The global pandemic of coronavirus disease 2019 (COVID‐19) is associated with the development of acute respiratory distress syndrome (ARDS), which requires ventilation in critically ill patients. The ...pathophysiology of ARDS results from acute inflammation within the alveolar space and prevention of normal gas exchange. The increase in proinflammatory cytokines within the lung leads to recruitment of leukocytes, further propagating the local inflammatory response. A consistent finding in ARDS is the deposition of fibrin in the air spaces and lung parenchyma. COVID‐19 patients show elevated D‐dimers and fibrinogen. Fibrin deposits are found in the lungs of patients due to the dysregulation of the coagulation and fibrinolytic systems. Tissue factor (TF) is exposed on damaged alveolar endothelial cells and on the surface of leukocytes promoting fibrin deposition, while significantly elevated levels of plasminogen activator inhibitor 1 (PAI‐1) from lung epithelium and endothelial cells create a hypofibrinolytic state. Prophylaxis treatment of COVID‐19 patients with low molecular weight heparin (LMWH) is important to limit coagulopathy. However, to degrade pre‐existing fibrin in the lung it is essential to promote local fibrinolysis. In this review, we discuss the repurposing of fibrinolytic drugs, namely tissue‐type plasminogen activator (tPA), to treat COVID‐19 associated ARDS. tPA is an approved intravenous thrombolytic treatment, and the nebulizer form has been shown to be effective in plastic bronchitis and is currently in Phase II clinical trial. Nebulizer plasminogen activators may provide a targeted approach in COVID‐19 patients to degrade fibrin and improving oxygenation in critically ill patients.
Platelet-Mediated Modulation of Fibrinolysis Whyte, Claire S; Mitchell, Joanne L; Mutch, Nicola J
Seminars in thrombosis and hemostasis,
03/2017, Volume:
43, Issue:
2
Journal Article
Peer reviewed
Open access
Platelets are crucial to the hemostatic response. Their role in coagulation is well documented and they have been considered for some time to promote resistance of thrombi to fibrinolysis. Platelets ...confer resistance to lysis by promoting clot retraction of the immediate fibrin network and through release of plasminogen activator inhibitor-1 from their α-granules. However, recent developments in the field indicate that the role of platelets in fibrinolysis is much more diverse. Indeed, novel studies suggest that platelets form different subpopulations upon activation that play varied roles in regulating hemostasis. Likewise the developments in our understanding of thrombus formation, architecture, and changes in fibrin deposition and composition suggest that these different subpopulations of platelets may populate distinct areas within thrombi and potentially dictate the local hemostatic balance in these areas. This review will discuss the diverse roles of platelets in fibrinolysis and highlight the recent developments in the field and the contribution of both the intracellular pool of modulators as well as the membrane surface in regulating these processes.
A subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in ...severely ill patients with COVID-19 are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesized that platelets might be susceptible to activation by anti–severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies and might contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike immunoglobulin G enhanced platelet-mediated thrombosis on von Willebrand factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcγRIIA, and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by the therapeutic small molecules R406 (fostamatinib) and ibrutinib, which inhibit tyrosine kinases Syk and Btk, respectively, or by the P2Y12 antagonist cangrelor.
•Aberrant glycosylation of anti-SARS-CoV-2 spike IgG immune complexes increases platelet thrombus formation on VWF.•Inhibition of Syk, Btk, P2Y12, or FcγRIIA reverses enhancement of thrombus formation mediated by anti-SARS-CoV-2 spike immune complexes.
