Platelet function under systemic oxidative stress

Neben ihrer zentralen physiologischen Bedeutung für die primäre Hämostase und die plasmatische Blutgerinnung spielen Thrombozyten und ihr Aktivierungszustand eine entscheidende Rolle in der Entstehung und dem Fortschreiten atherosklerotischer Erkrankungen. Eine Vielzahl an Befunden weist darauf hin, dass Lipoproteine - speziell LDL und HDL – imstande sind, die Thrombozytenaktivierung direkt zu beeinflussen. Hohe Plasmakonzentrationen von HDL gehen mit einer verminderten Thrombozytenreaktivität einher, während erhöhte Plasmakonzentrationen von LDL eng mit Thrombozytenhyperreaktivität korrelieren. Da jedoch Lipoproteine von hypercholesterinämischen Patienten eine erhöhte Anfälligkeit für oxidative Modifizierung zeigen bleibt unklar, ob die in diesen Patienten zu beobachtende Thrombozytenaktivierung primär eine Folge der erhöhten LDL Konzentrationen oder eine Konsequenz der Lipoproteinoxidation ist. In dieser Arbeit konnte gezeigt werden, dass nicht nur native HDL sondern auch native LDL einen inhibierenden Effekt auf Thrombozyten ausüben. Beide Lipoproteinklassen sind im Stande die durch verschiedene Agonisten verursachte Thrombozytenaggregation und Degranulierung abzuschwächen. Nach Oxidation der Lipoproteine durch das in vivo vorkommende Oxidans Hypochlorit verlieren sowohl LDL als auch HDL ihren hemmenden Einfluss auf die Thrombozytenfunktion und entwickeln die Fähigkeit unabhängig von anderen Agonisten Thrombozytenaggregation, Degranulierung und GPIIb/IIIa Aktivierung hervorzurufen, wie auch die intrazelluläre VASP Phosphorylierung zu reduzieren. Darüber hinaus sind Hypochlorit-oxidierte LDL im Stande, die Expression von CD40L an der Thrombozytenoberfläche zu induzieren. Dieser Befund ist auch insofern von großem Interesse, als CD40L Entzündungen und prothrombotische Antworten hervorruft, welche für das Fortschreiten von atherosklerotischen Geschehnissen mitverantwortlich sind. Die in dieser Arbeit präsentierten Daten weisen darauf hin, dass der Scavenger Rezeptors CD36 eine zentrale Rolle in der Interaktion von Thrombozyten mit oxidierten Lipoproteinen spielt, da das Blocken dieses Rezeptors zu einer starken Abschwächung der biologischen Wirkung oxidierter Lipoproteine auf die Thrombozyten führt. Die hier vorgestellten Befunde unterstützen das Modell, dass oxidativer Stress in engem Zusammenhang mit (potentiell) pro-thrombotischen und pro-inflammatorischen Folgereaktionen steht und dass hierbei Thrombozyten wie auch Lipoproteine eine entscheidende Rolle einnehmen.Besides their important function in primary haemostasis and coagulation, platelets and their activation state play a pivotal role in the initiation and progression of atherosclerotic disease. A wealth of evidence indicates that lipoproteins, especially low density lipoproteins (LDL) and high density lipoproteins (HDL), directly influence the activity state of platelets. It is generally accepted that high plasma levels of HDL inversely correlate with platelet hyperreactivity, whereat plasma levels of LDL show the opposite effect. Since hypercholesterolemic patients also show enhanced susceptibility to oxidative lipoprotein modification, it remains unclear if the underlying mechanism of platelet activation in these patients is a consequence of lipoprotein oxidation. Results of this study indicate that not only native HDL but also native LDL have inhibitory effects on platelet activation. Both classes of lipoproteins are able to impair agonist-induced platelet aggregation and degranulation. Upon oxidative modification of lipoproteins, which was performed by the in vivo occurring oxidant hypochlorite, not only LDL but also HDL invert their function and acquire the ability to independently trigger platelet aggregation, degranulation, GPIIb/IIIa activation and decrease of VASP phosphorylation. Moreover, hypochlorite-oxidised LDL upregulate expression of platelet CD40L. The latter is of special interest since (soluble) CD40L elicits inflammatory and pro-thrombotic responses that favour and accelerate the progression of atherosclerosis. The results shown within this work clearly argue for a central role of scavenger receptor CD36 in the interaction of platelets with oxidised lipoproteins, since blocking of this receptor leads to a strong attenuation of all platelet-activating effects. These novel findings further support the model that oxidative stress is closely related to potentially atherogenic events and that the interaction of platelets and lipoproteins might therefore play a pivotal role in the progression of these events

Cell Type-Specific Roles of NF-κB Linking Inflammation and Thrombosis

The transcription factor NF-κB is a central mediator of inflammation with multiple links to thrombotic processes. In this review, we focus on the role of NF-κB signaling in cell types within the vasculature and the circulation that are involved in thrombo-inflammatory processes. All these cells express NF-κB, which mediates important functions in cellular interactions, cell survival and differentiation, as well as expression of cytokines, chemokines, and coagulation factors. Even platelets, as anucleated cells, contain NF-κB family members and their corresponding signaling molecules, which are involved in platelet activation, as well as secondary feedback circuits. The response of endothelial cells to inflammation and NF-κB activation is characterized by the induction of adhesion molecules promoting binding and transmigration of leukocytes, while simultaneously increasing their thrombogenic potential. Paracrine signaling from endothelial cells activates NF-κB in vascular smooth muscle cells and causes a phenotypic switch to a “synthetic” state associated with a decrease in contractile proteins. Monocytes react to inflammatory situations with enforced expression of tissue factor and after differentiation to macrophages with altered polarization. Neutrophils respond with an extension of their life span—and upon full activation they can expel their DNA thereby forming so-called neutrophil extracellular traps (NETs), which exert antibacterial functions, but also induce a strong coagulatory response. This may cause formation of microthrombi that are important for the immobilization of pathogens, a process designated as immunothrombosis. However, deregulation of the complex cellular links between inflammation and thrombosis by unrestrained NET formation or the loss of the endothelial layer due to mechanical rupture or erosion can result in rapid activation and aggregation of platelets and the manifestation of thrombo-inflammatory diseases. Sepsis is an important example of such a disorder caused by a dysregulated host response to infection finally leading to severe coagulopathies. NF-κB is critically involved in these pathophysiological processes as it induces both inflammatory and thrombotic responses

