In Covid-19 Infection, Plasma Extracellular Vesicle Tissue Factor Activity Does Not Correlate with D-Dimer Levels
Authors: Girard Thomas J. , Eldem Irem , Remy Kenneth E , Mazer Monty , Di Paola Jorge 
Published: November 2021

Abstract
Objective: Identify a plasma-based activity, or biomarker, that defines the mechanism(s) by which Covid-19 disease triggers excessive coagulation.
Introduction: While acute respiratory syndrome is the fundamental feature of severe Covid-19 disease, having a high level of the coagulation biomarker D-dimer upon admission is associated with increased thrombosis and mortality. As such, hospitalized patients are often placed on anticoagulant heparins. How Covid-19 triggers excessive coagulation is unresolved. Sars-CoV-2 infection could expose existing tissue factor (TF) to blood or, via cytokines, induce TF expression on cells that are in direct contact with blood. Extracellular vesicles (EV) are lipid bound microparticles released by all types of healthy and damaged cell and Covid-19 patient plasma EV TF activity has been recently reported. Cellular activation and damage due to SARS-CoV-2 could also release polyanionic nucleic acids and polyphosphates and generate neutrophil extracellular traps as contact surfaces for clot formation.
Methods:
Study 1. We attempted to identify excessive coagulation pathway activities in Covid-19 plasma-based, Ca++-induced thrombin generation assays. Assays were performed in the absence and presence of selective extrinsic (TF) and intrinsic (contact activation) pathway inhibitors (n=296 plasma samples). D-dimer levels were also determined. In a smaller study, Covid-19 patient samples were collected directly into citrate or citrate plus corn trypsin inhibitor, then processed for analysis.
Study 2. We conducted studies to evaluate the extent to which EV TF activity contributes to the Covid-19-associated coagulopathies. Plasma EVs were isolated and EV TF activity determined by the difference in FXa activity in the absence vs presence of anti-TF antibody. D-dimer and tissue factor pathway inhibitor a (TFPIa) antigen levels were measured. Data from 232 samples collected from 96 Covid-19 positive patients and 18 samples from 14 healthy controls were analyzed. For each study analysis, patient samples were organized into groups based on the disease severity outcomes as follows: hospitalization (Hospitalization; n=37); intensive care (ICU; n=16); mechanical ventilation (Ventilation; n=22); or fatality (Deceased; n=22).
Result:
Study 1. Covid-19 samples showed considerable thrombin generation variability with some samples failing to generate thrombin; pathway selective inhibitors reduced thrombin generation while heparinase treatment increased thrombin generation. Upon analysis, thrombin generation parameters showed no significant correlations to either D-dimer levels or disease severity. Instead, plasma prepared from blood collected directly into corn trypsin inhibitor revealed that contact activation that occurred post-sample collection dominates procoagulant activity.
Study 2. Figure 1, shows EV TF activities, D-dimer and TFPIα levels obtained for Covid-19 samples, with data segregated based on disease severity outcomes. Statistically significant difference versus the Hospitalized group are shown. TFPIa levels were highest in heparin IV patients (24.4+1.5 nM) vs Heparin-SQ (12.8+0.9 nM) vs enoxaparin (10.8 +0.7 nM) (p value:&lt;0.0001). It is known that heparin treatment increases circulating TFPIα, however an increase in TFPIα might also further increase circulating TF/FVIIa/XaTFPI inhibitory complex, which would dissociate in citrated plasma, and might account for the increase in EV TF in other studies.
Conclusions: Contact activation that occurs post-sample collection is sufficient to obscure endogenous triggers of coagulation, if present, in Covid-19 patients' plasma. D-dimer and TFPIα strongly correlate with disease severity although the latter is likely affected by heparin treatment. The most severe Covid-19 patients with high D-dimer did not show detectible plasma EV TF activity. Plasma EV TF activity does not appear to adequately represent the mechanism responsible for elevated D-dimer levels in Covid-19 cases.
Figure 1 Figure 1.

Disclosures
Di Paola: CSL Behring: Consultancy, Honoraria.