Routine clinical anti-platelet agents have limited efficacy in modulating hypershear-mediated platelet activation associated with mechanical circulatory support.

INTRODUCTION: Continuous flow ventricular assist devices (cfVADs) continue to be limited by thrombotic complications associated with disruptive flow patterns and supraphysiologic shear stresses. Patients are prescribed complex antiplatelet therapies, which do not fully prevent recurrent thromboembolic events. This is partially due to limited data on antiplatelet efficacy under cfVAD-associated shear conditions. MATERIALS AND METHODS: We investigated the efficacy of antiplatelet drugs directly acting on three pathways: (1) cyclooxygenase (aspirin), (2) phosphodiesterase (dipyridamole, pentoxifylline, cilostazol), and (3) glycoprotein IIb-IIIa (eptifibatide). Gel-filtered platelets treated with these drugs were exposed for 10min to either constant shear stresses (30dyne/cm2 and 70dyne/cm2) or dynamic shear stress profiles extracted from simulated platelet trajectories through a cfVAD (Micromed DeBakey). Platelet activation state (PAS) was measured using a modified prothrombinase-based assay, with drug efficacy quantified based on PAS reduction compared to untreated controls. RESULTS AND CONCLUSIONS: Significant PAS reduction was observed for all drugs after exposure to 30dyne/cm2 constant shear stress, and all drugs but dipyridamole after exposure to the 30th percentile shear stress waveform of the cfVAD. However, only cilostazol was significantly effective after 70dyne/cm2 constant shear stress exposure, though no significant reduction was observed upon exposure to median shear stress conditions in the cfVAD. These results, coupled with the persistence of reported clinical thrombotic complication, suggest the need for the development of new classes of drugs that are especially designed to mitigate thrombosis in cfVAD patients, while reducing or eliminating the risk of bleeding.

Platelet activation is a preoperative risk factor for the development of thromboembolic complications in patients with continuous-flow left ventricular assist device.

AIMS: To correlate the dynamics of platelet activation with the development of thromboembolic events in patients with continuous-flow left ventricular assist device (cf-LVAD). METHODS AND RESULTS: The platelet activity state (PAS) assay was utilized to evaluate platelet activation in 68 cf-LVAD patients implanted with the HeartMate II (n = 15, 22%), HeartMate 3 (n = 15, 22%), or HeartWare HVAD (n = 38, 56%). PAS was measured preoperatively, early post-implant, and at long-term follow-up (1, 3, 6, 12, 18, and 24 months post-implant). PAS was also measured at the occurrence of adverse events in patients who developed thrombotic complications. Data on patient demographics, medical history, antithrombotic therapy, and coagulation parameters were also analysed. Over a median follow-up of 602 (234-942) days, PAS values did not increase over time in the overall population (P = 0.15). However, PAS measured at event was 15-fold higher in the six patients (9%) who suffered pump thrombosis (n = 2) or ischaemic stroke (n = 4) vs. the rest of the population [6.67% (5.59%-11.98%) vs. 0.45% (0.33%-0.75%); P = 0.012], despite comparable coagulation profile. Pre-implant PAS values were 4.5-fold higher in these patients [1.90% (1.24%-3.17%) vs. 0.42% (0.32%-0.72%); P = 0.006]. Neither preoperative variables nor the type of the pump or the antiplatelet strategy were associated with a higher risk of complications. CONCLUSIONS: Thrombotic events are associated with altered PAS values. Moreover, baseline elevated PAS values in patients who developed thrombotic events suggest patient-specific tendency to post-implant thromboembolic complications. Prospectively, systematic monitoring of PAS might guide the development of refined patient-tailored antithrombotic strategies and the technological improvement of LVAD design.

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.

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 125 µM) to limit shear-mediated platelet activation under conditions of: 1) constant shear stress (30 dynes/cm(2) and 70 dynes/cm(2)); 2) dynamic shear stress, and 3) initial high shear exposure (70 dynes/cm(2)) 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.

On the Use of the Platelet Activity State Assay for the In Vitro Quantification of Platelet Activation in Blood Recirculating Devices for Extracorporeal Circulation.

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinent flow conditions (500 mL/min), while providing straightforward information on shear-mediated PA dynamics in ECC devices. Being that the latter is intimately dependent on local flow dynamics, according to our results, the rate of thrombin production as measured by the PAS assay is a valuable biochemical marker of the selective contribution of PA in BRDs induced by device design features. Thus, we recommend the use of PAS assay as a means of evaluating the effect of modification of specific device geometrical features and/or different design solutions for developing ECC devices providing flow conditions with reduced thrombogenic impact.

