COVID-19-associated Coagulopathy Research Articles

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

Cost-effectiveness and Side Effects of Using Heparin and Enoxaparin as a Treatment for COVID-19-associated Coagulopathy

Objective: To analyze the cost-effectiveness and side effects of Heparin and Enoxaparin anti-coagulants in patients with COVID-19-associated coagulopathy (CAC). Materials and Methods: This was an observational study with a retrospective design of CAC patients from May 2020 to May 2022 in Surabaya, Indonesia, who received Heparin and Enoxaparin. Effectiveness was comprehensively evaluated as clinical outcomes including mortality, length of stay (LOS), laboratory results, and side effects. Pharmacoeconomic evaluation was analyzed by constructing Decision Tree modeling followed by Cost-Effectiveness Analysis (CEA) considering Quality-Adjusted Life Year (QALY), and Incremental Cost-Effectiveness Ratio (ICER). In addition, probability sensitivity analysis was performed to consider the cost-effective intervention. Results: A number of 274 samples were included from Medical Records and finance reports. Heparin has a higher mortality rate compared to Enoxaparin (13.9% vs. 23.9; p = 0.040), with no difference in the side effects ( p = 0.056). D-Dimer reported a significant change in values after receiving Heparin (2271.01 ± 4595.50 ng/mL) and Enoxaparin (2140.95 ± 5681.98 ng/mL), p = 0.019. Enoxaparin was more cost-effective in pharmacoeconomic analysis, with a US$130.58/QALY ACER value, while Heparin was US$138.67/QALY. Conclusion: Enoxaparin therapy has better effectiveness, while side effects and costs are similar to Heparin. However, Enoxaparin is far more cost-effective to use against CAC conditions than Heparin.

Independent risk factors for COVID-19-associated coagulopathy.

Past three years since the beginning of the outbreak, we have obtained satisfactory data on COVID-19. However, data on risk factors of COVID-19-associated coagulopathy (CAC) are extremely limited. Prediction of CAC might be a game changer since it is related to poor prognosis. Seeking independent risk factors for CAC was the main aim of the study. 510 hospitalized COVID-19 patients were retrospectively screened. Forty-eight of them were excluded due to irrelevant D-dimer or ferritin elevation. The remaining patients were stratified into three groups as overt coagulopathy, significant pulmonary microthrombosis, and patients without coagulopathy. The overt coagulopathy group included cases with macrothrombosis or disseminated intravascular coagulation (DIC). The significant pulmonary microthrombosis group covered the cases that had clinical deterioration with simultaneous marked D-dimer elevation. The group of patients without coagulopathy included the asymptomatic patients with normal or elevated D-dimer levels. Overt coagulopathy developed in 3.2% and significant pulmonary microthrombosis in 10.1% of the patients. In the multivariate analysis, not receiving low molecular weight heparin (LMWH) (p=0.002), a level of D-dimer >15,000 U/ml (p=0.013) were associated with overt coagulopathy. In addition, levels of initial LDH >480 IU/L (p=0.022) and initial ferritin >1,000 ng/ml (p=0.036) were associated with significant pulmonary microthrombosis. Not receiving LMWH (p=0.001) was also associated with significant pulmonary microthrombosis, when multivariate analysis was performed by the parameters with a p-value <0.1 in the univariate analysis. Furthermore, all cases with DIC had Gram-negative bacterial sepsis. Not receiving LMWH, high levels of D-dimer, initial LDH, and initial ferritin are independent risk factors for CAC. DIC does not appear to develop based on COVID-19.

