Cuyun Recipe ameliorates pregnancy loss by regulating macrophage polarization and hypercoagulable state during the peri-implantation period in an ovarian hyperstimulation mouse model.

BACKGROUND: The Chinese herbal prescription Cuyun Recipe (CYR) has been widely used to treat clinical infertility and has shown good efficacy. Animal experiments have shown that CYR can promote implantation in mice, however, the exact mechanism underlying the implantation has not been elucidated. PURPOSE: To investigate the effect and mechanism of CYR on regulating macrophage polarization and hypercoagulability during the peri-implantation period in mice with ovarian hyperstimulation. METHODS: An ovarian hyperstimulation mouse model was developed, followed by treatment with CYR. Mice were sacrificed on day (D)4.5, D6, or D8 of gestation. The number of implantation sites, the pathological changes of the uterus and ovaries were assessed. The polarization of monocytes/macrophages in the spleen and endometrium, the expression and localization of cytokines were further detected. Furthermore, analyses of hypercoagulable state of the blood were also performed. RESULTS: Treatment with CYR increased the average number of implantation sites, promoted angiogenesis in endometrial, and regulated monocytes/macrophages and the cytokine levels. Moreover, CYR downregulated the overexpression of D-dimer and fgl2 after ovarian hyperstimulation. CONCLUSION: CYR facilitates embryo implantation by alleviating ovarian hyperstimulation, promoting endometrial decidualization and angiogenesis, regulating macrophage polarization, and reversing the hypercoagulable state of the blood.

An interaction between the inflammatory condition and the hypercoagulable condition occurs in primary Sjögren syndrome.

This study aimed to assess the D-dimer level in patients with primary Sjögren syndrome (pSS), uncover its relationship with clinical symptoms, and appraise its predictive value in discriminating disease activity. The laboratory parameters of 101 consecutive patients with pSS and 101 healthy controls were analyzed and compared. Patients were divided into two subgroups according to their D-dimer levels, for the comparison of clinical features. Pearson's correlations were used to measure the relationships between D-dimer levels and other variables. The area under the curve (AUC) was calculated to predict disease activity. The erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (hsCRP) level, and D-dimer level were each higher in patients with pSS than in healthy controls. Compared with the low-D-dimer-level patients, those with elevated D-dimer levels exhibited higher ESRs (p < 0.0001) and higher levels of hsCRP (p < 0.0001), fibrinogen (p < 0.0001), and immunoglobulin A (p = 0.002). Cases with elevated D-dimer levels were prone to be more severe, based on ESSDAI evaluation (p < 0.0001). Patients with higher D-dimer levels had more articular involvement (p < 0.0001), which was significantly correlated with both the ESR (r = 0.21, p = 0.03) and hsCRP level (r = 0.56, p = 0.001). The D-dimer level may help to discriminate low disease activity from moderate/high disease activity (AUC = 0.754). The D-dimer level was correlated positively with both the ESR and hsCRP level in patients with pSS. The ESR and levels of hsCRP, fibrinogen, and disease activity were higher in the elevated D-dimer level group. The D-dimer level was demonstrated to have predictive value in differentiating pSS disease activity. Key Points •D-Dimer was higher in patients with pSS. •D-Dimer may help for predicting the disease activity in patients with pSS.

Serum amyloid A1 and pregnancy zone protein in pregnancy complications and correlation with markers of placental dysfunction.

