Initiation Of Thrombus Formation Research Articles

Antiplatelet effects of hydroxychloroquine in patients with systemic lupus erythematosus evaluated by the total thrombus-formation analysis system (T-TAS)

ObjectivesHydroxychloroquine (HCQ) has been shown to reduce thrombotic events in patients with SLE. However, the antiplatelet effects of HCQ are only supported by the platelet aggregation assay, which is a...

Apoptotic Pathway Inhibition in Cold-Stored Platelet Concentrates Facilitates Retained Ability to Form Thrombi

Introduction: Platelet concentrates (PCs) are used to treat or prevent bleeding in patients with impaired platelet function or after injury. However, the standard storage of PCs at room-temperature (RT) increases the risk of bacterial contamination and is associated with reduced platelet functionality due to storage lesions. Our group reported that cold-stored PCs have better functionality compared to RT-stored PCs but reduced in vivo circulation time due to cold-induced apoptosis. In order to investigate cold-stored PCs hemostatic functionality with and without apoptosis inhibition, we established an ex vivo model simulating physiological conditions of blood flow to evaluate the contribution of cold-stored PCs in thrombus formation. Methods: Apheresis-derived PCs were stored for 1, 4, 7 and 10 days under agitation at 4°C or RT either with or without the apoptosis inhibitor G04 (RhoA GTPase inhibitor). Next, PCs were stained with CD41 antibody, recalcified and incubated with TRAP6 (thrombin receptor-activating peptide 6) to initiate thrombus formation. Platelet-depleted whole blood samples from healthy donors were spiked-in with PCs and administered to the microfluidic channels of the ex vivo system (Bioflux), under physiological shear pressure. After five minutes of perfusion the resulting thrombi were imaged and analyzed. Ability to form thrombi was further assessed by thromboelastography and impedance aggregometry. Results: Platelets from 24h storage PCs showed stable thrombus formation upon activation with TRAP6. Unstimulated platelets did not form any thrombi during the entire period of perfusion. To evaluate the effect of cold storage on platelet functions we analyzed the extent of thrombus formation of PCs stored at RT and 4°C upon CD41 staining. Our results indicate that cold-stored platelets form larger clots under flow conditions and maintain higher functionality compared to PCs stored at RT. Moreover, incubation of cold-stored PCs with the apoptosis inhibitor G04 improved ex vivo thrombus formation compared to PCs stored without the inhibitor, without inducing any adverse effects (Figure 1). Conclusion: The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is suitable to test the hemostatic functions of PCs under physiological flow conditions. We demonstrated that PCs stored at 4°C show better thrombus formation ability upon stimulation by agonists compared to RT-stored PCs and that the ability to form clots under shear stress is still preserved after incubation with the apoptosis inhibitor. To conclude, cold-storage of PCs in combination with apoptosis inhibition is a promising stragedy to reduce the risk of bacterial contamination and prolong in vivo survival time while preserving platelet's hemostatic functionality.

A Multiscale Model for Shear-Mediated Platelet Adhesion Dynamics: Correlating In Silico with In Vitro Results.

Platelet adhesion to blood vessel walls is a key initial event in thrombus formation in both vascular disease processes and prosthetic cardiovascular devices. We extended a deformable multiscale model (MSM) of flowing platelets, incorporating Dissipative Particle Dynamics (DPD) and Coarse-Grained Molecular Dynamics (CGMD) describing molecular-scale intraplatelet constituents and their interaction with surrounding flow, to predict platelet adhesion dynamics under physiological flow shear stresses. Binding of platelet glycoprotein receptor Ibα (GPIbα) to von Willebrand factor (vWF) on the blood vessel wall was modeled by a molecular-level hybrid force field and validated with in vitro microchannel experiments of flowing platelets at 30 dyne/cm2. High frame rate videos of flipping platelets were analyzed with a Semi-Unsupervised Learning System (SULS) machine learning-guided imaging approach to segment platelet geometries and quantify adhesion dynamics parameters. In silico flipping dynamics followed in vitro measurements at 15 and 45dyne/cm2 with high fidelity, predicting GPIbα-vWF bonding and debonding processes, distribution of bonds strength, and providing a biomechanical insight into initiation of the complex platelet adhesion process. The adhesion model and simulation framework can be further integrated with our established MSMs of platelet activation and aggregation to simulate initial mural thrombus formation on blood vessel walls.

