Atherosclerotic Material Research Articles

The Btk inhibitor AB‐95‐LH34 potently inhibits atherosclerotic plaque–induced thrombus formation and platelet procoagulant activity

BackgroundNew antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque–mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non–Btk‐mediated effects. Btk‐deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy. ObjectivesTo compare the antithrombotic effects of the highly selective Btk inhibitor AB‐95‐LH34 (LH34) with ibrutinib. MethodsGlycoprotein VI and G‐protein coupled receptor‐mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi‐aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro. ResultsLH34 showed similar inhibition of Btk‐Y223 phosphorylation as ibrutinib, but had no off‐target inhibition of Src‐Y418 phosphorylation. Similar dose‐dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2β1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P‐selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque‐induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib. ConclusionsLH34 potently inhibits atherosclerotic plaque–induced thrombus formation and procoagulant platelet activity in vitro, with less off‐target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects.

The effect of Bruton's tyrosine kinase inhibitor ibrutinib on atherothrombus formation under stenotic flow conditions

BackgroundBruton's kinase (Btk) is critical for collagen-triggered platelet signal transduction. The Btk inhibitor ibrutinib has been shown to selectively block platelet adhesion to atherosclerotic plaque material under laminar arterial flow. However, this has not been studied under a shear gradient, which is characteristic for atherothrombosis. ObjectiveTo determine the effect of ibrutinib treatment on in vitro thrombus formation on collagen and atherosclerotic plaque material in the absence or presence of a shear gradient. MethodsBlood was obtained from patients with chronic lymphocytic leukemia, mantle-cell lymphoma and Waldenström macroglobulinemia with and without ibrutinib treatment and perfused through a microfluidic channel with(out) 60% stenosis over Horm type I collagen or human atherosclerotic plaque homogenate. ResultsAt a constant shear rate of 1500 s−1, platelet deposition was significantly decreased in blood from haematological malignancy patients treated with ibrutinib as compared to untreated patients, on atherosclerotic plaque material but not on collagen. However, thrombus size, stability, and height, were reduced on both plaque material and collagen. An increase in shear rate up to 3900 s−1, as induced by 60% stenosis, resulted in decreased platelet deposition and thrombus parameters on plaque material but not on collagen when compared to a laminar shear of 1500 s−1. Ibrutinib treatment decreased platelet deposition and thrombus parameters even further around the stenosis. ConclusionTreatment of patients with haematological disorders with the Btk inhibitor ibrutinib reduces in vitro platelet deposition, thrombus size and contraction on human atherosclerotic plaque around a stenosis when compared to patients not receiving ibrutinib.

Multicomponent material property characterization of atherosclerotic human carotid arteries through a Bayesian Optimization based inverse finite element approach

ObjectivePlaque rupture in atherosclerotic carotid arteries is a main cause of ischemic stroke and it is correlated with high plaque stresses. Hence, analyzing stress patterns is essential for plaque specific rupture risk assessment. However, the critical information of the multicomponent material properties of atherosclerotic carotid arteries is still lacking greatly. This work aims to characterize component-wise material properties of atherosclerotic human carotid arteries under (almost) physiological loading conditions. MethodsAn inverse finite element modeling (iFEM) framework was developed to characterize fibrous intima and vessel wall material properties of 13 cross sections from five carotids. The novel pipeline comprised ex-vivo inflation testing, pre-clinical high frequency ultrasound for deriving plaque deformations, pre-clinical high-magnetic field magnetic resonance imaging, finite element modeling, and a sample efficient machine learning based Bayesian Optimization. ResultsThe nonlinear Yeoh constants for the fibrous intima and wall layers were successfully obtained. The optimization scheme of the iFEM reached the global minimum with a mean error of 3.8% in 133 iterations on average. The uniqueness of the results were confirmed with the inverted Gaussian Process (GP) model trained during the iFEM protocol. ConclusionThe developed iFEM approach combined with the inverted GP model successfully predicted component-wise material properties of intact atherosclerotic human carotids ex-vivo under physiological-like loading conditions. SignificanceWe developed a novel iFEM framework for the nonlinear, component-wise material characterization of atherosclerotic arteries and utilized it to obtain human atherosclerotic carotid material properties. The developed iFEM framework has great potential to be advanced for patient-specific in-vivo application.

