Sodium bicarbonate as a local adjunctive agent for limiting platelet activation, aggregation, and adhesion within cardiovascular therapeutic devices.

Cardiovascular therapeutic devices (CTDs) remain limited by thrombotic adverse events. Current antithrombotic agents limit thrombosis partially, often adding to bleeding. The Impella® blood pump utilizes heparin in 5% dextrose (D5W) as an internal purge to limit thrombosis. While effective, exogenous heparin often complicates overall anticoagulation management, increasing bleeding tendency. Recent clinical studies suggest sodium bicarbonate (bicarb) may be an effective alternative to heparin for local anti-thrombosis. We examined the effect of sodium bicarbonate on human platelet morphology and function to better understand its translational utility. Human platelets were incubated (60:40) with D5W + 25 mEq/L, 50 mEq/L, or 100 mEq/L sodium bicarbonate versus D5W or D5W + Heparin 50 U/mL as controls. pH of platelet-bicarbonate solutions mixtures was measured. Platelet morphology was examined via transmission electron microscopy; activation assessed via P-selectin expression, phosphatidylserine exposure and thrombin generation; and aggregation with TRAP-6, calcium ionophore, ADP and collagen quantified; adhesion to glass measured via fluorescence microscopy. Sodium bicarbonate did not alter platelet morphology but did significantly inhibit activation, aggregation, and adhesion. Phosphatidylserine exposure and thrombin generation were both reduced in a concentration-dependent manner-between 26.6 ± 8.2% (p = 0.01) and 70.7 ± 5.6% (p < 0.0001); and 14.0 ± 6.2% (p = 0.15) and 41.7 ± 6.8% (p = 0.03), respectively, compared to D5W control. Platelet aggregation via all agonists was also reduced, particularly at higher concentrations of bicarb. Platelet adhesion to glass was similarly reduced, between 0.04 ± 0.03% (p = 0.61) and 0.11 ± 0.04% (p = 0.05). Sodium bicarbonate has direct, local, dose-dependent effects limiting platelet activation and adhesion. Our results highlight the potential utility of sodium bicarbonate as a locally acting agent to limit device thrombosis.

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.

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.

DNA Origami-Platelet Adducts: Nanoconstruct Binding without Platelet Activation.

Objective. Platelets are small, mechanosensitive blood cells responsible for maintaining vascular integrity and activatable on demand to limit bleeding and facilitate thrombosis. While circulating in the blood, platelets are exposed to a range of mechanical and chemical stimuli, with the platelet membrane being the primary interface and transducer of outside-in signaling. Sensing and modulating these interface signals would be useful to study mechanochemical interactions; yet, to date, no methods have been defined to attach adducts for sensor fabrication to platelets without triggering platelet activation. We hypothesized that DNA origami, and methods for its attachment, could be optimized to enable nonactivating instrumentation of the platelet membrane. Approach and Results. We designed and fabricated multivalent DNA origami nanotile constructs to investigate nanotile hybridization to membrane-embedded single-stranded DNA-tetraethylene glycol cholesteryl linkers. Two hybridization protocols were developed and validated (Methods I and II) for rendering high-density binding of DNA origami nanotiles to human platelets. Using quantitative flow cytometry, we showed that DNA origami binding efficacy was significantly improved when the number of binding overhangs was increased from two to six. However, no additional binding benefit was observed when increasing the number of nanotile overhangs further to 12. Using flow cytometry and transmission electron microscopy, we verified that hybridization with DNA origami constructs did not cause alterations in the platelet morphology, activation, aggregation, or generation of platelet-derived microparticles. Conclusions. Herein, we demonstrate that platelets can be successfully instrumented with DNA origami constructs with no or minimal effect on the platelet morphology and function. Our protocol allows for efficient high-density binding of DNA origami to platelets using low quantities of the DNA material to label a large number of platelets in a timely manner. Nonactivating platelet-nanotile adducts afford a path for advancing the development of DNA origami nanoconstructs for cell-adherent mechanosensing and therapeutic agent delivery.

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.

Electrical impedance vs. light transmission aggregometry: Testing platelet reactivity to antiplatelet drugs using the MICELI POC impedance aggregometer as compared to a commercial predecessor.

BACKGROUND: Patients' responses to antiplatelet therapy significantly vary, with individuals showing high residual platelet reactivity associated with thrombosis. To personalize thrombosis management, platelet function testing has been suggested as a promising tool able to monitor the antithrombotic effect of antiplatelet agents in real-time. We have prototyped the MICELI, a miniature and easy-to-use electrical impedance aggregometer (EIA), measuring platelet aggregation in whole blood. Here, we tested the capability of the MICELI aggregometer to quantify platelet reactivity on antiplatelet agents, as compared with conventional light-transmission aggregometry (LTA). METHODS: Platelet aggregation in ACD-anticoagulated whole blood and platelet-rich plasma of healthy donors (n = 30) was evaluated. The effect of clopidogrel, ticagrelor, cangrelor, cilostazol, and tirofiban on ADP-induced aggregation was tested, while aspirin was evaluated with arachidonic acid and collagen. Platelet aggregation was recorded using the MICELI or BioData PAP-8E (Bio/Data Corp.) aggregometers. RESULTS: The MICELI aggregometer detected an adequate and comparable dose-dependent decrease of platelet aggregation in response to increments of drugs' concentrations, as compared to LTA (the inter-device R2 = 0.79-0.93). Platelet aggregation in platelet-rich plasma recorded by LTA showed higher sensitivity to antiplatelet agents, but it couldn't distinguish between different drug doses as indicated by saturation of the aggregatory response. CONCLUSION: Platelet aggregation in whole blood as recorded by EIA represents a better model system for evaluation of platelet reactivity as compared with platelet aggregation in platelet-rich plasma as recorded by LTA, since EIA takes into consideration the modulatory effect of other blood cells on platelet hemostatic function and pharmacodynamics of antiplatelet drugs in vivo. As such, the MICELI impedance aggregometer could be potentially employed for the point-of-care monitoring of platelet function in patients on-treatment for personalized tailoring of their antiplatelet regimen.