BACKGROUND: Hypertensive disorders of pregnancy (preeclampsia, gestational hypertension, and chronic hypertension), diabetes mellitus, and placental dysfunction confer an increased risk of long-term maternal cardiovascular disease. Preeclampsia is also associated with acute atherosis that involves lesions of uteroplacental spiral arteries, resembling early stages of atherosclerosis. Serum amyloid A1 is involved in hypercoagulability and atherosclerosis and may aggregate into amyloid-aggregations of misfolded proteins. Pregnancy zone protein may inhibit amyloid aggregation. Amyloid is involved in Alzheimer's disease and cardiovascular disease; it has been identified in preeclampsia, but its role in preeclampsia pathophysiology is unclear. OBJECTIVE: We hypothesized that serum amyloid A1 would be increased and pregnancy zone protein decreased in hypertensive disorders of pregnancy and diabetic pregnancies and that serum amyloid A1 and pregnancy zone protein would correlate with placental dysfunction markers (fetal growth restriction and dysregulated angiogenic biomarkers) and acute atherosis. STUDY DESIGN: Serum amyloid A1 is measurable in both the serum and plasma. In our study, plasma from 549 pregnancies (normotensive, euglycemic controls: 258; early-onset preeclampsia: 71; late-onset preeclampsia: 98; gestational hypertension: 30; chronic hypertension: 9; diabetes mellitus: 83) was assayed for serum amyloid A1 and pregnancy zone protein. The serum levels of angiogenic biomarkers soluble fms-like tyrosine kinase-1 and placental growth factor were available for 547 pregnancies, and the results of acute atherosis evaluation were available for 313 pregnancies. The clinical characteristics and circulating biomarkers were compared between the pregnancy groups using the Mann-Whitney U, chi-squared, or Fisher exact test as appropriate. Spearman's rho was calculated for assessing correlations. RESULTS: In early-onset preeclampsia, serum amyloid A1 was increased compared with controls (17.1 vs 5.1 µg/mL, P<.001), whereas pregnancy zone protein was decreased (590 vs 892 µg/mL, P=.002). Pregnancy zone protein was also decreased in diabetes compared with controls (683 vs 892 µg/mL, P=.01). Serum amyloid A1 was associated with placental dysfunction (fetal growth restriction, elevated soluble fms-like tyrosine kinase-1 to placental growth factor ratio). Pregnancy zone protein correlated negatively with soluble fms-like tyrosine kinase-1 to placental growth factor ratio in all study groups. Acute atherosis was not associated with serum amyloid A1 or pregnancy zone protein. CONCLUSION: Proteins involved in atherosclerosis, hypercoagulability, and protein misfolding are dysregulated in early-onset preeclampsia and placental dysfunction, which links them and potentially contributes to future maternal cardiovascular disease.

Neutrophil extracellular traps and cancer-associated thrombosis.

The hypercoagulable state associated with malignancy is well described. However, the mechanisms by which tumors cause this hypercoagulable state are yet to be fully understood. This review summarizes the available literature of human and animal studies examining NETs and cancer-associated thrombosis. The methods for detecting and quantifying NET formation are growing but are not yet standardized in practice. Furthermore, it is important to distinguish between measuring neutrophil activation and NET formation, as the former can be present without the latter. Citrullination of histones by peptidylarginine deiminase 4 (PAD4) is considered one of the key pathways leading to NET formation. Cancer cells can prime neutrophils toward NET formation through the release of soluble mediators, such as interleukin-8, and activation of platelets, and may cause excess NET formation. Dismantling NETs through exogenous deoxyribonuclease has been shown to degrade NETs and reduce thrombus formation in vitro but may simultaneously release prothrombotic NET components, such as DNA and histones. Inhibiting PAD4 is far from clinical trials, but animal models show promising results with a potentially favorable safety profile. Interestingly, results from animal studies suggest that several therapies approved for other indications, such as interleukin-1 receptor blockade and JAK inhibition, may mitigate excessive NET formation or the prothrombotic effects of NETs in cancer. It is yet to be determined if inhibition of NET formation reduces cancer-associated thrombosis also in the clinical setting.

Cost-Effective Use of the Protein S Algorithm in Thrombophilia Testing.

