key: cord-0839824-ew9dlfdj
authors: Henderson, Michael W; Lima, Franciele; Moraes, Carla Roberta Peachizepi; Ilich, Anton; Huber, Stephany Cares; Barbosa, Mayck Silva; Santos, Irene; Palma, Andre C; Nunes, Thyago Alves; Ulaf, Raisa Gusso; Ribeiro, Luciana Costa; Bernardes, Ana Flavia; Bombassaro, Bruna; Dertkigil, Sergio San Juan; Moretti, Maria Luiza; Strickland, Sidney; Annichino-Bizzacchi, Joyce M; Orsi, Fernanda Andrade; Mansour, Eli; Velloso, Licio A; Key, Nigel S; De Paula, Erich Vinicius
title: Contact and intrinsic coagulation pathways are activated and associated with adverse clinical outcomes in COVID-19
date: 2022-03-03
journal: Blood Adv
DOI: 10.1182/bloodadvances.2021006620
sha: 2d5f31232b3ecc61b9b610c3a69b75c42d95021f
doc_id: 839824
cord_uid: ew9dlfdj

Coagulation activation is a prominent feature ofSARS-CoV-2infection (COVID-19). Activation of the contact system and intrinsic pathway has increasingly been implicated in the prothrombotic state observed in both sterile and infectious inflammatory conditions. We thus sought to assess activation of the contact system and intrinsic pathway in subjects with COVID-19 infection. Baseline plasma levels of protease:serpin complexes indicative of activation of the contact and intrinsic pathwayswere measured in samples from inpatients with COVID-19 andhealthy individuals. Cleaved kininogen, a surrogate for bradykinin release, was measured by ELISA, while extrinsic pathway activation was assessed by microvesicle tissue factor-mediated factor Xa generation (MVTF). Samples were collected within 24 hours of COVID-19 diagnosis. Thirty patients with COVID-19 and thirty age- and sex-matched controls were enrolled. Contact system and intrinsic pathway activation in COVID-19 were demonstrated by increased plasma levels offactor XIIa:C1 esterase inhibitor (FXIIa:C1), kallikrein:C1, FXIa:C1, FXIa:α1antitrypsin, and FIXa:antithrombin (FIXa:AT). MVTF levels were also increased in COVID-19 subjects. Since FIXa:AT levels were associated with both contact/intrinsic pathway complexes and MVTF, activation of FIX likely occurs through both the contact/intrinsic and extrinsic pathways. Among the protease:serpin complexes measured, FIXa:AT complexes were uniquely associated with clinical indices of disease severity, specificallythe total length of hospitalization, length of ICU stay, and extent of lung CT changes.We conclude that the contact/intrinsic pathway may contribute to the pathogenesis of the prothrombotic state in COVID-19. Larger prospective studies are required to confirm whether FIXa:AT complexes are a clinically useful biomarker of adverse clinical outcomes.

The critical role of coagulation pathways in the pathogenesis of SARS-CoV-2 disease (COVID-19) is supported by a growing number of studies demonstrating alterations of several biomarkers of coagulation activation, some of which have been associated with disease severity 1,2 , an elevated risk of venous thromboembolism 3, 4 , and/or the presence of pulmonary microvascular thrombi 5, 6 . Activation of coagulation in the context of infectious diseases has been long recognized as an integral part of the host response to pathogens 7, 8 , and involves a complex interplay of immune cells, coagulation factors and other bioactive compounds, that has been recently termed immunothrombosis. In the early phase of the SARS-CoV-2 pandemic, D-dimer was identified as a biomarker that correlates with disease severity 9, 10 . However, plasma Ddimer generation is dependent upon the both the coagulation and fibrinolytic systems, and as such, it does not address the mechanism of the coagulopathy in COVID-19. Tissue factor (TF) is a principal trigger of immunothrombosis in acute respiratory distress syndrome 11 , and plasma microvesicle-associated tissue factor activity (MVTF) has been demonstrated to correlate with mortality in respiratory viral infections 12 .