An APRI+ALBI Based Multivariable Model as Preoperative Predictor for Posthepatectomy Liver Failure.

OBJECTIVE AND BACKGROUND Clinically significant posthepatectomy liver failure (PHLF B+C) remains the main cause of mortality after major hepatic resection. This study aimed to establish an APRI+ALBI, aspartate aminotransferase to platelet ratio (APRI) combined with albumin-bilirubin grade (ALBI), based multivariable model (MVM) to predict PHLF and compare its performance to indocyanine green clearance (ICG-R15 or ICG-PDR) and albumin-ICG evaluation (ALICE). METHODS 12,056 patients from the National Surgical Quality Improvement Program (NSQIP) database were used to generate a MVM to predict PHLF B+C. The model was determined using stepwise backwards elimination. Performance of the model was tested using receiver operating characteristic curve analysis and validated in an international cohort of 2,525 patients. In 620 patients, the APRI+ALBI MVM, trained in the NSQIP cohort, was compared with MVM's based on other liver function tests (ICG clearance, ALICE) by comparing the areas under the curve (AUC). RESULTS A MVM including APRI+ALBI, age, sex, tumor type and extent of resection was found to predict PHLF B+C with an AUC of 0.77, with comparable performance in the validation cohort (AUC 0.74). In direct comparison with other MVM's based on more expensive and time-consuming liver function tests (ICG clearance, ALICE), the APRI+ALBI MVM demonstrated equal predictive potential for PHLF B+C. A smartphone application for calculation of the APRI+ALBI MVM was designed. CONCLUSION Risk assessment via the APRI+ALBI MVM for PHLF B+C increases preoperative predictive accuracy and represents an universally available and cost-effective risk assessment prior to hepatectomy, facilitated by a freely available smartphone app

Platelet-Leukocyte Interplay in Cancer Development and Progression

Beyond their crucial role in hemostasis, platelets are increasingly recognized as regulators of inflammation. Via modulation of the immune system by direct and indirect interactions with leukocytes, platelets regulate several aspects of tumor-associated pathology. They influence inflammatory processes in cancer at various stages: platelets alter the activation status of the endothelium, recruit leukocytes to tumor sites and attune the inflammatory milieu at sites of primary and metastatic tumors. Patients with cancer show systemic changes of platelet activation. Tumor-associated platelet activation facilitates initiation of the coagulation cascade and constitutes a significant risk for thrombosis. Tumor-activated platelets further contribute to cancer progression by promoting critical processes such as angiogenesis and metastasis. Platelets modulate innate leukocyte effector functions such as antigen presentation by dendritic cells, monocyte recruitment and differentiation or neutrophil extracellular trap formation, which sculpture immune responses but also promote thrombosis and metastasis. On the other hand, responses of the adaptive immune system are also regulated by platelets. They are also involved in T-helper cell 17 differentiation, which represents a double-edged sword in cancer progression, as these cells propagate angiogenesis and immunosuppressive activities but are also involved in recruiting immune cells into tumors and stimulating effector CD8+ T cells. Moreover, platelets fine-tune tumor surveillance processes by modulating natural killer cell-mediated cancer cell recognition and effector functions. This review aims at summarizing the role of platelet-leukocyte interactions in the development and progression of cancer and puts its focus on cancer-related alterations of platelet and leukocyte functions and their impact on cancer pathology

Native High Density Lipoproteins (HDL) Interfere with Platelet Activation Induced by Oxidized Low Density Lipoproteins (OxLDL)

Platelets and lipoproteins play a crucial role in atherogenesis, in part by their ability to modulate inflammation and oxidative stress. While oxidized low density lipoproteins (OxLDL) play a central role in the development of this disease, high density lipoproteins (HDL) represent an atheroprotective factor of utmost importance. As platelet function is remarkably sensitive to the influence of plasma lipoproteins, it was the aim of this study to clarify if HDL are able to counteract the stimulating effects of OxLDL with special emphasis on aspects of platelet function that are relevant to inflammation. Therefore, HDL were tested for their ability to interfere with pro-thrombotic and pro-inflammatory aspects of platelet function. We are able to show that HDL significantly impaired OxLDL-induced platelet aggregation and adhesion. In gel-filtered platelets, HDL decreased both the formation of reactive oxygen species and CD40L expression. Furthermore, HDL strongly interfered with OxLDL-induced formation of platelet-neutrophil aggregates in whole blood, suggesting that platelets represent a relevant and sensitive target for HDL. The finding that HDL effectively competed with the binding of OxLDL to the platelet surface might contribute to their atheroprotective and antithrombotic properties

Editorial: Platelet and megakaryocyte dysfunctions in infectious diseases

International audienc

A Novel, Rapid Method to Quantify Intraplatelet Calcium Dynamics by Ratiometric Flow Cytometry

<div><p>Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar⁺-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.</p></div