Characterization of Atherosclerotic Plaque Coating for Thrombosis Microfluidics Assays

IntroductionStudying arterial thrombus formation by in vitro flow assays is a widely used approach. Incorporating human atherosclerotic plaque material as a thrombogenic surface in these assays represents a method to model the pathophysiological environment of thrombus formation upon plaque disruption. Up until now, achieving a homogeneous coating of plaque material and subsequent reproducible platelet adhesion has been challenging. Here, we characterized a novel method for coating of plaque material on glass coverslips for use in thrombosis microfluidic assays.MethodsA homogenate of human atherosclerotic plaques was coated on glass coverslips by conventional manual droplet coating or by spin coating. Prior to coating, a subset of coverslips was plasma treated. Water contact angle measurements were performed as an indicator for the hydrophilicity of the coverslips. Homogeneity of plaque coatings was determined using profilometric analysis and scanning electron microscopy. Thrombogenicity of the plaque material was assessed in real time by microscopic imaging while perfusing whole blood at a shear rate of 1500 s−1 over the plaque material.ResultsPlasma treatment of glass coverslips, prior to spin coating with plaque material, increased the hydrophilicity of the coverslip compared to no plasma treatment. The most homogeneous plaque coating and highest platelet adhesion was obtained upon plasma treatment followed by spin coating of the plaque material. Manual plaque coating on non-plasma treated coverslips yielded lowest coating homogeneity and platelet adhesion and activation.ConclusionSpin coating of atherosclerotic plaque material on plasma treated coverslips leads to a more homogenous coating and improved platelet adhesion to the plaque when compared to conventional droplet coating on non-plasma treated coverslips. These properties are beneficial in ensuring the quality and reproducibility of flow experiments.

GPVI inhibition by glenzocimab synergistically inhibits atherosclerotic plaque-induced platelet activation when combined with conventional dual antiplatelet therapy

Abstract Introduction Aspirin and a potent platelet P2Y12 inhibitor, such as prasugrel or ticagrelor, are not always sufficient to prevent thrombus formation in patients with ST-elevation MI (STEMI), leading to “slow flow” or “no reflow” effects after stenting. GPIIb/IIIa inhibitors, such as eptifibatide, may help in this setting, but are not used routinely due to their bleeding risk. GPVI has critical roles in thrombosis and a minimal role in haemostasis. Here we tested whether depletion of GPVI has effects on thrombus formation after MI in an animal model and investigated the effects of a novel platelet GPVI inhibitor, glenzocimab (a Fab fragment of a monoclonal antibody), on platelet activation and thrombus formation when combined with aspirin and ticagrelor. Methods We used intravital microscopy in a murine model of ST-elevation myocardial infarction and ischaemia-reperfusion injury to investigate microvascular thrombosis. We investigated the antithrombotic effects of adding glenzocimab (previously known as ACT017) to blood from healthy donors and 20 patients with ACS treated with aspirin and ticagrelor. We compared the effect of glenzocimab with the GPIIb/IIIa inhibitor eptifibatide ex-vivo. We stimulated platelets with collagen and atherosclerotic plaque material that was sourced from patients undergoing carotid endarterectomy. We investigated effects on platelet aggregation, spreading, signalling, adhesion, thrombin generation, thrombus formation and clot stability ex vivo. Results Genetic depletion of GPVI in an animal model of myocardial infarction reduced microvascular thrombosis. Ex vivo, aspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation (assessed by multiple electrode aggregometry) by 48% compared to control (34±3 vs. 65±4 U; P<0.001; Figure 1). Atherosclerotic plaque-induced platelet aggregation, adhesion, secretion and activation were critically dependent on platelet GPVI activation and were potently inhibited by glenzocimab. Glenzocimab alone reduced atherosclerotic plaque-induced platelet aggregation by 75% compared to control (16±4 vs. 65±4 U; P<0.001; Figure 1) and by over 95% when combined with aspirin and ticagrelor (3±1 vs 65±4 U; P<0.001; Figure 1). Furthermore, glenzocimab provided multiple synergistic antithrombotic effects when added to the blood of aspirin and ticagrelor-treated patients with ACS ex vivo. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, had many similar antithrombotic effects but glenzocimab had less effect on mechanisms of general haemostasis compared to eptifibatide, as assessed by ROTEM (Figure 2). Conclusions The addition of glenzocimab to aspirin and ticagrelor provides synergistic inhibition of multiple critical mechanisms of atherothrombosis. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, share many similar antithrombotic effects, although glenzocimab has less impact on mechanisms involved in haemostasis compared to eptifibatide. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Academy of Medical Sciences UK Clinical Lecturer Starter GrantRoyal Embassy of Saudi Arabia