Recently, the association of MVTF with COVID-19 severity 13 has been reported, suggesting a role for extrinsic activation in this disease. Yet, due to the lack of an international TF standard, standardization of this assay has not been possible 14 . In addition, the assay operates at the limits of sensitivity with undetectable levels in many healthy subjects. For these reasons, its use has been limited to research laboratories.

Components of the contact system (namely, factors XII and XI) have increasingly been recognized as key mediators in the prothrombotic states observed in multiple clinical contexts such as sepsis and cancer, despite their lesser roles in hemostasis [15] [16] [17] . Both factor XIa (FXIa) and kallikrein (PKa) can activate factor IX (FIX) following contact activation [18] [19] [20] , while the TF:FVIIa complex can also directly activate FIX 21 . Similarly, FXI may be activated not only by FXIIa, but also by thrombin. FXIa is irreversibly inactivated by several serpins, including C1 Inhibitor, α1-antitrypsin and (in the presence of heparin) antithrombin, while kallikrein is primarily inactivated by C1. In turn, FIXa is primarily inactivated by antithrombin, forming a stable covalent FIXa:antithrombin (FIXa:AT) complex. Meticulous sampling technique and processing must be considered when evaluating plasma for these complexes, as the principal activator of the contact pathway, factor XII (FXII), undergoes accelerated autoactivation upon contact with an anionic surface 22 . While the growing interest in the contact pathway has raised the question of targeting its component proteins for the prevention of thrombotic complications 23, 24 , the laboratory characterization of these complexes as indices of coagulation activation continues to be challenging 15 .

In order to address the mechanism of coagulation activation in subjects presenting with COVID-19, we employed a recently described strategy to assess activation of the contact and intrinsic pathways 25 , and also measured MVTF levels as a biomarker of extrinsic pathway activation 26 . In a preliminary secondary analysis, we then sought to associate levels of these biomarkers with adverse clinical outcomes in a well characterized study cohort.

Study population: The study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards of the University of Campinas and the University of North Carolina at Chapel Hill. All patients (or their legal representatives) and all healthy volunteers signed a written informed consent before any study procedure. The study population consisted of patients admitted to a single academic hospital with a diagnosis of COVID-19 between April 23 rd and June 14 th 2020 who were enrolled in a clinical trial focused on bradykinin inhibition in COVID-19 (Trial  registration  at  the  Brazilian  Clinical  Trials Registry: https://ensaiosclinicos.gov.br/rg/RBR-5s2mqg). In brief, 30 patients with severe COVID-19 were randomized to one of the following 3 treatment arms: standard care, icatibant (a bradykinin receptor 2 inhibitor) or C1 esterase inhibitor 27 . Samples used in our study were obtained before any trial intervention (hereafter referred to as 'baseline' samples). Inclusion criteria included: age ≥ 18 years old, symptom duration ≤ 12 days, a positive RT-PCR for SARS-CoV-2, presence of typical COVID-19 pneumonia on lung CT scan, sat O 2 ≤ 94% in ambient air or PaO 2 /FiO 2 < 300mmHg. Exclusion criteria were pregnancy, severe renal or liver disease, HIV infection or other immunodeficiency state, previous diagnosis of cancer, ischemic myocardial disease, history of thromboembolic events, hereditary angioedema or use of any experimental treatment for SARS-CoV-2 infection. Age and sex-matched healthy individuals from the same geographic regions were recruited among healthcare employees of the local blood bank, observing the same exclusion criteria.

Sample collection and processing: after ensuring satisfactory venipuncture, blood samples were collected into 3.2% sodium citrate or EDTA K2 tubes immediately after enrollment in the clinical trial (median time from admission: 1 day; range 0 -3 days). Samples were processed within 2 hours of collection by double centrifugation at 1,800g for 15 minutes at 22 o C to obtain platelet free plasma (PFP). EDTA-K2 samples were only submitted to a single centrifugation step. 300 µL aliquots were immediately frozen at -80 o C until analysis.

