Coagulation Complex Research Articles

Isolation and characterization of a novel inhibitor of coagulation complexes from the venom of the black-necked spitting cobra, Naja nigricollis

Isolation and characterization of a novel inhibitor of coagulation complexes from the venom of the black-necked spitting cobra, Naja nigricollis

Spontaneous intracerebral hemorrhage: a review of the prevalence, trigger factors, therapeutic and surgical treatment

Spontaneous intracerebral hemorrhage is the second most common subtype of stroke and is a significant cause of mortality and morbidity worldwide. Trigger factors that “accelerate” disease onset may include hypertension, excessive alcohol consumption, constant smoking, hypocholesterolemia, medications, male sex, advanced age, living in an underdeveloped country, ethnicity, chronic kidney disease, cerebral amyloid angiopathy, and microhemorrhages. Therapeutic solutions for spontaneous intracerebral hemorrhage aimed at stopping the progression of hemorrhage, reducing the clot volume of intraventricular and parenchymal hematoma, combating perihematomal edema and inflammation, rapidly reducing blood pressure, and providing hemostatic therapy with transfusion of platelets and other coagulation complexes. Clinical trials on minimally invasive methods of surgical evacuation (endoscopic surgery, stereotactic aspiration, and invasive craniopuncture) are ongoing and may provide positive results because of the shorter operation time and use of local anesthesia. Minimally invasive surgery methods can theoretically improve the outcomes of open surgery by reducing damage to collateral tissues and reducing anesthesia time; however, they cannot be a full-fledged alternative.

Economic analysis of the use of drugs for the treatment of hemophilia A, B and Willebrand's disease

Relevance. Therapy of hemophilia A, B and Willebrand's disease is carried out under the program of 14 VZN due to the use of federal budget funds. The use of funding is increasing in line with the growth of the total number of patients. In 2022,85.99 billion rubles were allocated, which corresponds to a 1.5‑fold increase in funding compared to 2018. The aim of the study was to conduct an economic analysis of the use of drugs for the therapy of hemophilia A, B and Willebrand's disease. Methods. The materials for the analysis were the dosing regimen of LPs selected on the basis of clinical recommendations, the availability of LPs in the 14 VZN program, and the prices for LPs from the State Register of maximum selling prices for VED drugs. Costs of on demand prophylaxis and treatment regimens for hemophilia A, B and Willebrand's disease according to clinical recommendations for each INN for all trade names (TN) were assessed. Results. For hemophilia A the number of submitted drugs by INN is 7;IN — 14; of them domestic drugs were submitted — 1. The minimum cost of LPs for prophylaxis is 2 584 764,00 rubles. Coit-DVI (Grifols Therapeutics LLC, USA), and the maximum cost is RUR 9,955,517.21. Advait (Takeda Manufacturing AG, Austria). Forhemophilia B, the number of represented PL — 2 INN; INN — 6; domestic drugs — 2. The minimum cost is RUR 1,559,376.00. Innonafactor (AO Generium, Russia), maximum — 3 079 319,88 rubles for a year course of prophylaxis LP Immunin (TakedaManufacturing, Austria). To stop bleeding in patients with inhibitor form use anti-inhibitor coagulant complex (Feiba, Austria), which is necessary on average 9100 units per 1 patient, and the average cost of application — 430 863,52 rubles or eptacog alpha(activated) (Coagil-VII, Russia) at multiple administration to the patient 6 times a day — maximum cost — 2 739 803,64 rubles or once a day — 293 550,35 rubles. Conclusion. In the structure of drug supply for patients with hemophilia, domestic drugs are represented by 5 names: Octofactor (JSC "Generium", Russia); Agemfil B (FGBU "NMIC Hematology" of the Ministry of Health of Russia, Russia) and Innonafactor (JSC "Generium", Russia); ArioSaven (LLC "PSK Pharma", Russia) and Coagil-VII (JSC "SG Biotech", Russia). There are no domestic drugs available for the therapy of Willebrand's disease.

Biomarkers of sustained systemic inflammation and microvascular dysfunction associated with post-COVID-19 condition symptoms at 24 months after SARS-CoV-2-infection.