Helical Structures for Medical Applications

The latest advances in the field of microrobotics assisted medical experts in the treatment of some of human diseases especially intravascular diseases such as atherosclerosis. In this research, a phantom for the atherosclerotic plaque material is fabricated using Gelatine and Nano Hydroxyapatite in a catheter tube to mimic the condition in the human body in vitro. For plaque penetration a helical microrobot is designed to achieve the optimum plaque clearing efficiency. The robot is a remotely controlled micro helical magnetic based metallic robot which is inserted in the catheter to penetrate the phantom by rotational motion along its axis. The motion of the robot during mechanical grinding of the plaque phantom is simulated using MATLAB where the distance of the robot’s journey inside the plaque phantom is plotted against the time consumed by the robot.

Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin.

Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kβ, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.

MiR-223-3p and miR-122-5p as circulating biomarkers for plaque instability.

BackgroundIn this study, we discovered and validated candidate microRNA (miRNA) biomarkers for coronary artery disease (CAD).MethodCandidate tissue-derived miRNAs from atherosclerotic plaque material in patients with stable coronary artery disease (SCAD) (n=14) and unstable coronary artery disease (UCAD) (n=25) were discovered by qPCR-based arrays. We validated differentially expressed miRNAs, along with seven promising CAD-associated miRNAs from the literature, in the serum of two large cohorts (n=395 and n=1000) of patients with SCAD and UCAD and subclinical atherosclerosis (SubA) and controls, respectively.ResultFrom plaque materials (discovery phase), miR-125b-5p and miR-193b-3p were most upregulated in SCAD, whereas miR-223-3p and miR-142-3p were most upregulated in patients with UCAD. Subsequent validation in serum from patients with UCAD, SCAD, SubA and controls demonstrated significant upregulation of miR-223-3p, miR-133a-3p, miR-146-3p and miR-155-5p. The ischaemia-related miR-499-5p was also highly upregulated in patients with UCAD compared with the other groups (SCAD OR 20.63 (95% CI 11.16 to 38.15), SubA OR 96.10 (95% CI 40.13 to 230.14) and controls OR 15.73 (95% CI 7.80 to 31.72)). However, no significant difference in miR-499-5p expression was observed across SCAD, SubA and controls. MiR-122-5p was the only miRNA to be significantly upregulated in the serum of both patients with UCAD and SCAD.ConclusionIn conclusion, miR-122-5p and miR-223-3p might be markers of plaque instability.