BACKGROUND: One of the most complex risk factors for the laboratory assessment of thrombophilia is Protein S (PS). The testing algorithm for PS employs the plasma-based assays of free PS antigen, total PS antigen, and PS activity creating a complex diagnostic scheme that can lead to misdiagnosis if incorrectly used, and a potential waste of resources and money. CONTENT: This paper compares the recently published evidence-based algorithm from the International Society for Hemostasis and Thrombosis (ISTH) with several commonly performed nonevidence-based testing schemes, to demonstrate the efficiency of the evidence-based algorithm for diagnostic efficiency with improved patient care and increased cost savings for the laboratory. SUMMARY: Significant savings (31%-60%) can be realized when the evidence-based algorithm is used in place of other testing modalities of initial PS activity testing (31%) or testing with all 3 assays simultaneously (60%). This study utilizing the PS testing evidence-based algorithm as part of a thrombophilia evaluation demonstrates that the appropriate testing methods can be used to limit wasteful practices while achieving the maximum level of information in this time of limited resources and need for increase monetary savings.

Anticoagulation targeting membrane-bound anionic phospholipids improves outcomes of traumatic brain injury in mice.

Severe traumatic brain injury (TBI) often causes an acute systemic hypercoagulable state that rapidly develops into consumptive coagulopathy. We have recently demonstrated that TBI-induced coagulopathy (TBI-IC) is initiated and disseminated by brain-derived extracellular vesicles (BDEVs) and propagated by extracellular vesicles (EVs) from endothelial cells and platelets. Here, we present results from a study designed to test the hypothesis that anticoagulation targeting anionic phospholipid-expressing EVs prevents TBI-IC and improves the outcomes of mice subjected to severe TBI. We evaluated the effects of a fusion protein (ANV-6L15) for improving the outcomes of TBI in mouse models combined with in vitro experiments. ANV-6L15 combines the phosphatidylserine (PS)-binding annexin V (ANV) with a peptide anticoagulant modified to preferentially target extrinsic coagulation. We found that ANV-6L15 reduced intracranial hematoma by 70.2%, improved neurological function, and reduced death by 56.8% in mice subjected to fluid percussion injury at 1.9 atm. It protected the TBI mice by preventing vascular leakage, tissue edema, and the TBI-induced hypercoagulable state. We further showed that the extrinsic tenase complex was formed on the surfaces of circulating EVs, with the highest level found on BDEVs. The phospholipidomic analysis detected the highest levels of PS on BDEVs, as compared with EVs from endothelial cells and platelets (79.1, 15.2, and 3.5 nM/mg of protein, respectively). These findings demonstrate that TBI-IC results from a trauma-induced hypercoagulable state and may be treated by anticoagulation targeting on the anionic phospholipid-expressing membrane of EVs from the brain and other cells.

Biomarkers of Coagulation and Inflammation in COVID-19-Associated Ischemic Stroke.

[Figure: see text].

Protein S: function, regulation, and clinical perspectives.

PURPOSE OF REVIEW: Protein S (PS) is an essential natural anticoagulant. PS deficiency is a major contributor to acquired hypercoagulability. Acquired hypercoagulability causes myocardial infarction, stroke, and deep vein thrombosis in millions of individuals. Yet, despite its importance in hemostasis, PS is the least understood anticoagulant. Even after 40 years since PS was first described, we are still uncovering information about how PS functions. The purpose of this review is to highlight recent findings that advance our understanding of the functions of PS and explain hypercoagulability caused by severe PS deficiency. RECENT FINDINGS: PS has long been described as a cofactor for Activated Protein C (APC) and Tissue Factor Pathway Inhibitor (TFPI). However, a recent report describes direct inhibition of Factor IXa (FIXa) by PS, an activity of PS that had been completely overlooked. Thrombophilia is becoming a more frequently reported disorder. Hereditary PS deficiency is an anticoagulant deficiency that results eventually in thrombophilia. In addition, PS deficiency is a predisposing factor for venous thromboembolism (VTE), but an effect of PS deficiency in arterial thrombosis, such as arterial ischemic stroke, is uncertain. Plasma PS concentration decreases in pregnant women. Inherited thrombophilias are important etiologies for recurrent pregnancy loss, and anticoagulation therapy is of benefit to women with recurrent pregnancy loss who had documented only PS deficiency.Hypoxia is a risk factor for VTE, and hypoxia downregulates plasma PS level. Importantly, COVID-19 can lead to hypoxemia because of lung damage from IL6-driven inflammatory responses to the viral infection. Because hypoxia decreases the abundance of the key anticoagulant PS, we surmise that the IL6-induced cytokine explosion combined with hypoxemia causes a drop in PS level that exacerbates the thrombotic risk in COVID-19 patients. SUMMARY: This review is intended to advance understanding of the anticoagulant function of an important plasma protein, PS. Despite 40+ years of research, we have not had a complete description of PS biology as it pertains to control of blood coagulation. However, the picture of PS function has become sharper with the recent discovery of FIXa inhibition by PS. Hemostasis mediated by PS now includes regulation of FIXa activity alongside the cofactor activities of PS in the TFPI/APC pathways. In addition, the direct inhibition of FIXa by PS suggests that PS, particularly a small derivative of PS, could be used to treat individuals with PS deficiencies or abnormalities that cause thrombotic complications.

Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS mice.

BACKGROUND: Alveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PURPOSE: The aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. METHODS: 50 µl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 µg/kg, 10 µg/kg, 50 µg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 µg/kg, 10 µg/kg, 50 µg/kg) were intraperitoneally injected or 10 µg/50 µl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. RESULTS: LPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/ß and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. CONCLUSIONS: TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

Relationship between oxidant-antioxidant status and hypercoagulobility indices in children with iron deficiency anaemia.

Oxidative stress is a potential mechanism involved in the pathogenesis of iron deficiency anaemia (IDA). Although a tendency for hypercoagulability has been reported in IDA, its underlying mechanism is yet to be elucidated. This study investigated the probable relationship between oxidative stress and hypercoagulability in children with IDA. This study included 57 children diagnosed with IDA (IDA group) between October 2016 and October 2017 in addition to 48 healthy children (control group). The maximum clot firmness (MCF) index, and clot formation time (CFT) index, which are indicators of hypercoagulability in rotational thromboelastometry assays [intrinsic TEM (INTEM) and extrinsic TEM (EXTEM)] derived from our previous study, were recorded. Total oxidant status (TOS), total antioxidant capacity (TAC) and oxidative stress index (OSI) were analysed from serum samples of the individuals. In IDA group, OSI and TOS levels were higher and TAC level was lower compared to the control group (P < 0.001, for all). The EXTEM and INTEM MCF in the IDA group was higher than in the control group, while the INTEM CFT was lower than in the control group (P < 0.001, P < 0.001, P < 0.05, published data).TOS and OSI had a negative correlation with INTEM CFT (r:-0.361, P < 0.001 and r:-0.333, P = 0.001) and a positive correlation with INTEM MCF (r:+0.420, P < 0.001 and r:+0.367, P < 0.001) and EXTEM MCF (r:+0.476, P < 0.001 and r:+0.403, P < 0.001). However, TAC demonstrated no correlation with CFT and MCF index. The oxidant-antioxidant balance is disrupted in favour of oxidative stress in children with IDA. In addition, TOS and OSI, which are parameters of oxidative stress, are correlated with CFT and MCF indices. Oxidative stress appears to be an important factor for the development of tendency to hypercoagulability in IDA.

Shedding light on vitamin D: the shared mechanistic and pathophysiological role between hypovitaminosis D and COVID-19 risk factors and complications.