Comprehensive studies investigating sustained hypercoagulability, endothelial function, and/or inflammation in relation to post-COVID-19 (PCC) symptoms with a prolonged follow-up are currently lacking. Therefore, the aim of this single-centre cohort study was to investigate serum biomarkers of coagulation activation, microvascular dysfunction, and inflammation in relation to persisting symptoms two years after acute COVID-19. Patients diagnosed with acute SARS-CoV-2 infection between February and June 2020 were recruited. Outcome measures included the CORona Follow-Up (CORFU) questionnaire, which is based on an internationally developed and partially validated basic questionnaire on persistent PCC symptoms. Additionally, plasma biomarkers reflecting coagulation activation, endothelial dysfunction and systemic inflammation were measured. 167 individuals were approached of which 148 (89%) completed the CORFU questionnaire. At 24 months after acute infection, fatigue was the most prevalent PCC symptom (84.5%). Over 50% of the patients experienced symptoms related to breathing, cognition, sleep or mobility; 30.3% still experienced at least one severe or extreme (4 or 5 on a 5-point scale) PCC symptom. Multiple correlations were found between several PCC symptoms and markers of endothelial dysfunction (endothelin-1 and von Willebrand factor) and systemic inflammation (Interleukin-1 Receptor antagonist). No positive correlations were found between PCC symptoms and coagulation complexes. In conclusion, this study shows that at 24 months after acute COVID-19 infection patients experience a high prevalence of PCC symptoms which correlate with inflammatory cytokine IL-1Ra and markers of endothelial dysfunction, especially endothelin-1. Our data may provide a rationale for the selection of treatment strategies for further clinical studies. This study was performed in collaboration with the CORona Follow-Up (CORFU) study (NCT05240742, https://clinicaltrials.gov/ct2/show/ NCT05240742).

Effects of Antiplatelet Drugs on Platelet-Dependent Coagulation Reactions.

Activated platelets are involved in blood coagulation by exposing phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes. Platelets accelerate fibrin formation and thrombin generation, two final reactions of the coagulation cascade. We investigated the effects of antiplatelet drugs on platelet impact in these reactions and platelet ability to expose PS. Washed human platelets were incubated with acetylsalicylic acid (ASA), ticagrelor, ASA in combination with ticagrelor, ruciromab (glycoprotein IIb-IIIa antagonist), or prostaglandin E1 (PGE1). Platelets were not activated or activated by collagen and sedimented in multiwell plates, and plasma was added after supernatant removal. Fibrin formation (clotting) was monitored in a recalcification assay by light absorbance and thrombin generation in a fluorogenic test. PS exposure was assessed by annexin V staining using flow cytometry. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, prolonged the lag phase and decreased the maximum rate of plasma clotting and decreased the peak and maximum rate of thrombin generation. Inhibition was observed when platelets were not treated with exogenous agonists (activation by endogenous thrombin) and pretreated with collagen. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, decreased PS exposure on washed platelets activated by thrombin and by thrombin + collagen. PS exposure on activated platelets in whole blood was lower in patients with acute coronary syndrome receiving ticagrelor + ASA in comparison with donors free of medications. These results indicate that antiplatelet drugs are able to suppress platelet coagulation activity not only in vitro but also after administration to patients.

Effects of platelets activated by different agonists on fibrin formation and thrombin generation

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.

Laparoscopic appendectomy in a child with hemophilia A with inhibitors receiving prophylactic treatment with emicizumab

Surgeries in patients treated prophylactically with emicizumab (including surgical procedures with a high risk of postoperative bleeding) are not associated with higher risks or technical difficulties compared with operations in patients receiving standard replacement therapy. On the contrary, the presence of emicizumab in the blood and its maintenance of permanent basic hemostasis allow for the reduction of doses and infusion time of bypassing agents. Patients undergoing surgical procedures with a high risk of postoperative bleeding require an additional hemostatic treatment during the first two or three days after the surgery. Then the duration of hemostatic treatment should be determined individually. We recommend the use of recombinant activated factor VII for the prevention/control of postoperative bleeding in patients with hemophilia A with inhibitors who are currently receiving emicizumab or have received it in the past 6 months. If treatment with anti-inhibitor coagulant complex is required, the daily dose should not exceed 100 units/kg. Here we report the first case of laparoscopic appendectomy in an 8-year-old child with hemophilia A with inhibitors receiving prophylactic treatment with emicizumab. The patient's parents gave their consent to the use of their child's data, including photographs, for research purposes and in publications.