Covid-19 associated coagulopathy (CAC): Global research output, 2020-2022

Objective. This study examined the Covid-19 associated coagulopathy (CAC) publication and citation trends, focusing on the top authors, countries, organizations, journals, collaboration, subject areas, keywords, and high-cited articles. Design/Methodology/Approach. Publications data on Covid-19 associated coagulopathy (CAC) were identified and extracted from the Scopus database. Data analysis has been performed using VOS viewer software, Biblioshiny, and MS Excel Software. Results/Discussion. The study identified 1740 articles on Covid-19 associated coagulopathy from the Scopus database, which received 47,502 citations, averaging 27.3 citations per paper. In all, 905 authors from 601 organizations originating in 96 countries participated in research and published in 776 journals. United States, Italy, and China contributed the most publications (498, 258, and 164 papers). While China (73.43 and 2.69), France (57.60 and 2.11,) and Germany (50.13 and 1.84) registered the highest citation impact by CPP and RCI. Harvard Medical School, USA, Huazhong University of S&amp;T, China, and Tongji Medical College, China, lead with most of the publications (4,4, 41, and 39 papers). In contrast, Tongji Medical College, China (198.31 and 7.26), Huazhong University of S&amp;T, China (198.15 and 7.26), and University College Hospital NHS Foundation Trust, U.K (129.23 and 4.73) lead in citation impact. J.H. Levy, T. Iba and M. Levy contributed to most of the publications (27, 25, and 20), while N. Tag (804.75 and 29.48), A. Tripodi (157.83 and 5.78), and J. Thachil (154.17 and 5.65) registered the highest citation impact by CPP and RCI. Journal of Thrombosis and Haemostasis, Journal of Thrombosis and Thrombolysis, and Thrombosis Research were the most productive ones (with 55, 51, and 46 publications). At the same time, Medical Hypotheses (1222), Blood (355.71) and Thrombosis Journal (233.86) were the most impactful journals. The top 9 keywords were Covid-19 (1681 occurrences), Blood Clotting (952), D Dimer (652), C (628), Coagulopathy (628), Thrombosis (584), blood (506), Anticoagulants (499), Blood Coagulation (426) and Fibrinogen (376). Conclusions. Global research on Covid-19 associated coagulopathy since the pandemic was considered for scientometric assessment for the first time, combining the productivity and citation measures to present an overall picture of the literature in this area. Such an analysis will provide scholars and policy-makers with a meaningful reference for further exploration of topical issues and research trends in the field.

COVID-19 associated coagulopathy: A bibliometric investigation

Infection with SARS-CoV-2 initiates an immune-hemostatic response. While both systems are intimately connected and necessary for an efficient immune response to contain the infection, excessive coagulation activation might exceed the valuable benefits by causing thrombotic consequences and excessive inflammation. This biological response is new to clinicians and researchers, and accordingly, tremendous studies have been conducted on coagulopathy and its relationship to COVID-19 disease during this pandemic. Therefore, it takes a research insight from a bibliometric perspective to determine research hotspots and trends of COVID-19 associated coagulopathy (C19-CA). The analysis relies on the Scopus database for bibliographic content and Visualization of Similarities viewer software to map bibliometric data of C19-CA. Our study finds the most eminent authors, journals, institutions, funding organizations, and countries that publish in the C19-CA. Additionally; this research employs bibliometric analysis of co-authorship, co-citations, bibliographic coupling, and co-occurrence of keywords. A total of 2242 studies were retrieved, and the number of annual publications of C19-CA showed remarkable growth. The top-publishing authors on C19-CA are Smadja, D.M., Diehl, J.L., and Gendron, N (France). The total number of articles published in English in these three years was 1241, with the original article accounting for 99.8% and conference papers accounting for 0.2%. Huazhong University of Science and Technology (China) is the top-productive institution, with the US being the top-publishing country. Journal of Thrombosis and Thrombolysis received the highest number of original articles. The research results were mainly published in the fields of Medicine, Biochemistry, Genetics, and Molecular Biology, Immunology and Microbiology. Yuanyuan Li, who is (China), is the top-collaborating author. China and its authors have the highest number of citations. Keywords' co-occurrence analyses of the authors and all keywords revealed the following themes in C19-CA; abnormal coagulation parameters, pulmonary coagulopathy, venous and arterial thrombotic disorders, distinct features of coagulopathy, inflammation, and thrombosis in COVID-19, and anticoagulants and thrombolytic therapies. By combining bibliometric analysis with VOSviewer software, we identified C19-CA's leaders, collaborating institutions, and research hotspots, as well as give references for future research paths.

Platelet-neutrophil interaction in COVID-19 and vaccine-induced thrombotic thrombocytopenia.

