Gene editing of human iPSCs rescues thrombophilia in hereditary antithrombin deficiency in mice.

Hereditary antithrombin deficiency is caused by SERPINC1 gene mutations and predisposes to recurrent venous thromboembolism that can be life-threatening. Therefore, lifelong anticoagulation is required, which has side effects and may not be effective. In this study, peripheral blood mononuclear cells from a patient with severe antithrombin deficiency were reprogrammed into induced pluripotent stem cells (iPSCs). The mutation was corrected using CRISPR-Cas9 and Cre/LoxP genome editing. iPSCs were differentiated into hepatocytes, which were injected into the spleen of antithrombin knockout mice to restore the activity of antithrombin and reduce the thrombophilic state. Human iPSC-differentiated hepatocytes colonized mice and secreted antithrombin stably, normalizing antithrombin in plasma (activity: from 46.8 ± 5.7% to 88.6 ± 7.6%, P < 0.0001; antigen: from 146.9 ± 19.5 nanograms per milliliter to 390.7 ± 16.1 nanograms per milliliter, P < 0.0001). In venous thrombosis model, the rate of thrombosis in mice treated with edited hepatocytes, parental hepatocytes, and wild-type mice were 60, 90, and 70%, respectively. The thrombus weight was much lighter in mice treated with edited hepatocytes compared with parental hepatocytes (7.25 ± 2.00 milligrams versus 15.32 ± 2.87 milligrams, P = 0.0025) and showed no notable difference compared with that in wild-type mice (10.41 ± 2.91 milligrams). The activity and concentration of antithrombin remained high for 3 weeks after injection. The liver and kidney function markers showed no obvious abnormality during the observation period. This study provides a proof of principle for correction of mutations in patient-derived iPSCs and potential therapeutic applications for hereditary thrombophilia.

Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus).

BACKGROUND: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. OBJECTIVES: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. METHODS: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. RESULTS: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. CONCLUSIONS: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.

Waterpipe smoke-induced hypercoagulability and cardiac injury in mice: Influence of cessation of exposure.

Waterpipe tobacco smoking has gained worldwide popularity, particularly among youths. Several clinical and experimental studies have reported that waterpipe smoking (WPS) injures the cardiovascular system. However, the impact of smoking cessation (CS) on the cardiovascular toxicity induced by WPS received scant attention. Hence, we assessed, in C57BL/6 mice, the cardiovascular effects of WPS exposure for 3 months followed by 3 months of SC, as compared with mice exposed for either 3 months to WPS or air (control). WPS exposure induced hypertension, prothrombotic events both in vivo and in vitro and increased the plasma concentrations of tissue factor, fibrinogen and plasminogen activator inhibitor-1. These effects were significantly alleviated by SC. In heart tissue, the levels of troponin I, creatine kinase, lipid peroxidation, 8-isoprostane, tumor necrosis factor α, inteleukin 6, DNA damage and cleaved caspase-3 were significantly increased by WPS exposure. These actions were significantly reduced in the group of mice exposed to WPS followed by SC. Similarly, the increase in the level of nuclear factor κ-ß induced by WPS exposure was significantly mitigated by SC. Immunohistochemical analysis of the hearts showed that WPS exposure increased the expression of nuclear factor erythroid-derived 2-like 2 by cardiomyocytes. The latter effect was significantly reduced by SC. Taken together, our data show that SC is associated with amelioration of WPS induced hypertension, prothrombotic events and cardiac oxidative stress, inflammation, DNA damage and apoptosis.

Targeting hypercoagulation to alleviate Alzheimer's disease progression in metabolic syndrome.

INTRODUCTION: Metabolic Syndrome (MetS) constitutes an important risk factor for Alzheimer's disease (AD); however, the mechanism linking these two disorders has not been completely elucidated. Hence, hypercoagulation may account for the missing hallmark connecting MetS and AD. The present review proposes how hemostatic imbalance triggered in MetS advances in the context of AD. MetS causes interruption of insulin signaling and inflammation, inciting insulin resistance in the brain. Subsequently, neuroinflammation and brain endothelial dysfunction are prompted that further intensify the exorbitant infiltration of circulating lipids and platelet aggregation, thereby causing hypercoagulable state, impairing fibrinolysis and eventually inducing prothrombic state in the brain leading to neurodegeneration. OBJECTIVE: This study aims to understand the role of hypercoagulation in triggering the progression of neurodegeneration in MetS. It also offers a few interventions to prevent the progression of AD in MetS targeting hypercoagulation. METHODS: Literature studies based on MetS related neurodegeneration, the impact of coagulation on aggravating obesity and AD via the mechanisms of BBB disruption, neuroinflammation, and hypofibrinolysis. CONCLUSION: The present paper proposes the hypothesis that hypercoagulation might amplify MetS associated insulin resistance, neuroinflammation, BBB disruption, and amyloid beta accumulation which eventually leads to AD.