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Formyl peptide receptors (FPRs) are members of seven transmembrane G protein-coupled receptors superfamily that exhibit different responses based on the nature of stimulating ligand type. FPRs have ...been shown to be present in platelets and regulate their function. However, the effect of formyl peptide receptor 2 (FPR2/ALX) lipid ligands on platelets has not yet been addressed. Hence, we sought to study the role of FPR2/ALX ligand and lipoxin A4 lipid analogue, BML-111, in the modulation of platelet function and thrombus formation. Immunofluorescence microscopy showed subcellular distribution and peripheral mobilisation of FPR2/ALX in stimulated platelets. This variation in distribution was further confirmed using flow cytometry. BML-111 inhibited a range of platelet activities in a dose-dependent manner in response to several platelet agonists. This included aggregation, fibrinogen binding to integrin αIIbβ3, α-granule secretion, dense granule secretion, Ca
mobilisation and integrin αIIbβ3-mediated outside-in signaling. The selectivity of BML-111 for FPR2/ALX was confirmed using FPR2/ALX deficient mice in flow cytometry assays. In vitro thrombus formation was also inhibited by various concentrations of BML-111. Moreover, the levels of vasodilator stimulated phosphorylation (VASP-S157) increased significantly after BML-111 treatment in resting and stimulated platelets via protein kinase A (PKA) independently of cyclic adenosine monophosphate (cAMP) signaling. Together, our findings demonstrate the significance of BML-111 as a modulator of platelet function via FPR2/ALX and unravel the thrombo-protective potentials of BML-111 induced signaling against thrombo-inflammatory diseases.
Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking α2-antiplasmin (α2AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of ...this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIII-depleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to α2AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was α2AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking α2AP to fibrin.
•Factor XIII-A is exposed in protruding caps on the activated platelet surface.•Platelet FXIII-A exerts antifibrinolytic function by cross-linking α2AP to fibrin.
Platelets release platelet-derived extracellular vesicles (PDEVs) upon activation – in a process that is regulated by generation of reactive oxygen species (ROS). Platelet NADPH oxidase-1 (Nox-1) ...contributes to ROS generation and thrombus formation downstream of the collagen receptor GPVI.
We aimed to investigate whether PDEVs contain Nox-1 and whether this is relevant for PDEV-induced platelet activation.
PDEVs were isolated through serial centrifugation after platelet activation with thrombin receptor agonist TRAP-6 (activated PDEVs) or in the absence of agonist (resting PDEVs). The physical properties of PDEVs were analyzed through nanoparticle tracking analysis. Nox-1 levels, fibrinogen binding and P-selectin exposure were measured using flow cytometry, and protein levels quantified by immunoblot analysis. ROS were quantified using DCF fluorescence and electron paramagnetic resonance.
Nox-1 was found to be increased on the platelet outer membrane upon activation and was present in PDEVs. PDEVs induced platelet activation, while co-addition of GPVI agonist collagen-related peptide (CRP) did not potentiate this response. PDEVs were shown to be able to generate superoxide in a process at least partially mediated by Nox-1, while Nox-1 inhibition with ML171 (also known as 2-APT) did not influence PDEV production. Finally, inhibition of Nox-1 abrogated PDEV-mediated platelet activation.
PDEVs are able to generate superoxide, bind to and activate platelets in a process mediated by Nox-1. These data provide novel mechanisms by which Nox-1 potentiates platelet responses, thus proposing Nox-1 inhibition as a feasible strategy to treat and prevent thrombotic diseases.
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•Activated platelets have increased NADPH oxidase-1 (Nox-1) exposure on the outer membrane.•Platelet-derived extracellular vesicles (PDEVs) express Nox-1 and generate superoxide in a process mediated by Nox-1.•PDEVs bind and activate platelets in a Nox-1-dependent manner.
Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish ...oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs.
We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs.
A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation.
n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay.
Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis.
This trial was registered at clinicaltrials.gov as NCT03203512.
The response of platelets to activating stimuli and pharmaceutical agents varies greatly within the normal population. Current platelet function tests are used to measure end-point levels of platelet ...activation without taking the speed at which platelets activate into account, potentially missing vital metrics to characterize platelet reactivity.
To identify variability, to agonists and among individuals, in platelet activation kinetics and assess the impact of this on thrombus formation.
We have developed a bespoke real-time flow cytometry assay and analysis package to measure the rate of platelet activation over time using 2 parameters of platelet activation, fibrinogen binding and P-selectin exposure.
The rate of platelet activation varied considerably within the normal population but did not correlate with maximal platelet activation, demonstrating that platelet activation rate is a separate and novel metric to describe platelet reactivity. The relative rate of platelet response between agonists was strongly correlated, suggesting that a central control mechanism regulates the rate of platelet response to all agonists.
