Connective tissue disorders in COVID-19: Reply to "People with a connective tissue disorder may be especially vulnerable to the endothelial damage that characterizes long COVID due to the fragility of their vasculature and slow wound healing".

Connective tissue serves as a framework for other tissues and organs, supporting their functions, shielding them from harmful factors, and aiding repair. In COVID-19, damaged endothelial cells (ECs), increased endothelial permeability, and thrombi contribute to the connective tissue disorders. Even post-recovery, the damage to ECs and connective tissues persists, resulting in long COVID. Individuals with connective tissue disorders are prone to developing severe COVID-19 and experiencing long COVID symptoms. It is advised that these patients receive at least three vaccine doses, undergo early prophylactic antithrombotic therapy during acute COVID-19, and maintain prophylactic anticoagulant treatment in cases of long COVID.

Damage to endothelial barriers and its contribution to long COVID.

The world continues to contend with COVID-19, fueled by the emergence of viral variants. At the same time, a subset of convalescent individuals continues to experience persistent and prolonged sequelae, known as long COVID. Clinical, autopsy, animal and in vitro studies all reveal endothelial injury in acute COVID-19 and convalescent patients. Endothelial dysfunction is now recognized as a central factor in COVID-19 progression and long COVID development. Different organs contain different types of endothelia, each with specific features, forming different endothelial barriers and executing different physiological functions. Endothelial injury results in contraction of cell margins (increased permeability), shedding of glycocalyx, extension of phosphatidylserine-rich filopods, and barrier damage. During acute SARS-CoV-2 infection, damaged endothelial cells promote diffuse microthrombi and destroy the endothelial (including blood-air, blood-brain, glomerular filtration and intestinal-blood) barriers, leading to multiple organ dysfunction. During the convalescence period, a subset of patients is unable to fully recover due to persistent endothelial dysfunction, contributing to long COVID. There is still an important knowledge gap between endothelial barrier damage in different organs and COVID-19 sequelae. In this article, we mainly focus on these endothelial barriers and their contribution to long COVID.

Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid.

Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine-binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia.

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation.

Phosphatidylserine-exposing blood and endothelial cells contribute to the hypercoagulable state in essential thrombocythemia patients.

The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P < 0.001)] than those in controls. Among ET patients, those with JAK2 mutations showed higher levels of PS-positive erythrocytes, platelets, neutrophils, and serum-cultured ECs than TN patients or those with CALR mutations, which show similar levels. Coagulation function assays showed that higher levels of PS-positive blood cells and serum-cultured ECs led to markedly shortened coagulation time and dramatically increased levels of FXa, thrombin, and fibrin production. This procoagulant activity could be largely blocked by addition of lactadherin (approx. 70% inhibition). Confocal microscopy showed that the FVa/FXa complex and fibrin fibrils colocalized with PS on ET serum-cultured ECs. Additionally, we found a relationship between D-dimer, prothrombin fragment F1 + 2, and PS exposure. Our study reveals a previously unrecognized link between hypercoagulability and exposed PS on cells, which might also be associated with distinct thrombotic risks among mutational subtypes in ET. Thus, blocking PS-binding sites may represent a new therapeutic target for preventing thrombosis in ET.

Phosphatidylserine on microparticles and associated cells contributes to the hypercoagulable state in diabetic kidney disease.

Background: Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with diabetic kidney disease (DKD). This study was designed to evaluate whether exposed PS on microparticles (MPs) and MP-origin cells were involved in the hypercoagulability in DKD patients. Methods: DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate, defined as normoalbuminuria (No-A) (<30 mg/24 h), microalbuminuria (Mi-A) (30-299 mg/24 h) or macroalbuminuria (Ma-A) (>300 mg/24 h), and compared with healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells (BCs) and human umbilical vein endothelial cells (HUVECs) were treated with 25, 5 or 2.5 mmol/L glucose as well as 3-12 mg/dL uric acid and cells were evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure and fibrin strands were observed using confocal microscopy. Results: Using flow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and BCs were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with Ma-A was significantly higher than in patients with No-A. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with Ma-A versus serum from patients with Mi-A or normoalbuminuria. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated colocalization of fibrin with PS on HUVECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by ∼80%. In vitro, BCs and endothelial cells exposed more PS in hypoglycemia or hyperglycemia. Interestingly, reconstitution experiments showed that hypoglycemia-treated cells could be further activated or injured when recovery is obtained reaching hyperglycemia. Moreover, uric acid induced PS exposure on cells (excluding platelets) at concentrations >6 mg/dL. Linear regression analysis showed that levels of PS+ BCs and microparticles were positively correlated with uric acid and proteinuria, but negatively correlated with glomerular filtration rate. Conclusions: Our results suggest that PS+ MPs and MP-origin cells play procoagulant roles in patients with DKD. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine.