How to recognize and manage COVID-19-associated coagulopathy.

COVID-19 is frequently associated with abnormalities on coagulation testing and a coagulopathy driven by inflammation, intravascular coagulation activation, and microvascular thrombosis. Elevated D-dimer is the most common finding and is a predictor of adverse outcomes including thrombosis, critical illness, and death. Although COVID-19-associated coagulopathy has some similarities to disseminated intravascular coagulation, the platelet count is usually preserved, coagulation times are usually normal or minimally prolonged, and thrombosis is more common than bleeding, at least in noncritically ill patients. Bleeding is uncommon but may be a significant problem in critically ill patients, including those who may develop a consumptive coagulopathy with frank disseminated intravascular coagulation and those on extracorporeal membrane oxygenation. Blood product support to correct coagulopathy is reserved for bleeding patients or those requiring invasive procedures. Current recommendations suggest that all hospitalized patients should receive at least a prophylactic dose of anticoagulation. Results from a multiplatform randomized clinical trial suggest that therapeutically dosed anticoagulation may improve outcomes, including the need for organ support and mortality in moderately ill patients but not in those requiring critical care. The results of ongoing trials evaluating the impact of different antithrombotic strategies (therapeutic agents and intensity) on COVID-19 outcomes are eagerly awaited and are expected to have important implications for patient management. We also discuss COVID-19 vaccine-associated cytopenias and bleeding as well as vaccine-induced thrombotic thrombocytopenia, in which thrombosis is associated with thrombocytopenia, elevated D-dimer, and, frequently, hypofibrinogenemia.

Clinical guidance for peripartum management of patients with hereditary thrombophilia.

Hereditary thrombophilia is a condition in which individuals are susceptible to the formation of thrombi due to a hereditary deficiency in anticoagulant factors, antithrombin (AT), protein C (PC), or protein S (PS). Many Japanese thrombophilia patients have PS deficiency, especially PS p.K196E (also called as PS Tokushima), which is exclusive to the Japanese population, and thrombosis sometimes occurs during pregnancy. At present, no management guidelines for pregnancy and delivery in thrombophilia patients have been developed. The Study Group for Hereditary Thrombophilia, one of the research groups of blood coagulation abnormalities in the Research Program on Rare and Intractable Diseases supported with the Research Grants of the Ministry of Health, Labour and Welfare Science, has therefore developed this clinical guidance to provide healthcare workers with necessary information on safe pregnancy, parturition and neonatal management, adopting a format of responses to seven clinical questions (CQ). At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Chinese expert consensus on diagnosis and treatment of trauma-induced hypercoagulopathy.

Trauma-induced coagulopathy (TIC) is caused by post-traumatic tissue injury and manifests as hypercoagulability that leads to thromboembolism or hypocoagulability that leads to uncontrollable massive hemorrhage. Previous studies on TIC have mainly focused on hemorrhagic coagulopathy caused by the hypocoagulable phenotype of TIC, while recent studies have found that trauma-induced hypercoagulopathy can occur in as many as 22.2-85.1% of trauma patients, in whom it can increase the risk of thrombotic events and mortality by 2- to 4-fold. Therefore, the Chinese People's Liberation Army Professional Committee of Critical Care Medicine and the Chinese Society of Thrombosis, Hemostasis and Critical Care, Chinese Medicine Education Association jointly formulated this Chinese Expert Consensus comprising 15 recommendations for the definition, pathophysiological mechanism, assessment, prevention, and treatment of trauma-induced hypercoagulopathy.

Fibrinolysis Shutdown in COVID-19: Clinical Manifestations, Molecular Mechanisms, and Therapeutic Implications.

The COVID-19 pandemic has introduced a global public health threat unparalleled in our history. The most severe cases are marked by ARDS attributed to microvascular thrombosis. Hypercoagulability, resulting in a profoundly prothrombotic state, is a distinct feature of COVID-19 and is accentuated by a high incidence of fibrinolysis shutdown. The aims of this review were to describe the manifestations of fibrinolysis shutdown in COVID-19 and its associated outcomes, review the molecular mechanisms of dysregulated fibrinolysis associated with COVID-19, and discuss potential implications and therapeutic targets for patients with severe COVID-19.