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

Predicting Platelet Age Using Artificial Intelligence Techniques

Background: Platelets are small, enucleated cells with their primary physiological role to repair vascular damage (hemostasis) and initiate thrombus formation in response to vascular injury. Low platelet count (thrombocytopenia) is associated with increased bleeding risk and is caused by decreased platelet production and/or increased platelet clearance. Platelet transfusion is a routinely used lifesaving procedure to control or prevent bleeding in patients with thrombocytopenia or platelet dysfunction. During storage platelet function deteriorates significantly over time and regulation of platelet granular content is essential in maintaining a healthy platelet function. Currently no in vitro tests are available to predict platelet age, which may be used to diagnose the underlying cause of thrombocytopenia, or to determine storage time of platelet concentrates (PC). Using confocal and super-resolution microscopy we could quantify loss of platelet granule content during aging. In this study we used Artificial intelligence techniques (AIT) to derive exact platelet age from imaging data. Aim: To develop AIT-based algorithms that can determine platelet age from images of platelets stored in plasma-rich-plasma (PRP) and in platelets concentrates (PC) measured by confocal microscopy. Methods: Platelets were stored in PRP for 8 hours at room temperature. Platelets were isolated and stained with antibodies for alpha-tubulin, von Willebrand Factor (VWF), SPARC or alpha-tubulin and imaged at T=0, 4 and 8 hours using confocal microscopy. Additionally 3 single donor PC in plasma were stored for 10 days at standard blood bank conditions. Platelet samples were pooled and analyzed at day 1,2,3,6,7,8,9 and 10. We trained a neural network consisting of multiple convolutional layers to classify age (time after donation) of platelets that were stored in either PRP or PC. Results: During storage in PRP as well as of PC we used for each time point 1334 62µm x 62µm x 4µm 3D images, containing 50 to 150 platelets per image, for training. When staining with markers VWF, SPARC or alpha-tubulin alone as training set we could already classify mean platelet age up to 78%, 85%, 88% respectively. When combining all three markers a maximum accuracy of 95% for platelets stored in PRP up to 8 hours was reached (Figure 1). The additional effect of combining these three markers highlights that the model used information in the images synergistically. For PC, an accuracy of 90% was reached for classifying mean platelet age of platelets stored up to 8 days. The model could not be trained to discriminate between days 9 and 10 resulting in an overfitted model. However the model was able to distinguish day between day 1, 3, 6 and 10 (Figure 2). As negative control a model trained on a randomized sample was made. This control model was not able to assign a category correctly, indicating an actual difference in platelet characteristics during storage was detected. By using visualization of the weight-gradient (e.g. where in the images the model assigns the most weight) we additionally confirmed that the model used separate individual platelets to characterize the age of the complete platelet sample. Conclusion: Combining confocal platelet images with a convolutional neural network, we trained a model that could synergistically integrate different stainings and information of individual platelets to predict with >95% accuracy platelet age in vitro as well as in PC up to 10 days of storage. Additionally our model could distinguish "young" platelets (stored 1, 3 or 6 days) from "old" platelets (stored 10 days). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Binding to Platelets Differs Among Recombinant Factor VIII Products in Vitro