Severe acute respiratory syndrome coronavirus (SARS-COV-2) is the culprit of the Coronavirus Disease (COVID-19), which has infected approximately 173 million people and killed more than 3.73 million. At risk groups including diabetic and obese patients are more vulnerable to COVID-19-related complications and poor outcomes. Substantial evidence points to hypovitaminosis D as a risk factor for severe disease, the need for ICU, and mortality. 1,25(OH)D, a key regulator of calcium homeostasis, is believed to have various immune-regulatory roles including; promoting anti-inflammatory cytokines, down regulating pro-inflammatory cytokines, dampening entry and replication of SARS-COV-2, and the production of antimicrobial peptides. In addition, there are strong connections which suggest that dysregulated 1,25(OH)D levels play a mechanistic and pathophysiologic role in several disease processes that are shared with COVID-19 including: diabetes, obesity, acute respiratory distress syndrome (ARDS), cytokine storm, and even hypercoagulable states. With evidence continuing to grow for the case that low vitamin D status is a risk factor for COVID-19 disease and poor outcomes, there is a need now to address the public health efforts set in place to minimize infection, such as lock down orders, which may have inadvertently increased hypovitaminosis D in the general population and those already at risk (elderly, obese, and disabled). Moreover, there is a need to address the implications of this evidence and how we may apply the use of cheaply available supplementation, which has yet to overcome the near global concern of hypovitaminosis D. In our review, we exhaustively scope these shared pathophysiologic connections between COVID-19 and hypovitaminosis D.

Microparticles: a link to increased thrombin generation.

Circulating microparticles in human plasma may play a significant role in thrombogenesis because they carry the initiator of blood coagulation, tissue factor. Microparticles in blood are derived from diverse cell types, including erythrocytes, endothelial cells and platelets. Thrombin generation is an important part of the coagulation system and might be influenced by the presence of microparticles in the circulation. With this study, we determined the contribution of microparticles to increased thrombin generation in plasma samples received for thrombophilia workup and compare that with normal plasma. Microparticles were isolated from 50 plasma samples with increased thrombin generation and 20 plasma samples with normal thrombin generation, using filtration. Thrombin generation assay were performed by adding a low concentration of tissue factor-containing phospholipids and a fluorescence substrate for thrombin formation to plasma samples and measuring fluorescence at 1-min intervals over a period of 90 min on all samples (with and without the presence of microparticles). The peak thrombin, velocity-index and area under the curve were calculated. Microparticles contribute to the different parameters in samples with increased thrombin generation as follows: 50 ±â€Š19% for peak thrombin, 58 ±â€Š24% for velocity-index and 35 ±â€Š13% for area under the curve. Microparticles did not contribute to thrombin generation in plasma samples with normal thrombin generation. Microparticles play a significant role in coagulation and contribute largely to increased thrombin generation in plasma; however, microparticles do not contribute to coagulation in the plasma of participants with normal thrombin generation.

Clinical characteristics, genes identification and follow-up study of a patient with central venous thrombosis from a protein S deficiency pedigree.

OBJECTIVE: To explore the clinical and prognostic features of CVT caused by PROS1 gene mutations and to provide clinical experience for new oral anticoagulants, such as rivaroxaban, in the treatment of CVT with a high risk of thrombosis. PATIENTS AND METHODS: The CVT patient's clinical symptoms were described, and the brain imaging and blood coagulation tests were performed to confirm the diagnosis of CVT. The patient's family members were recruited to receive blood coagulation tests and ultrasonic examination of lower limb vessels. Genetic analysis on the pedigree was carried out to identify the responsible gene for PS deficiency. We followed-up with this patient for 24 months to evaluate the clinical outcomes, laboratory results and imaging performances of CVT. RESULTS: The patient presented with typical CVT symptoms, including headache and epilepsy. Brain CT showed hemorrhage in the bilateral frontal lobe and left occipital lobe, while MRV demonstrated that thrombus had occurred. It was reviewed that the patient and his mother had a history of bilateral leg deep vein thrombosis. Gene tests revealed that the patient and two family members carried a heterozygous mutation of PROS1 (c.751_752delAT, p.M251Vfs*17). During 24 months of follow-up study, the patient was treated with rivaroxaban continuously and recovered well, supported by an mRS score that remained below 2. Blood coagulation tests were within normal limits, and MRV revealed partial recanalization of the cerebral venous sinus. CONCLUSIONS: The frame shift mutation in the PROS1 gene (c.751_752delAT) may greatly affect the function of protein S and lead to a severe phenotype of CVT. Rivaroxaban showed a satisfying therapeutic effect in this CVT patient with hereditary thrombophilia.