Coronavirus disease 2019 (COVID-19) is known to commonly induce a thrombotic diathesis, particularly in severely affected individuals. So far, this COVID-19-associated coagulopathy (CAC) has been partially explained by hyperactivated platelets as well as by the prothrombotic effects of neutrophil extracellular traps (NETs) released from neutrophils. However, precise insight into the bidirectional relationship between platelets and neutrophils in the pathophysiology of CAC still lags behind. Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare autoimmune disorder caused by auto-antibody formation in response to immunization with adenoviral vector vaccines. VITT is associated with life-threatening thromboembolic events and thus, high fatality rates. Our concept of the thrombophilia observed in VITT is relatively new, hence a better understanding could help in the management of such patients with the potential to also prevent VITT. In this review we aim to summarize the current knowledge on platelet-neutrophil interplay in COVID-19 and VITT.

Interrelationship between COVID-19 and Coagulopathy: Pathophysiological and Clinical Evidence.

Since the first description of COVID-19 infection, among clinical manifestations of the disease, including fever, dyspnea, cough, and fatigue, it was observed a high incidence of thromboembolic events potentially evolving towards acute respiratory distress syndrome (ARDS) and COVID-19-associated-coagulopathy (CAC). The hypercoagulation state is based on an interaction between thrombosis and inflammation. The so-called CAC represents a key aspect in the genesis of organ damage from SARS-CoV-2. The prothrombotic status of COVID-19 can be explained by the increase in coagulation levels of D-dimer, lymphocytes, fibrinogen, interleukin 6 (IL-6), and prothrombin time. Several mechanisms have been hypothesized to explain this hypercoagulable process such as inflammatory cytokine storm, platelet activation, endothelial dysfunction, and stasis for a long time. The purpose of this narrative review is to provide an overview of the current knowledge on the pathogenic mechanisms of coagulopathy that may characterize COVID-19 infection and inform on new areas of research. New vascular therapeutic strategies are also reviewed.

COVID-19, Blood Lipid Changes, and Thrombosis

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A2 that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A2 IIA (sPLA2-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA2-IIA levels together with eicosanoids in the sera of COVID patients. sPLA2 could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A2, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA2-IIA in COVID-19-associated coagulopathy (CAC).

An investigation to find the correlation between lupus anticoagulant and coagulation abnormalities in COVID-19 patients; a narrative review

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19) which is associated with nonspecific respiratory syndromes, varying from mild symptoms of upper airway to required mechanical ventilation hypoxemia. A unique feature of this disease is COVID-19-associated coagulopathy (CAC) that linked with disease severity and hospital mortality. These patients have a profound hypercoagulable state and in patients with severe type arterial and venous thrombotic events are frequent. Abnormal coagulation parameters such as activated partial thromboplastin time (aPTT) were observed in patients with COVID-19. Regarding the above finding it could be considered as a reason to avoid anticoagulation at the both doses of therapeutic and prophylactic. A prolonged aPTT may indicate a coagulation factor deficiency or inhibitor of coagulation, which can be specific (antibody to factor VIII) or nonspecific (lupus anticoagulant, LA). LA can affect laboratory tests of blood coagulation, but is not usually associated with bleeding; however, it can be associated with thrombotic risk as a part of the antiphospholipid syndrome. In a phospholipid concentration-dependent manner LA recognizes a type of antiphospholipid antibodies (aPLs) that prolong clotting tests. In patients with antiphospholipid syndrome (APS) LA is considered as one of the laboratory criteria representing a significant risk factor of both thrombosis and pregnancy morbidity. Similarities between some of the pathophysiological features of COVID-19 and APS has been focused in several reports particularly in the most severe form, catastrophic APS. This study aimed to evaluate the effect of LA on the incidence of thrombophilia in patients with COVID-19, as well as its impact on the inflammation and finally the mortality final rate.

Missense Variants of von Willebrand Factor in the Background of COVID-19 Associated Coagulopathy.

