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Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase \(A_2\) (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.
Effects of cocoa genotypes on coat color, platelets and coagulation parameters in French Bulldogs
(2021)
A nonsense variant in HPS3, c.2420G>A or p.Trp807*, was recently discovered as the cause for a brown coat color termed cocoa in French Bulldogs. Here, we studied the genotype–phenotype correlation regarding coat color in HPS3 mutant dogs that carried various combinations of mutant alleles at other coat color genes. Different combinations of HPS3, MLPH and TYRP1 genotypes resulted in subtly different shades of brown coat colors. As HPS3 variants in humans cause the Hermansky–Pudlak syndrome type 3, which in addition to oculocutaneous albinism is characterized by a storage pool deficiency leading to bleeding tendency, we also investigated the phenotypic consequences of the HPS3 variant in French Bulldogs on hematological parameters. HPS3 mutant dogs had a significantly lowered platelet dense granules abundance. However, no increased bleeding tendencies in daily routine were reported by dog owners. We therefore conclude that in dogs, the phenotypic effect of the HPS3 variant is largely restricted to pigmentation. While an effect on platelet morphology is evident, we did not obtain any indications for major health problems associated with the cocoa coat color in French Bulldogs. Further studies will be necessary to definitely rule out very subtle effects on visual acuity or a clinically relevant bleeding disorder.
Die vorliegende Arbeit beschreibt die günstigen Auswirkungen der chronischen Aktivierung der löslichen Guanylatcyclase durch HMR 1766 auf die Regulation der Thrombozytenaktivität im experimentellen Diabetes mellitus. Es konnte die zugrunde liegende Hypothese eines aktivierten Zustands von Thrombozyten im Diabetes bestätigt und darüber hinaus die Reversibilität dieser Aktivierung durch medikamentöse, direkte, NO-unabhängige, chronische Stimulation der sGC, dem Schlüsselenzym zur Vermittlung endogener NO-Wirkung, gezeigt werden. Hierzu wurden mit Hilfe durchflusszytometrischer Bestimmungen in frisch entnommenem Vollblut verschiedene thrombozytäre Marker analysiert. Zum einen deuteten die Ergebnisse auf eine verminderte NO-Bioverfügbarkeit beziehungsweise eine verringerte Aktivität des Effektorenzyms im Diabetes und eine gesteigerte Aktivität des Enzyms bei HMR 1766-behandelten Versuchstieren hin, zum anderen konnte ein Rückgang der Expression thrombozytärer Aktivierungsparameter in Richtung des Niveaus gesunder Kontrollen bei Thrombozyten der Tiere gezeigt werden, die den GC-Aktivator erhielten. Darüber hinaus stellte sich eine verminderte Aktivierbarkeit auf externe Stimuli bei in vitro Versuchen in Vollblut als auch in PRP heraus. Bei aggregometrischen Untersuchungen mit PRP zeigte sich eine verminderte Aggregation als Reaktion auf Fractalkine-Inkubation und anschließende direkte Stimulation mit ADP bei dem Material HMR 1766 behandelter Tiere. Zur Untersuchung von Akut-Effekten der Substanz wurden Proben gesunder beziehungsweise diabetischer Tiere teils direkt mit dem Medikament inkubiert, und es wurde einmalig medikamentös behandelten Tieren Blut entnommen. Hier stellte sich eine verminderte ADP-induzierte Aggregation bei behandelten Proben heraus, zudem zeigten sich positive Akut-Effekte hinsichtlich der basalen als auch der mit einem NO-Donor stimulierten VASP-Phosphorylierung. Bei der Betrachtung hämatologischer und metabolischer Parameter fiel bei sonst unauffälligen Ergebnissen das MPV auf, das im Diabetes erhöht, nach Behandlung mit dem GC-Aktivator jedoch wieder auf Kontrollwerte reduziert war. Die vielversprechenden Ergebnisse könnten ein Schritt zur Etablierung dieser Substanz als zukünftiges Medikament zur Prävention beziehungsweise Behandlung kardiovaskulärer Komplikationen bei Diabetes sein.
Endothelial–platelet interactions in influenza‐induced pneumonia: A potential therapeutic target
(2020)
Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro‐thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza‐mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza‐induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.