Clinical and laboratory outcomes: clinical and laboratory data were obtained from electronic medical records and from case report forms of the clinical trial. The extent of lung disease on admission was based on CT scans performed upon enrollment, and scored according to previously published criteria 27 .

Laboratory evaluation of classical hemostatic parameters: coagulation screening assays (PT, aPTT), coagulation factor (F) activities (fibrinogen, FVIII:C, FIX:C, FX:C, FXI:C and FXII:C), vWF antigen, vW ristocetin cofactor activity and antithrombin levels were measured in an automated coagulometer (ACL TOP 550 CTS, Instrumentation Laboratory, USA) using commercially available assays from the same manufacturer (HemosIL reagents). D-dimer was measured using an immunoturbidimetric assay (Innovance D-Dimer, Siemens Healthcare). P-selectin, u-PAR (urokinase-type plasminogen activator receptor) and PAI-1 (plasminogen activator inhibitor 1) levels were measured using a customized Luminex immunoassay (Procarta Plex multiplex panel, Thermo-Fischer Scientific) in a Bioplex 200 instrument (Bio-Rad). Plasminantiplasmin levels were measured using a commercially available ELISA (Technozym). All assays were performed in citrate-anticoagulated plasma, except for the Luminex assays that were performed in EDTA-anticoagulated samples.

Protease:serpin complexes: Activation of the contact system and the intrinsic coagulation pathway was determined by immunological assays to detect complexes of proteases and their respective serpin inhibitors, as previously described 25 . Briefly, citrated test plasma was thawed at 37°C for 10 minutes before dilution into buffer containing benzamidine and FPR-chloromethylketone to quench any remaining enzymatic activity. Samples were assayed in triplicate, and calculated against a standard curve. Complex standards were diluted into deficient plasma for the respective assay; for example, FXIIa:C1 complexes were serially diluted into FXIIdeficient plasma.

Measurement of intact and cleaved HK: The immunoassay for detecting intact and cleaved kininogen was performed as previously described 28 .

Measurement of microvesicle tissue factor (MVTF) activity: Assessment of procoagulant capacity of tissue-factor bearing microvesicles was performed as previously described, using an assay based on FXa generation by isolated microvesicles in the presence of added FX and FVIIa 29 .

Detection and enumeration of platelet-derived microvesicles (PDMV): PDMV were detected and enumerated by flow cytometry, using a CytoFLEX cytometer (Beckman Coulter, USA) as previously described 30 . Briefly, 50μL of platelet free plasma was thawed at 37°C for 3 minutes and incubated on ice for 30 minutes with 20 μL of a reagent mix containing 1 μL calcein Violet AM (Thermo), 1.5 μL bovine lactadherin FITC (Haematologic Tech), 1 μL anti-CD41 APC (eBioscience), and 16.5 μL filtered sterile PBS buffer. Sterile PBS (1,930μL) was added and the solution was centrifuged at 20,000 g for 30 minutes at 4°C. The resulting MV pellet was re-suspended in 1,000 μL of PBS buffer, and 100μL aliquots were subjected to flow cytometric analysis. PDMV were identified as events that were positive for lactadherin, calcein and anti-CD41. Polystyrene spherical particles <2,000 nm (Rosetta calibration beads, Exometry, Netherlands) were used for the quantification of events. PBS buffer was used as negative control and PDMV were expressed per events per mL.

Statistical analysis: Data are presented as mean ± standard deviation (SD) or as medians and interquartile range (IQR), as indicated. Differences in continuous variables were analyzed using Student's t-test or Mann-Whitney test according to data distribution, assessed by the D'Agostino & Pearson normality test. Correlation was calculated using the Pearson or Spearman correlation coefficient. P value  0.05 was considered significant. All statistical analyses were performed using SPSS version 26 (IBM) or GraphPad Prism 7.0 Software (GraphPad Inc).

Data sharing statement: For original data please contact nigel_key@med.unc.edu or erich@unicamp.br.