Introduction: Platelets are activated in response to injury and play a key role in blood clotting. In primary hemostasis, platelets adhere to exposed subendothelial matrix proteins (e.g., collagen) and subsequent downstream signaling pathways induce granule secretion and platelet aggregation. In secondary hemostasis, procoagulant platelets in the periphery activate clotting factors, leading to a more stable fibrin clot. Binding of factor VIII (FVIII) to procoagulant platelets ensures efficient production and localization of thrombin at the site of injury. However, it is less clear how platelet-FVIII interactions spatiotemporally control the initial thrombus formation and pave the way for successful tissue repair. In patients with hemophilia A, replacement FVIII therapy is used to treat and prevent bleeds. A number of recombinant FVIII (rFVIII) products are indicated for use in patients with hemophilia A. Simoctocog alfa (Nuwiq®) is a chemically unmodified rFVIII with no fusion protein produced in a human cell line. Damoctocog alfa pegol (Jivi®) and rurioctocog alfa pegol (Adynovate®) are pegylated rFVIII products, while efmoroctocog alfa (Eloctate®) consists of FVIII fused with a Fc protein. It is not clear whether such modifications impact the platelet binding potential of FVIII. This study aims to examine platelet binding of different rFVIII products in vitro. Methods: Platelets were isolated from healthy donors, activated using thrombin and collagen-related peptide (CRP-XL) and incubated with individual rFVIII concentrates. After 30 min, the reaction was stopped by dilution. Binding was quantified by flow cytometry using one of two approaches: rFVIII immunostaining with a fluorescent (Alexa Fluor 647; AF647) anti-FVIII antibody; or using rFVIII molecules that had been pre-labeled with AF647. For comparison among products, experiments were performed at equimolar concentrations (17.6 nM) or equal activities of rFVIII concentrates. Values were normalized to simoctocog alfa. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc testing. Results: Platelets were obtained from 10 healthy donors. At equimolar concentrations, efmoroctocog alfa and damoctocog alfa pegol bound significantly (p < 0.001) less than simoctocog alfa to stimulated platelets, as visualized via immunostaining (n = 10). Binding to platelets was significantly lower with damoctocog alfa pegol than efmoroctocog alfa (p < 0.001). Similar results were obtained from experiments with pre-labelled rFVIII (n = 6) or when using equal activities of rFVIII concentrates. In experiments with platelets from 2 healthy donors, rurioctocog alfa pegol exhibited lower platelet binding compared with simoctocog alfa, when tested using the immunostaining method and equimolar concentrations of rFVIII. Conclusions: Simoctocog alfa showed increased binding to procoagulant platelets compared with other rFVIII concentrates tested. Further studies, including additional functional platelet assays, will be performed using samples from a larger number of healthy donors and from patients with hemophilia A. The results of this study may provide new insights into the function and efficacy of different rFVIII products in patients with hemophilia A.

Continuum modeling of thrombus formation and growth under different shear rates

Obstruction of blood flow due to thrombosis is a major cause of ischemic stroke, myocardial infarction, and in severe cases, mortality. In particular, in blood wetting medical devices, thrombosis is a common reason for failure. The prediction of thrombosis by understanding signaling pathways using computational models, lead to identify the risk of thrombus formation in blood-contacting devices and design improvements. In this study, a mathematical model of thrombus formation and growth is presented. A biochemical model of platelet activation and aggregation is developed to predict thrombus size and shape at the site of vascular injury. Computational fluid dynamics using the finite volume method is employed to compute the velocity and pressure fields which are influenced by the growing thrombi. The passive transport of platelets, agonists, the platelet activation kinetics, their adhesion to the growing thrombi and embolization of platelets are solved by a fully coupled set of convection–diffusion–reaction equations. The thrombogenic surface representing blood-contacting material or injured blood vessel was incorporated into the model as a surface flux boundary condition to initiate thrombus formation. The blood is considered as a Newtonian fluid, while the thrombus is treated as a porous medium. The results are compared with in vitro experiments of a microfluidic chamber at an initial inlet venous shear rate of 200s−1 using a pressure–inlet boundary condition. The thrombus development due to agonist concentrations and change in the shear rate as well as thromboembolism for this benchmark problem is successfully computed. Furthermore, to extend the current model to a physiologically relevant configuration, thrombus formation in a blood tube is simulated. Two different heterogeneous reaction rates for platelet aggregation are used to simulate thrombus growth under a constant inlet flow rate. The findings show that the thrombus shape can be substantially altered by the hemodynamic conditions, increase in the shear rate and due to the combined effects of shear induced platelet activation and the heterogeneous reaction rates. It is also concluded that the model is able to predict thrombus formation in different physiological and pathological hemodynamics.

Synthesis and evaluation of novel and potent protease activated receptor 4 (PAR4) antagonists based on a quinazolin-4(3H)-one scaffold.