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.

Characterization of the competing role of surface-contact and shear stress on platelet activation in the setting of blood contacting devices.

Supraphysiological shear stress and surface-contact are recognized as driving mechanisms of platelet activation (PA) in blood contacting devices (BCDs). However, the competing role of these mechanisms in triggering thrombogenic events is poorly understood. Here, we characterized the dynamics of PA in response to the combined effect of shear stress and material exposure. Human platelets were stimulated with different levels of shear stress (500, 750, 1000 dynes/cm2) over a range of exposure times (10, 20, and 30 min) within capillary tubes made of various polymeric materials. Polyethylene (PE), polytetrafluoroethylene (PTFE), ethylene tetrafluoroethylene (ETFE), and polyether ether ketone (PEEK), used for BCDs fabrication, were investigated as compared to glass and thromboresistant Sigma™-coated glass. PA was quantified using the Platelet Activity State assay. Our results indicate that mechanical stimulation and polymer surface-contact both significantly contribute to PA. Notably, the contribution of the mechanical stimulus ranges between +36% and +43%, while that associated with polymer surface-contact ranges from +48% to +59%, depending on the exposure time. In more detail, our results indicate that: (i) PA increases with increasing shear stress magnitude; (ii) PA has a non-linear, time-dependent relationship to exposure time; (iii) PA is largely influenced by biomaterials, with PE and PEEK having respectively the lowest and highest prothrombotic potential; (iv) the effects of polymer surface-contact and shear stress are not correlated and can be studied separately. Our results suggest the importance of incorporating the evaluation of platelet activation driven by the combined effect of shear stress and polymer surface-contact for the comprehensive assessment, and eventually minimization, of BCDs thrombogenic potential.

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

[Figure: see text].

Impella 5.5 Versus Centrimag: A Head-to-Head Comparison of Device Hemocompatibility.

Despite growing use of mechanical circulatory support, limitations remain related to hemocompatibility. Here, we performed a head-to-head comparison of the hemocompatibility of a centrifugal cardiac assist system-the Centrimag, with that of the latest generation of an intravascular microaxial system-the Impella 5.5. Specifically, hemolysis, platelet activation, microparticle (MP) generation, and von Willebrand factor (vWF) degradation were evaluated for both devices. Freshly obtained porcine blood was recirculated within device propelled mock loops for 4 hours, and alteration of the hemocompatibility parameters was monitored over time. We found that the Impella 5.5 and Centrimag exhibited low levels of hemolysis, as indicated by minor increase in plasma free hemoglobin. Both devices did not induce platelet degranulation, as no alteration of ß-thromboglobulin and P-selectin in plasma occurred, rather minor downregulation of platelet surface P-selectin was detected. Furthermore, blood exposure to shear stress via both Centrimag and Impella 5.5 resulted in a minor decrease of platelet count with associated ejection of procoagulant MPs, and a decrease of vWF functional activity (but not plasma level of vWF-antigen). Greater MP generation was observed with the Centrimag relative to the Impella 5.5. Thus, the Impella 5.5 despite having a lower profile and higher impeller rotational speed demonstrated good and equivalent hemocompatibility, in comparison with the predicate Centrimag, with the advantage of lower generation of MPs.

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.

The MICELI (MICrofluidic, ELectrical, Impedance): Prototyping a Point-of-Care Impedance Platelet Aggregometer.

As key cellular elements of hemostasis, platelets represent a primary target for thrombosis and bleeding management. Currently, therapeutic manipulations of platelet function (antithrombotic drugs) and count (platelet transfusion) are performed with limited or no real-time monitoring of the desired outcome at the point-of-care. To address the need, we have designed and fabricated an easy-to-use, accurate, and portable impedance aggregometer called "MICELI" (MICrofluidic, ELectrical, Impedance). It improves on current platelet aggregation technology by decreasing footprint, assay complexity, and time to obtain results. The current study aimed to optimize the MICELI protocol; validate sensitivity to aggregation agonists and key blood parameters, i.e., platelet count and hematocrit; and verify the MICELI operational performance as compared to commercial impedance aggregometry. We demonstrated that the MICELI aggregometer could detect platelet aggregation in 250 µL of whole blood or platelet-rich plasma, stimulated by ADP, TRAP-6, collagen, epinephrine, and calcium ionophore. Using hirudin as blood anticoagulant allowed higher aggregation values. Aggregation values obtained by the MICELI strongly correlated with platelet count and were not affected by hematocrit. The operational performance comparison of the MICELI and the Multiplate® Analyzer demonstrated strong correlation and similar interdonor distribution of aggregation values obtained between these devices. With the proven reliability of the data obtained by the MICELI aggregometer, it can be further translated into a point-of-care diagnostic device aimed at monitoring platelet function in order to guide pharmacological hemostasis management and platelet transfusions.

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.

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.