Protein C and Protein S Levels in ß-Thalassemia Major Patients in Erbil, Kurdistan Region.

Oxygen is transported in the blood through red blood cells and a protein called hemoglobin. The protein consists of two alpha and two beta chains. The lack of any of these chains is caused by the malfunction of the genes that produce them, and can lead to a genetic disease called thalassemia. In ß-thalassemia, hemoglobin does not produce enough beta protein. According to mild to severe effects on the body, ß-thalassemia is divided into three types minor, interstitial and major thalassemia. There are increasing risks for thrombosis complications in thalassemia major. The purpose of this study was to evaluate protein C and protein S levels in ß-thalassemia major and their association to the hypercoagulable state. Seventy patients with ß-thalassemia major and 35 apparently healthy subjects as a control group were investigated for protein C and protein S. The mean of protein C (71.31%) and protein S (34.3%) levels were significantly reduced in ß- thalassemia major patients in comparison with control subjects (p-value <0.001). Mean of fibrinogen level (2.42) g/l was significantly decreased in ß-thalassemia major patients while the mean of D dimer level (0.43) µg/ml was significantly increased in comparison to control subjects (p-value 0.001). This study demonstrates a chronic hypercoagulable state in B- thalassemia major patients.

Morphometric Parameters of the Lung Tissue in Isolated Musculoskeletal Injuries under Conditions of Moderate Hyperhomocysteinemia.

The combination of inherited and acquired factors of increased thrombosis complicates establishing the cause-effect relationships between mechanical injury and death during forensic medical examination. Morphometric parameters of the lung tissue and pulmonary vessels in isolated mechanical trauma under conditions of moderate hyperhomocysteinemia were studied in an experiment on laboratory animals. A regularity in changes of the morphometry parameters of the pulmonary vessels was found. Morphometric changes in the wall of pulmonary parenchyma veins can be used as an additional marker to assess the causal relationship between isolated mechanical injury and fatal complication in the form of pulmonary embolism.

Thrombosis in Coronavirus disease 2019 (COVID-19) through the prism of Virchow's triad.

The pathogenesis of Coronavirus disease 2019 (COVID-19) is gradually being comprehended. A high number of thrombotic episodes are reported, along with the mortality benefits of heparin. COVID-19 can be viewed as a prothrombotic disease. We overviewed the available evidence to explore this possibility. We identified various histopathology reports and clinical case series reporting thromboses in COVID-19. Also, multiple coagulation markers support this. COVID-19 can be regarded as a risk factor for thrombosis. Applying the principles of Virchow's triad, we described abnormalities in the vascular endothelium, altered blood flow, and platelet function abnormalities that lead to venous and arterial thromboses in COVID-19. Endothelial dysfunction, activation of the renin-angiotensin-aldosterone system (RAAS) with the release of procoagulant plasminogen activator inhibitor (PAI-1), and hyperimmune response with activated platelets seem to be significant contributors to thrombogenesis in COVID-19. Stratifying risk of COVID-19 thromboses should be based on age, presence of comorbidities, D-dimer, CT scoring, and various blood cell ratios. Isolated heparin therapy may not be sufficient to combat thrombosis in this disease. There is an urgent need to explore newer avenues like activated protein C, PAI-1 antagonists, and tissue plasminogen activators (tPA). These should be augmented with therapies targeting RAAS, antiplatelet drugs, repurposed antiinflammatory, and antirheumatic drugs. Key Points • Venous and arterial thromboses in COVID-19 can be viewed through the prism of Virchow's triad. • Endothelial dysfunction, platelet activation, hyperviscosity, and blood flow abnormalities due to hypoxia, immune reactions, and hypercoagulability lead to thrombogenesis in COVID-19. • There is an urgent need to stratify COVID-19 patients at risk for thrombosis using age, comorbidities, D-dimer, and CT scoring. • Patients with COVID-19 at high risk for thrombosis should be put on high dose heparin therapy.