Protease activated receptor 4 (PAR4) is an important target in antiplatelet therapy to reduce the risk of heart attack and thrombotic complications in stroke. PAR4 antagonists can prevent harmful and stable thrombus growth, while retaining initial thrombus formation, by acting on the late diffusion stage of platelet aggregation, and may provide a safer alternative to other antiplatelet agents. To date, only two PAR4 antagonists, BMS-986120 and BMS-986141 have entered clinical trials for thrombosis. Thus, the development of a potent and selective PAR4 antagonist with a novel chemotype is highly desirable. In this study, we explored the activity of quinazolin-4(3H)-one-based PAR4 antagonists, beginning with their IDT analogues. By repeated structural optimisation, we developed a series of highly selective PAR4 antagonists with nanomolar potency on human platelets. Of these, 13 and 30g, with an 8-benzo[d]thiazol-2-yl-substituted quinazolin-4(3H)-one structure, showed optimal activity (h. PAR4-AP PRP IC50=19.6nM and 6.59nM, respectively) on human platelets. Furthermore, 13 and 30g showed excellent selectivity for PAR4 versus PAR1 and other receptors (IC50s>10μM) on human platelets. And 13 and 30g were lack of cross-reactivity for PAR1 or PAR2 (PAR1 AP FLIPR IC50>3162nM, PAR2 AP FLIPR IC50>1000nM) in the calcium mobilization assays. Metabolic stability assays and cytotoxicity tests of 13 and 30g indicated that these compounds could sever as promising drug candidates for the development of novel PAR4 antagonists. In summary, the quinazolin-4(3H)-one-based analogues are the first reported chemotypes with excellent activity and selectivity against PAR4, and, in the current study, we expanded the structural diversity of PAR4 antagonists. The two compounds, 13 and 30g, found in our study could be promising starting points with great potential for further research in antiplatelet therapy.

Antithrombotic Therapy in Acute Coronary Syndromes: Current Evidence and Ongoing Issues Regarding Early and Late Management.

A few decades ago, the understanding of the pathophysiological processes involved in the coronary artery thrombus formation has placed anticoagulant and antiplatelet agents at the core of the management of acute coronary syndrome (ACS). Increasingly potent antithrombotic agents have since been evaluated, in various association, timing, or dosage, in numerous randomized controlled trials to interrupt the initial thrombus formation, prevent ischemic complications, and ultimately improve survival. Primary percutaneous coronary intervention, initial parenteral anticoagulation, and dual antiplatelet therapy with potent P2Y12 inhibitors have become the hallmark of ACS management revolutionizing its prognosis. Despite these many improvements, much more remains to be done to optimize the onset of action of the various antithrombotic therapies, for further treating and preventing thrombotic events without exposing the patients to an unbearable hemorrhagic risk. The availability of various potent P2Y12 inhibitors has opened the door for individualized therapeutic strategies based on the clinical setting as well as the ischemic and bleeding risk of the patients, while the added value of aspirin has been recently challenged. The strategy of dual-pathway inhibition with P2Y12 inhibitors and low-dose non-vitamin K antagonist oral anticoagulant has brought promising results for the early and late management of patients presenting with ACS with and without indication for oral anticoagulation. In this updated review, we aimed at describing the evidence supporting the current gold standard of antithrombotic management of ACS. More importantly, we provide an overview of some of the ongoing issues and promising therapeutic strategies of this ever-evolving topic.

Transcatheter Aortic Valve Thrombogenesis: A Foreign Materials Perspective.

The initiation of thrombus formation in transcatheter aortic valves (TAVs) is not well understood. The foreign material components of a TAV may play a key role in TAV thrombogenesis. The goal of this study was to evaluate the thrombogenic potential of a TAV (entire valve) and its stent (with skirt). Blood was collected from eight human donors with citrate anticoagulation and later reconstituted with calcium chloride. A low-volume steady flow loop (flow rate = 0.8 L/min) was designed to facilitate three separate conditions (experimental duration = 1h) per donor blood: (1) control (n = 8), (2) stent-with-skirt (leaflets removed from a 23mm SAPIEN XT valve; n = 8) and (3) entire valve (an intact 23mm SAPIEN XT valve; n = 8). Samples were collected at the start and end of each experiment. Serum D-Dimer and thrombin-antithrombin (TAT) concentrations were measured as markers of thrombogenicity. There was no significant change in serum D-Dimer and TAT concentration with time for the control group. An increasing trend in D-Dimer and TAT concentration was observed with time for the stent-with-skirt group. Interestingly, there was a decreasing trend in serum D-Dimer and TAT concentration with time for the entire valve (leaflet dominating) group. Moreover, changes in D-Dimer and TAT concentration were significantly different between the stent-with-skirt and entire valve (leaflet dominating) groups. Stent-with-skirt was found to impart the most prominent thrombogenic effect, indicating the significance of blood-stent and blood-skirt interactions in TAV thrombosis.