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbß3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.

Phosphatidylserine on blood cells and endothelial cells contributes to the hypercoagulable state in cirrhosis.

BACKGROUND & AIMS: The mechanism of thrombogenicity in cirrhosis is largely unknown. Our objective was to study the relationship between phosphatidylserine on blood cells and endothelial cells and the hypercoagulable state in cirrhotic patients. METHODS: Patients with cirrhosis and healthy controls were studied. Lactadherin was used to quantify phosphatidylserine exposure on blood cells and endothelial cells. Procoagulant activity of cells was evaluated using clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure, fibrin strands and FVa/Xa binding on cells were observed using confocal microscopy. RESULTS: Our study showed that phosphatidylserine exposure on erythrocytes, platelets and leucocytes in cirrhotic patients increased progressively with Child-Pugh categories. In addition, we found that endothelial cells treated with cirrhotic serum in vitro exposed more phosphatidylserine than those exposed to healthy serum. The exposed phosphatidylserine supported a shorter coagulation time and increased FXa, thrombin and fibrin formation. Notably, phosphatidylserine+ erythrocytes also promoted shorter coagulation times and more fibrin generation in cirrhotic microparticle-depleted plasma, regardless of Child-Pugh categories. Confocal microscopy data showed that the FVa/FXa complex and fibrin fibrils colocalized with phosphatidylserine on endothelial cells. Lactadherin significantly inhibited FXa and thrombin generation and consequently decreased fibrin production in normal or cirrhotic plasma. CONCLUSIONS: These results lead us to believe that exposed phosphatidylserine on activated or injured erythrocytes, platelets, leucocytes and endothelial cells plays an important role in the hypercoagulable state in cirrhotic patients. Thus, blocking phosphatidylserine binding sites might be a new therapeutic target for preventing thrombosis.

Increased phosphatidylserine-exposing microparticles and their originating cells are associated with the coagulation process in patients with IgA nephropathy.

BACKGROUND: Relatively little information is available about phosphatidylserine positive (PS(+)) microparticles (MPs) and their originating cells in IgA nephropathy (IgAN) despite well-established intraglomerular coagulation. Our objectives were to detect PS exposure on MP membranes and MP-origin cells and to evaluate its role in procoagulant activity (PCA) and fibrin formation and their association with pathological lesions in the disease. METHODS: Patients with IgAN and healthy controls were studied. Lactadherin was used to quantify PS exposure on MPs and MP-origin cells. PCA of MPs and MP-origin cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure, fibrin strands and FVa/Xa binding were observed on MPs/cells using confocal microscopy. RESULTS: Using flow cytometry, we found that IgAN patients had high levels of PS(+) MPs derived from lymphocytes, monocytes, neutrophils, platelets, erythrocytes and endothelial cells (ECs). The PS exposure on MP-origin cells also increased in these patients. MPs and MP-origin cells (leukocytes, platelets and erythrocytes) isolated from IgAN patients and ECs cultured with IgAN serum had a significantly shorter median coagulation time (P < 0.001), higher median intrinsic FXa (P < 0.001) and higher thrombin (P < 0.001) generation than controls. These coagulation functional assays were associated with the glomerular lesions. The lesions were also correlated with glomerular fibrin deposition (all P < 0.05). In the presence of patient MPs or their related cells, fibrin formation peaked faster with a higher maximum turbidity when compared with healthy controls. Blocking PS with lactadherin in the IgAN group prolonged coagulation time to control levels, inhibited the PCA up to 80% and markedly reduced fibrin formation. More importantly, we observed that fibrin strands formed on MPs and ECs in the same regions that bound lactadherin, similar to the FVa/Xa costaining. CONCLUSIONS: We find that high levels of PS(+) MPs and the MP-origin cells are associated with the coagulation process in IgAN, and this may provide a previously unrecognized contribution to intraglomerular coagulation.

Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity.

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV(MTTS/Y), had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV(MTTS/Y) showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.

Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1-derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.

The intersection of obesity and (long) COVID-19: Hypoxia, thrombotic inflammation, and vascular endothelial injury.

The role of hypoxia, vascular endothelial injury, and thrombotic inflammation in worsening COVID-19 symptoms has been generally recognized. Damaged vascular endothelium plays a crucial role in forming in situ thrombosis, pulmonary dysfunction, and hypoxemia. Thrombotic inflammation can further aggravate local vascular endothelial injury and affect ventilation and blood flow ratio. According to the results of many studies, obesity is an independent risk factor for a variety of severe respiratory diseases and contributes to high mechanical ventilation rate, high mortality, and slow recovery in COVID-19 patients. This review will explore the mechanisms by which obesity may aggravate the acute phase of COVID-19 and delay long COVID recovery by affecting hypoxia, vascular endothelial injury, and thrombotic inflammation. A systematic search of PubMed database was conducted for papers published since January 2020, using the medical subject headings of "COVID-19" and "long COVID" combined with the following keywords: "obesity," "thrombosis," "endothelial injury," "inflammation," "hypoxia," "treatment," and "anticoagulation." In patients with obesity, the accumulation of central fat restricts the expansion of alveoli, exacerbating the pulmonary dysfunction caused by SARS-CoV-2 invasion, inflammatory damage, and lung edema. Abnormal fat secretion and immune impairment further aggravate the original tissue damage and inflammation diffusion. Obesity weakens baseline vascular endothelium function leading to an early injury and pre-thrombotic state after infection. Enhanced procoagulant activity and microthrombi promote early obstruction of the vascular. Obesity also prolongs the duration of symptoms and increases the risk of sequelae after hospital discharge. Persistent viral presence, long-term inflammation, microclots, and hypoxia may contribute to the development of persistent symptoms, suggesting that patients with obesity are uniquely susceptible to long COVID. Early interventions, including supplemental oxygen, comprehensive antithrombotic therapy, and anti-inflammatory drugs, show effectiveness in many studies in the prevention of serious hypoxia, thromboembolic events, and systemic inflammation, and are therefore recommended to reduce intensive care unit admission, mortality, and sequelae.

Microparticle Phosphatidylserine Mediates Coagulation: Involvement in Tumor Progression and Metastasis.

Tumor progression and cancer metastasis has been linked to the release of microparticles (MPs), which are shed upon cell activation or apoptosis and display parental cell antigens, phospholipids such as phosphatidylserine (PS), and nucleic acids on their external surfaces. In this review, we highlight the biogenesis of MPs as well as the pathophysiological processes of PS externalization and its involvement in coagulation activation. We review the available evidence, suggesting that coagulation factors (mainly tissue factor, thrombin, and fibrin) assist in multiple steps of tumor dissemination, including epithelial-mesenchymal transition, extracellular matrix remodeling, immune escape, and tumor angiogenesis to support the formation of the pre-metastatic niche. Platelets are not just bystander cells in circulation but are functional players in primary tumor growth and metastasis. Tumor-induced platelet aggregation protects circulating tumor cells (CTCs) from the blood flow shear forces and immune cell attack while also promoting the binding of CTCs to endothelial cells and extravasation, which activates tumor invasion and sustains metastasis. Finally, in terms of therapy, lactadherin can inhibit coagulation by competing effectively with coagulation factors for PS binding sites and may similarly delay tumor progression. Furthermore, we also investigate the therapeutic potential of coagulation factor inhibitors within the context of cancer treatment. The development of multiple therapies targeting platelet activation and platelet-tumor cell interactions may not only reduce the lethal consequences of thrombosis but also impede tumor growth and spread.