COVID-19 associated coagulopathy (CAC), characterized by endothelial dysfunction and hypercoagulability, evokes pulmonary immunothrombosis in advanced COVID-19 cases. Elevated von Willebrand factor (vWF) levels and reduced activities of the ADAMTS13 protease are common in CAC. Here, we aimed to determine whether common genetic variants of these proteins might be associated with COVID-19 severity and hemostatic parameters. A set of single nucleotide polymorphisms (SNPs) in the vWF (rs216311, rs216321, rs1063856, rs1800378, rs1800383) and ADAMTS13 genes (rs2301612, rs28729234, rs34024143) were genotyped in 72 COVID-19 patients. Cross-sectional cohort analysis revealed no association of any polymorphism with disease severity. On the other hand, analysis of variance (ANOVA) uncovered associations with the following clinical parameters: (1) the rs216311 T allele with enhanced INR (international normalized ratio); (2) the rs1800383 C allele with elevated fibrinogen levels; and (3) the rs1063856 C allele with increased red blood cell count, hemoglobin, and creatinine levels. No association could be observed between the phenotypic data and the polymorphisms in the ADAMTS13 gene. Importantly, in silico protein conformational analysis predicted that these missense variants would display global conformational alterations, which might affect the stability and plasma levels of vWF. Our results imply that missense vWF variants might modulate the thrombotic risk in COVID-19.

Coagulation Disorders in Sepsis and COVID-19-Two Sides of the Same Coin? A Review of Inflammation-Coagulation Crosstalk in Bacterial Sepsis and COVID-19.

Sepsis is a major cause of morbidity and mortality worldwide. Sepsis-associated coagulation disorders are involved in the pathogenesis of multiorgan failure and lead to a subsequently worsening prognosis. Alongside the global impact of the COVID-19 pandemic, a great number of research papers have focused on SARS-CoV-2 pathogenesis and treatment. Significant progress has been made in this regard and coagulation disturbances were once again found to underlie some of the most serious adverse outcomes of SARS-CoV-2 infection, such as acute lung injury and multiorgan dysfunction. In the attempt of untangling the mechanisms behind COVID-19-associated coagulopathy (CAC), a series of similarities with sepsis-induced coagulopathy (SIC) became apparent. Whether they are, in fact, the same disease has not been established yet. The clinical picture of CAC shows the unique feature of an initial phase of intravascular coagulation confined to the respiratory system. Only later on, patients can develop a clinically significant form of systemic coagulopathy, possibly with a consumptive pattern, but, unlike SIC, it is not a key feature. Deepening our understanding of CAC pathogenesis has to remain a major goal for the research community, in order to design and validate accurate definitions and classification criteria.

Značaj serumskog amiloida A za tok i ishod SARS-CoV-2 infekcije

The occurrence of a new coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), quickly became a global threat after it had spread across the continents in just a few months. Over the next three years, it caused infections in over 646.6 million people and resulted in over 6.6 million deaths. As a novel disease, Coronavirus Disease 19 (COVID-19) became the subject of intensive research. Due to various clinical manifestations of the infection with possible fatal outcomes, it became evident that a finer understanding of COVID-19 pathogenesis, clinical manifestations, and complications is necessary. Investigation of acute-phase reaction as a component of the immune system response to infection can be very helpful. Serum amyloid A (SAA) was investigated for this purpose as one of the acute-phase reactants primarily synthesized by the hepatocytes in response to pro-inflammatory cytokines. It has been found that elevated SAA levels were independent factors for gastrointestinal manifestations and liver injury during COVID-19 but also one of the factors in COVID19-associated coagulopathy. Studies showed that SAA levels positively correlate with disease severity and prognosis. Patients with severe infection demonstrated significantly higher levels of SAA. Higher SAA levels were observed in COVID-19 patients with chronic diseases such as diabetes mellitus, hypertension, cerebrovascular diseases, and obesity, all recognized as independent risk factors for critical disease and poor prognosis. Patients with COVID-19 who died had higher levels of SAA than survivors. This short review will summarize current studies and knowledge about SSA in COVID-19, its role in the pathogenesis of SARS-CoV-2 infection, and its clinical usefulness in COVID-19 patients.

A Review of the Prevalence of Thromboembolic Complications among Pregnant Women Infected with COVID-19.