Thirty patients with COVID-19 and thirty healthy individuals were included in this study. Basic demographic and hematologic characteristics of the study population are shown in Table 1 , while patient co-morbidities and medications are shown in Supplemental Table 1 . Clinical characteristics of the COVID-19 patients are shown in Table 2 . As expected, at the time of admission, significantly higher levels of several coagulation and fibrinolysis biomarkers were observed in patients at presentation (Table 3) . These included elevated levels of multiple contact and intrinsic pathway coagulation factors (factors XII, XI, IX and VIII) measured by one-stage clotting assays. In addition, as reported by others, von Willebrand factor and fibrinolytic parameters were elevated 1, 9, 10 . However, neither antithrombin chromogenic activity nor P-selectin antigen levels differed between patients and controls. Of note, neither baseline differences, nor the clinical outcomes evaluated in the study were affected by treatment allocation in the three arms of the clinical trial from which baseline samples were obtained for this study (Supplemental Table 2 ) 27 . Moreover, the standard patient care protocol did not differ by treatment allocation (Supplemental Table 3 ).

Circulating levels of protease:serpin complexes in patients and controls at baseline are shown in Figure 1 . The levels of all complexes in the contact and intrinsic pathways, with the exception of FXIa:AT, were significantly elevated in COVID-19 patients compared to healthy individuals. These results are indicative of systemic activation of the contact and intrinsic pathways. FIXa:AT levels correlated with other protease:serpin complexes in the contact pathway, particularly FXIIa:C1 (r s =0.54, P<.001) and FXIa:C1 (r s =0.41, P=.001), supporting the hypothesis that activation of the contact pathway contributes at least in part to the 'downstream' activation of FIX ( Figure 2 ). Of note, no differences were observed in FIXa:AT levels between patients allocated to any of the three treatment arms 27 , either on admission or at the end of treatment with study drug (day +4) (Supplemental Figure 1 ). These data support the fact that the experimental treatment arms had no influence on FIXa:AT levels. Lastly, we noted that 14 of the 30 enrolled patients received a single prophylactic 40 mg dose of enoxaparin before the baseline labs were obtained. However, neither the serine protease:serpin levels, nor any of the other biomarkers demonstrated any trends when comparing those who did vs. those did not receive enoxaparin (data not shown).

Next, we evaluated whether classical biomarkers of thromboinflammation correlated with circulating FIXa:AT complexes. The correlation coefficient (Rs) between FIXa:AT levels and D-dimer was 0.481, P=<0.001. A commonly measured prognostic biomarker in COVID-19, C-reactive protein (CRP), was also moderately correlated with D-dimer (r s =0.51, P=.005) but only to a moderate to low degree with FIXa:AT (r s =0.37, P=.06). Soluble P-selectin, which has been reported to be elevated in patients with COVID-19 31 , was moderately correlated with CRP (r s =0.64, P<.0001), D-dimer (r s =0.52, P=.003), and FIXa:AT (r s =0.41, P=.002). We posited that participation of FIXa in the phospholipid-dependent 'intrinsic tenase complex' on activated platelets might contribute to thrombin-dependent activation of platelets and enhanced release of platelet (CD41+)-derived microvesicles. Indeed, as shown in Figure 3 , FIXa:AT complexes were moderately correlated with platelet-derived microvesicle numbers in plasma (r s =0.45, P=.0004).

Recent reports have demonstrated that tissue factor-bearing microvesicles are correlated with disease severity and mortality in COVID-19 13, 32 . In order to explore whether increased extrinsic pathway activation could also contribute to FIXa generation in COVID-19, we measured MVTF activity in plasma. Figure 4A demonstrates that MVTF activity was significantly elevated in COVID-19 patients compared to healthy individuals, albeit with significant overlap at lower or undetectable concentrations. However, MVTF activity was moderately, but significantly, correlated with circulating FIXa:AT complexes (R=0.45; P = 0.0006) ( Figure 4B ). The latter observation suggests that TF:FVIIa complexes may also contribute to FIX activation in vivo.