Protease activated receptor 4 (PAR4) antagonists: Research progress on small molecules in the field of antiplatelet agents.

Protease activated receptor 4 (PAR4) is a key target in antiplatelet medication to reduce the risk of heart attack and thrombotic complications in stroke. PAR4 antagonists can prevent harmful and stable thrombus growth while retaining initial thrombus formation by acting on the late diffusion stage of platelet activation, which may provide a safer alternative than other antiplatelet agents. Currently, research on PAR4 antagonists is of increasing interest in the field of antiplatelet agents. This article provides an overview of the discovery and development of small-molecule antagonists of PAR4 as novel antiplatelet agents, including structure-activity relationship (SAR) analysis, progress of structure and bioassay optimization, and the latest structural and/or clinical information of representative small-molecule antagonists of PAR4.

EFFECT OF CISPLATIN ON HEMOSTASIS IN EXPERIMENTAL ANIMALS

Introduction. Thrombotic complications caused by the tumor and consequences of its treatment are the leading causes of death in cancer patients. The development of a model for the pathology of hemostasis, in particular the excessive pathological clot formation, in the laboratory animals receiving antitumor agents, could help find new pharmacological methods for correcting hemostatic disorders.The purpose of the study was to study the effect of cisplatin on the blood coagulation system in mice and rats.Results. An experiment using outbred mice showed that the levels of PT-INR and aPTT were decreased and the level of fibrinogen was increased on day 10 after administration of cisplatin in the maximum tolerated dose of 10 mg/kg. A significant decrease in the PT-INR and aPTT levels was observed on day 15 after cisplatin injection only in female mice. The cisplatin injection at a dose of 4 mg/kg resulted in a decrease in the PT-INR, and aPTT levels and an increase in fibrinogen concentration on day 10. In rats, a significant decrease in the PT and aPTT levels was observed in both females and males on day 15 after cisplatin injection.Conclusion. A change in the PT and NIR, aPTT levels towards decrease and fibrinogen concentration towards increase indicates the initiation of thrombus formation.

Novel antiplatelet targets in the treatment of acute coronary syndromes

Acute coronary syndromes (ACS) are a global cause of mortality and morbidity that affect millions of lives worldwide. Following atherosclerotic plaque rupture, platelet activation and aggregation are the two major elements that initiate thrombus formation inside a coronary artery, which can obstruct blood flow and cause myocardial ischemia; ergo, antiplatelet therapy forms a major part of the treatment strategy for ACS. Patients with ACS routinely receive dual antiplatelet therapy (DAPT), which consists of aspirin and a platelet P2Y12 inhibitor to both treat and prevent atherothrombosis. Use of platelet glycoprotein (GP) IIb/IIIa inhibitors is now limited due to the risk of severe bleeding and thrombocytopenia. Thus, administration of GPIIb/IIIa inhibitors is generally restricted to bail out thrombotic events associated with PCI. Furthermore, current antiplatelet medications mainly rely on thromboxane A2 and P2Y12 inhibition, which have broad-acting effects on platelets and are known to cause bleeding, which especially limits the long-term use of these agents. In addition, not all ACS patients treated with current antiplatelet treatments are protected from recurrence of arterial thrombosis, since many platelet mechanisms and activation pathways remain uninhibited by current antiplatelet therapy. Pharmacological antagonism of novel targets involved in platelet function could shape future antiplatelet therapies that could ultimately lead to more effective or safer therapeutic approaches. In this article, we focus on inhibitors of promising targets that have not yet been introduced into clinical practice, including inhibitors of GPVI, protease-activated receptor (PAR)-4, GPIb, 5-hydroxytryptamine receptor subtype 2A (5-HT2A), protein disulfide isomerase, P-selectin and phosphoinositide 3-kinase β.