Physiologic and biochemical rationale for treating COVID-19 patients with hyperbaric oxygen.

The SARS-Cov-2 (COVID-19) pandemic remains a major worldwide public health issue. Initially, improved supportive and anti-inflammatory intervention, often employing known drugs or technologies, provided measurable improvement in management. We have recently seen advances in specific therapeutic interventions and in vaccines. Nevertheless, it will be months before most of the world's population can be vaccinated to achieve herd immunity. In the interim, hyperbaric oxygen (HBO2) treatment offers several potentially beneficial therapeutic effects. Three small published series, one with a propensity-score-matched control group, have demonstrated safety and initial efficacy. Additional anecdotal reports are consistent with these publications. HBO2 delivers oxygen in extreme conditions of hypoxemia and tissue hypoxia, even in the presence of lung pathology. It provides anti-inflammatory and anti-proinflammatory effects likely to ameliorate the overexuberant immune response common to COVID-19. Unlike steroids, it exerts these effects without immune suppression. One study suggests HBO2 may reduce the hypercoagulability seen in COVID patients. Also, hyperbaric oxygen offers a likely successful intervention to address the oxygen debt expected to arise from a prolonged period of hypoxemia and tissue hypoxia. To date, 11 studies designed to investigate the impact of HBO2 on patients infected with SARS-Cov-2 have been posted on clinicaltrials.gov. This paper describes the promising physiologic and biochemical effects of hyperbaric oxygen in COVID-19 and potentially in other disorders with similar pathologic mechanisms.

Pediatric Otogenic Cerebral Sinus Vein Thrombosis and Thrombophilia.

BACKGROUND: Otogenic cerebral sinus vein thrombosis (CSVT) is a rare but severe complication of otitis media in children. To date, the role of prothrombotic evaluation is still controversial. OBJECTIVES: To report the clinical manifestations, prothrombotic evaluation, and current management of CSVT. METHODS: We performed a retrospective study of nine pediatric patients with otogenic CSVT who underwent prothrombotic evaluation between 2008 and 2018. RESULTS: Prominent clinical features included persistent otorrhea (88.8%), signs of mastoiditis (88.8%), high fever ≥ 38.3°C (100%), a classic spiking fever pattern (55.5%), and neurological signs (55.5%). A subperiosteal abscess (66.6%) was the most common otitis media complication associated with mastoiditis and CSVT. No microorganism was identified in 55.5% of patients. Cultures collected from ear secretions had a low yield (6.25%). However, PCR assays had a high detection rate (100%; n=3). The prothrombotic evaluation demonstrated an abnormal LAC-dRVVT ratio (6/9), elevated Factor VIII (5/8) (and a combination of both in four patients), antiphospholipid antibodies (2/8), and high homocysteine levels (1/5).The surgical intervention of choice included one-sided mastoidectomy with myringotomy and ventilation-tube placement on the affected side (77.7%). There were no mortalities and no long-term sequela except chronic otitis media (22.2%). CONCLUSIONS: Our findings demonstrate good outcomes for otogenic CSVT treatment with intravenous antibiotics, anticoagulation, and conservative surgical intervention, which supports the current trend in management. The prothrombotic evaluation revealed transient inflammation-related risk factors but did not alter management. Further prospective multicenter studies are needed to determine its relevance.

The roles and potential therapeutic implications of C5a in the pathogenesis of COVID-19-associated coagulopathy.

Emerging evidence has documented that multisystem organ failure in coronavirus disease 2019 (COVID-19) patients is strongly associated with various coagulopathies. Treatments for COVID-19-associated coagulopathy are still a clinical challenge. An advancement in the knowledge of mechanisms of the excessive or inappropriate activation of the complement cascade involved in the genesis of COVID-19-associated coagulopathy might be a fundamental approach for developing novel classes of anticoagulant drugs. In this context, there is emerging evidence indicating that C5a, a component of the complement system, and its receptors (C5aRs) play a critical role in the genesis of the COVID-19-associated hypercoagulable state. Thus, this review describes the mechanisms by which C5a/C5aR signaling participates in the cascade of events involved in the pathophysiology of COVID-19-associated coagulopathy. Furthermore, it highlights the current possibilities for the development of a novel therapeutic approach for COVID-19 patients that targets C5a/C5aRs signaling.

Evaluation and Management of Coagulopathies and Thrombophilias in Pediatric Patients.

