Platelet Activation By Collagen Can Initiate Coagulation, Independent Of Factor Xii And Tissue Factor

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.

Download Full-text

Activation of human factor VII during clotting in vitro

Blood ◽

10.1182/blood.v65.1.218.218 ◽

1985 ◽

Vol 65 (1) ◽

pp. 218-226 ◽

Cited By ~ 19

Author(s):

LV Rao ◽

SP Bajaj ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Glass Tube ◽

Factor Vii ◽

Clotting Factor ◽

Activate Factor ◽

Factor Xii ◽

Factor X ◽

Normal Plasma

Abstract We have studied factor VII activation by measuring the ratio of factor VII clotting to coupled amidolytic activity (VIIc/VIIam) and cleavage of 125I-factor VII. In purified systems, a low concentration of Xa or a higher concentration of IXa rapidly activated 125I-factor VII, yielding a VIIc/VIIam ratio of 25 and similar gel profiles of heavy and light chain peaks of VIIa. On further incubation, VIIa activity diminished and a third 125I-peak appeared. When normal blood containing added 125I- factor VII was clotted in a glass tube, the VIIc/VIIam ratio rose fivefold, and 20% of the 125I-factor VII was cleaved. Clotting normal plasma in an activated partial thromboplastin time (APTT) system yielded a VIIc/VIIam ratio of 25 and over 90% cleavage of 125I-factor VII. Clotting factor XII-deficient plasma preincubated with antibodies to factor X in an APTT system with added XIa yielded a VIIc/VIIam ratio of 19 and about 60% cleavage, which indicates that IXa, at a concentration achievable in plasma, can effectively activate factor VII. Clotting normal plasma with undiluted tissue factor yielded a VIIc/VIIam ratio of 15 to 20 and 60% cleavage of 125I-factor VII, whereas clotting plasma with diluted tissue factor activated factor VII only minimally. We conclude that both Xa and IXa can function as significant activators of factor VII in in vitro clotting mixtures but believe that only small amounts of factor VII may be activated in vivo during hemostasis.

Download Full-text

Factor XII-independent activation of factor XI in plasma: effects of sulfatides on tissue factor-induced coagulation

Blood ◽

10.1182/blood.v82.3.813.813 ◽

1993 ◽

Vol 82 (3) ◽

pp. 813-819 ◽

Cited By ~ 37

Author(s):

D Gailani ◽

GJ Jr Broze

Keyword(s):

Tissue Factor ◽

Gel Electrophoresis ◽

Dextran Sulfate ◽

Polyacrylamide Gel Electrophoresis ◽

Factor Xii ◽

Factor Xi ◽

Enhancing Effect ◽

Peptide Release ◽

Plasma Effects ◽

Release Assay

Abstract Factor XI (FXI) may be activated in a purified system by thrombin and by autoactivation in the presence of negatively charged substances such as dextran sulfate or sulfatides. The current studies were performed to determine if these processes occur during the coagulation of plasma. FXII--deficient plasma was supplemented with 125I-FXI and clot formation was induced with tissue factor and/or sulfatides. Cleavage of FXI was studied by standard polyacrylamide gel electrophoresis and autoradiography. Activated FXI (FXIa) was detected after 20 minutes of incubation with sulfatides alone and this process was markedly accelerated by the addition of tissue factor (TF). The enhancing effect of TF was blocked by hirudin, which indicated thrombin involvement in FXI activation. The contribution of FXIa to FIX activation in this system was studied using a 3H-FIX activation peptide release assay. Sulfatides increased FIX activation about twofold in plasma induced to clot with TF but had no effect if the plasma was immunodepleted of FXI. FIX activation was also increased in plasma induced to clot with FXa if sulfatides were present. The enhanced generation of FIXa was dependent on FXI and was blocked by hirudin. Some activation was seen in the reactions with sulfatides and hirudin and is likely solely caused by FXI autoactivation. The data indicate that during the coagulation of plasma in the presence of sulfatides, FXI is activated by a mechanism that is thrombin dependent and does not require FXII.

