SARS-CoV-2 infection induces the activation of tissue factor-mediated coagulation via activation of acid sphingomyelinase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte-derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2-SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2-SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration-approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.

An oxidase road to platelet adhesion.

Platelet adhesion to collagen via collagen receptors is an important part of thrombosis. In this issue of Blood, Matsuura et al identify collagen receptors as previously unrecognized targets of the extracellular enzyme lysyl oxidase (LOX), the level of which is increased in myeloproliferative neoplasms (MPNs) and other conditions associated with pathological thromboses.

Lysyl oxidase is associated with increased thrombosis and platelet reactivity.

Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2ß1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

Abnormal megakaryopoiesis and platelet function in cyclooxygenase-2-deficient mice.

Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.

Prevalence of JAK2V617F mutation in deep venous thrombosis patients and its clinical significance as a thrombophilic risk factor: Indian perspective.

Venous thromboembolism is known to be a complex interaction of genetic and acquired factors leading to thrombosis. JAK2V617F mutation is believed to contribute to a thrombophilic phenotype, possibly through enhanced leukocyte-platelet interactions in myeloproliferative neoplasms (MPNs). Several studies have focused on the importance of screening for JAK2V617F mutation in patients with splanchnic venous thrombosis (VT) for the detection of nonovert MPNs. The role of JAK2V617F mutation in VT outside the splanchnic region is still widely unsettled. The primary aim of this study was to find out the prevalence of JAK2V617F mutation in patients with deep venous thrombosis (DVT), its clinical significance as a prothrombotic risk factor, and its possible interactions with other genetic thrombophilic risk factors. A total of 148 patients with idiopathic, symptomatic DVT were evaluated. Median age of presentation was 32 years (range 15-71 years) with a sex ratio of 1.3:1. Overall, the most common genetic prothrombotic factor was factor V Leiden mutation, found in 10.8% (16 of 148) of patients who also showed strong association with increased risk of thrombosis (odds ratio 5.94, confidence interval 1.33-26.4, P = .019). Deficiencies in protein C, protein S, and antithrombin were seen in 8 (5.4%), 10 (6.7%), and 8 (5.4%) patients, respectively. It was observed that the frequency of JAK2V617F mutation was lower in Indian patients, and it also showed weaker association with risk of thrombosis, at least in cases of venous thrombosis outside the splanchnic region.

[Recurrent deep vein thrombosis and myeloproliferative syndrom: emergence of JAK2 mutation five years after the initial event]. / Thromboses veineuses à répétition et syndrome myéloprolifératif : positivité secondaire d'une mutation JAK2 cinq ans après l'événement initial.

JAK 2 mutation is the molecular event responsible for 95% of polycythemia cases and 50% of thrombocythemia vera and myelofibrosis cases. It can be used as a tool for the diagnosis of myeloproliferative disorders. We report a case illustrating the fact that a negative result does not definitively eliminate the diagnosis. A 40-year old woman, with a medical history of familial deep vein thrombosis, developed thrombosis of the inferior vena cava with extension to the suprahepatic veins and pulmonary embolism. No constitutional or acquired thrombophilia was diagnosed; search for JAK 2 mutation was negative. The patient was treated with fluindione. Five years later, she relapsed with popliteo-femoral and vena cava deep vein thrombosis. The etiological work-up included a PET scan which revealed diffuse uptake in bones and suspected neoplasic bone marrow invasion. Progenitor cell cultures were positive and JAK 2 mutation was confirmed. The bone marrow aspirate had the cytologic appearance of a myeloproliferative disorder. This case illustrates the fact that JAK 2 mutation can be identified several years after onset of a latent myeloproliferative disorder. Cases with a high clinical likelihood should lead to renewed search for this mutation. Secondary discovery of this mutation can be explained by a higher proportion of mutation expressing clones.

Left atrial myxoma presenting as pulmonary embolism: potential role of heme oxygenase-1.