Management of stent under-expansion

Stent under-expansion represents a negative prognostic factor, because of the risk to develop intrastent restenosis and stent thrombosis. It is often due to excessive calcium burden at the target lesion level. The list of devices available for the treatment of stent under-expansion includes super high-pressure non-compliant (NC) balloons, excimer laser and intravascular lithotripsy.NC OPNTM balloons (SIS Medical AG, Winterthur, Switzerland) reach much higher pressures than those of standard NC balloons (up to 35 atm), so they are able to overcome the resistance given by the calcific plaque. The excimer laser catheter instead generates ultraviolet light pulses with low penetration power. This one fragments the atherosclerotic material surrounding the implanted stent. This technology has proven to be extremely effective, however it requires specific operator experience to reduce the risk of serious complications. Intravascular lithotripsy is the most recent method, and it has shown very promising results so far. The Shockwave Medical device (S-IVL; Shockwave Medical Inc, Santa Clara, CA, USA) emits sound waves that act selectively on the calcific component, breaking it up and making the vessel more compliant.

Rivaroxaban Reduces Arterial Thrombosis by Inhibition of FXa-Driven Platelet Activation via Protease Activated Receptor-1.

A reduced rate of myocardial infarction has been reported in patients with atrial fibrillation treated with FXa (factor Xa) inhibitors including rivaroxaban compared with vitamin K antagonists. At the same time, low-dose rivaroxaban has been shown to reduce mortality and atherothrombotic events in patients with coronary artery disease. Yet, the mechanisms underlying this reduction remain unknown. In this study, we hypothesized that rivaroxaban's antithrombotic potential is linked to a hitherto unknown rivaroxaban effect that impacts on platelet reactivity and arterial thrombosis. In this study, we identified FXa as potent, direct agonist of the PAR-1 (protease-activated receptor 1), leading to platelet activation and thrombus formation, which can be inhibited by rivaroxaban. We found that rivaroxaban reduced arterial thrombus stability in a mouse model of arterial thrombosis using intravital microscopy. For in vitro studies, atrial fibrillation patients on permanent rivaroxaban treatment for stroke prevention, respective controls, and patients with new-onset atrial fibrillation before and after first intake of rivaroxaban (time series analysis) were recruited. Platelet aggregation responses, as well as thrombus formation under arterial flow conditions on collagen and atherosclerotic plaque material, were attenuated by rivaroxaban. We show that rivaroxaban's antiplatelet effect is plasma dependent but independent of thrombin and rivaroxaban's anticoagulatory capacity. Here, we identified FXa as potent platelet agonist that acts through PAR-1. Therefore, rivaroxaban exerts an antiplatelet effect that together with its well-known potent anticoagulatory capacity might lead to reduced frequency of atherothrombotic events and improved outcome in patients.

Optimizing Platelet GPVI Inhibition versus Haemostatic Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and Evobrutinib.

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.

MRI-based patient-specific human carotid atherosclerotic vessel material property variations in patients, vessel location and long-term follow up.

BackgroundImage-based computational models are widely used to determine atherosclerotic plaque stress/strain conditions and investigate their association with plaque progression and rupture. However, patient-specific vessel material properties are in general lacking in those models, limiting the accuracy of their stress/strain measurements. A noninvasive approach of combining in vivo 3D multi-contrast and Cine magnetic resonance imaging (MRI) and computational modeling was introduced to quantify patient-specific carotid plaque material properties for potential plaque model improvements. Vessel material property variation in patients, along vessel segment, and between baseline and follow up were investigated.MethodsIn vivo 3D multi-contrast and Cine MRI carotid plaque data were acquired from 8 patients with follow-up (18 months) with written informed consent obtained. 3D thin-layer models and an established iterative procedure were used to determine parameter values of the Mooney-Rivlin models for the 81slices from 16 plaque samples. Effective Young’s Modulus (YM) values were calculated for comparison and analysis.ResultsAverage Effective Young’s Modulus (YM) and circumferential shrinkage rate (C-Shrink) value of the 81 slices was 411kPa and 5.62%, respectively. Slice YM value varied from 70 kPa (softest) to 1284 kPa (stiffest), a 1734% difference. Average slice YM values by vessel varied from 109 kPa (softest) to 922 kPa (stiffest), a 746% difference. Location-wise, the maximum slice YM variation rate within a vessel was 311% (149 kPa vs. 613 kPa). The average slice YM variation rate for the 16 vessels was 134%. The average variation of YM values for all patients from baseline to follow up was 61.0%. The range of the variation of YM values was [-28.4%, 215%]. For plaque progression study, YM at follow-up showed negative correlation with plaque progression measured by wall thickness increase (WTI) (r = -0.7764, p = 0.0235). Wall thickness at baseline correlated with WTI negatively, with r = -0.5253 (p = 0.1813). Plaque burden at baseline correlated with YM change between baseline and follow-up, with r = 0.5939 (p = 0.1205).ConclusionIn vivo carotid vessel material properties have large variations from patient to patient, along the diseased segment within a patient, and with time. The use of patient-specific, location specific and time-specific material properties in plaque models could potentially improve the accuracy of model stress/strain calculations.