Download Full-text

Involvement Of Human Factor IX In Tissue Thromboplastin Induced Coagulation

10.1055/s-0038-1652050 ◽

1981 ◽

Author(s):

A M H P van den Besselaar ◽

I E Ram ◽

R M Bertina

Keyword(s):

Tissue Factor ◽

Polyacrylamide Gel Electrophoresis ◽

Fold Increase ◽

Factor Ix ◽

Factor Vii ◽

Factor Xii ◽

Free System ◽

Plasma Coagulation ◽

Human Factor Ix ◽

Tissue Thromboplastin

This study is concerned with the question whether activation of factor IX by factor VII - tissue thromboplastin contributes to the rate of plasma coagulation. The protein component of tissue factor was partially purified from human brain. Its molecular weight as deduced from SDS - polyacrylamide gel electrophoresis was about 48,000. Reconstitution of thromboplastin activity was obtained by mixing apoprotein and phospholipids in the presence of Triton X-100 and subsequent removal of Triton by adsorption to Biobeads SM-2. Reconstituted tissue factor greatly accelerated the activation of factor IX by isolated factor VII in the presence of calcium ions. In a contact free system (plasma from a patient with congenital factor XII deficiency; factor XII<0.001 Unit/ml) plasma coagulation times (tc) were determined as a function of apoprotein concentration (at constant phospholipid) both in the presence and absence of factor IX. At high apoprotein concentration tc showed to be independent of factor IX, whereas at low apoprotein concentration the removal of factor IX resulted in a 2 - 3 fold increase of tc. The involvement of the tissue factor - factor VII complex in this phenomenon was evaluated using a specific anti-factor VII serum. The results indicate that activation of factor IX by factor VII - tissue thromboplastin does not significantly contribute to the rate of plasma coagulation.

Download Full-text

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Blood ◽

10.1182/blood.2020007252 ◽

2020 ◽

Vol 136 (11) ◽

pp. 1330-1341 ◽

Cited By ~ 43

Author(s):

Eugenio D. Hottz ◽

Isaclaudia G. Azevedo-Quintanilha ◽

Lohanna Palhinha ◽

Lívia Teixeira ◽

Ester A. Barreto ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Activation ◽

Ex Vivo ◽

Invasive Mechanical Ventilation ◽

The Novel ◽

Aggregate Formation ◽

Effector Cells ◽

Life Threatening ◽

Emergent Pathogen ◽

Novel Coronavirus

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/β3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.

Download Full-text

Breast-cancer extracellular vesicles induce platelet activation and aggregation by tissue factor-independent and -dependent mechanisms

Thrombosis Research ◽

10.1016/j.thromres.2017.09.019 ◽

2017 ◽

Vol 159 ◽

pp. 24-32 ◽

Cited By ~ 26

Author(s):

Fausto G. Gomes ◽

Vanessa Sandim ◽

Vitor H. Almeida ◽

Araci M.R. Rondon ◽

Barbara B. Succar ◽

...

Keyword(s):

Breast Cancer ◽

Tissue Factor ◽

Extracellular Vesicles ◽

Platelet Activation

Download Full-text

The Effect of BIBT 986, a Novel Small Molecule Dual Inhibitor of Thrombin and Factor Xa, on Coagulation in a Model of Endotoxin Induced, Tissue Factor Triggered Coagulation Activation in Humans.

Blood ◽

10.1182/blood.v108.11.912.912 ◽

2006 ◽

Vol 108 (11) ◽

pp. 912-912 ◽

Cited By ~ 2

Author(s):

Bernd Jilma ◽

Judith M. Leitner ◽

Francesco Cardona ◽

Florian B. Mayr ◽

Christa Firbas ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Activation ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Thrombin Time ◽