OC4. Abstract Title: Thrombus Stability Explains the Factor V Leiden Paradox: A Mouse Model

<h3>Background</h3> Factor V Leiden (FVL) is an inherited autosomal dominant condition that is identified in 20%-50% of patients with venous thromboembolic diseases and 5-8% of the population in Canada. People with FVL are often called hypercoagulable, and have a five-fold increase in the risk of venous thrombosis. There is a low prevalence of FVL among patients with fatal pulmonary embolism (PE) and a higher incidence of deep vein thrombosis (DVT) than PE in patients with FVL. This has been referred to as the FVL paradox in thrombosis. We wished to test whether FVL and normal patients are at the same risk of initial thrombus formation, expect FVL patients present with symptomatic DVT while normal patients do not present, because the small asymptomatic DVT in normal patients embolizes to become an asymptomatic PE. <h3>Aim</h3> Therefore, to explain why patients with FVL are more likely to present with a DVT rather than a PE we will study DVT stability in FVL mice using a modification of our previously published mouse model of venous thrombus stability. <h3>Methods</h3> Platelets were fluorescently labeled using CD41 fab fragments conjugated to an Alexa Fluor 488. DVT was induced using a ferric chloride saturated filter paper on the femoral vein of female mice. We chose conditions such that only a small thrombus developed in wild type (WT) mice, and then compared them to thrombi in FVL heterozygous (F5L/+) and FVL homozygous (F5L/L) mice. Intravital videomicroscopy recorded embolic events leaving the thrombus and the thrombus sizes every 10 minutes for 2 hours. Lungs were harvested sectioned and stained for presence of PE. <h3>Results</h3> Although thrombus size remained small in WT mice, it significantly increased over time in F5L/+ and F5L/L mice. The number of total and large embolic events, and the percent of thrombus that embolized was significantly decreased in F5L/+ and F5L/L mice compared to WT mice. Previously we have correlated increased large embolic events with increased PE burden. FVL mice had significantly reduced PE burden compared to WT mice. Mice with F5L had higher F1.2 levels than in WT, consistent with increased thrombin generation. <h3>Conclusion</h3> This suggests that in noncarriers (reflected by WT), a minor insult initially resulting in a small DVT tends to remain small and asymptomatic, due to the embolization. Alternatively, the same insult in people with FVL (reflected by F5L/L) leads to thrombus growth due to less embolization and thus development of symptomatic DVT, but no PE.

A Multiscale Model for Recruitment Aggregation of Platelets by Correlating with In Vitro Results.

We developed a multiscale model to simulate the dynamics of platelet aggregation by recruitment of unactivated platelets flowing in viscous shear flows by an activated platelet deposited onto a blood vessel wall. This model uses coarse grained molecular dynamics (CGMD) for platelets at the microscale and dissipative particle dynamics (DPD) for the shear flow at the macroscale. Under conditions of relatively low shear, aggregation is mediated by fibrinogen via αIIbβ3 receptors. The binding of αIIbβ3 and fibrinogen is modeled by a molecular-level hybrid force field consisting of Morse potential and Hooke law for the nonbonded and bonded interactions, respectively. The force field, parametrized in two different interaction scales, is calculated by correlating with the platelet contact area measured in vitro and the detaching force between αIIbβ3 and fibrinogen. Using our model, we derived, the relationship between recruitment force and distance between the centers of mass of two platelets, by integrating the molecular-scale inter-platelet interactions during recruitment aggregation in shear flows. Our model indicates that assuming a rigid-platelet model, underestimates the contact area by 89% and the detaching force by 93% as compared to a model that takes into account the platelet deformability leading to a prediction of a significantly lower attachment during recruitment. The molecular-level predictive capability of our model sheds a light on differences observed between transient and permanent platelet aggregation patterns. The model and simulation framework can be further adapted to simulate initial thrombus formation involving multiple flowing platelets as well as deposition and adhesion onto blood vessels.