Background: Thrombotic conditions triggered by SARS-CoV-2 virus can result in high mortality, especially in pregnant women as they are already in a hypercoagulability state. This thereby leads to excessive inflammation that will increase the risk of thromboembolic (TE) complications. Objective: The aim of this study is to review the prevalence of thromboembolic complications such as deep venous thrombosis, pulmonary embolism, and intervillous thrombosis, and their preventive strategies among pregnant women infected with COVID-19. Method: The articles were retrieved from online databases PubMed and ScienceDirect published from February 2020 to April 2022. Findings: A total of 5249 participants including 5128 pregnant women and 121 placentas from 19 studies were identified for having TE complications after being infected with COVID-19. The types of TE complications that developed within pregnant women were disseminated intravascular coagulation (DIC) (n = 44, 0.86%), unmentioned thromboembolic complications (TE) (n = 14, 0.27%), intervillous thrombosis (IVT) (n = 9, 0.18%), pulmonary embolism (PE) (n = 6, 0.12%), COVID-19 associated coagulopathy (CAC) (n = 5, 0.10%), and deep venous thrombosis (DVT) (n = 2, 0.04%). Whereas the prevalence of TE complications reported from studies focusing on placenta were IVT (n = 27, 22.3%), subchorionic thrombus (SCT) (n = 9, 7.44%), and placental thrombosis (n = 5, 4.13%). Thromboprophylaxis agent used among pregnant women include low molecular weight heparin (LMWH) at prophylactic dose (n = 9). Conclusions: The prevalence of thromboembolic complications among pregnant women infected by COVID-19 is low with DIC being the most common form and placental thrombosis being the least common form of TE complications that occurred within pregnant women infected with COVID-19. Anticoagulation, in particular LMWH (variable dose), is frequently used to prevent TE complications.

Coagulopathy during COVID-19 infection: a brief review.

The COVID-19 pandemic caused by SARS-CoV-2 continues to spread rapidly due to its virulence and ability to be transmitted by asymptomatic infected persons. If they are present, the symptoms of COVID-19 may include rhinorrhea (runny nose), headache, cough, and fever. Up to 5% of affected persons may experience more severe COVID-19 illness, including severe coagulopathy, acute respiratory distress syndrome (ARDS) characterized by respiratory failure that requires supplementary oxygen and mechanical ventilation, and multi-organ failure. Interestingly, clinical evidence has highlighted the distinction between COVID-19-associated coagulopathy (CAC) and disseminated intravascular coagulation (DIC). Patients with CAC exhibit different laboratory values than DIC patients for activated partial thromboplastin time (aPTT) and prothrombin time (PT) which may be normal or shortened, varying platelet counts, altered red blood cell morphology, unique bleeding complications, a lack of schistocytes in the peripheral blood, and no decrease in fibrinogen levels. In this review, we consider the search for 1) laboratory results that can diagnose or predict development of CAC, including serum levels of D-dimers, fibrinogen, interleukin-6 (IL-6) and the growth factor angiopoietin-2 (Ang-2), 2) mechanisms of CAC induction, and 3) novel therapeutic regimens that will successfully treat COVID-19 before development of CAC.

Understanding COVID-19-associated coagulopathy.

COVID-19-associated coagulopathy (CAC) is a life-threatening complication of SARS-CoV-2 infection. However, the underlying cellular and molecular mechanisms driving this condition are unclear. Evidence supports the concept that CAC involves complex interactions between the innate immune response, the coagulation and fibrinolytic pathways, and the vascular endothelium, resulting in a procoagulant condition. Understanding of the pathogenesis of this condition at the genomic, molecular and cellular levels is needed in order to mitigate thrombosis formation in at-risk patients. In this Perspective, we categorize our current understanding of CAC into three main pathological mechanisms: first, vascular endothelial cell dysfunction; second, a hyper-inflammatory immune response; and last, hypercoagulability. Furthermore, we pose key questions and identify research gaps that need to be addressed to better understand CAC, facilitate improved diagnostics and aid in therapeutic development. Finally, we consider the suitability of different animal models to study CAC.

Immuno-Thrombotic Complications of COVID-19: Implications for Timing of Surgery and Anticoagulation.