Coagulation Activation ◽

Dual Inhibitor ◽

Activation Markers ◽

Markers Of Inflammation ◽

Prolonged Aptt

Abstract Background: BIBT 986 is a novel potent anticoagulant that dually inhibits Factors Xa and IIa. We hypothesized that BIBT 986 would dose-dependently decrease endotoxin-induced, tissue factor triggered coagulation activation. Hence it was the aim of the study to compare with placebo the anticoagulant activity of three dosages of BIBT 986 on parameters of coagulation, platelet activation and inflammation and to examine the safety of BIBT 986 in this setting. Methods: This study was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants were randomised to receive bolus primed continuous infusions of one of the three doses of BIBT 986 or placebo. All of them received a bolus infusion of 2ng/kg body weight lipopolysaccharide (LPS). Results: BIBT dose-dependently increased anti-Xa activity, activated partial thromboplastin time (APTT), ecarin clotting time (ECT), thrombin time (TT) and the international normalisation ratio (INR). Importantly, BIBT 986 dose-dependently blocked the LPS-induced coagulation as assessed by the in vivo markers of thrombin generation and action: BIBT 986 doses that prolonged APTT by 25% were already effective. The BIBT dose that prolonged APTT by 100%, completely suppressed the increase in prothrombin fragment (F1+2), thrombin-antithrombin complexes (TAT) and D-dimer. BIBT 986 had no influence on activation markers of inflammation, fibrinolysis, endothelial or platelet activation. Conclusion: Infusion of BIBT 986 was safe and well tolerated. BIBT 986 specifically and dose-dependently blocked LPS-induced, tissue factor trigger coagulation. When compared to different anticoagulants tested previously in this standardized model, BIBT 986 was more effective in suppressing thrombin generation (F1+2 levels) than standard doses of danaparoid, dalteparin or lepirudin. BIBT 986 represents the first drug of a new class of dual FXa and FIIa inhibitors, and displays high potency.

Download Full-text

Protein Disulfide Isomerase Is Required for Fibrin Generation and Platelet Thrombus Formation In Vivo.

Blood ◽

10.1182/blood.v110.11.292.292 ◽

2007 ◽

Vol 110 (11) ◽

pp. 292-292 ◽

Cited By ~ 1

Author(s):

Jaehyung Cho ◽

Barbara C. Furie ◽

Shaun R. Coughlin ◽

Bruce Furie

Keyword(s):

Tissue Factor ◽

Platelet Activation ◽

Thrombus Formation ◽

Null Mouse ◽

Fibrin Formation ◽

Platelet Thrombus ◽

Wall Injury ◽

Protein Disulfide ◽

Dose Dependent

Abstract Thiol isomerases catalyze disulfide oxidation, reduction and isomerization, playing an important role during protein synthesis. Recent studies suggest a role for protein disulfide isomerase (PDI), a prototype of the thiol isomerase family, in platelet function and regulation of tissue factor activity (Essex and Li. Curr Drug Targets. 2006; Chen and Hogg. J Thromb Haemost. 2006). To determine the role of intravascular PDI during thrombus formation, PDI expression, platelet accumulation, and fibrin generation were monitored following laser-induced arteriolar injury in the mouse cremaster muscle by intravital fluorescence microscopy. PDI antigen exhibited a time-dependent increase in the developing thrombus after vessel wall injury and remained associated with the thrombus. Infusion of bacitracin, a non-specific inhibitor of thiol isomerases, into the circulation inhibited platelet thrombus formation and fibrin generation in a dose-dependent manner. Infusion of a function-blocking monoclonal antibody to PDI (RL90) into the circulation of a wild type mouse also resulted in dose-dependent inhibition of platelet accumulation and fibrin generation. To determine whether PDI inhibits fibrin formation by blocking tissue factor activation, or by preventing platelet activation and the development of the membrane surface that is required for assembly of the tenase and the prothrombinase complex in vivo, we explored fibrin formation in mice lacking protease-activated receptor-4 (Par4). Although there is no stable accumulation of platelets and no platelet activation, fibrin formation is normal in the Par4 null mouse (Vandendries et al, Proc Natl Acad Sci USA. 2007), suggesting that fibrin generation in the laser-induced vessel injury model is independent of platelet activation. Infusion of the function-blocking anti-PDI antibody (RL90) into the circulation of a Par4 null mouse prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required to generate tissue factor in a form that leads to thrombin generation and fibrin formation during thrombus development and is required for thrombus formation.

Download Full-text