Laparoscopic appendectomy in a child with hemophilia A with inhibitors receiving prophylactic treatment with emicizumab

Surgeries in patients treated prophylactically with emicizumab (including surgical procedures with a high risk of postoperative bleeding) are not associated with higher risks or technical difficulties compared with operations in patients receiving standard replacement therapy. On the contrary, the presence of emicizumab in the blood and its maintenance of permanent basic hemostasis allow for the reduction of doses and infusion time of bypassing agents. Patients undergoing surgical procedures with a high risk of postoperative bleeding require an additional hemostatic treatment during the first two or three days after the surgery. Then the duration of hemostatic treatment should be determined individually. We recommend the use of recombinant activated factor VII for the prevention/control of postoperative bleeding in patients with hemophilia A with inhibitors who are currently receiving emicizumab or have received it in the past 6 months. If treatment with anti-inhibitor coagulant complex is required, the daily dose should not exceed 100 units/kg. Here we report the first case of laparoscopic appendectomy in an 8-year-old child with hemophilia A with inhibitors receiving prophylactic treatment with emicizumab. The patient's parents gave their consent to the use of their child's data, including photographs, for research purposes and in publications.

Inflammatory cytokines induce neutrophil extracellular traps interaction with activated platelets and endothelial cells exacerbate coagulation in moderate and severe essential hypertension.

Essential hypertension (EH) patients suffer from paradoxically thrombotic rather than haemorrhagic, although the exact mechanism remains elusive. Our aim is to explore whether and how neutrophil extracellular traps (NETs) play the procoagulant role in EH patients, as well as evaluated whether the NET releasing were triggered by inflammatory cytokines. The concentration of plasma NETs components were detected by ELISA. The morphology of cells and NETs formation were analysed using immunofluorescence. Procoagulant activity was analysed by clotting time, purified coagulation complex and fibrin generation assays. Phosphatidylserine (PS) exposure on endothelial cells (ECs) was analysed with flow cytometry. Moderate to severe EH patients plasma NETs levels were significantly higher compared to mild EH patients or controls. Furthermore, inflammatory cytokines can induce NETs generation, depleting these patients plasma inflammatory cytokines led to a reduction in NET releasing. NETs from moderate to severe EH patients neutrophils led to significantly decreased clotting time (CT), increased potency to generate thrombin and fibrin (all P < 0.05). These procoagulant effects were markedly attenuated by approximately 70% using DNase I. Additionally, high concentrations NETs exerted a strong cytotoxic effect on ECs, conferring them a procoagulant phenotype. Our study reveals that EH drives a systemic inflammatory environment, which, in turn, drives neutrophils to prime and NET releasing, and found a link between hypercoagulability and NETs levels in moderate to severe EH patients. Therefore, anti-inflammatory combined with block the generation of NETs may represent a new therapeutic target for preventing thrombosis in EH patients.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

Extracorporeal membrane oxygenator as a bridge to heart–liver en bloc transplant in a patient with Fontan circulation

Extracorporeal membrane oxygenator as a bridge to heart–liver en bloc transplant in a patient with Fontan circulation

Analysis of nationwide hemophilia care: A cohort study using two Japanese healthcare claims databases.

Background and aimsIn many developed countries, hemophilia care is provided by specialized centers which can offer standardized high‐quality care for patients and collect data for patient registries. However, in countries with less centralized provision of hemophilia care, registry data lacks accuracy and medical care is inconsistent among providers. Claims databases can be an alternative for obtaining nationwide data on hemophilia care, and we applied this approach to evaluate inequalities in hemophilia care in Japan.MethodsMedical records of hemophilia A patients were collected by a combination of ICD‐10 code (D66) and prescribed coagulation factors from two major Japanese claims databases (JMDC and Medical Data Vision [MDV]). Patient records with an anti‐inhibitor coagulant complex were excluded.Based on the annual number of hemophilia A patients, medical facilities were categorized into specialized facilities (SP, ≥5 patients) and nonspecialized facilities (N‐SP, <5 patients). Patient age, comorbidities, diagnostic testing, prescribed drugs and their dosages were compared between facility types.ResultsThe JMDC and MDV databases included 274 and 1266 hemophilia A patients, respectively. In the MDV database, SP facilities prescribed extended half‐life factor VIII (FVIII) products for more patients (31.8% vs 24.3%) than N‐SP. The mean annual FVIII consumption per patient was higher in SP facilities (240 333 IU [international units] vs 210 334 IU), and the mean FVIII dosage was higher in SP facilities for all types of FVIII products. The proportion of patients who received diagnostic blood tests was higher in SP (75.7% vs 56.2%).ConclusionThe MDV database revealed disparities in hemophilia A care between SP and N‐SP facilities in types of FVIII products prescribed, FVIII consumption, and frequency of the relevant management such as blood tests. Claims databases can be an alternative for the assessment of nationwide hemophilia care patterns in countries without a well‐established registry.