Early in the coronavirus disease 2019 (COVID-19) pandemic, global governing bodies prioritized transmissibility-based precautions and hospital capacity as the foundation for delay of elective procedures. As elective surgical volumes increased, convalescent COVID-19 patients faced increased postoperative morbidity and mortality and clinicians had limited evidence for stratifying individual risk in this population. Clear evidence now demonstrates that those recovering from COVID-19 have increased postoperative morbidity and mortality. These data—in conjunction with the recent American Society of Anesthesiologists guidelines—offer the evidence necessary to expand the early pandemic guidelines and guide the surgeon’s preoperative risk assessment. Here, we argue elective surgeries should still be delayed on a personalized basis to maximize postoperative outcomes. We outline a framework for stratifying the individual COVID-19 patient’s fitness for surgery based on the symptoms and severity of acute or convalescent COVID-19 illness, coagulopathy assessment, and acuity of the surgical procedure. Although the most common manifestation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19 pneumonitis, every system in the body is potentially afflicted by an endotheliitis. This endothelial derangement most often manifests as a hypercoagulable state on admission with associated occult and symptomatic venous and arterial thromboembolisms. The delicate balance between hyper and hypocoagulable states is defined by the local immune-thrombotic crosstalk that results commonly in a hemostatic derangement known as fibrinolytic shutdown. In tandem, the hemostatic derangements that occur during acute COVID-19 infection affect not only the timing of surgical procedures, but also the incidence of postoperative hemostatic complications related to COVID-19-associated coagulopathy (CAC). Traditional methods of thromboprophylaxis and treatment of thromboses after surgery require a tailored approach guided by an understanding of the pathophysiologic underpinnings of the COVID-19 patient. Likewise, a prolonged period of risk for developing hemostatic complications following hospitalization due to COVID-19 has resulted in guidelines from differing societies that recommend varying periods of delay following SARS-CoV-2 infection. In conclusion, we propose the perioperative, personalized assessment of COVID-19 patients’ CAC using viscoelastic hemostatic assays and fluorescent microclot analysis.

Longitudinal Trend of Plasma Concentrations of Extracellular Vesicles in Patients Hospitalized for COVID-19

Plasma concentrations of extracellular vesicles (EVs) originating from cells involved in COVID-19-associated coagulopathy (CAC), their longitudinal trend and association with clinical outcomes were evaluated. Blood samples of consecutive COVID-19 patients admitted to a medical Unit were longitudinally collected within 48 h of admission, at discharge and 30 days post-discharge. EVs were analyzed using high sensitivity flow cytometry and phospholipid-dependent clotting time (PPL). The following EVs were measured: endothelium-, platelet-, leukocyte-derived, bearing tissue factor (TF)+, angiotensin-converting enzyme (ACE2)+, platelet-derived growth factor receptor-β (PDGF-β)+ and SARS-CoV-2-nucleoprotein (NP)+. 91 patients were recruited for baseline EV analysis (mean age 67 ± 14 years, 50.5% male) and 48 underwent the longitudinal evaluation. From baseline to 30-days post-discharge, we observed significantly decreased plasma concentrations of endothelium-derived EVs (E-Selectin+), endothelium-derived bearing TF (E-Selectin+ TF+), endothelium-derived bearing ACE2 (E-Selectin+ACE2+) and leukocyte-EVs bearing TF (CD45+TF+), p < 0.001, p = 0.03, p = 0.001, p = 0.001, respectively. Conversely, platelet-derived (P-Selectin+) and leukocyte-derived EVs (CD45+) increased from baseline to 30-days post-discharge (p = 0.038 and 0.032, respectively). EVs TF+, ACE2+, PDGF-β+, and SARS-CoV-2-NP+ did not significantly change during the monitoring. PPL increased from baseline to 30-days post-discharge (+ 6.3 s, p = 0.006). P-Selectin + EVs >1,054/µL were associated with thrombosis (p = 0.024), E-Selectin + EVs ≤531/µL with worsening/death (p 0.026) and 30-days P-Selectin+ and CD45 + EVs with persistent symptoms (p < 0.0001). We confirmed increased EVs originating from cells involved in CAC at admission and discharge. EVs derived from activated pericytes and expressing SARS-CoV-2-NP were also detected. 30-days post-discharge, endothelium-EVs decreased, while platelet- and leukocyte-EVs further increased, indicating that cellular activation persists long after the acute phase.

