Platelet Activation Potential Research Articles

Megakaryocytes possess a STING pathway that is transferred to platelets to potentiate activation.

Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor protein downstream of the DNA sensor cyclic GMP-AMP synthase (cGAS) that is activated by cytosolic microbial and self-DNA during infections, and in the context of loss of cellular integrity, to instigate the production of type-I IFN and pro-inflammatory cytokines. To date, whether the cGAS-STING pathway is present in platelets and contributes to platelet functions is not defined. Using a combination of pharmacological and genetic approaches, we demonstrate here that megakaryocytes and platelets possess a functional cGAS-STING pathway. Our results suggest that in megakaryocytes, STING stimulation activates a type-I IFN response, and during thrombopoiesis, cGAS and STING are transferred to proplatelets. Finally, we show that both murine and human platelets contain cGAS and STING proteins, and the cGAS-STING pathway contributes to potentiation of platelet activation and aggregation. Taken together, these observations establish for the first time a novel role of the cGAS-STING DNA sensing axis in the megakaryocyte and platelet lineage.

Computational investigation of outflow graft variation impact on hemocompatibility profile in LVADs.

Hemocompatibility-related adverse events (HRAE) occur commonly in patients with left ventricular assist devices (LVADs) and add to morbidity and mortality. It is unclear whether the outflow graft orientation can impact flow conditions leading to HRAE. This study presents a simulation-based approach using exact patient anatomy from medical images to investigate the influence of outflow cannula orientation in modulating flow conditions leading to HRAEs. A 3D model of a proximal aorta and outflow graft was reconstructed from a computed tomography (CT) scan of an LVAD patient and virtually modified to model multiple cannula orientations (n = 10) by varying polar (cranio-caudal) (n = 5) and off-set (anterior-posterior) (n = 2) angles. Time-dependent computational flow simulations were then performed for each anatomical orientation. Qualitative and quantitative hemodynamics metrics of thrombogenicity including time-averaged wall shear stress (TAWSS), oscillatory shear index (OSI), endothelial cell platelet activation potential (ECAP), particle residence time (PRT), and platelet activation potential (PLAP) were analyzed. Within the simulations performed, endothelial cell activation potential (ECAP) and particle residence time (PRT) were found to be lowest with a polar angle of 85°, regardless of offset angle. However, polar angles that produced parameters at levels least associated with thrombosis varied when the offset angle was changed from 0° to 12°. For offset angles of 0° and 12° respectively, flow shear was lowest at 65° and 75°, time averaged wall shear stress (TAWSS) was highest at 85° and 35°, and platelet activation potential (PLAP) was lowest at 65° and 45°. This study suggests that computational fluid dynamic modeling based on patient-specific anatomy can be a powerful analytical tool when identifying optimal positioning of an LVAD. Contrary to previous work, our findings suggest that there may be an "ideal" outflow cannula for each individual patient based on a CFD-based hemocompatibility profile.

Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation

BackgroundEspecially in disease conditions, platelets can encounter activating agents in circulation. ObjectivesTo investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation. MethodsShort-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation. ResultsAfter 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in αIIbβ3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when αIIbβ3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow. ConclusionThis work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation.

Effect of melatonin on selected parameters of platelet activation, clot formation and overall potential of fibrinolysis

Introduction: Melatonin produced in the pineal gland plays a key role in regulating sleep and wake hours. Synthetic melatonin is used as an adjunct to treat sleep disorders, regulate the sleep-wake rhythm and prevent ailments related to changing time zones or shift work. Its other applications are more widely described, including antioxidant and immunomodulatory properties – therefore melatonin supplementation may be beneficial in alleviating symptoms associated with the occurrence of COVID-19. However, reports on the influence of exogenous melatonin on the platelet, plasma and vascular hemostasis are ambiguous. Aim: The aim of the study was to evaluate the in vitro influence of melatonin on spontaneous and ADP-induced adhesion of platelets to fibrinogen, kinetic parameters of ADP-induced aggregation and selected elements of plasma haemostasis: general potential for clot formation and fibrinolysis, as well as kinetic parameters of the clot formation process, its stabilization and fibrinolysis. Material and methods: The study were performed with the use of the previously described research model, which includes the method of assessing platelet adhesion, a multi-parameter test for assessing platelets aggregation and a test that enables kinetic assessment of the clot formation process, the period of fibrin stabilization and its lysis. Results: Our preliminary studies indicated that melatonin at concentrations: 0.2-10 nmol/L does not show a significant and direct impact on the assessed kinetic parameters of the studied processes, important for platelet and plasma hemostasis. Conclusions: The pleiotropic effects of melatonin are increasingly applied, especially its antioxidant and immunomodulating properties, therefore further and in-depth in vitro as well as in vivo hemostasis studies followed by clinical observations of patients using melatonin are needed.