Plasma analytes as a potential marker of disease severity Finally, we explored whether circulating level(s) of one or more protease:serpin complexes and/or other biomarkers might be associated with clinically-relevant adverse outcomes, specifically, the total length of hospital stay, length of ICU stay, and progression of lung disease based on the radiologic CT score. As shown in Table 4 , while a number of analytes were significantly associated with one or more of these outcomes, only a few were associated with all 3 outcomes. These included several candidate biomarkers that have previously been implicated as progostic biomarkers, including C-reactive protein 33 , D-dimer 34 , and PAI-1 35 . In addition, plasma levels of FIXa:AT complexes were also associated with all the recorded adverse clinical outcomes (Table 4 and Figure 5 ). In sum, these data suggest that FIXa generation, a specific marker of coagulation activation, may be a novel biomarker of clinically significant adverse outcomes in COVID-19.

The mechanism underlying coagulation activation and its contribution to adverse outcomes in severe SARS-CoV-2 disease (COVID-19) remains elusive. In this exploratory study, circulating levels of protease:serpin complexes indicative of contact and intrinsic pathway activation were observed to be elevated in COVID-19 patients. We demonstrated that levels of the various FXIa:serpin complexes, and to a lesser extent PKa:C1 complexes, were positively correlated with FIXa:AT levels, suggesting that both FXIa and PKa might be contributing to FIX activation in vivo [18] [19] [20] . Furthermore, in agreement with previous reports 13, 32 , we observed that MVTF levels were also elevated at baseline and also correlated with levels of FIXa:AT, suggesting that the activation of FIX occurs via both the contact/intrinsic and extrinsic pathways. Whatever the mechanism of initial FIX activation, the resultant downstream thrombin generation may then activate FXI, thereby promoting further FIX activation 36 .

While activation of the contact pathway by bacterial infection has been described in humans and in animal models 24, 37 , few studies have investigated whether this pathway is activated in viral infection 38, 39 . Hyperinflammation associated with COVID-19 results in activation of innate immune cells (neutrophils and monocytes) that interact with platelets and the coagulation cascade, ultimately leading to micro-and macrothrombosis. The generation of neutrophil extracellular traps (NETs) 40, 41 and/or release of platelet-derived polyphosphate 42 may promote activation of the contact pathway in COVID-19 during immunothrombosis in the microvasculature of the lung and other vascular beds. In addition to its contribution to thrombin generation, contact activation leads to an inflammatory cacade mediated by bradykinin as a product of PKa cleavage of HK. Indeed, the phrase 'bradykinin storm' was coined to describe the dysregulated generation of BK (and its metabolite des-Arg9-BK) in COVID-19 infection that may enhance pulmonary inflammation and increase vascular permeability 43, 44 . While the very short half-life of BK makes it challenging to measure directly in plasma, BK generation can be inferred by quantitation of the more stable cleaved form of HK (HKc) 28 . Our data suggest that in the early stages of infection, levels of HKc are significantly increased in COVID compared to healthy individuals, but this is accompanied by an increased concentration of the intact form of HK (HKi), likely as a compensatory response. This interpretation is compatible with other data shown here that increased zymogen levels of the other contact factors are also associated with elevated levels of the respective serine protease:serpin (for example, elevated FXII:C and increased FXIIa:C1 complexes were observed in the same samples).