Pattern of Conventional Coagulation and Thromboelastographic Parameters in Patients with COVID-19 Diseases and Association of COVID-Associated Coagulopathy with Mortality in Intensive Care Unit.

Background:Knowledge of underlying pathophysiology of coagulopathy is evolving and the pattern of coagulation parameters in coronavirus disease 2019 (COVID-19)–associated diseases is still not very clear.Aims:In the present study, we aimed to find out the pattern and distribution of conventional coagulation parameters and thromboelastographic (TEG) parameters in COVID-19–associated coagulopathy (CAC) in survivors and nonsurvivors at 28 days.Setting and Design:The present prospective observational study was conducted at a tertiary care COVID-19 intensive care unit (ICU) facility from March 21, 2020, to July 15, 2021.Materials and Methods:Admission clinical and laboratory data (conventional coagulation, inflammatory and TEG parameters, and disease severity parameters) of 64 COVID-19 patients admitted to the ICU were collected. Patients were divided into two groups, i.e., survivors and nonsurvivors.Statistical Analysis:Data were compared between two groups, i.e., survivors versus no survivors on 28 days using Student's t-test/Mann–Whitney U-test or Chi-square test/Fisher's exact test.Results:Admission mean plasma fibrinogen levels (474.82 ± 167.41 mg.dL−1) and D-dimer were elevated (1.78 [0.66, 3.62] mg.mL−1) in the COVID-19 ICU patients. Overall, COVID-19 patients had mean lower normal platelet count (150 ± 50 × 103 cells.mm−3), with marginally elevated prothrombin time (16.25 ± 3.76 s) and activated partial thromboplastin time (38.22 ± 16.72 s). A 65.6% (42/64) TEG profile analysis showed a normal coagulation profile, and the rest 21.9% (14/64) and 12.5% (8/64) had hypercoagulable and hypocoagulable states, respectively. Plasma D-dimer level was markedly elevated in nonsurvivors compared to survivors (P < 0.05), while no other conventional coagulation parameters and TEG profile demonstrated statistically significant between the two groups.Conclusion:Markedly elevated plasma D-dimer level was observed in nonsurvivors of COVID-19 ICU patients. A large portion of COVID-19 ICU patients had a normal TEG profile. Conventional coagulation parameters and TEG profile were similar between survivors and nonsurvivors.

Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3“-galloyl-4”,6“-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh , inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

349 COVID Induced Acute Ischaemic Limb

The coronavirus pandemic has overwhelmed healthcare systems worldwide, with more than 40 million affected and 1 million deaths. In addition to the typical symptoms, less clinically obvious was the underlying hypercoagulable state, termed COVID-19 associated coagulopathy (CAC). The exact mechanism of CAC is unknown, but it is suspected to involve all three aspects of Virchow’s Triad: endothelial injury, stasis, and a hypercoagulable state. Here we report the case of a woman in her 60’s who was admitted with COVID-19 and developed CAC with subsequent complications. She presented with a 3-day history of fevers, severe shortness of breath, and cough. Past medical history was significant for hypertension, poorly controlled insulin dependant diabetes, and obesity. She was admitted with COVID induced type 2 respiratory failure (pO2 of 5.5 on 15L), DKA (ketones 2.1), AKI (Cr 163), and a hypercoagulable state (D-dimer >20 000). She was intubated and commenced on an insulin sliding scale, therapeutic anticoagulation, and antibiotics. On day 2 she became anuric, with CT-angio demonstrating thrombus extending from renal arteries down to the right popliteal artery. She underwent an embolectomy and 4 compartment fasciotomy and was started on renal replacement therapy. Unfortunately, her leg was not deemed viable, and due to persistent hyperkalaemia required amputation 2 days later. She continued to deteriorate despite maximal multiple organ support and was palliated 15 days later. CAC can have life-threatening sequelae, and patients with COVID-19 infection should be commenced on anticoagulation upon diagnosis.