (SMPA) by supra-physiological blood flow found within MCS is a main driver and contributor of device-related thrombosis. Thus, antiplatelet drugs targeting the main pathways of platelet activation are routinely prescribed. We have shown, that aspirin and GP IIbIIIa blockers have limited ability in mitigating SMPA, and do NOT protect platelets from integrin shedding and microparticle (MP) generation. In contrast, ticagrelor, a direct P2Y12 purinergic receptor antagonist, drastically inhibits platelet prothrombinase activity induced by high shear. The aim of the study was to define the role of the P2Y12 pathway in SMPA and evaluate the effect of P2Y12 antagonists on platelet procoagulant activity and MP generation induced by high shear stress. Methods The following P2Y12 antagonists were used: clopidogrel active metabolite (10 µM), cangrelor (1 µM) and ticagrelor (1 µM). After 10 min incubation with the agent, human platelets were exposed to continuous high shear stress (70 dyn/cm2, 10 min) in a hemodynamic shearing device. Platelet procoagulant activity was assessed using the annexin V binding assay, detecting procoagulant surface exposure, and platelet activity state (PAS) assay measuring thrombin generation. MPs were quantified by flow cytometry as CD41+/AnV+ particles distinguished from platelets by their FS/SS. Results Ticagrelor and cangrelor showed similar inhibitory effect, consistently decreasing both platelet procoagulant activity and microparticle generation induced by shear stress. The number of AnV+ platelets, AnV+ MPs, and PAS levels were decreased by 26%, 27%, and 30%, respectively, as compared with non-treated controls subjected to shear. Clopidogrel active metabolite, known for its low affinity to the targeted receptor, reduced PAS by 15%, while annexin V binding was diminished in more than 10 times for platelets and 2 times for MPs, indicating a significant reduction of platelet procoagulant surface exposure but limited inhibition of thrombin generation. Conclusion P2Y12 purinergic receptor antagonists inhibit shear-mediated platelet procoagulant surface formation and microparticle generation, both contributors in overall platelet prothrombotic activity induced by high shear stress within MCS. Our results suggest that P2Y12 pathway is involved in the regulation of SMPA.

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

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. 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. 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). 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.

Microfluidic flow-based platforms for induction and analysis of dynamic shear-mediated platelet activation-Initial validation versus the standardized hemodynamic shearing device.

A microfluidic flow-based platform (μFP), able to stimulate platelets via exposure of shear stress patterns pertinent to cardiovascular devices and prostheses, was compared to the Hemodynamic Shearing Device (HSD)-a state-of-the-art bench-top system for exposure of platelets to defined levels and patterns of shear. Platelets were exposed to time-varying shear stress patterns in the two systems; in detail, platelets were recirculated in the μFP or stimulated in the HSD to replicate comparable exposure time. Shear-mediated platelet activation was evaluated via (i) the platelet activity state assay, allowing the measurement of platelet-mediated thrombin generation and associated prothrombotic tendencies, (ii) scanning electron microscopy to evaluate morphological changes of sheared platelets, and (iii) flow cytometry for the determination of platelet phosphatidylserine exposure as a marker of shear activation. The results revealed good matching and comparability between the two systems, with similar trends of platelet activation, formation of microaggregates, and analogous trends of activation marker exposure for both the HSD and microfluidic-stimulated samples. These findings support future translation of the microfluidic platform as a Point-of-Care facsimile system for the diagnosis of thrombotic risk in patients implanted with cardiovascular devices.

MCS Shear-Medicated Platelet Activation Does not Cause Integrin GPIIbIIIa Activation, Rather Shedding and Microparticle Generation

Shear-mediated platelet activation (SMPA) is a prime driver of thrombus formation in mechanical circulatory support (MCS). We have shown that current anti-platelet agents, including anti-GPIIbIIIa rescue, fail to prevent or resolve post-implant thrombosis, frequently leading to bleeding. Here we examine the effect of physiologically relevant and elevated shear (ES) on platelet integrin GPIIbIIIa surface expression, activation, and function as platelet aggregation response to shear and biochemical agonists.

Ticagrelor but not Aspirin Limits Shear-Mediated Platelet Activation

Ticagrelor but not Aspirin Limits Shear-Mediated Platelet Activation

High Frequency Components of Hemodynamic Shear Stress Profiles are a Major Determinant of Shear-Mediated Platelet Activation in Therapeutic Blood Recirculating Devices

We systematically analyzed the relative contributions of frequency component elements of hemodynamic shear stress waveforms encountered in cardiovascular blood recirculating devices as to overall platelet activation over time. We demonstrated that high frequency oscillations are the major determinants for priming, triggering and yielding activated “prothrombotic behavior” for stimulated platelets, even if the imparted shear stress has low magnitude and brief exposure time. Conversely, the low frequency components of the stress signal, with limited oscillations over time, did not induce significant activation, despite being of high magnitude and/or exposure time. In vitro data were compared with numerical predictions computed according to a recently proposed numerical model of shear-mediated platelet activation. The numerical model effectively resolved the correlation between platelet activation and the various frequency components examined. However, numerical predictions exhibited a different activation trend compared to experimental results for different time points of a stress activation sequence. With this study we provide a more fundamental understanding for the mechanobiological responsiveness of circulating platelets to the hemodynamic environment of cardiovascular devices, and the importance of these environments in mediating life-threatening thromboembolic complications associated with shear-mediated platelet activation. Experimental data will guide further optimization of the thromboresistance of cardiovascular implantable therapeutic devices.