We present the case of a patient with left atrial myxoma that presented with pulmonary embolism. The patient did not have any intracardiac communication between right and left sides of the heart. Using thrombelastography, the patient was determined to have an abnormally large velocity of plasma thrombus growth and strength with reduced vulnerability to lysis. Critically, increased carboxyhemoglobin concentrations were present, likely secondary to hemolysis from the tumor and engagement of systemic heme oxygenase-1. It was determined that the patient's plasmatic hypercoagulability was in part due to carboxyhemefibrinogen formation via a thrombelastographic method. In addition to circulating hypercoagulability, the patient also had an area of chronic venous stasis in his left ankle that had not changed for over a decade prior to this thrombophilic episode. In conclusion, we present the first case of paradoxical pulmonary embolism in the presence of a left atrial myxoma, potentially secondary to a combination of hemolysis, heme oxygenase-1 up-regulation, systemic hypercoagulability/hypofibrinolysis, and regional venous stasis.

Thoracic tumor effects on plasmatic coagulation: role of hemeoxygenase-1.

OBJECTIVES: Lung cancer is an important health threat worldwide, and is associated with a 3.8-13.9% incidence of thrombophilia. Of interest, patients with lung tumors have been noted to have an increase in endogenous carbon monoxide production via upregulation of hemeoxygenase-1 activity. Given that it has been demonstrated that carbon monoxide enhances plasmatic coagulation in vitro and in vivo via formation of carboxyhemefibrinogen, we sought to determine if patients with thoracic tumors undergoing lung resection/pneumonectomy had an increase in endogenous carbon monoxide and concurrent plasmatic hypercoagulability. MATERIALS AND METHODS: Nonsmoking patients with thoracic tumors (n=19) had preoperative carboxyhemoglobin (a measure of carbon monoxide production) determined, and a thromboelastometric method to assess citrated plasma coagulation kinetics and the formation of carboxyhemefibrinogen was utilized. Thoracic tumor patient coagulation kinetics was compared with normal subject (n=30) plasma samples. RESULTS AND CONCLUSION: Patients with thoracic tumors were determined to have an abnormally increased carboxyhemoglobin concentration of 2.1±0.6%, indicative of hemeoxygenase-1 upregulation. It was found that 84% of thoracic tumor patients had plasma clot strength that exceeded the 95% confidence interval value observed in normal subjects, and 44% of this hypercoagulable subgroup had carboxyhemefibrinogen formation. Future investigation of the role played by plasmatic hypercoagulability and hemeoxygenase-1 derived carboxyhemefibrinogen in the pathogenesis of thoracic tumor related thrombophilia is warranted.

Hemeoxygenase-1 mediated hypercoagulability in a patient with thyroid cancer.

Thyroid cancers can cause significant regional thrombotic morbidity and mortality. Of interest, thyroid cancer cell lines can have upregulation of the carbon monoxide-producing enzyme, hemeoxygenase-1. Carbon monoxide has been demonstrated to markedly enhance plasmatic coagulation in vitro and in vivo via enhancement of fibrinogen's substrate properties by binding to a fibrinogen-associated heme group(s). We present a patient undergoing removal of a malignant thyroid tumour who was serendipitously found to have abnormally increased carboxyhaemoglobin concentration (2.4%) and plasmatic hypercoagulability with a carbon monoxide-mediated clot strength as determined by a thrombelastographic method. This initial observation serves as a rationale to further investigate the role played by hemeoxygenase-1 upregulation in the setting of cancers associated with increased endogenous carbon monoxide production.

Investigation of inherited thrombophilias in patients with pulmonary embolism.

Inherited thrombophilias are thought to play an important role in the cause of pulmonary embolism and its recurrence. Ninety of 281 patients objectively diagnosed as pulmonary embolism between 2006 and 2009 were included in the study. The screening for thrombophilia included mutations of factor V Leiden (FVL), prothrombin (PTM) G20210A, methylene tetrahydrofolate reductase C677T-A1298C, the serum levels of antithrombin III, protein C, protein S, factor VIII and activated protein C resistance. Forty-two male (46.7%) and 48 female (53.3%) patients had a mean age of 62.6 ±â€Š13.4 years. Patients with common thrombophilias comprised 30% of all cases (FVL: 19.1%, PTM G20210A: 3.4%, antithrombin III deficiency: 1.1%, protein C deficiency: 5.7%, protein S deficiency: 13.6%). A significant association between recurrence of pulmonary embolism (10 patients, 12.2%) and protein S deficiency was established (P = 0.040). Serum level of protein C was also significantly lower in the subgroup of recurrent pulmonary embolism (P = 0.049). FVL and PTM mutations were high in cancer patients; the presence of inherited thrombophilia was low with risk factors of surgery and immobilization. Genetic risk factors were high in patients with pulmonary embolism. Protein C and S deficiencies may play a role in pulmonary embolism recurrence. DVT or family history of pulmonary embolism was not found to be related to inherited thrombophilias. Surgery and immobilization were thought not to have priorities for detection of genetic risk factors. The high percentages of FVL and PTM mutations in cancer patients should be considered.