Noncanonical Sonic Hedgehog signaling amplifies platelet reactivity and thrombogenicity

AbstractSonic Hedgehog (Shh) is a morphogen in vertebrate embryos that is also associated with organ homeostasis in adults. We report here that human platelets, though enucleate, synthesize Shh from preexisting mRNAs upon agonist stimulation, and mobilize it for surface expression and release on extracellular vesicles, thus alluding to its putative role in platelet activation. Shh, in turn, induced a wave of noncanonical signaling in platelets leading to activation of small GTPase Ras homolog family member A and phosphorylation of myosin light chain in activated protein kinase-dependent manner. Remarkably, agonist-induced thrombogenic responses in platelets, which include platelet aggregation, granule secretion, and spreading on immobilized fibrinogen, were significantly attenuated by inhibition of Hedgehog signaling, thus, implicating inputs from Shh in potentiation of agonist-mediated platelet activation. In consistence, inhibition of the Shh pathway significantly impaired arterial thrombosis in mice. Taken together, the above observations strongly support a feed-forward loop of platelet stimulation triggered locally by Shh, similar to ADP and thromboxane A2, that contributes significantly to the stability of occlusive arterial thrombus and that can be investigated as a potential therapeutic target in thrombotic disorders.

The Supera Interwoven Nitinol Stent as a Flow Diverting Device in Popliteal Aneurysms

PurposeThe feasibility of using a compressed interwoven Supera stent as a flow diverting device for popliteal aneurysms was recently demonstrated in patients. It is unclear, however, what the optimal flow diverting strategy is, because of the fusiform shape of popliteal aneurysms and their exposure to triphasic flow. To assess this flow diverting strategy for popliteal aneurysms, flow profiles and thrombus formation likelihood were investigated in popliteal aneurysm models.Materials and MethodsSix popliteal aneurysm models were created and integrated into a pulsatile flow set-up. These models covered a bent and a straight anatomy in three configurations: control, single-lined and dual-lined Supera stents. Two-dimensional flow velocities were visualized by laser particle image velocimetry. In addition, the efficacy of the stent configurations for promoting aneurysm thrombosis was assessed by simulations of residence time and platelet activation.ResultsOn average for the two anatomies, the Supera stent led to a twofold reduction of velocities in the aneurysm for single-lined stents, and a fourfold reduction for dual-lined stents. Forward flow was optimally diverted, whereas backward flow was generally deflected into the aneurysm. The dual-lined configuration led to residence times of 15–20 s, compared to 5–15 s for the single stent configurations. Platelet activation potential was not increased by the flow diverting stents.ConclusionA compressed Supera stent was successfully able to divert flow in a popliteal aneurysm phantom. A dual-lined configuration demonstrated superior hemodynamic characteristics compared to its single-lined counterpart.