Oxidative status of maternal blood in pregnancies burdened by inherited thrombophilias.

Oxidative status of maternal blood represents an important parameter of pregnancy that is involved in both, regulation of physiological processes and (if significantly altered) development of different pregnancy complications. Inherited thrombophilias represent genetic disorders that increase the risk of thromboembolism in pregnancy. Little is known about the impact of thrombophilia on the oxidative status of maternal blood. In this study, we analyzed oxidative status of blood of 56 women with pregnancies burdened by inherited thrombophilias. The status was established at three different trimesters using biochemical assays and electrochemical measurements, and it was compared to 10 age- and trimester-matching controls. Activities of superoxide dismutase, catalase, and glutathione reductase in the 1st and the 2nd trimester of thrombophilic pregnancy were lower than controls. Also, there was less oxidation in the plasma, according to higher concentration of reduced thiols and lower oxidation-reduction potential. Therefore, it appears that thrombophilic mothers do not experience oxidative stress in the circulation in the first two trimesters. However, the rise in GPx, GR and SOD activities in the 3rd trimester of thrombophilic pregnancy implies that the risk of oxidative stress is increased during the late pregnancy. These results are important for developing antioxidative treatment that could tackle thrombophilia-related pregnancy complications.

SN50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome mice through inhibiting NF-κB p65 translocation.

BACKGROUND: It has been confirmed that NF-κB p65 signaling pathway is involved in the regulation of alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS). Whether SN50, a NF-κB cell permeable inhibitor, could attenuate alveolar hypercoagulation and fibrinolysis inhibition in ARDS remains to be elucidated. PURPOSE: We explored the efficacy and potential mechanism of SN50 on alveolar hypercoagulation and fibrinolysis inhibition in ARDS in mice. MATERIALS AND METHODS: Mouse ARDS was made by 50 µl of lipopolysaccharide (LPS) (4 mg/ml) inhalation. Male BALB/c mice were intraperitoneally injected with different does of SN50 1 h before LPS inhalation. Lung tissues were collected for hematoxylin-eosin (HE) staining, wet/dry ratio. Pulmonary expressions of tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), collagen III, as well as phosphorylated p65 (p-p65), p65 in nucleus (p'-p65), IκBα and IKKα/ß were measured. Bronchoalveolar lavage fluid (BALF) was gathered to test the concentrations of TF, PAI-1, activated protein C (APC) and thrombinantithrombin complex (TAT). DNA binding activity of NF-κB p65 was also determined. RESULTS: After LPS stimulation, pulmonary edema and exudation and alveolar collapse occured. LPS also stimulated higher expressions of TF and PAI-1 in lung tissues, and higher secretions of TF, PAI-1, TAT and low level of APC in BALF. Pulmonary collagen III expression was obviously enhanced after LPS inhalation. At same time, NF-κB signaling pathway was activated with LPS injury, shown by higher expressions of p-p65, p'-p65, p-IKKα/ß, p-Iκα in pulmonary tissue and higher level p65 DNA binding activity. SN50 dose-dependently inhibited TF, PAI-1 and collagen IIIexpressions, and decreased TF, PAI-1, TAT but increased APC in BALF. SN50 treatment attenuated pulmonary edema, exudation and reduced lung tissue damage as well. SN50 application significantly reduced p'-p65 expression and weakened p65 DNA binding activity, but expressions of p-p65, p-IKKα/ß, p-Iκα in cytoplasm of pulmonary tissue were not affected. CONCLUSIONS: SN 50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in ARDS via inhibition of NF-κB p65 translocation. Our data demonstrates that NF-κB p65 pathway is a viable new therapeutic target for ARDS treatment.

The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor.

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.