The diagnosis of coagulopathy or thrombophilia in pediatric patients can be challenging. Congenital coagulopathies often present in the pediatric period and require appropriate work-up for diagnosis and ongoing management. Acquired coagulopathies of childhood are frequently encountered in hospitalized children and warrant appropriate coagulation testing for goal-directed therapy. The incidence of thrombosis is increasing in pediatric patients. After identifying the presence of thrombus, acute management includes initiating therapeutic anticoagulation. Choice of anticoagulant depends on patient's clinical status, along with availability of the anticoagulant. Thrombophilia evaluation is performed when children present with spontaneous thrombosis. Thrombophilia tests are inaccurate during acute illness.

Recurrent implantation failure - an overview of current research.

BACKGROUND: Recurrent implantation failure (RIF) can be defined as a failure to achieve a clinical pregnancy after transfer of at least four embryos of good quality in a minimum of three fresh or frozen cycles in women under the age of 40. RIF is often a complex problem with a wide variety of etiologies and mechanisms as well as treatment options. SUMMARY: Anatomical conditions of the uterus, thrombophilia, genetic abnormalities, or immunological factors are only a few examples which could be responsible for RIF. The recommendations for women with RIF vary depending on the source of their problem. There is not just one treatment option, but many depending on the etiology and the severity of the problem. KEY MESSAGE: However, it would help to establish a set of standardized examinations and tests to use, in order to do a preliminary evaluation on each patient, which would then hopefully direct the approach of treatment for each individual couple.

COVID-19 Associated Hypercoagulability: Manifestations, Mechanisms, and Management.

ABSTRACT: Patients with severe coronavirus disease-2019 (COVID-19) frequently have hypercoagulability caused by the immune response to the severe acute respiratory syndrome coronavirus-2 infection. The pathophysiology of COVID-19 associated hypercoagulability is not fully understood, but characteristic changes include: increased fibrinogen concentration, increased Factor VIII activity, increased circulating von Willebrand factor, and exhausted fibrinolysis. Anticoagulant therapy improves outcomes in mechanically ventilated patients with COVID-19 and viscoelastic coagulation testing offers an opportunity to tailor anticoagulant therapy based on an individual patient's coagulation status. In this narrative review, we summarize clinical manifestations of COVID-19, mechanisms, monitoring considerations, and anticoagulant therapy. We also review unique considerations for COVID-19 patients who are on extracorporeal membrane oxygenation.

"TEG talk": expanding clinical roles for thromboelastography and rotational thromboelastometry.

Viscoelastic assays (VEAs) that include thromboelastography and rotational thromboelastometry add value to the investigation of coagulopathies and goal-directed management of bleeding by providing a complete picture of clot formation, strength, and lysis in whole blood that includes the contribution of platelets, fibrinogen, and coagulation factors. Conventional coagulation assays have several limitations, such as their lack of correlation with bleeding and hypercoagulability; their inability to reflect the contribution of platelets, factor XIII, and plasmin during clot formation and lysis; and their slow turnaround times. VEA-guided transfusion algorithms may reduce allogeneic blood exposure during and after cardiac surgery and in the emergency management of trauma-induced coagulopathy and hemorrhage. However, the popularity of VEAs for other indications is driven largely by extrapolation of evidence from cardiac surgery, by the drawbacks of conventional coagulation assays, and by institution-specific preferences. Robust diagnostic studies validating and standardizing diagnostic cutoffs for VEA parameters and randomized trials comparing VEA-guided algorithms with standard care on clinical outcomes are urgently needed. Lack of such studies represents the biggest barrier to defining the role and impact of VEA in clinical care.

Viscoelastic testing in liver transplantation.

Despite the lack of large randomized clinical studies, viscoelastic tests (VETs) have been a critical armamentarium for hemostatic control in liver transplantation (LT) since the 1960s. Many transplant institutions have adopted VETs in their clinical practice. Several small-size randomized clinical trials on LT patients have suggested that VET-guided hemostatic treatment algorithms have led to decreased indications for and amounts of transfused blood products, especially fresh-frozen plasma, compared to standard laboratory-based hemostatic management. VETs have also been reported to offer insight into the diagnosis and prediction of LT patients' development of hypercoagulability-related morbidity and mortality. There is still a need for VET device-specific hemostatic algorithms in LT, and clinicians must take into account the tendency to underestimate the coagulation capacity of VETs in patients with end-stage liver disease where hemostasis is rebalanced.