Essentiell für die Blutstillung (Haemostase) ist die Thrombozyten- oder Blutplaettchen-Adhaesion und die Thrombus-Bildung. Beide Vorgaenge werden hauptsaechlich durch den Thrombozyten-Rezeptor Integrin alphaIIb-beta3 vermittelt. Nach Bindung des Liganden Fibrinogen aendert sich die Rezeptor-Konformation, Integrine assoziieren und ein intrazellulaeres Signalnetzwerk wird aktiviert, welches die Organisation des Aktin-Zytoskeletts steuert. Diese Zytoskelett-Reorganisationen sind Grundlage für zellulaere Adhaesions- und Aggregations-Prozesse. Die Signalvermittlung vom Integrin zum Zytoskelett wird durch die Protein-Tyrosinkinase Src eingeleitet, deren Aktivitaetszustand den Signalweg reguliert. Bei der Src-Aktivierung wird Tyrosin 418 durch Autokatalyse phosphoryliert. Die Kinase muss jedoch wieder inaktiviert werden. Dies übernimmt in Plaettchen ausschliesslich die Tyrosinkinase Csk (C-terminale Src Kinase) durch Phosphorylierung von Tyrosin 529 im C-terminalen Ende des Proteins. Die Csk-vermittelte Inaktivierung von Src stellt den entscheidenden Kontrollschritt des alphaIIb-beta3-vermittelten Signalwegs dar. Obwohl bekannt ist, dass die Src-Aktivierung bei der Zelladhaesion an den Zellraendern der Lamellipodien geschieht und man den Mechanismus und die Kinetik der Src-Csk Interaktion genauer versteht, ist bislang immer noch unbekannt, wo und wie Src inaktiviert wird bzw. welche Rolle der Src-Inaktivierung genau zukommt. FRET (Fluoreszenz-Resonanz-Energie-Transfer) ist ein physikalischer Effekt, mit dem Interaktionen beliebiger fluoreszenzmarkierter Proteine mikroskopisch detektiert werden koennen. Diese Technik wurde genutzt, um die Src-Csk-Interaktion waehrend der alphaIIb-beta3-vermittelten Fibrinogen-Adhaesion in einer etablierten Thrombozyten-Modellzelllinie (A5-CHO) direkt visualisierbar zu machen. Es zeigten sich starke Src-Csk Interaktionen (FRET-Signale) an den Zellraendern aktiver Lamellipodien und zusaetzlich in Fokalkontakten, wo beide Proteine mit Vinculin, einem Fokalkontakte-Marker, co-lokalisierten. Die Proteininteraktionen folgten einem hochdynamischen Ablauf. Nach der Akkumulation der Src-Csk Komplexe an den Zellraendern wanderten sie in Abstaenden von 2-3 Minuten nach innen, fragmentierten und bildeten schliesslich stabile Fokal-Adhaesionen. FRET-Signale an den Zellraendern fanden sich vor allem in ruhenden Lamellipodien bzw., waehrend des Lamellipodien-Rückzugs, in wachsenden Lamellipodien traten die FRET-Signale dort dagegen nicht auf. In unabhaengigen biochemischen Tests im Zeitfenster der FRET-Beobachtungen wurde ein spezifischer Anstieg der Src-Tyr529-Phosphorylierung (Inaktivierung) und eine parallele Abnahme der Src-Tyr418-Phosphorylierung (Aktivierung) gemessen. Weiterführende Ergebnisse lieferten Versuche mit Src- und Csk-Mutanten. Die Co-Expression von Wildtyp-Src mit Kinase-inaktivem CskK222R hatte weder einen Effekt auf die Adhaesion und Ausbreitung der Zellen noch auf die Praesenz von FRET, es aenderte sich jedoch drastisch die zellulaere Verteilung der FRET-Signale sowie das Wachstum und die Form der Lamellipodien. Die Co-Expression von Wildtyp-Csk mit konstitutiv aktivem SrcY529F verursachte dagegen eine stark verringerte Adhaesionsfaehigkeit und Hemmung der Lamellipodien-Bildung. Die Fokal-Adhaesionspunkte in diesen Zellen waren sehr schwach und ueberdimensioniert und lagen ungeordnet verteilt in der Adhaesionsebene. Zusaetzlich verursachte SrcY529F eine starke Ueberaktivierung des Zytoskeletts und das fast vollstaendige Verschwinden der FRET-Signale. Die ermittelten Daten zeigen, dass die enge Kontrolle der Src-Aktivitaet durch Csk eine bedeutende Rolle für die funktionelle Zell-Adhaesion and -Ausbreitung spielt. Co-Immunpraezipitations-Resultate und Messungen der Menge an markiertem Protein in Zellen, in welchen FRET detektierbar war, untermauern zusaetzlich unsere These, zum ersten Mal die Src-Regulation durch Csk in lebenden Zellen direkt beobachtbar gemacht zu haben. Dieser neue FRET-Ansatz kann auch als Reporter-System für Prozesse der Src-Inaktivierung in anderen Signalwegen und Zellen angewendet werden. Das Messprinzip kann weiterhin auf das Studium der Inaktivierung weiterer Mitglieder der Familie der Src-Kinasen (in verschiedensten Signalwegen) erweitert werden.
