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.

A Novel Multiparametric Score for the Detection and Grading of Prosthetic Mitral Valve Obstruction in Cases With Different Disc Motion Abnormalities.

Prosthetic mechanical valves are the elective choice in mitral valve (MV) replacement, because of their reliability and easiness of implantation. However, these prostheses can suffer from complications, the major one being prosthetic mitral valve thrombosis (PMVT). In these cases, transthoracic doppler echocardiogram (TDE) is the standard diagnostic workup for diagnosis of valve malfunction. The American Society of Echocardiography (ASE) indicates the possible TDE-derived indexes, which can help in identifying insurgence of MV replacement complications. Unfortunately, in some cases, it is not possible to detect PMVT based on these criteria. In these cases, we speak of Doppler silent thrombosis and only more accurate and invasive analyses, such as fluoroscopy, allow for a correct diagnosis. In this work, computational fluid dynamic models were implemented to simulate valve fluid dynamics in different clinical scenarios in order to improve the reliability of PMVT diagnosis based on TDE. In detail, seven mechanical valve configurations, associated to different potential thrombotic conditions (symmetric and asymmetric stenosis), were designed and tested using five pathologic transmitral velocity profile, extracted from real TDE images; to obtain the flow rate profiles, each TDE velocity profile was scaled to yield a mean flow rate (MFR) of 4, 5 and 6 L/min, respectively. As a result, 105 (7 × 5 × 3) synthetic cases, accounting for different velocity profiles, MFRs and valve configurations, were simulated. TDE-derived indexes were calculated according to the ASE guidelines that were extracted. Advanced statistical methods were applied to propose a new diagnostic algorithm for detecting PMVT. Our results showed that there isn't any significant difference between symmetric and asymmetric stenosis, probe location and flow rate waveform and confirmed that the single modality diagnostic is not able to predict thrombosis in a relevant number of cases, referable to mild and mild-severe stenosis cases. To overcome the problem, a novel multi-parametric discrete score based on the designed diagnostic algorithm was attained and tested; the percentage of stenosis (POS) was predicted with an accuracy rate of 90.5%. Even more interestingly, the error rate of 9.5% is related to four false positive cases corresponding to mild stenosis (POS = 15%) which were erroneously classified as mild-severe stenosis. No false negatives were obtained. Our results suggest that a reliable estimation must take into account the mean flow rate as well as the transmitral velocity profile in order to provide a correct diagnosis.

Fluid-Structure Interaction and In Vitro Analysis of a Real Bileaflet Mitral Prosthetic Valve to Gain Insight Into Doppler-Silent Thrombosis.

Prosthetic valve thrombosis (PVT) is a serious complication affecting prosthetic heart valves. The transvalvular mean pressure gradient (MPG) derived by Doppler echocardiography is a crucial index to diagnose PVT but may result in false negatives mainly in case of bileaflet mechanical valves (BMVs) in mitral position. This may happen because MPG estimation relies on simplifying assumptions on the transvalvular fluid dynamics or because Doppler examination is manual and operator dependent. A deeper understanding of these issues may allow for improving PVT diagnosis and management. To this aim, we used in vitro and fluid-structure interaction (FSI) modeling to simulate the function of a real mitral BMV in different configurations: normally functioning and stenotic with symmetric and completely asymmetric leaflet opening, respectively. In each condition, the MPG was measured in vitro, computed directly from FSI simulations and derived from the corresponding velocity field through a Doppler-like postprocessing approach. Following verification versus in vitro data, MPG computational data were analyzed to test their dependency on the severity of fluid-dynamic derangements and on the measurement site. Computed MPG clearly discriminated between normally functioning and stenotic configurations. They did not depend markedly on the site of measurement, yet differences below 3 mmHg were found between MPG values at the central and lateral orifices of the BMV. This evidence suggests a mild uncertainty of the Doppler-based evaluation of the MPG due to probe positioning, which yet may lead to false negatives when analyzing subjects with almost normal MPG.

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.

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.

Microfludic platforms for the evaluation of anti-platelet agent efficacy under hyper-shear conditions associated with ventricular assist devices.