An exploratory aim of this study was to evaluate associations between established and novel biomarkers of coagulation activation and adverse clinical outcomes. Along with D-dimer, CRP and PAI-1, the concentration of FIXa:AT complexes upon admission was associated with the total duration of hospitalization, duration of intensive care, and degree of lung injury assessed by the CT score. Although involvement of the contact pathway in SARS-CoV2 infection was suggested early in the pandemic [45] [46] [47] [48] , there are limited data linking activation of this pathway with patient outcomes 49, 50 This study has several limitations. First, the sample size is relatively small, although the differences in the levels of measured analytes between patients and healthy subjects are rather profound. Moreover, consecutive enrollment of a well-defined patient population, standardized sample collection with control of pre-analytical variables, and structured obtention of clinical and laboratory data in the setting of a randomized controlled trial served to minimize the inherent heterogeneity of observational studies that were conducted in the early phases of the pandemic. Second, we did not measure longitudinal trends in the selected analytes; however, our primary objective was to assess baseline levels of the contact activation markers and to preliminarily evaluate their clinical prognostic potential. Furthermore, we wished to avoid any confounding by treatment allocation that may have been operative at subsequent time points. Finally, while our study did not include a control group of patients with non-COVID pneumonia, it should be noted that the primary objective was not to determine whether any observed changes were specific to COVID-19. Rather, we sought to identify whether one or more of the measured analytes might be a novel prognostic indicator of adverse clinical outcomes. Indeed, our preliminary analysis suggests that baseline FIXa:AT complexes may have such prognostic significance. However, since it is difficult to conclude that a meaningful association exists between these laboratory parameters and clinical outcomes when the latter are <20, a larger appropriately powered prospective study is needed to validate this hypothesis.

MWH: performed protease:serpin complex assays and microvesicle assays, contributed to data analysis and drafted the manuscript; revised and approved the manuscript. FL: obtained and processed samples, performed coagulation factor assays and contributed to data analysis; revised and approved the manuscript. CRPM: obtained and processes samples and performed multiplex assays; revised and approved the manuscript. AI: performed protease:serpin complexes assays and contributed to data analysis; revised and approved the manuscript. SCH, MSB and IS: processed samples for microvesicle quantification, designed the strategy and performed flow cytometry assays; revised and approved the manuscript. ACP, TAN, RGU, AFB and BB: recruited and managed patients and obtained patient/outcome data; revised and approved the manuscript. SSJD: analyzed and scored lung tomographies; revised and approved the manuscript. SS: provided resources for measurement of high molecular weight kininogen; revised and approved the manuscript. JMA: provided laboratory support and infra-structure for classical coagulation assays; revised and approved the manuscript. FAO: contribute to study design and data analysis; revised and approved the manuscript. MLM, EM and LAV: designed and conducted the clinical trial from which patients were recruited; revised and approved the manuscript. NSK contributed to study design, oversaw and provided resources and infra-structure for protease:serpin complexes assays, contributed to data analysis and drafted the manuscript; ; revised and approved the manuscript. EVDP: designed the study, obtained and processed samples, oversaw and provided resources and infra-structure for coagulation and microvesicle analysis, contributed to data analysis and drafted the manuscript; revised and approved the manuscript.

All authors declare no competing financial interests. Mean  SD or median (horizontal bars) are depicted for gaussian and non-gaussian data, respectively; similarly, P values are from t-test or Mann-Whitney test according to data distribution (n=28-30 per group). Notably, because the standard curve typically spans 4 logs for FXIa:1-antitrypsin complexes, many patient samples exceeded the upper limit of detection. Samples from two patients were not processed due to critical pre-analytical issues (low volume). <0.001 * Median (interquartile range); † mean  SD; ‡ Mann-Whitney test or t-test for nonparametric and parametric data respectively. PT: prothrombin time; aPTT: activated partial thromboplastin time; VWF: Von Willebrand Factor; Rist: ristocetin; uPAR: urokinase-type plasminogen activator receptor; PAI-1: plasminogen activator inhibitor-1; two sodium citrate samples from patients were not processed due to critical preanalytical issues (low volume). 

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Authors would like to thank staff members from the hospital and research institutions in which this study was conducted, and to patients and healthy volunteers who agreed to participate. This study was funded by the Sao Paulo Research Foundation (FAPESP), grants 2016/14172-6 and 2020/05985-9, FAEPEX-Unicamp grant 2404/2020; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior -Brasil (CAPES), finance code 001. The project was also supported by the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number UL1TR002489. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.