This work summarizes the results of studies on three major aspects of platelet signaling and of the pathogenesis of immune thrombocytopenia. Therefore, this thesis is divided into three parts. i) Platelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also trigger myocardial infarction and stroke. The initial capture of flowing platelets to the injured vessel wall is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor (vWF) immobilized on the exposed subendothelial extracellular matrix (ECM). The central importance of GPIb for platelet adhesion is well established, whereas GPV is generally considered to be of minor relevance for platelet physiology and thrombus formation. This study intended to clarify the relevance of this receptor during thrombus formation using Gp5-/- mice and mice with different double-deficiencies in GPV and in other platelet receptors. It was found that GPV and the collagen receptor integrin a2b1 have partially redundant functions in collagentriggered platelet aggregation. Further, it was revealed that GPV limits thrombus formation and impairs hemostasis in vivo. The data presented here demonstrate that the protective effect of GPVI-deficiency (another platelet collagen receptor) in arterial thrombosis and ischemic stroke depends on the expression of GPV. Moreover, it was demonstrated that lack of GPV restores the hemostatic function of mice lacking both GPVI and a2b1 or mice lacking GPVI and the C-type lectin receptor 2 (CLEC-2). Conclusively, GPV-depletion or blockade might have the potential to treat hemorrhagic disease states. ii) Platelets contain the two phospholipase (PL) D isoforms, PLD1 and PLD2, both of which presumably become activated upon platelet stimulation. However, the function of PLD in the process of platelet activation and aggregation has not been definitively explored. Thus, PLD-deficient mice were analyzed. Mice lacking PLD1 or PLD2 were viable, fertile and had normal platelet counts. PLD1 was found to be responsible for the inducible PLD-activity in platelets and to contribute to efficient integrin activation under static conditions. Moreover, flow adhesion experiments revealed that PLD1 is essential for efficient GPIb-mediated integrin activation. Consequently, Pld1-/- mice were protected from arterial thrombosis and ischemic brain infarction without affecting tail bleeding times. Hence, inhibition of PLD1 might be a novel approach for antithrombotic therapy. iii) Cellular activation of platelets or immune cells results in increased cytosolic calcium (Ca2+) levels. Store-operated calcium entry (SOCE) via the STIM1-Orai1 axis is the main route of Ca2+ entry downstream of immunoreceptor tyrosine-based activating motif (ITAM) receptor stimulation in mast cells and T cells. However, the requirement of Ca2+-mobilization in Fcg receptor (FcgR)-signaling and the relevance of STIM2 for T cell SOCE have been unclear. To address these questions, genetically modified mice lacking central molecules of the SOCE machinery were analyzed. Ca2+-measurements revealed that both STIM isoforms contribute to Ca2+-mobilization downstream of T cell receptor activation. Additionally, it was found that FcgR stimulation results in SOCE and is mediated by STIM1 and probably Orai1. Animal models of immune thrombocytopenia (ITP) revealed that SOCE is essential for platelet clearance and that both STIM isoforms contribute to the pathology of ITP. Moreover, in this work it was also demonstrated that STIM1 and Orai1 are essential in IgG-mediated systemic anaphylaxis. STIM2 contributes to IgG-mediated, but not to IgE-mediated anaphylaxis. The data indicate that interference with SOCE might become a new strategy to prevent or treat IgG-dependent autoimmune diseases.
Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed “reperfusion injury.” Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.
The pancreas is the key organ for the maintenance of euglycemia. This is regulated in particular by α-cell-derived glucagon and β-cell-derived insulin, which are released in response to nutrient deficiency and elevated glucose levels, respectively. Although glucose is the main regulator of insulin secretion, it is significantly enhanced by various potentiators.
Platelets are anucleate cell fragments in the bloodstream that are essential for hemostasis to prevent and stop bleeding events. Besides their classical role, platelets were implemented to be crucial for other physiological and pathophysiological processes, such as cancer progression, immune defense, and angiogenesis. Platelets from diabetic patients often present increased reactivity and basal activation. Interestingly, platelets store and release several substances that have been reported to potentiate insulin secretion by β-cells. For these reasons, the impact of platelets on β-cell functioning was investigated in this thesis.
Here it was shown that both glucose and a β-cell-derived substance/s promote platelet activation and binding to collagen. Additionally, platelet adhesion specifically to the microvasculature of pancreatic islets was revealed, supporting the hypothesis of their influence on glucose homeostasis. Genetic or pharmacological ablation of platelet functioning and platelet depletion consistently resulted in reduced insulin secretion and associated glucose intolerance. Further, the platelet-derived lipid fraction was found to enhance glucose-stimulated insulin secretion, with 20-hydroxyeicosatetraenoic acid (20-HETE) and possibly also lyso-precursor of platelet-activating factor (lysoPAF) being identified as crucial factors. However, the acute platelet-stimulated insulin secretion was found to decline with age, as did the levels of platelet-derived 20-HETE. In addition to their direct stimulatory effect on insulin secretion, specific defects in platelet activation have also been shown to affect glucose homeostasis by potentially influencing islet vascular development. Taking together, the results of this thesis suggest a direct and indirect mechanism of platelets in the regulation of insulin secretion that ensures glucose homeostasis, especially in young individuals.
Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s\(^{−1}\)). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A\(_2\) (TxA\(_2\)), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.