Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1α and Cathepsin G

Abstract Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

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Factor Vila and other Haemostatic Variables following Bone Marrow Transplantation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648806 ◽

1994 ◽

Vol 72 (01) ◽

pp. 028-032 ◽

Cited By ~ 21

Author(s):

P Collins ◽

A Roderick ◽

D O’Brien ◽

E Tuddenham ◽

A O’Driscoll ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cell ◽

Bone Marrow Transplantation ◽

Tissue Factor ◽

Marrow Transplantation ◽

General Circulation ◽

Cell Activation ◽

Convincing Evidence ◽

Endothelial Cell Activation

SummaryHepatic venocclusive disease causes considerable morbidity and mortality following bone marrow transplantation. There are two hypotheses regarding the aetiology of this syndrome; firstly that changes in plasma coagulation factors and natural anticoagulants lead to a prothrombotic state and secondly that endothelial cell activation stimulates intravascular deposition of fibrin. We have investigated these mechanisms by measuring the changes in proteins C and S and factors VII and X in the post transplant period and by using the plasma concentration of factor Vila as an in vivo marker of potential endothelial cell tissue factor expression. Protein C fell in both allograft and autograft patients but more so in the allografts. Similar results were found for factors VII and X. These changes were predominantly due to hepatic dysfunction induced by the chemo-radiotherapy. Factor Vila levels were unchanged in both the allograft and autograft patients. We conclude that there is no convincing evidence for a procoagulant state following BMT as there are both anticoagulant and procoagulant changes. The absence of any changes in factor Vila levels suggests that tissue factor was not exposed to the general circulation following BMT but does not exclude focal expression at the sites of thrombosis.

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Laser-induced endothelial cell activation supports fibrin formation

Blood ◽

10.1182/blood-2010-05-283986 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4675-4683 ◽

Cited By ~ 54

Author(s):

Ben T. Atkinson ◽

Reema Jasuja ◽

Vivien M. Chen ◽

Prathima Nandivada ◽

Bruce Furie ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Tissue Factor ◽

Cell Activation ◽

Umbilical Vein ◽

Calcium Mobilization ◽

Endothelial Cell Activation ◽

Human Umbilical Vein ◽

Laser Injury ◽

Umbilical Vein Endothelial Cells

Laser-induced vessel wall injury leads to rapid thrombus formation in an animal thrombosis model. The target of laser injury is the endothelium. We monitored calcium mobilization to assess activation of the laser-targeted cells. Infusion of Fluo-4 AM, a calcium-sensitive fluorochrome, into the mouse circulation resulted in dye uptake in the endothelium and circulating hematopoietic cells. Laser injury in mice treated with eptifibatide to inhibit platelet accumulation resulted in rapid calcium mobilization within the endothelium. Calcium mobilization correlated with the secretion of lysosomal-associated membrane protein 1, a marker of endothelium activation. In the absence of eptifibatide, endothelium activation preceded platelet accumu-lation. Laser activation of human umbilical vein endothelial cells loaded with Fluo-4 resulted in a rapid increase in calcium mobilization associated cell fluorescence similar to that induced by adenosine diphosphate (10μM) or thrombin (1 U/mL). Laser activation of human umbilical vein endothelial cells in the presence of corn trypsin inhibitor treated human plasma devoid of platelets and cell microparticles led to fibrin for-mation that was inhibited by an inhibitory monoclonal anti–tissue factor antibody. Thus laser injury leads to rapid endothelial cell activation. The laser activated endothelial cells can support formation of tenase and prothrombinase and may be a source of activated tissue factor as well.