Computational Investigation of Outflow Graft Variation Impact on Hemocompatibility Profile in LVADs

<h3>Purpose</h3> Hemocompatibility related adverse events (HRAE) such as pump thrombosis, stroke, and gastrointestinal (GI) bleeding occur commonly in patients with left ventricular assist devices (LVADs). It is unclear whether the outflow graft orientation can impact flow conditions leading to HRAE. This study, used a simulation-based approach to investigate the influence that the cannula orientation has in modulating flow conditions leading to HRAEs. <h3>Methods</h3> A 3D model of a proximal aorta and outflow graft was reconstructed from a computer tomography (CT) scan of an LVAD patient and virtually modified to account for multiple cannula orientations (n=10) by varying polar (n=5) and off-set (n=2) angles. Time-dependent computer flow simulations were then performed and flow metrics as time-averaged wall shear stress (TAWSS), oscillatory shear index (OSI), endothelial cell activation potential (ECAP), flow shear (S), particle residence time (PRT), and platelet activation potential (PLAP) estimated. <h3>Results</h3> Flow shear was highest and lowest at the proximal aorta closer to the anastomosis and aortic sinus, respectively, in all configurations. Among all angles, time and space-averaged flow shear values were lowest at polar angles of 65° and 75° for 0° and 12° off-set angles, respectively. Flow metrics associated to thrombosis such as low TAWSS and high PRT, OSI, and ECAP were high at the aorta proximal to the sinus. <h3>Conclusion</h3> Acuter polar angles are commonly associated to flow conditions leading to fewer HRAEs. Using a time-depend flow simulation, this study suggests that an "ideal acute" rather than an acuter polar angle might lead to such flow conditions.

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

Platelet procoagulant potential is reduced in platelet concentrates ex vivo but appears restored following transfusion.

The procoagulant profile of platelet concentrates (PCs) following transfusion has been difficult to evaluate due to lack of specific markers. This study aimed to characterize procoagulant platelets in PCs and the effect of transfusion. Buffy coat-derived PCs from 12 donors were pooled, split, then stored conventionally, cold (2-6°C) or cryopreserved (-80°C). Procoagulant platelet profiles were assessed by flow cytometry (GSAO+ /P-selectin+ ), lactadherin-binding, and calibrated automated thrombogram, during storage, unstimulated, or after thrombin and collagen stimulation and compared with blood from healthy volunteers. Platelet activation (P-selectin) and procoagulant platelet formation potential were measured (flow cytometry) in patients receiving clinically indicated conventional PC transfusion. Independent of significant increases with storage, procoagulant platelet proportions with and without agonist stimulation were significantly blunted in conventionally stored PCs (stimulated day 5 conventional PC 4.2 ± 1.3%, healthy volunteer blood 11.1 ± 2.9%; p< .0001). Cryopreserved PCs contained the highest proportion of procoagulant platelets (unstimulated: cryopreserved 25.6 ± 1.8% vs. day 5 conventional 0.5 ± 0.1% vs. day 14 cold-stored 5.8 ± 1.0%, p< .0001), but demonstrated minimal increase with agonist. Transfusion of PCs was associated with an increase in procoagulant platelets (2.2 ± 1.4% vs. 0.6 ± 0.2%; p= .004) and reversal of the blunted agonist response (15.8 ± 5.9% vs. 4.0 ± 1.6%; p< .0001). Procoagulant responses post-transfusion were significantly higher than healthy controls, suggesting a priming effect. The P-selectin agonist response was not restored upon transfusion (79.4 ± 13.9% vs. 82.0 ± 2.5%). Storage blunts the procoagulant platelet response to agonist stimulation in PCs. Despite this, conventionally stored PCs have high procoagulant potential following transfusion, with a discordant, persistent reduction in P-selectin response.

Patient-Specific Computational Analysis of the Impact of Fenestrated and Chimney Endovascular Aortic Repair on Haemodynamics in Renal Arteries

