Acquired Platelet GPVI Dysfunction As Possible Predictor for Early Sepsis Diagnosis and Poor Outcome
Lukas J. WeißGeorgi ManukjanNils NaglerMarkus KredelThiên‐Trí LâmBernhard NieswandtDirk WeismannHarald Schulze
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GPVI
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BackgroundCollagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation.ObjectivesTo determine the importance of GPVI clusters for thrombus formation in whole blood under shear.MethodsWe utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials.ResultsAutomated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation.ConclusionLabeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.
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Background: Severe sepsis is defined as sepsis plus organ dysfunction. There is a need to quantify this dysfunction, and several scoring systems have been developed. Method: Review of the pertinent English-language literature. Results: Early scoring of organ failure simply counted the number of failing organs, but the degree of dysfunction is an important variable. The first system to grade dysfunction was the multiple organ dysfunction score (MODS). The drawback of MODS is the variable used to quantify cardiovascular failure. To alleviate this limitation, the Brussels score was developed. However, it, too, was suboptimal and was replaced by the sequential or sepsis-related organ failure assessment (SOFA) score. There is a clear correlation between the total SOFA score and the mortality rate, and in certain studies, the total SOFA score was at least as good as traditional methods of predicting death. A key advantage of these simple scores is that they can be repeated to follow the course of organ dysfunction. The evolution of the SOFA score has been used to demonstrate the effects of various therapeutic interventions. Conclusion: Quantifying organ dysfunction in patients with sepsis can assist in assessing prognosis and determining treatment effectiveness. The simplicity, reliability, and reproducibility of current scores facilitate their widespread use.
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Glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. Clinical studies suggest that the plasma level of soluble GPVI (sGPVI) is a highly specific and useful platelet activation marker. However, many properties of sGPVI have not been fully characterized, such as its sensitivity in detecting platelet activation and its elimination rate from the blood. In this study we established a sandwich enzyme-linked immunosorbent assay for human sGPVI, which cross-reacts to cynomolgus monkey sGPVI, and evaluated the time course of sGPVI production in a cynomolgus monkey model of lipopolysaccharide (LPS)-induced thrombocytopenia. The sGPVI levels in this model were dramatically elevated and returned to baseline by 24 hours after LPS injection, the change was more pronounced than the existing platelet activation biomarker, soluble P-selectin (sP-selectin) levels. The elimination half-life of recombinant human sGPVI was about 2.5 hours following intravenous administration to monkeys. These results suggest that plasma sGPVI closely reflects platelet activation in the bloodstream and has a short half-life. sGPVI would be a useful biomarker for disorders marked by platelet activation and for monitoring anti-platelet therapy.
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Platelet activation and subsequent thrombus formation are important for preventing excessive blood loss at sites of vascular injury, a process termed haemostasis. However, excessive platelet activation at sites of atherosclerotic plaque rupture can lead to thrombus formation, which may occlude the vessel and cause heart attack or stroke. The platelet collagen receptor GPVI is essential for thrombus formation, but is largely dispensable for haemostasis.
Tetraspanins are transmembrane proteins which compartmentalise the membrane through formation of dynamic tetraspanin-enriched microdomains. Due to regulation of a wide range of partner proteins, tetraspanins have been implicated in many cellular processes, including platelet activation, though most platelet tetraspanins have not been characterised.
The aim of this thesis was to investigate the novel platelet tetraspanin Tspan18, using the Tspan18 knockout mouse. Tspan18 was shown to have a role in platelet activation and platelet Ca\(^2\)\(^+\) signalling specifically downstream of GPVI. Tspan18 also appeared to have a role in haemostasis, as Tspan18 deficient mice displayed a severe bleeding phenotype. The bleeding was shown to be driven by non-haematopoietic cells and is therefore unlikely to be platelet-driven. Additionally, Tspan18-induced Ca\(^2\)\(^+\) mobilisation was shown to be dependant on functioning Orai1 Ca\(^2\)\(^+\) channels and a novel interaction between Tspan18 and the Orai family was identified. Together, these findings suggest a role for Tspan18 in platelet activation and regulation of Ca\(^2\)\(^+\) mobilisation, potentially via interaction with Orai proteins.
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The present analysis investigated the role of GPVI for platelet activation and platelet-mediated endothelial activation in patients with type 2 diabetes. The major findings of the present study are: a) patients with type 2 diabetes have an enhanced platelet surface expression of FcgRIIA that correlates with GPVI expression compared with non-diabetic patients; b) stimulation of GPVI results in substantial secretion of CD40L in normal control platelets, GPVI-dependent CD40L release is enhanced in type 2 diabetes as compared with non-diabetes; c) soluble GPVI inhibits GPVI-induced secretion of platelet CD40L; d) co-incubation of cultured endothelial cells with GPVI/ligation-stimulated platelets induces substantial endothelial activation.
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Upon vascular injury, platelets interact with exposed ligands, whereafter they become activated and form a platelet plug, to minimize blood loss. However, unwanted platelet activation can lead to arterial thrombosis, hart infarcts and stroke. There is still an unmet clinical need for the development of novel antiplatelet drugs, which inhibit thrombosis, without increasing the bleeding risk. This thesis investigated acute and persistent mechanisms of platelet activation, mainly via the platelet receptors GPVI and PAR1, which are interesting and potential targets for novel antiplatelet therapy. The novel insights into the mechanisms involved in platelet activation via GPVI and PAR1 contribute to a better understanding of the complex platelet activation mechanisms in thrombosis and haemostasis.
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Objectives Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. Methods 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). Results The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer ( P <0.0001) as well as demonstrating higher resting P-selectin exposure ( P <0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r 2 = 0.88, P <0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission ( P <0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls ( P <0.0001). Conclusions Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.
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