Evaluation of Tissue Factor -603 A/G Promoter Gene Polymorphism in Thrombotic Processes in Patients with Antiphospholipid Syndrome

<strong>Objective</strong>: Major clinical manifestations of Antiphospholipid syndrome (APS) are trombosis and thrombosis related complications. Tissue factor (TF) serves primary initiatory of the in vivo coagulation. TF gene expression may be induced in endothelial cells to initiate thrombus formation. In this study, we investigated the frequency of TF 603A/G promoter gene polymorphism in patients with antiphospholipid syndrome (APS) and its effects on TF expression and thrombotic events. <br> <strong>Materials and Methods</strong>: This study included 64 patients with APS (APS patient with thrombosis arterial and venous (n=38), antiphospholipid antibody-positive patients without thrombosis (n=26) and 44 healthy controls (HCs). Plasma TF levels and antiphospholipid antibodies were measured by ELISA method. Lupus anticoagulant was measured by activated partial thromboplastin time and kaolin clotting time. TF-603 A/G polymorphism was investigated by PCR method. <br> <strong>Results</strong>: Our results have showed that patients with APS, TF levels were found in patients significantly higher than the healthy control group (p: 0.006, p&lt;0.001, respectively). Compared between the two patient groups, TF level was significantly higher in the patient with thrombosis group compared to the patient without thrombosis group (p: 0.027). <br> <strong>Conclusion</strong>: In this study, we suggest that thrombotic complications observed in patients with APS are associated with increased TF expression.&nbsp;

OR04-5 Stimulation of Protein Disulfide Isomerase Activity by Activation of The Renin-Angiotensin System

Understanding the molecular and cellular mechanisms that are affected by activation of the renin-angiotensin system (RAS) are important for development of novel therapeutic strategies to more specifically treat hypertension and reduce cardiovascular risk. RAS regulates blood pressure (BP) in part via its effector molecule, angiotensin II (AngII), a potent vasopressor that stimulates the production of reactive oxygen species (ROS). Protein disulfide isomerase (PDI) is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum and regulates ROS production. Also, PDI is present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of RAS and PDI remain unclear. We hypothesized that RAS activation would lead to increased PDI levels thus contributing to oxidative stress. First, we studied the in vitro effects of AngII on EA.hy926, a human endothelial cell line, and measured PDI activity. Our results show that AngII dose and time dependently increased extracellular PDI activity (p<0.05, n=6); an event that was reduced to baseline levels by preincubation with 0.5 uM losartan, an AngII type 1 receptor antagonist (ARB). Consistent with these results, AngII stimulated: 1) a 3-fold rise of extracellular PDI levels as determined by ELISA (p<0.05, n=4); 2) a 2.5-fold increase in PDI mRNA levels by RT-PCR (p<0.05, n=4); and 3) increased PDI protein levels by western blot analyses that were blocked by losartan. We also studied the effect of AngII on extracellular PDI activity in differentiated HL-60 cells, a human polymorphonuclear neutrophil cell line, and observed significantly increased extracellular PDI activity following incubation with AngII (p<0.05, n=4). We then characterized the in vivo effects of exogenous AngII infusion on PDI in Sprague-Dawley rats, a model of increased BP, inflammation and target organ damage. We studied three conditions: (1) control; (2) AngII infused (80 ng/min x 28 days); (3) AngII + ARB (10 mg losartan/kg/day x 21 days). AngII infusion led to significant increases in plasma PDI levels that were reduced by 20% following ARB treatment (p<0.05, n=5/group). We then studied a model of naturally increased AngII and hypertension, the Otsuka Long Evans Tokushima Fatty rats (OLETF) and their strain controls that were randomly assigned to the following groups: (1) untreated strain controls; (2) untreated OLETF; (3) OLETF + ARB. OLETF rats had increased BP and significantly greater circulating PDI activity than control rats that was likewise blocked by ARB treatment (p<0.05; n=6/group). Thus, RAS activation represents a novel mechanism for increased PDI levels and activity that may contribute to the deleterious effects of disordered RAS in cardiovascular disease.