Introduction: Fenestrated and chimney endovascular aortic repair with renal artery vascularization enables minimally invasive repair of juxtarenal aortic aneurysms. However, there are concerns regarding postoperative kidney function. The aim was to analyze the hemodynamic impact of fenestrations and chimneys on renal arteries (RA) in 2 patient-specific series of computer models using Computational Fluid Dynamics (CFD) simulations. Methods: We included 2 patients who had juxta-renal abdominal endovascular aortic repair (EVAR): 1 fenestrated-EVAR (F-EVAR) and 1 chimney-EVAR (Ch-EVAR). For each patient, 3 models were built from CT-scan in the software CRIMSON: preoperative, real postoperative and virtual postoperative models. The virtual model was an alternative scenario with replacement of renal fenestrated stentgrafts with renal chimneys in the first patient, and vice versa in the second patient. Patient-specific boundary conditions remained constant in all models for each patient. CFD analysis of hemodynamics was performed using CRIMSON. Pressure, flow, velocity, time-averaged wall shear stress (TAWSS) and platelet activation potential (PLAP) were compared in all models. Results: Aortic and RA flows were constant in preoperative and postoperative models for each patient. Peak velocity was higher in postoperative models compared to preoperative ones in both patients. Higher vortices were observed in both F-EVAR models and lower vortices in both Ch-EVAR. Flow recirculation zones were located below and above the aortic protruding proximal part of the renal stentgraft in both F-EVAR. Aortic inlet systolic pressures were higher in F-EVAR compared to preoperative and Ch-EVAR models. Renal systolic pressures were higher in both F-EVAR compared to Ch-EVAR. Renal mean pressure was the lowest in Ch-EVAR. TAWSS was lower in F-EVAR RA compared to Ch-EVAR in both patients. TAWSS was the highest in both Ch-EVAR. Both Ch-EVAR seem to carry higher risk of thrombosis in RA as the PLAP increase relative to the preoperative model was the highest at Ch-EVAR RA, around 40 %-increase in Ch-EVAR versus 20% in F-EVAR. Conclusion: Complex EVAR resulted in aortorenal flow disturbances that potentially contribute to renal stent-graft complications during follow-up. This intra-individual numerical comparison of the hemodynamic impact of complex EVAR on RA showed that Ch-EVAR carried the highest risk of RA thrombosis. F-EVAR increased renal and inlet pressures exposing the vascular wall to remodeling and wall thickening. Preoperative planning of endovascular repair could help physician to make the best choice between different treatment but further studies with more patient specific computational analysis of hemodynamics need to be performed. Disclosure: Nothing to disclose

Numerical investigation on the effect of bileaflet mechanical heart valve's implantation tilting angle and aortic root geometry on intermittent regurgitation and platelet activation.

Platelet activation induced by shear stresses and non-physiological flow field generated by bileaflet mechanical heart valves (BMHVs) leads to thromboembolism, which can cause fatal consequences. One of the causes of platelet activation could be intermittent regurgitation, which arises due to asynchronous movement and rebound of BMHV leaflets during the valve closing phase. In this numerical study, the effect of intermittent regurgitation on the platelet activation potential of BMHVs was quantified by modeling a BMHV in the straight and anatomic aorta at implantation tilt angles 0°, 5°, 10°, and 20°. A fully implicit Arbitrary Lagrangian-Eulerian-based Fluid-Structure Interaction formulation was adopted with blood modeled as a multiphase, non-Newtonian fluid. Results showed that the intermittent regurgitation and consequently the platelet activation level increases with the increasing implantation tilt of BMHV. For the straight aorta, the leaflet of the 20° tilted BMHV underwent a rebound of approximately 20° after initially closing, whereas the leaflet of the 10°, 5°, and 0° tilted BMHVs underwent a rebound of 8.5°, 3°, and 0°, respectively. For the anatomic aorta, the leaflet of the 20° tilted BMHV underwent a rebound of approximately 24° after initially closing, whereas the leaflet of the 10°, 5°, and 0° tilted BMHVs underwent a rebound of 14°, 10°, and 7°, respectively. For all the implantation orientations of BMHVs, intermittent regurgitation and platelet activation were always higher in the anatomic aorta than in the straight aorta. The study concludes that the pivot axis of BMHV must be implanted parallel to the aortic root's curvature to minimize intermittent regurgitation and platelet activation.