(6) - Platelet Membrane-Related Morphologic Alterations: An Early Marker of Supra-Physiologic Shear-Mediated Platelet Activation Associated with VADs

(6) - Platelet Membrane-Related Morphologic Alterations: An Early Marker of Supra-Physiologic Shear-Mediated Platelet Activation Associated with VADs

Shear-mediated platelet activation in the free flow: Perspectives on the emerging spectrum of cell mechanobiological mechanisms mediating cardiovascular implant thrombosis

Shear-mediated platelet activation (SMPA) is central in thrombosis of implantable cardiovascular therapeutic devices. Despite the morbidity and mortality associated with thrombosis of these devices, our understanding of mechanisms operative in SMPA, particularly in free flowing blood, remains limited. Herein we present and discuss a range of emerging mechanisms for consideration for “free flow” activation under supraphysiologic shear. Further definition and manipulation of these mechanisms will afford opportunities for novel pharmacologic and mechanical strategies to limit SMPA and enhance overall implant device safety.

Aspirin has limited ability to modulate shear-mediated platelet activation associated with elevated shear stress of ventricular assist devices

Continuous flow ventricular assist devices (cfVADs) while effective in advanced heart failure, remain plagued by thrombosis related to abnormal flows and elevated shear stress. To limit cfVAD thrombosis, patients utilize complex anti-thrombotic regimens built upon a foundation of aspirin (ASA). While much data exists on ASA as a modulator of biochemically-mediated platelet activation, limited data exists as to the efficacy of ASA as a means of limiting shear-mediated platelet activation, particularly under elevated shear stress common within cfVADs. We investigated the ability of ASA (20, 25 and 125μM) to limit shear-mediated platelet activation under conditions of: 1) constant shear stress (30dynes/cm2 and 70dynes/cm2); 2) dynamic shear stress, and 3) initial high shear exposure (70dynes/cm2) followed by low shear exposure – i.e. a platelet sensitization protocol, utilizing a hemodynamic shearing device providing uniform shear stress in vitro. The efficacy of ASA to limit platelet activation mediated via passage through a clinical cfVAD system (DeBakey Micromed) in vitro was also studied. ASA reduced platelet activation only under conditions of low shear stress (38% reduction compared to control, n=10, p<0.004), with minimal protection at higher shear stress and under dynamic conditions (n=10, p>0.5) with no limitation of platelet sensitization. ASA had limited ability (25.6% reduction in platelet activation rate) to modulate shear-mediated platelet activation induced via cfVAD passage. These findings, while performed under “deconstructed” non-clinical conditions by utilizing purified platelets alone in vitro, provide a potential contributory mechanistic explanation for the persistent thrombosis rates experienced clinically in cfVAD patients despite ASA therapy. An opportunity exists to develop enhanced pharmacologic strategies to limit shear-mediated platelet activation at elevated shear levels associated with mechanical circulatory support devices.

Microfluidic emulation of mechanical circulatory support device shear-mediated platelet activation.

Thrombosis of ventricular assist devices (VADs) compromises their performance, with associated risks of systemic embolization, stroke, pump stop and possible death. Anti-thrombotic (AT) drugs, utilized to limit thrombosis, are largely dosed empirically, with limited testing of their efficacy. Further, such testing, if performed, typically examines efficacy under static conditions, which is not reflective of actual shear-mediated flow. Here we adopted our previously developed Device Thrombogenicity Emulation methodology to design microfluidic platforms able to emulate representative shear stress profiles of mechanical circulatory support (MCS) devices. Our long-term goal is to utilize these systems for point-of-care (POC) personalized testing of AT efficacy under specific, individual shear profiles. First, we designed different types of microfluidic channels able to replicate sample shear stress patterns observed in MCS devices. Second, we explored the flexibility of microfluidic technology in generating dynamic shear stress profiles by modulating the geometrical features of the channels. Finally, we designed microfluidic channel systems able to emulate the shear stress profiles of two commercial VADs. From CFD analyses, the VAD-emulating microfluidic systems were able to replicate the main characteristics of the shear stress waveforms of the macroscale VADs (i.e., shear stress peaks and duration). Our results establish the basis for development of a lab-on-chip POC system able to perform device-specific and patient-specific platelet activation state assays.

Laser-Energized Plasmonics for Nanopatterning Medical Devices

A scalable, prototype plasmonic nanomanufacturing system was designed, built, and tested for patterning nanostructures on the surfaces of drug-eluting stents (DES), the objective being to prevent the late-stent thrombosis (LST). Nanopatterning, unlike micro/macropatterning, of DES has proven to provide optimal, rapid, and preferential endothelial cell (EC) attachment (antithrombosis) while not significantly affecting shear-mediated platelet activation (prothrombosis). In this work, laser-induced, high-density surface plasmon polaritons (SPPs) were generated and utilized to produce nanostructures on the surfaces of DES by electric field enhancement mechanism. The scalability aspects such as downsizing the feature, improving the precision, increasing the throughput, and reducing the cost were investigated. Results indicated fairly uniform nanostructures; high throughput; excellent repeatability and resolution; significant cost savings; and potential for high retention of drug dose in the stent. The work represents an unprecedented area in nanomanufacturing where the basic science contribution is to harness the energy from plasmon polaritons by effectively “customizing” and “controlling” their propagation, while the engineering contribution is a scalability approach to reliably nanopattern medical devices in high volume with nanometer resolution. The nanomanufacturing system developed in this study may be an enabling technology to strongly impact other fields such as semiconductors, organic solar cells, and nano-electromechanical systems (NEMS).