Activation of the Mineralocorticoid Receptor Leads to Increases in Protein Disulfide Isomerase Activity

Activation of the mineralocorticoid receptor (MR), a critical component of the Renin‐Angiotensin‐Aldosterone (ALDO) System (RAAS), plays an important role in inflammatory and vascular endothelial responses in addition to its well‐described effects on sodium and water homeostasis. Activation of endothelial cells and leukocytes leads to, among other factors, increased protein disulfide isomerase (PDI) release. PDI is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum, is also present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of ALDO, MR activation and PDI remain unclear. We hypothesized that MR activation would lead to increased PDI levels thus contributing to inflammation and oxidative stress. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10 −8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT‐PCR (p&lt;0.01, n=5), an event that was blocked by pre‐incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (p&lt;0.05, n=5). Consistent with these results, measurements of PDI levels by ELISA confirmed a rise in PDI that was likewise blocked by CA (p&lt;0.01, n=4). Also, we measured PDI activity in the supernatant of ALDO‐stimulated EA cells using a Di‐E‐GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10^−8 M) incubation when compared to vehicle treatment (p&lt;0.05, n=5). We then studied the effects of ALDO on HL‐60 cells, a human promyelocytic cell line that was induced to differentiate into neutrophil‐like leukocytes by incubation for 5 days with 1.3% DMSO. In these cells, we detected the presence of MR by western blot analyses and observed that ALDO (10^−8 M) stimulated a rise in PDI activity that was blocked by CA (p&lt;0.05, n=6). To test the in vivo effects of MR activation on PDI activity, we characterized a well‐described rodent model of RAAS‐mediated increased BP, inflammation and target organ damage using exogenous angiotensin II (AngII) infusion in Sprague–Dawley rats. Rats were randomized to three conditions: (1) control; (2) AngII infused (80 ng/min × 28 days); (3) AngII + eplerenone (100 mg eplerenone/kg/day × 21 days), an MR antagonist. Consistent with our in vitro data, AngII infusion led to significant increases in plasma PDI activity that was significantly reduced by approximately 65% following eplerenone treatment (p&lt;0.05, n=5/group). Thus, MR activation represents a novel mechanism for PDI regulation that may contribute to the deleterious effects of disordered RAAS activity in cardiovascular disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

12. Abstract Title: Factor V Leiden Mutation Stabilizes Thrombi and Reduces Embolization in Mice

Background: Factor V Leiden (FVL) is an inherited autosomal dominant condition that is identified in 20%-50% of patients with venous thromboembolic diseases and 5-8% of the population in Canada. People with FVL are often called hypercoagulable and have a five-fold increase in the risk of venous thrombosis. There is a low prevalence of FVL among patients with fatal pulmonary embolism (PE) and a higher incidence of deep vein thrombosis (DVT) than PE in patients with FVL. This has been referred to as the FVL paradox in thrombosis. We wished to test whether FVL and normal patients are at the same risk of initial thrombus formation, expect FVL patients present with symptomatic DVT while normal patients do not present, because the small asymptomatic DVT in normal patients embolizes to become an asymptomatic PE.

Kounis Syndrome: A Case Report and Literature Review of Pre-Hospital Treatment

IntroductionKounis syndrome is an uncommon clinical presentation of acute coronary syndrome secondary to an allergic or hypersensitivity reaction, especially anaphylaxis. It results when inflammatory mediators are released following mast cell activation, some of these mediators cause coronary artery vasospasm and may initiate thrombus formation in susceptible individuals. Although Kounis syndrome is becoming more widely known, many clinicians are still unaware of its existence. We present a case report and a literature review of the pre-hospital treatment of Kounis syndrome by emergency medical services.MethodsA literature search of the EMBASE, MEDLINE and PubMed electronic medical databases was conducted using the terms ‘Kounis syndrome’, ‘allergic acute coronary syndrome’ and ‘allergic myocardial infarction’. The purpose of the literature search was to identify the pre-hospital treatment of Kounis syndrome by emergency medical services. We included case reports of Kounis syndrome that described the medical treatment provided by emergency medical services, published any time up to October, 2017.ResultsAnaphylaxis is the most commonly treated component of Kounis syndrome by emergency medical services (66% of reported cases). Both components of Kounis syndrome, anaphylaxis and acute coronary syndrome, were treated in 16% of reported cases. No specific treatment was provided for either component of Kounis syndrome in 16% of reported cases.ConclusionThe pre-hospital treatment of Kounis syndrome by emergency medical services is infrequently reported in the literature. Kounis syndrome involves two distinct clinical conditions, both of which should be considered during treatment.