Determinants of stroke following percutaneous coronary intervention in acute myocardial infarction (from ORPKI Polish National Registry)

Both hemorrhagic and ischemic cerebrovascular events are rare but serious complications after acute myocardial infarction (MI) and coronary angiography/percutaneous coronary intervention (PCI). The most threatening one is intracranial hemorrhage related to antiplatelet and antithrombotic treatment of acute MI. In up to 10% of patients with acute MI ischemic stroke can result from embolisation of intraventricular or intraatrial thrombi [ [1] Kleczynski P. Dziewierz A. Rakowski T. et al. Cardioembolic acute myocardial infarction and stroke in a patient with persistent atrial fibrillation. Int. J. Cardiol. 2012; 161: e46-e47 Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar ]. It may occur within the first 2 weeks, but also as early as in the first 24 h. On the contrary, ischemic stroke observed during or early after coronary angiography/PCI is frequently iatrogenic and may be caused by embolisation of atherosclerotic or thrombotic material from the ascending aorta, carotid artery dissection, or air embolism [ 2 Hamon M. Baron J.C. Viader F. Hamon M. Periprocedural stroke and cardiac catheterization. Circulation. 2008; 118: 678-683 Crossref PubMed Scopus (82) Google Scholar , 3 Cohen M.G. Ohman E.M. Should the benefit of transradial access still be questioned?. JACC. Cardiovasc. Interv. 2016; 9: 908-910 Crossref PubMed Scopus (4) Google Scholar ]. There are some ambiguous data from observational and randomized studies on the higher rate of stroke associated with the radial access [ 3 Cohen M.G. Ohman E.M. Should the benefit of transradial access still be questioned?. JACC. Cardiovasc. Interv. 2016; 9: 908-910 Crossref PubMed Scopus (4) Google Scholar , 5 Patel V.G. Brayton K.M. Kumbhani D.J. Banerjee S. Brilakis E.S. Meta-analysis of stroke after transradial versus transfemoral artery catheterization. Int. J. Cardiol. 2013; 168: 5234-5238 Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar ]. In addition, an increased risk of stroke was suggested for primary PCI with the use of thrombectomy devices [ 4 Kwok C.S. Kontopantelis E. Myint P.K. et al. Stroke following percutaneous coronary intervention: type-specific incidence, outcomes and determinants seen by the British Cardiovascular Intervention Society 2007–12. Eur. Heart J. 2015; 36: 1618-1628 Crossref PubMed Scopus (61) Google Scholar , 6 Jolly S.S. Cairns J.A. Yusuf S. et al. Stroke in the TOTAL trial: a randomized trial of routine thrombectomy vs. percutaneous coronary intervention alone in ST elevation myocardial infarction. Eur. Heart J. 2015; 36: 2364-2372 Crossref PubMed Scopus (82) Google Scholar , 7 Barkagan M. Steinvil A. Berchenko Y. et al. Impact of routine manual aspiration thrombectomy on outcomes of patients undergoing primary percutaneous coronary intervention for acute myocardial infarction: a meta-analysis. Int. J. Cardiol. 2016; 204: 189-195 Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar ]. However, these findings were not confirmed by other studies. Thus, we sought to identify the predictors of periprocedural stroke in a large, unselected cohort of patients presenting with acute MI enrolled in the Polish National Registry of PCI (ORPKI) from 2014 to 2015.