Membrane curvature and PS localize coagulation proteins to filopodia and retraction fibers of endothelial cells.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α-stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

Membrane-binding properties of the Factor VIII C2 domain.

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa-Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.

Persistent Lung Injury and Prothrombotic State in Long COVID.

Lung injury may persist during the recovery period of COVID-19 as shown through imaging, six-minute walk, and lung function tests. The pathophysiological mechanisms leading to long COVID have not been adequately explained. Our aim is to investigate the basis of pulmonary susceptibility during sequelae and the possibility that prothrombotic states may influence long-term pulmonary symptoms of COVID-19. The patient's lungs remain vulnerable during the recovery stage due to persistent shedding of the virus, the inflammatory environment, the prothrombotic state, and injury and subsequent repair of the blood-air barrier. The transformation of inflammation to proliferation and fibrosis, hypoxia-involved vascular remodeling, vascular endothelial cell damage, phosphatidylserine-involved hypercoagulability, and continuous changes in serological markers all contribute to post-discharge lung injury. Considering the important role of microthrombus and arteriovenous thrombus in the process of pulmonary functional lesions to organic lesions, we further study the possibility that prothrombotic states, including pulmonary vascular endothelial cell activation and hypercoagulability, may affect long-term pulmonary symptoms in long COVID. Early use of combined anticoagulant and antiplatelet therapy is a promising approach to reduce the incidence of pulmonary sequelae. Essentially, early treatment can block the occurrence of thrombotic events. Because impeded pulmonary circulation causes large pressure imbalances over the alveolar membrane leading to the infiltration of plasma into the alveolar cavity, inhibition of thrombotic events can prevent pulmonary hypertension, formation of lung hyaline membranes, and lung consolidation.

Pathophysiological mechanisms of thrombosis in acute and long COVID-19.

COVID-19 patients have a high incidence of thrombosis, and thromboembolic complications are associated with severe COVID-19 and high mortality. COVID-19 disease is associated with a hyper-inflammatory response (cytokine storm) mediated by the immune system. However, the role of the inflammatory response in thrombosis remains incompletely understood. In this review, we investigate the crosstalk between inflammation and thrombosis in the context of COVID-19, focusing on the contributions of inflammation to the pathogenesis of thrombosis, and propose combined use of anti-inflammatory and anticoagulant therapeutics. Under inflammatory conditions, the interactions between neutrophils and platelets, platelet activation, monocyte tissue factor expression, microparticle release, and phosphatidylserine (PS) externalization as well as complement activation are collectively involved in immune-thrombosis. Inflammation results in the activation and apoptosis of blood cells, leading to microparticle release and PS externalization on blood cells and microparticles, which significantly enhances the catalytic efficiency of the tenase and prothrombinase complexes, and promotes thrombin-mediated fibrin generation and local blood clot formation. Given the risk of thrombosis in the COVID-19, the importance of antithrombotic therapies has been generally recognized, but certain deficiencies and treatment gaps in remain. Antiplatelet drugs are not in combination with anticoagulant treatments, thus fail to dampen platelet procoagulant activity. Current treatments also do not propose an optimal time for anticoagulation. The efficacy of anticoagulant treatments depends on the time of therapy initiation. The best time for antithrombotic therapy is as early as possible after diagnosis, ideally in the early stage of the disease. We also elaborate on the possible mechanisms of long COVID thromboembolic complications, including persistent inflammation, endothelial injury and dysfunction, and coagulation abnormalities. The above-mentioned contents provide therapeutic strategies for COVID-19 patients and further improve patient outcomes.

Long COVID: The Nature of Thrombotic Sequelae Determines the Necessity of Early Anticoagulation.