Procoagulant Platelet Potential Is Inversely Correlated with Bleeding and Joint Disease in Severe Hemophilia A

The variability of clinical phenotype in patients with severe hemophilia A (factor VIII activity < 1%) has been well established, currently and in the era prior to wide-spread prophylaxis. The introduction of prophylaxis as a treatment modifier of the natural history of severe hemophilia A has led to the introduction of composite scores to assist in the assessment of disease severity. Perhaps the most prominent of these is that proposed by Schulman et al (J Thromb Haem 2008;6(7):1113-21) which utilizes three domains of phenotypic presentation to characterize a patient's clinical phenotype, acute (annualized bleeding rate), chronic (joint status), and treatment intensity (normalized factor usage). However, the biologic mechanisms driving this variability in clinical phenotype continue to remain largely uncertain. Procoagulant platelet potential is an individual's ability to generate procoagulant platelets in response to a standard concentration of agonist. This response, which generates platelets capable of intensely supporting coagulation, tends to remain relatively constant and varies substantially between individuals. A few small case-control studies have investigated the role of procoagulant platelet potential or platelet-supported thrombin generation in mediating the phenotypic variability with discordant results.The relationship of procoagulant platelet potential and platelet-supported thrombin generation to phenotypic severity was investigated in a single-center cohort of 92 pediatric and adult severe hemophilia A patients without active inhibitors. Procoagulant platelet potential (defined as the ratio of procoagulant platelets / all activated platelets following stimulation with thrombin (0.5 units/mL) and convulxin (250 ng/mL)) and the expression of other platelet activation markers, including P-selectin (granule release) and PAC-1 (integrin activation) were investigated in washed platelets. Platelet-dependent thrombin generation was investigated in a limited cohort (45) of these patients. Phenotypic data was collected in regard to reported bleeding events over the past 5 years, joint health using the hemophilia joint health score, intensity of factor therapy, and therapeutic intent (prophylaxis or on-demand). Correlations were assessed as continuous variables using regression analysis with p <0.05 deemed significant.Procoagulant platelet potential was inversely correlated with both acute (annualized bleeding rate) (r = -0.47, p = 0.0006) and chronic measures (joint score) (r = -0.38, p = 0.0001) of phenotypic severity. In contrast, when platelet-dependent thrombin generation or other markers of thrombin or GPVI-dependent platelet activation potential were assessed i.e. P-selectin expression or PAC-1, no correlation was identified with either acute or chronic measures of phenotypic severity or with the HSS composite score. Surprisingly, when the composite score of HSS as defined by Schulman (acute + chronic + factor utilization) was utilized to assess a potential relationship this correlation was not observed. Further analysis revealed that this lack of correlation was due to the fact that therapeutic intensity (factor utilization) was directlycorrelated with procoagulant platelet potential (r = 0.54, p = 0.0006).Here we have identified a potentially critical role for procoagulant platelets as biologic modifiers of hemophilia A. Furthermore, we have identified a risk associated with the use of a composite score in assessing disease severity in hemophilia. Utilization of a composite score as the sole measure would have missed the significant effects noted in each of the individual domains. Procoagulant platelets both support thrombin generation and inflammatory cell recruitment. Thus, one potential hypothesis for these opposing effects on the domains of the composite score is that increased procoagulant platelet formation supports stable hemostatic plug formation (reduced bleeding rate) or increases inflammatory presentation resulting in therapy intensification or both. Finally, our results here point towards new tools and pathways for risk stratification and therapeutic modulation in patients with hemophilia A based on assessment of procoagulant platelet potential. DisclosuresJobe:Shire: Consultancy; Octapharma: Consultancy; CSL: Consultancy.

Reducing the effects of compressibility in DPD-based blood flow simulations through severe stenotic microchannels