Thrombus formation is a major adverse event affecting patients implanted with ventricular assist devices (VADs). Despite anti-thrombotic drug administration, thrombotic events remain frequent within the first year post-implantation. Platelet activation (PA) is an essential process underling thrombotic adverse events in VAD systems. Indeed, abnormal shear forces, correlating with specific flow trajectories of VADs, are strong agonists mediating PA. To date, the ability to determine efficacy of anti-platelet (AP) agents under shear stress conditions is limited. Here, we present a novel microfluidic platform designed to replicate shear stress patterns of a clinical VAD, and use it to compare the efficacy of two AP agents in vitro. Gel-filtered platelets were incubated with i) acetylsalicylic acid (ASA) and ii) ticagrelor, at two different concentrations (ASA: 125 and 250 µM; ticagrelor: 250 and 500 nM) and were circulated in the VAD-emulating microfluidic platform using a peristaltic pump. GFP was collected after 4 and 52 repetitions of exposure to the VAD shear pattern and tested for shear-mediated PA. ASA significantly inhibited PA only at 2-fold higher concentration (250 µM) than therapeutic dose (125 µM). The effect of ticagrelor was not dependent on drug concentration, and did not show significant inhibition with respect to untreated control. This study demonstrates the potential use of microfluidic platforms as means of testing platelet responsiveness and AP drug efficacy under complex and realistic VAD-like shear stress conditions.

Impact of different aortic valve calcification patterns on the outcome of transcatheter aortic valve implantation: A finite element study.

Transcatheter aortic valve implantation (TAVI) can treat symptomatic patients with calcific aortic stenosis. However, the severity and distribution of the calcification of valve leaflets can impair the TAVI efficacy. Here we tackle this issue from a biomechanical standpoint, by finite element simulation of a widely adopted balloon-expandable TAVI in three models representing the aortic root with different scenarios of calcific aortic stenosis. We developed a modeling approach realistically accounting for aortic root pressurization and complex anatomy, detailed calcification patterns, and for the actual stent deployment through balloon-expansion. Numerical results highlighted the dependency on the specific calcification pattern of the "dog-boning" of the stent. Also, local stent distortions were associated with leaflet calcifications, and led to localized gaps between the TAVI stent and the aortic tissues, with potential implications in terms of paravalvular leakage. High stresses were found on calcium deposits, which may be a risk factor for stroke; their magnitude and the extent of the affected regions substantially increased for the case of an "arc-shaped" calcification, running from commissure to commissure. Moreover, high stresses due to the interaction between the aortic wall and the leaflet calcifications were computed in the annular region, suggesting an increased risk for annular damage. Our analyses suggest a relation between the alteration of the stresses in the native anatomical components and prosthetic implant with the presence and distribution of relevant calcifications. This alteration is dependent on the patient-specific features of the calcific aortic stenosis and may be a relevant indicator of suboptimal TAVI results.

Microfluidic approaches for the assessment of blood cell trauma: a focus on thrombotic risk in mechanical circulatory support devices.

INTRODUCTION: Mechanical circulatory support devices (MCSDs) are emerging as a valuable therapeutic option for the management of end-stage heart failure. However, although recipients are routinely administered with anti-thrombotic (AT) drugs, thrombosis persists as a severe post-implant complication. Conventional clinical assays and coagulation markers demonstrate partial ability in preventing the onset of thrombosis. Through years, different laboratory techniques have been proposed as potential tools for the evaluation of platelets' hemostatic response in MCSD recipients. Most rely on platelet aggregation tests; they are performed in static or low shear conditions, neglecting the prominent contribution of MCSD shear-induced mechanical load in enhancing platelet activation (PA). On the other hand, those tests able to account for shear-induced PA have limited possibility of effective clinical translation. AIMS AND METHODS: Advances on this side have been addressed by microfluidic technology. Microfluidic devices have been developed for AT drug monitoring under flow, able to replicate physiological and/or constant shear flow conditions in vitro. In this paper, we present a newly developed microfluidic platform able to expose platelets to MCSD-specific dynamic shear stress patterns. We performed in vitro tests circulating human platelets in the microfluidic platform and quantifying the dynamics of PA by means of the Platelet Activity State (PAS) assay. RESULTS: Our results prove the feasibility of using microfluidics for the diagnosis of MCSD-related thrombotic risk. This study paves the way for the development of a miniaturized point-of-care device for monitoring AT drug regimen. Such a system may have significant impact on limiting the incidence of thrombosis in MCSD recipients.

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.