For the first time, we have shown that platelet response rate corresponds to thrombus size and structure, wherein faster responders form larger, more densely packed thrombi at arterial, but crucially not venous, shear. We have demonstrated that the rate of platelet activation is an important metric in stratifying individual platelet responses and will provide a novel focus for the design and development of antiplatelet therapy, targeting high-shear thrombosis without exacerbating bleeding at low shear.
•Current platelet function tests often overlook the kinetics of platelet activation.•Real-time flow cytometry assay and analysis package measures platelet activation kinetics.•The rate of platelet activation varies considerably within the normal population.•Platelet activation rate alters thrombus size and structure at arterial, but not venous, shear.
Risks and outcomes of myocardial infarction (MI) are different between men and women and some studies have demonstrated that the latter have a higher risk of mortality. Whilst there are many reasons ...for this, it may also partially be linked to stronger innate and adaptive immune responses mounted by females compared to males. However, little is known about how sex impacts the coronary microvessels, the site where inflammatory processes take place, after an MI. Intravital and laser speckle microscopy was used to image coronary microvessels and ventricular perfusion
in response to myocardial ischaemia-reperfusion (IR) injury in male and female mice. Interleukin-36 (IL-36) is the latest addition to the IL-1 superfamily of pro-inflammatory cytokines and has recently been shown to mediate inflammation in a number of non-cardiovascular diseases. Its role in mediating potential sex-related microcirculatiory pertubations in the heart are unknown. Therefore, the vasculoprotective efficacy of an IL-36 receptor antagonist (IL-36Ra) was also investigated.
Immunostaining and flow cytometry demonstrated higher expression of IL-36 and its receptor in female hearts, an observation confirmed in human samples. Intravital imaging of the anaesthetised mouse beating heart identified significantly greater neutrophil recruitment in female hearts, but a greater burden of thrombotic disease in male hearts. Male mice had reduced functional capillary density and were unable to restore perfusion to baseline values as effectively as females. However, female mice had significantly larger infarcts. Interestingly, IL-36Ra decreased inflammation, improved perfusion, and reduced infarct size in both sexes despite increasing platelet presence in male hearts. Mechanistically, this was explained by IL-36Ra attenuating endothelial oxidative damage and VCAM-1 expression. Importantly, IL-36Ra administration during ischaemia was critical for vasculoprotection to be realised.
This novel study identified notable sex-related differences in the coronary microcirculatory response to myocardial IR injury which may explain why some studies have noted poorer outcomes in women after MI. Whilst contemporary MI treatment focuses on anti-platelet strategies, the heightened presence of neutrophils in female IR injured coronary microvessels necessitates the development of an effective anti-inflammatory approach for treating female patients. We also emphasise the importance of early intervention during the ischaemic period in order to maximise therapeutic effectiveness.
Multiple myeloma (MM) and its precursor states, smoldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS) are associated with increased incidence of thrombosis, however ...the cause of this is unknown. Lenalidomide treatment of MM substantially improves patient survival, although significantly increases thrombotic risk by an unknown mechanism. This pilot study aimed to establish the impact of MM and its treatment with Lenalidomide on platelet function. We analyzed platelet function in MGUS, SM and MM compared to healthy controls. We report an increase in platelet reactivity in MGUS, SM, and MM where increases in fibrinogen binding, P-selectin exposure, altered receptor expression, elevated levels of aggregation and enhanced sensitivity to agonist stimulation were observed. We also demonstrate an increase in patient platelet reactivity post Lenalidomide treatment compared to pre-treatment. We show Lenalidomide treatment of platelets
increased reactivity that was associated with formation of larger thrombi at arterial shear rates but not venous shear rates. This study demonstrates a clear increase in platelet reactivity and prothrombotic potential in patients with MGUS, SM and MM which is elevated further upon treatment with Lenalidomide. Our observations suggest that more detailed studies are warranted to determine mechanisms of thrombotic complications to enable the development of new preventative strategies that specifically target platelets.
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