Coagulation activity of circulating membrane microparticles in patients with cardiovascular diseases

Membrane microparticles (MP) are released by activated or damaged cells and are able to accelerate blood clotting (coagulation). MP possess coagulation activity since all of them contain on their surface phosphatidylserine (PS), a substrate for the assembly of coagulation complexes, and some of them tissue factor (TF), the primary initiator of coagulation cascade reactions. We compared the coagulation activity and amount of MP in the blood of healthy donors (n=34) and patients with myocardial infarction (MI) (n=32), advanced atherosclerosis (AA) (n=32) and idiopathic pulmonary arterial hypertension (IPAH) (n=19). Total MP fraction was obtained from blood plasma by sedimentation at 20000 g, 30 min. The coagulation activity of PM isolated from 100 μl of donor and patient plasma was determined using a modified recalcification test. MP were added to substrate plasma devoid of endogenous MF, plasma was recalcified, and clotting was recorded by changes in optical density (A450), determining lag phase (min) and maximum rate (Vmax, %A450/min). MP were counted by flow cytometry as PS+ particles (lactadgerin-FITC staining) smaller than 1 μm and their concentration was expressed as 105 MP/μl plasma. MP in all patient groups accelerated plasma clotting more effectively than donor MP. Lag phase compared with donors (11.8 [11.0-13.1] median and interquartile range) was shorter in patients with AA (8.8 [7.0-10.3], p.

Neutrophil extracellular traps contribute to tissue plasminogen activator resistance in acute ischemic stroke.

Circulating neutrophil extracellular traps (NETs) resistant to t-PA have not been studied completely although NETs in thrombi may contribute to tissue plasminogen activator (t-PA) resistance. This research intended to elucidate whether circulating NETs are associated with t-PA resistance and the underlying mechanism. The levels of NETs were detected in the circulating neutrophils, ischemic brain tissue of acute ischemic stroke (AIS) patients, and transient middle cerebral artery occlusion (tMCAO) models. NET formation in blood, thrombi, and ischemic brain tissue of mice were analyzed by immunofluorescence. Exposed phosphatidylserine (PS) was assessed using flow cytometry and confocal microscopy. Procoagulant activity (PCA) was evaluated using fibrin formation assays, thrombin, and purified coagulation complex. The plasma levels of NETs in AIS patients were significantly higher than those in healthy individuals. After thrombolysis, a significant increase was noted in NET markers in no-improvement patients, while the changes in improvement patients were not significant. Importantly, NETs were decorated with von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) in the blood and thrombi, which could reverse the fibrinolytic effects. In addition, NETs activated platelets (PLTs) and endothelial cells (ECs), stimulating a procoagulant phenotype and facilitating vWF and PAI-1 release. DNase I, activated protein C (APC), and sivelestat markedly inhibited these effects. Furthermore, targeting NETs protected mice from tMCAO-induced cerebral ischemia, possibly by regulating vWF and PAI-1. In summary, NETs may contribute to t-PA resistance in AIS through activation of PLTs and ECs. Strategies against NETs may present a promising therapeutic approach to improve the thrombolysis efficiency of t-PA in AIS patients.