Hemolysate-mediated platelet aggregation: an additional risk mechanism contributing to thrombosis of continuous flow ventricular assist devices.

Despite the clinical success and growth in the utilization of continuous flow ventricular assist devices (cfVADs) for the treatment of advanced heart failure, hemolysis and thrombosis remain major limitations. Inadequate and/or ineffective anticoagulation regimens, combined with high pump speed and non-physiological flow patterns, can result in hemolysis which often is accompanied by pump thrombosis. An unexpected increase in cfVADs thrombosis was reported by multiple major VAD implanting centers in 2014, highlighting the association of hemolysis and a rise in lactate dehydrogenase (LDH) presaging thrombotic events. It is well established that thrombotic complications arise from the abnormal shear stresses generated by cfVADs. What remains unknown is the link between cfVAD-associated hemolysis and pump thrombosis. Can hemolysis of red blood cells (RBCs) contribute to platelet aggregation, thereby, facilitating prothrombotic complications in cfVADs? Herein, we examine the effect of RBC-hemolysate and selected major constituents, i.e., lactate dehydrogenase (LDH) and plasma free hemoglobin (pHb) on platelet aggregation, utilizing electrical resistance aggregometry. Our hypothesis is that elements of RBCs, released as a result of shear-mediated hemolysis, will contribute to platelet aggregation. We show that RBC hemolysate and pHb, but not LDH, are direct contributors to platelet aggregation, posing an additional risk mechanism for cfVAD thrombosis.

Shear-mediated platelet activation in patients implanted with continuous flow LVADs: A preliminary study utilizing the platelet activity state (PAS) assay.

Left ventricular assist devices (LVADs) have emerged as vital life-saving therapeutic systems for patients with advanced and end-stage heart failure (HF). Despite their efficacy, VAD systems remain limited by post-implantation thrombotic complications. Shear-mediated platelet activation is the major driver of such complications in these devices. Nowadays few platelet function assays are routinely utilized in assessing the degree of platelet activation in VAD implanted patients. No assays exist that specifically target shear-mediated platelet activation. The platelet activity state (PAS) is a novel assay that has been well validated in vitro, measuring thrombin release as a surrogate for shear-mediated platelet activation. To date limited data exist as to the utility of this assay in the clinical setting. In the present study we evaluated eight LVAD patients' platelet activation level using the PAS assay. Simultaneous measurements of conventional prothrombotic and hemolysis markers, - i.e. fibrinogen and lactate dehydrogenase (LDH) - were also performed. Trends as to alteration from baseline were studied. We observed that the PAS assay allowed detection of an abnormal level of platelet activation in one patient in our series who suffered from an overt thrombosis. Interestingly in the same patient no signal of major abnormality in fibrinogen or LDH was detected. Further for 7/8 patients who were free of thrombosis, no significant level of platelet activation was detected via PAS assay, while elevation in fibrinogen and LDH were observed. As such, from our observational series it appears that the PAS assay is a sensitive and specific indicator of shear-mediated platelet activation. Further patients' experience will help elucidate the role of this promising assay in the management of LVAD implanted patients.

Mobility timing for agent communities, a cue for advanced connectionist systems.

We introduce a wait-and-chase scheme that models the contact times between moving agents within a connectionist construct. The idea that elementary processors move within a network to get a proper position is borne out both by biological neurons in the brain morphogenesis and by agents within social networks. From the former, we take inspiration to devise a medium-term project for new artificial neural network training procedures where mobile neurons exchange data only when they are close to one another in a proper space (are in contact). From the latter, we accumulate mobility tracks experience. We focus on the preliminary step of characterizing the elapsed time between neuron contacts, which results from a spatial process fitting in the family of random processes with memory, where chasing neurons are stochastically driven by the goal of hitting target neurons. Thus, we add an unprecedented mobility model to the literature in the field, introducing a distribution law of the intercontact times that merges features of both negative exponential and Pareto distribution laws. We give a constructive description and implementation of our model, as well as a short analytical form whose parameters are suitably estimated in terms of confidence intervals from experimental data. Numerical experiments show the model and related inference tools to be sufficiently robust to cope with two main requisites for its exploitation in a neural network: the nonindependence of the observed intercontact times and the feasibility of the model inversion problem to infer suitable mobility parameters.