Viscous fluid flow simulations based on dissipative particle dynamics (DPD) may bear compressible flow effects when flowing through severe stenotic geometries. This is caused by the soft repulsive potential employed in the DPD force field, which limits the particle-based fluid system ability to sustain a large degree of compression. To mitigate this problem, a Morse potential was added to the DPD force field. We studied the fluid properties of the modified fluid model (DPD–Morse) and compared it with a previously published conventional DPD based fluid model. Our DPD–Morse model demonstrated reduced compressibility while preserving other fluid properties such as the fluid density and viscosity. We further investigated the fluid flow properties for a severe 3D stenotic microchannel with a 67% stenosis, using the two models. The DPD fluid model presented a significant density gradient along the flow direction, where the fluid density increased upstream towards the stenosis and decreased downstream from the stenosis before regaining its initial value. In contrast, the DPD–Morse model demonstrated a far better uniform fluid density distribution along the flow direction. We compared both solutions with CFD simulations. The DPD–Morse fluid resembled the behavior of the continuum fluid model whereas DPD fluid deviated from it. To estimate the effect that the difference between the two DPD formulations may have on the platelet activation potential, we have further embedded a platelet model within the flow field and investigated the shear stress accumulation along the platelet transport trajectory. In the stenotic section, the DPD fluid demonstrated a larger stress gradient than the DPD–Morse fluid. The platelet transport period was shorter for the DPD fluid as it generated a larger fluid density gradient that overestimated the acceleration of the platelet through the stenosis. With reduced fluid compressibility, our modified DPD–Morse fluid model was more accurate than the DPD fluid model when computing the platelet activation potential in a severe stenosis.

Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP

Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin αIIbβ3 through interactions with the KGD/KGE sequence motif in huPK. Integrin αIIbβ3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS473, ERK1/2, and p38 MAPK, and to Ca2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and αIIbβ3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis.

Computational Fluid Dynamics and Aortic Thrombus Formation Following Thoracic Endovascular Aortic Repair

We present the possible utility of computational fluid dynamics in the assessment of thrombus formation and virtual surgical planning illustrated in a patient with aortic thrombus in a kinked ascending aortic graft following thoracic endovascular aortic repair. A patient-specific three-dimensional model was built from computed tomography. Additionally, we modeled 3 virtual aortic interventions to assess their effect on thrombosis potential: (1) open surgical repair, (2) conformable endografting, and (3) single-branched endografting. Flow waveforms were extracted from echocardiography and used for the simulations. We used the computational index termed platelet activation potential (PLAP) representing accumulated shear rates of fluid particles within a fluid domain to assess thrombosis potential. The baseline model revealed high PLAP in the entire arch (119.8 ± 42.5), with significantly larger PLAP at the thrombus location (125.4 ± 41.2, p < 0.001). Surgical repair showed a 37% PLAP reduction at the thrombus location (78.6 ± 25.3, p < 0.001) and a 24% reduction in the arch (91.6 ± 28.9, p < 0.001). Single-branched endografting reduced PLAP in the thrombus region by 20% (99.7 ± 24.6, p < 0.001) and by 14% in the arch (103.8 ± 26.1, p < 0.001), whereas a more conformable endograft did not have a profound effect, resulting in a modest 4% PLAP increase (130.6 ± 43.7, p < 0.001) in the thrombus region relative to the baseline case. Regions of high PLAP were associated with aortic thrombus. Aortic repair resolved pathologic flow patterns, reducing PLAP. Branched endografting also relieved complex flow patterns reducing PLAP. Computational fluid dynamics may assist in the prediction of aortic thrombus formation in hemodynamically complex cases and help guide repair strategies.

Tamoxifen Directly Inhibits Platelet Activation, Angiogenic Potential and Platelet-Mediated Metastasis