JAK2 mutation status, hemostatic risk factors and thrombophilic factors in essential thrombocythemia (ET) patients.

The recently discovered JAK2 V617F point mutation, found in 50-60% of ET patients, has been reported to be associated with a higher risk of thrombotic events. In this study, we explored if JAK2 V617F mutation, or coexisting thrombophilic and hemostatic risk factors, contributed to these complications. We examined 32 patients with ET, and looked for pathogenetic JAK2 V617F mutation and prothrombotic genes mutations: factor V Leiden, prothrombin and MTHFR. We also evaluated plasma levels of fibrinogen, factors VIII and XII, AT, protein C, protein S and serum level of homocysteine. Urokinase concentration was assessed in patients' plasma as well as platelet lysates. There was no difference in the number of thrombotic complications between ET patients with and without JAK2 mutation. However, we found a number of thrombophilic and hemostatic risk factors that could contribute to thrombotic complications in ET patients.

JAK2 mutation and thrombosis in the myeloproliferative neoplasms.

The clinical course of the classic myeloproliferative neoplasms (MPNs) is burdened by an increased rate of cardiovascular events, which are the major cause of mortality. Age and history of thrombosis are the criteria used to stratify patients to the most appropriate therapeutic options. However, the mechanisms ultimately responsible for the increased thrombotic tendency have not yet been elucidated; abnormalities of blood cell count, neutrophil and platelet activation, and a state of hypercoagulability can all occur. Recurrent mutations in JAK2 or MPL have been described in MPNs and serve as disease markers. There is also evidence that a JAK2V617F mutant state represents an independent factor associated with thrombosis, and abnormalities of cell function attributable to JAK2V617F have been characterized. It is hoped that elucidation of the role mutant JAK2 plays in MPNs will improve our understanding of the pathophysiology of thrombosis and eventually result in improved patient treatment using molecularly targeted drugs.

Primary thrombophilia in México VII: the V617F mutation of JAK2 is not a frequent cause of thrombosis.

The study of the V617F JAK2 gene mutation has been used to identify the presence of an underlying myeloproliferative disorder (MPD) as the cause of unexplained thrombosis. In a group of 77 consecutive Mexican patients with a clinical marker of a primary thrombophilic condition, we looked for this JAK2 mutation and did not find any individual displaying it. Given these results, we conclude that an undetected MPD is a very improbable cause of thromboses in Mexican mestizos, a population where the prevalence of these disorders has been found to be lower than that found in Caucasian populations. Accordingly, it seems that the investigation for the V617F mutation of the JAK2 gene is not mandatory in all Mexican mestizo patients with unexplained thrombophilia and that this genetic study should be reserved for special cases, such as patients with thrombosis in uncommon sites or patients with cell counts suggesting the presence of an underlying MPD.

Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity.

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.

[TAME]-esterase--a new cardiovascular risk factor in smokers.

A mathematical model has been proposed to study the effect of [TAME] esterase on blood clotting time. Using this model, clotting time was found to decrease by 30% with increasing plasma [TAME] esterase activity in a group of smokers. It is hypothesized that [TAME] esterase through its effect on Hageman factor could affect clotting time. However mechanism of clotting by [TAME] esterase remains to be elucidated. It is concluded that [TAME] esterase is involved in the cascade of reactions leading to blood coagulation and increased [TAME] esterase activity could be an additional risk factor for possible cerebro-vascular accidents in smokers.