Outcomes of activated prothrombin complex concentrate for direct Xa inhibitor bleeding

Bleeding due to oral anticoagulant therapy is recognized as a high priority drug safety problem [ [1] FDA Adverse Events Reporting System (FAERS)Latest Quarterly Data Files (2017) Food and Drug Administration Web Site. 2021 Google Scholar ]. As the use of direct Xa inhibitors continues to expand, the management of bleeding is an important issue. Andexanet, a modified human factor Xa decoy reversal agent for apixaban or rivaroxaban associated bleeding, is available in some countries however concerns with its use have been expressed [ [2] Peled H. Dau N.Q. Lau H. Key points to consider when evaluating andexxa for formulary addition. Neurocrit. Care. 2020; 33: 20-24 Crossref PubMed Scopus (7) Google Scholar ]. Four factor prothrombin complex concentrate (4FPCC) is an alternative reversal agent which has been used off-label for direct Xa inhibitor bleeding [ 3 Steffel J. et al. The 2018 european heart rhythm association practical guide on the use of non-vitamin K antagonist oral anticoagulants in patients with atrial fibrillation. Eur. Heart J. 2018; 39: 1330-1393 Crossref PubMed Scopus (1060) Google Scholar , 4 Witt D.M. et al. American Society of Hematology 2018 guidelines for management of venous thromboembolism: optimal management of anticoagulation therapy. Blood Adv. 2018; 2: 3257-3291 Crossref PubMed Scopus (188) Google Scholar ]. Four factor PCC is available either as an activated or non-activated product. Limited clinical data exist evaluating activated 4FPCC, FEIBA [anti-inhibitor coagulant complex®, Baxalta US Inc., Lexington MA, USA] as a reversal agent [ 5 Engelbart J.M. et al. Safety and effectiveness of factor eight inhibitor bypassing activity for direct oral anticoagulant-related hemorrhage reversal. Am. J. Emerg. Med. 2019; 37: 214-219 Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar , 6 Mao G. et al. Factor eight inhibitor bypassing agent (FEIBA) for reversal of target-specific oral anticoagulants in life-threatening intracranial bleeding. J. Emerg. Med. 2017; 52: 731-737 Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar ]. As andexanet is not on formulary, the reversal protocol at our institution recommended the use of activated 4FPCC 25–50 IU/kg for direct Xa inhibitor bleeding for situations where reversal is desired. Our goal was to assess the patient characteristics, management and outcomes of activated 4FPCC for reversal of apixaban or rivaroxaban acute bleeding events.

Phosphatidylserine on Blood Cells and Microparticles Contributes to the Hyper-Coagulable State in Diabetic Kidney Disease

Introduction: Patients with diabetes mellitus (DM) are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially when microvascular complications of diabetes mellitus occur, such as diabetic kidney disease (DKD). Additionally, microthrombosis in glomerular capillaries could decrease the kidney blood flow and induce renal insufficiency. However, patients with DKD do not receive routine antithrombotic therapy. Therefore, more study is needed on the mechanisms of coagulation in DKD with the aim of identifying therapeutic targets. Recent studies have shown that phosphatidylserine (PS), a membrane constituent, plays an important role in the thrombosis. However, relatively little is known about the precise role of PS in the prothrombotic state of DKD. Our objectives were to study the effect of increased PS exposure on microparticles (MPs) and the outer membrane of MP-origin cells in DKD patients, and to evaluate its effect on procoagulant activity (PCA).Methods: DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate (UAER), defined as normoalbuminuria (<30 mg/24 h), microalbuminuria (30-299 mg/24 h), or macroalbuminuria (>300 mg/24 h), and compared to healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells and human umbilical vein endothelial cells (HUVECs) were treated with 25 mM, 5 mM or 2.5 mM glucose and then PS exposure was measured. PCA of MPs and cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure and fibrin strands were observed using confocal microscopy.Results: Usingflow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and blood cells (BCs) were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with macroalbuminuria was significantly higher than in patients with normoalbuminuria. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with macro-DKD versus serum from patients with micro-DKD or normo-DKD. 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 co-localization of fibrin with PS on ECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 80%. Additionally, both hyperglycemia (25 mmol/L) and hypoglycemia (2.5 mmol/L) induced increased PS exposure on cells compared to normoglycemia (5 mmol/L). Linear regression analysis showed that levels of PS+ BCs and MPs were positively correlated with uric acid and proteinuria, but negatively correlated with GFR.Conclusions: Our results suggested that PS+ MPs and blood cells play a prominent role in inducing the hypercoagulable and prothrombotic state in patients with DKD. The occurrence of abnormal glucose during the hypoglycemic therapy is associated with hypercoagulation in DKD. PS+ cells and MPs likely support thrombosis in renal vascular and result in renal dysfunction and disease progression. Our results demonstrate that PS provides binding sites for FXa and prothrombinase complexes and promotes thrombin formation. Thus, directly targeting FXa and prothrombinase complexes might decrease thrombotic and renal disease risk. The results from this study suggest that future research should focus on discovery and deployment of direct PS inhibitors.Disclosure of Interest: Jialan Shi has a patent for the use of lactadherin as a PS probe. DisclosuresNo relevant conflicts of interest to declare.