The influence of constitutive law choice used to characterise atherosclerotic tissue material properties on computing stress values in human carotid plaques

Calculating high stress concentration within carotid atherosclerotic plaques has been shown to be complementary to anatomical features in assessing vulnerability. Reliability of stress calculation may depend on the constitutive laws/strain energy density functions (SEDFs) used to characterize tissue material properties. Different SEDFs, including neo-Hookean, one-/two-term Ogden, Yeoh, 5-parameter Mooney–Rivlin, Demiray and modified Mooney–Rivlin, have been used to describe atherosclerotic tissue behavior. However, the capacity of SEDFs to fit experimental data and the difference in the stress calculation remains unexplored. In this study, seven SEDFs were used to fit the stress–stretch data points of media, fibrous cap, lipid and intraplaque hemorrhage/thrombus obtained from 21 human carotid plaques. Semi-analytic solution, 2D structure-only and 3D fully coupled fluid-structure interaction (FSI) analyses were used to quantify stress using different SEDFs and the related material stability examined. Results show that, except for neo-Hookean, all other six SEDFs fitted the experimental points well, with vessel stress distribution in the circumferential and radial directions being similar. 2D structural-only analysis was successful for all seven SEDFs, but 3D FSI were only possible with neo-Hookean, Demiray and modified Mooney–Rivlin models. Stresses calculated using Demiray and modified Mooney–Rivlin models were nearly identical. Further analyses indicated that the energy contours of one-/two-term Ogden and 5-parameter Mooney–Rivlin models were not strictly convex and the material stability indictors under homogeneous deformations were not always positive. In conclusion, considering the capacity in characterizing material properties and stabilities, Demiray and modified Mooney–Rivlin SEDF appear practical choices for mechanical analyses to predict the critical mechanical conditions within carotid atherosclerotic plaques.

Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

Platelet CD40L Modulates Thrombus Growth Via Phosphatidylinositol 3-Kinase β, and Not Via CD40 and IκB Kinase α.

To investigate the roles and signaling pathways of CD40L and CD40 in platelet-platelet interactions and thrombus formation under conditions relevant for atherothrombosis. Platelets from mice prone to atherosclerosis lacking CD40L (Cd40lg(-/-)Apoe(-/-)) showed diminished αIIbβ3 activation and α-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets (Cd40(-/-)Apoe(-/-)) were not decreased. Using blood from Cd40lg(-/-)Apoe(-/-) and Cd40(-/-)Apoe(-/-) mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe(-/-) blood. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI-induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cb(R/R) platelets or by inhibiting phosphatidylinositol 3-kinase β (PI3Kβ). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3Kβ. Finally, platelets from Chuk1(A/A)Apoe(-/-) mice deficient in IκB kinase α (IKKα), implicated in CD40 signaling to nuclear factor (NF) κB, showed unchanged responses to CD40L in aggregation or thrombus formation. Under atherogenic conditions, CD40L enhances collagen-induced platelet-platelet interactions by supporting integrin αIIbβ3 activation, secretion and thrombus growth via PI3Kβ, but not via CD40 and IKKα/NFκB. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formation.

The role of T and B cells in human atherosclerosis and atherothrombosis.