Objective - Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in tumor metastasis and neovascularization. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Platelets carry a plethora of potent angiogenic and metastatic mediators within their alpha-granules and exposure to breast tumor cells induces platelet activation, leading to release of these mediators. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer; its use is associated with a 50% reduction in the risk of invasive and noninvasive breast cancer in women who utilized the drug for at least 5 years. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown to directly impact platelet function. Because platelets are critical for metastatic spread, we posited that tamoxifen or its metabolites may exert anti-tumor effects through direct platelet inhibition. To test this hypothesis, we examined the impact of tamoxifen on platelet activation, angiogenic potential and metastasis both ex vivoand in breast cancer patients utilizing tamoxifen therapy.Approach and Results - We found that ex vivo pretreatment with tamoxifen or its metabolite 4-hydroxytamoxifen (4-OH) lead to a significant inhibition of platelet activation in response to TRAP, ADP or the breast tumor cell lines MDA-MB-231 and MCF-7 in platelets from healthy human donors. Platelets, known to promote tumor angiogenesis, are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Activated platelets exposed to tamoxifen or 4-OH released significantly lower amounts of the pro-angiogenic regulator VEGF in while anti-angiogenic endostatin release is unaffected, thus shifting the balance of angiogenic regulators that are released. To examine the impact of this alteration in angiogenic protein release, we performed functional angiogenesis assays using releasates generated from tamoxifen or 4-OH treated platelets. These in vitroangiogenesis assays confirmed that tamoxifen pretreatment led to dramatically diminished capillary tube formation and decreased endothelial migration. Tamoxifen also significantly inhibited the ability of platelets to promote metastasis in vitro, causing a dramatic decrease in breast tumor cell invasion and transendothelial migration. Next, we utilized membrane-based cytokine arrays to further interrogate the effect of tamoxifen on the release of stored platelet factors. Using this method, we identified several key proteins known to be associated with metastasis that were lower in releasate from tamoxifen treated platelets including TGF-b, IL-6 and IGF-1 while anti-angiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished VEGF release and lower angiogenic potential.Conclusions - Overall we demonstrate that tamoxifen directly influences the release of specific stored platelet factors leading to decreased tumor cell support. The mechanism is directly linked to tamoxifen's inhibitory role in platelet activation, causing altered of release of key platelet-derived angiogenic and metastatic factors including VEGF, angiopoietin-1, TGF-b, IL-6 and IGF-1 during tumor cell and platelet crosstalk. Furthermore, translational studies confirmed that platelet activation and angiogenic potential are significantly suppressed in breast cancer patients utilizing tamoxifen therapy. Our work stresses the importance of platelets for successful angiogenesis and metastatic spread and, ultimately, supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in malignancy. DisclosuresNo relevant conflicts of interest to declare.

Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations

Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion.

Mechanical Platelet Activation Potential in Abdominal Aortic Aneurysms

Intraluminal thrombus (ILT) in abdominal aortic aneurysms (AAA) has potential implications to aneurysm growth and rupture risk; yet, the mechanisms underlying its development remain poorly understood. Some researchers have proposed that ILT development may be driven by biomechanical platelet activation within the AAA, followed by adhesion in regions of low wall shear stress. Studies have investigated wall shear stress levels within AAA, but platelet activation potential (AP) has not been quantified. In this study, patient-specific computational fluid dynamic (CFD) models were used to analyze stress-induced AP within AAA under rest and exercise flow conditions. The analysis was conducted using Lagrangian particle-based and Eulerian continuum-based approaches, and the results were compared. Results indicated that biomechanical platelet activation is unlikely to play a significant role for the conditions considered. No consistent trend was observed in comparing rest and exercise conditions, but the functional dependence of AP on stress magnitude and exposure time can have a large impact on absolute levels of anticipated platelet AP. The Lagrangian method obtained higher peak AP values, although this difference was limited to a small percentage of particles that falls below reported levels of physiologic background platelet activation.

A review of fluid-structure interaction simulations of prosthetic heart valves.

Dysfunctional natural heart valves are replaced with prosthetic heart valves through surgery. However, prosthetic valves are far from ideal. Bioprosthetic heart valves (BHVs) suffer from early calcification and structural damages. Mechanical heart valves (MHVs) are durable but highly thrombogenic and require lifelong anticoagulant treatment. These complications are believed to be related to nonphysiologic flow patterns created by these valves. Fluid-structure interaction (FSI) simulations are essential in revealing the hemodynamics of these valves. By combining the three-dimensional (3D) flow field obtained from realistic FSI simulations with platelet activation models, nonphysiologic flow patterns can be identified. In this review paper, state-of-the-art methods for simulating FSI in heart valves are reviewed, and the flow physics uncovered by FSI simulations are discussed. Finally, the limitations of current methods are discussed, and future research directions are proposed as follows: (1) incorporation of realistic, image-based ventricle and atrium geometries; (2) comparing MHV and BHV under similar conditions to identify nonphysiologic flow patterns; (3) developing better models to estimate platelet activation potential to be incorporated into the simulations; and (4) identifying the optimum placement of the valves in both mitral and aortic positions.