Many discharged COVID-19 patients affected by sequelae experience reduced quality of life leading to an increased burden on the healthcare system, their families and society at large. Possible pathophysiological mechanisms of long COVID include: persistent viral replication, chronic hypoxia and inflammation. Ongoing vascular endothelial damage promotes platelet adhesion and coagulation, resulting in the impairment of various organ functions. Meanwhile, thrombosis will further aggravate vasculitis contributing to further deterioration. Thus, long COVID is essentially a thrombotic sequela. Unfortunately, there is currently no effective treatment for long COVID. This article summarizes the evidence for coagulation abnormalities in long COVID, with a focus on the pathophysiological mechanisms of thrombosis. Extracellular vesicles (EVs) released by various types of cells can carry SARS-CoV-2 through the circulation and attack distant tissues and organs. Furthermore, EVs express tissue factor and phosphatidylserine (PS) which aggravate thrombosis. Given the persistence of the virus, chronic inflammation and endothelial damage are inevitable. Pulmonary structural changes such as hypertension, embolism and fibrosis are common in long COVID. The resulting impaired lung function and chronic hypoxia again aggravates vascular inflammation and coagulation abnormalities. In this article, we also summarize recent research on antithrombotic therapy in COVID-19. There is increasing evidence that early anticoagulation can be effective in improving outcomes. In fact, persistent systemic vascular inflammation and dysfunction caused by thrombosis are key factors driving various complications of long COVID. Early prophylactic anticoagulation can prevent the release of or remove procoagulant substances, thereby protecting the vascular endothelium from damage, reducing thrombotic sequelae, and improving quality of life for long-COVID patients.

The cross-talk of lung and heart complications in COVID-19: Endothelial cells dysfunction, thrombosis, and treatment.

The pandemic respiratory illness SARS-CoV-2 has increasingly been shown to be a systemic disease that can also have profound impacts on the cardiovascular system. Although associated cardiopulmonary sequelae can persist after infection, the link between viral infection and these complications remains unclear. There is now a recognized link between endothelial cell dysfunction and thrombosis. Its role in stimulating platelet activation and thrombotic inflammation has been widely reported. However, the procoagulant role of microparticles (MPs) in COVID-19 seems to have been neglected. As membrane vesicles released after cell injury or apoptosis, MPs exert procoagulant activity mainly by exposing phosphatidylserine (PS) on their lipid membranes. It can provide a catalytic surface for the assembly of the prothrombinase complex. Therefore, inhibiting PS externalization is a potential therapeutic strategy. In this paper, we describe the pathophysiological mechanism by which SARS-CoV-2 induces lung and heart complications through injury of endothelial cells, emphasizing the procoagulant effect of MPs and PS, and demonstrate the importance of early antithrombotic therapy. In addition, we will detail the mechanisms underlying hypoxia, another serious pulmonary complication related to SARS-CoV-2-induced endothelial cells injury and discuss the use of oxygen therapy. In the case of SARS-CoV-2 infection, virus invades endothelial cells through direct infection, hypoxia, imbalance of the RAAS, and cytokine storm. These factors cause endothelial cells to release MPs, form MPs storm, and eventually lead to thrombosis. This, in turn, accelerates hypoxia and cytokine storms, forming a positive feedback loop. Given the important role of thrombosis in the disease, early antithrombotic therapy is an important tool for COVID-19. It may maintain normal blood circulation, accelerating the clearance of viruses, waning the formation of MPs storm, and avoiding disease progression.

Intestinal Damage in COVID-19: SARS-CoV-2 Infection and Intestinal Thrombosis.

The intestinal tract, with high expression of angiotensin-converting enzyme 2 (ACE2), is a major site of extrapulmonary infection in COVID-19. During pulmonary infection, the virus enters the bloodstream forming viremia, which infects and damages extrapulmonary organs. Uncontrolled viral infection induces cytokine storm and promotes a hypercoagulable state, leading to systemic microthrombi. Both viral infection and microthrombi can damage the gut-blood barrier, resulting in malabsorption, malnutrition, and intestinal flora entering the blood, ultimately increasing disease severity and mortality. Early prophylactic antithrombotic therapy can prevent these damages, thereby reducing mortality. In this review, we discuss the effects of SARS-CoV-2 infection and intestinal thrombosis on intestinal injury and disease severity, as well as corresponding treatment strategies.