Far from being merely a passive cholesterol accumulation within the arterial wall, the development of atherosclerosis is currently known to imply both inflammation and immune effector mechanisms. Adaptive immunity has been implicated in the process of disease initiation and progression interwined with traditional cardiovascular risk factors. Although the body of knowledge regarding the correlation between atherosclerosis and immunity in humans is growing rapidly, a relevant proportion of it derives from studies carried out in animal models of cardiovascular disease (CVD). However, while the mouse is a well-suited model, the results obtained therein are not fully transferrable to the human setting due to intrinsic genomic and environmental differences. In the present review, we will discuss mainly human findings, obtained either by examination of post-mortem and surgical atherosclerotic material or through the analysis of the immunological profile of peripheral blood cells. In particular, we will discuss the findings supporting a pro-atherogenic role of T cell subsets, such as effector memory T cells or the potential protective function of regulatory T cells. Recent studies suggest that traditional T cell-driven B2 cell responses appear to be atherogenic, while innate B1 cells appear to exert a protective action through the secretion of naturally occurring antibodies. The insights into the immune pathogenesis of atherosclerosis can provide new targets in the quest for novel therapeutic targets to abate CVD morbidity and mortality.

Peripheral embolization of aortic valve calcium following trans-femoral Sapien XT valve implantation requiring emergency surgical embolectomy

Trans-catheter Aortic Valve Implantation (TAVI) is a well-established, alternative, standard of care treatment option for inoperable and high risk patients with severe, symptomatic aortic stenosis (AS) [1]. Peri-procedural cerebrovascular events are a known complication of TAVI and comprise more than half of TAVI related strokes with a 30day incidence of 2–5% [2]. In the acute (<24hours) and sub-acute (1–30day) setting, post-TAVI stroke may be due to embolization of atherosclerotic or calcific material from the aortic valve, the risk of which is increased when valve embolization occurs or post-dilatation is carried out [3].

TCT-131 Tissue Removal Assessment with Ultrasound of the SFA and Popliteal Study (TRUTH): Orbital Atherectomy Acute Data and Intravascular Ultrasound Analysis

Percutaneous removal of atherosclerotic material using atherectomy devices may offer an alternative approach for improving procedural success. The TRUTH study (24 patients) evaluated the removal of plaque with the Orbital Atherectomy System (OAS) (Cardiovascular Systems, Inc., St. Paul, MN) by

Cross-clamping a porcelain aorta: an alternative technique for high-risk patients

Aortic cross-clamping in patients with porcelain aorta is associated with high mortality and morbidity rates. The aim is to establish a new approach to improve the outcome in this high-risk population. Between September 2007 and November 2012, 42 patients with an aortic (N.=33; 81.3±6.4 years) or mitral valve disease (N.=9; 80.3±5.7) combined with a porcelain aorta underwent aortic (AVR) or mitral valve replacement (MVR). After arterial cannulation via distal aortic arch or femoral artery, longitudinal aortotomy under total cardiopulmonary bypass (CPB) was performed. The aorta was slowly clamped, thus mobilized atherosclerotic material could leave the aorta through the open incision. Subsequent to the actual operation, the aorta was gradually unclamped. Again, plaques were flushed out via the still open aortotomy ("open proximal ascending aorta"). Intraoperatively, no technical no problems occurred. Mean CPB time was 92.2±27.9 min (AVR) and 92.3±36.3 min (MVR); cardiac ischemia time was 74.3±26.7 min (AVR) and 77.1±31.6 min (MVR). Surgical revision was necessary in three patients (7.1%) due to major bleedings. Two AVR-patients suffered from minor stroke and one MVR-patient from major stroke (neurological deficit rate =7.1%). Transient ischemic attacks occurred in three patients (7.1%), another three patients (7.1%) required temporary hemofiltration. Neither gastrointestinal disorders nor respiratory failure or valve-related problems were noted. 30-day mortality was 6.9%. Cross-clamping with "open proximal ascending aorta" is effective and the incidence of stroke and systemic embolization in patients with porcelain aorta is low compared to literature.