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Extracellular Vesicles Derived from HT-29 Colorectal Cancer Cells Induce Endothelial Cell Activation and Procoagulant Activity

Blood ◽

10.1182/blood.v126.23.3456.3456 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3456-3456

Author(s):

Yun Tian ◽

Meifang Wu ◽

Alok A. Khorana ◽

Keith R. McCrae

Keyword(s):

Colorectal Cancer ◽

Endothelial Cells ◽

Endothelial Cell ◽

Board Of Directors ◽

Tissue Factor ◽

Cell Activation ◽

Endothelial Cell Activation ◽

Advisory Committees ◽

Cell Monolayers ◽

Ht 29

Abstract Introduction: Hypercoagulability leading to thromboembolic complications is associated with significant morbidity and mortality in patients with colorectal cancer, however the underlying mechanisms are not well understood. Elevated levels of extracellular vesicles (EV), both microparticles (> 100 nm) and exosomes (< 100 nm), circulate in patients with colorectal cancer. While some studies suggest that EV-expressed tissue factor is associated with thrombosis in patients with cancer, overall results are inconsistent. However, EV contain many other mediators such as inflammatory cytokines and microRNAs (miRNA) that may be transferred to other cells and induce phenotypic changes. We hypothesized that EV derived from colorectal cancer cells in vitro would induce endothelial cell activation and promote endothelial cell procoagulant activity. Methods: Human colorectal adenocarcinoma cells (HT-29) were cultured at a density of 1.0×107 cells per T75 flask in EV-free media containing 2% FBS, and after removal of floating cells and cell debris, microparticles were collected by centrifugation at 20,000 x G for 15 minutes; each flask contained approximately 4.0 x 106 annexin V+ microparticles. Exosomes were collected by additional centrifugation of the supernatant at 100,000 x G for 90 minutes. Microparticles and exosomes, at different dilutions, were separately co-cultured with confluent endothelial cell monolayers in 6 or 96-well plates, for 6 hours, using TNF-α (4 ng/ml) as a positive control. Endothelial cell activation was assessed by measuring expression of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), Il-1β and tissue factor by quantitative RT-PCR (qPCR) and immunoblotting. We also analyzed the effects of microparticles and exosomes on endothelial cell procoagulant activity by layering plasma over confluent endothelial cell monolayers. Briefly, after incubating endothelial cell monolayers with microparticles or exosomes (or 4 ng/ml TNFα as a positive control) for 6 hours, monolayers were washed three times with 20mM HEPES buffer (pH 7.4) containing 5mM calcium chloride and then overlaid with 100 µl per well of pooled normal human plasma. Plasma was re-calcified by the addition of 11 ul 200 mM calcium chloride, and plates were then placed in a kinetic microtiter-plate reader (Synergy HT) maintained at 37°C, and fibrin formation measured as an increase in the optical density at 405 nm. Results: Microparticles and exosomes constitutively released by HT-29 cells induced activation of endothelial cells in a concentration-dependent manner. Significant increases in the expression of E-selectin, ICAM-1, VCAM-1, tissue factor and IL-1β mRNA and protein were observed (Figure 1). In general, microparticles were more potent in inducing endothelial cell activation than exosomes when the EV were derived from the same number of HT-29 cells, though quantification of small exosomes is imprecise. Interestingly, while the response of endothelial cells to microparticles and exosomes was generally similar to that of 4 ng/ml TNFα, both types of EV appeared more potent in the induction of endothelial cell tissue factor, particularly at the mRNA level. HT-29 cell-derived EV, both microparticles and exosomes, also significantly shortened the clotting time of recalcified plasma added to endothelial cell monolayers (Figure 2). Conclusions: EV constitutively released from HT-29 cells, both microparticles and exosomes, directly activate cultured endothelial cells leading to increased expression of cell adhesion molecules, elaboration of IL-1β, and expression of tissue factor. The change in endothelial phenotype is not due to simple transfer of biomolecules since EV stimulated changes at the mRNA and protein level. While responses to microparticles and exosomes were similar, some differences were observed, suggesting that different underlying mechanisms may contribute. Endothelial cell activation in response to EV leads to accelerated clotting of plasma on the endothelial cell monolayer. These findings suggest a novel effect of cancer cell-derived EV that may contribute to the hypercoagulability present in patients with malignancy. Disclosures Khorana: Daiichi Sankyo: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Leo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; sanofi: Consultancy, Honoraria. McCrae:Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy.

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