Neutrophil extracellular traps induced by pro-inflammatory cytokines enhance procoagulant activity in NASH patients

Nonalcoholic steatohepatitis (NASH) patients are at a high risk of developing venous thromboembolism, with a high rate of morbidity and mortality. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in patients with NASH remains unclear. Our study aimed to investigate the formation of NETs in NASH patients stimulated by specific pro-inflammatory factors. Moreover, we evaluated the pivotal role of NETs in the induction of hypercoagulability in NASH and the interaction between NETs and endothelial injury. The levels of the NETs biomarkers were evaluated in the plasma samples of 27 NASH patients and 18 healthy subjects. The formation of NETs was visualized using immunofluorescence microscopy. The PCA of the NETs was assessed using coagulation time, purified coagulation complex, and fibrin formation assays. Confocal microscopy was further used to evaluate the interactions between the NETs and HUVECs. The levels of NETs markers in the plasma of NASH patients were significantly higher than healthy controls. NETs derived from NASH enhanced thrombin and fibrin formation and significantly reduced CT (p<0.05). The mixture of IL-6 and TNF-α triggered the NETs release in the plasma rather than them alone. Additionally, the NETs exerted cytotoxic effects on the endothelial cells, converting them to a procoagulant and pro-inflammatory phenotype, and DNase I could reverse these effects. Our results revealed the primary role of NETs in promoting the hypercoagulable state in NASH patients. Methods that prevent the formation of NETs may be a novel approach for the prevention and treatment of NASH.

Microparticles in COVID-19 as a link between lung injury extension and thrombosis.

Procoagulant microparticles are associated with the extent of lung injuries in #COVID19 and pulmonary thrombosis https://bit.ly/3eX2LPc

Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy

AbstractInteraction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D′D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D′D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII–VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D′D3 is covalently linked to an Fc domain of a B domain–deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D′D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII–VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF–D′/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.

Hemostatic Balance in Pediatric Acute Liver Failure: Epidemiology of Bleeding and Thrombosis, Physiology, and Current Strategies.

Pediatric Acute Liver Failure (PALF) is a rapidly progressive clinical syndrome encountered in the pediatric ICU which may rapidly progress to multi-organ dysfunction, and on occasion to life threatening cerebral edema and hemorrhage. Pediatric Acute Liver Failure is defined as severe acute hepatic dysfunction accompanied by encephalopathy and liver-based coagulopathy defined as prolongation of International Normalized Ratio (INR) >1.5. However, coagulopathy in PALF is complex and warrants a deeper understanding of the hemostatic balance in acute liver failure. Although an INR value of >1.5 is accepted as the evidence of coagulopathy and has historically been viewed as a prognostic factor of PALF, it may not accurately reflect the bleeding risk in PALF since it only measures procoagulant factors. Paradoxically, despite the prolongation of INR, bleeding risk is lower than expected (around 5%). This is due to “rebalanced hemostasis” due to concurrent changes in procoagulant, anticoagulant and fibrinolytic systems. Since the liver is involved in both procoagulant (Factors II, V, IX, XI, and fibrinogen) and anticoagulant (Protein C, Protein S, and antithrombin) protein synthesis, PALF results in “rebalanced hemostasis” or even may shift toward a hypercoagulable state. In addition to rebalanced coagulation there is altered platelet production due to decreased thrombopoietin production by liver, increased von Willebrand factor from low grade endothelial cell activation, and hyperfibrinolysis and dysfibrinogenemia from altered synthetic liver dysfunction. All these alterations contribute to the multifactorial nature of coagulopathy in PALF. Over exuberant use of prophylactic blood products in patients with PALF may contribute to morbidities such as fluid overload, transfusion-associated lung injury, and increased thrombosis risk. It is essential to use caution when using INR values for plasma and factor administration. In this review we will summarize the complexity of coagulation in PALF, explore “rebalanced hemostasis,” and discuss the limitations of current coagulation tests. We will also review strategies to accurately diagnose the coagulopathy of PALF and targeted therapies.