Thrombin-antithrombin Complex Research Articles

Hemostatic profiles in dogs with sudden acquired retinal degeneration syndrome.

Sudden acquired retinal degeneration syndrome (SARDS) is a common cause of irreversible blindness in dogs. It bears clinical resemblance to hypercortisolism, which can be associated with hypercoagulability. The role of hypercoagulability in dogs with SARDS is unknown. Determine hemostatic profiles in dogs with SARDS. Prospective pilot study: Dogs with a history of SARDS (n = 12). Prospective case-control study: Dogs with recent onset of SARDS (n = 7) and age-, breed-, and sex-matched controls (n = 7). Prospective pilot study: We performed thromboelastography (TEG). Prospective case-control study: Dogs had CBC, serum biochemistry, urinalysis, TEG, fibrinogen concentration, antithrombin activity, D-dimers, thrombin-antithrombin complexes, and optical platelet aggregometry performed. Prospective pilot study: 9/12 dogs with a history of SARDS were hypercoagulable with increased TEG G value and 2/3 had hyperfibrinogenemia. Case-control study: All dogs with SARDS and 5/7 controls were hypercoagulable based on TEG G value. Dogs with SARDS had significantly higher G values (median, 12.7 kdynes/s; range, 11.2-25.4; P = .04) and plasma fibrinogen concentration (median, 463 mg/dL; range, 391-680; P < .001) compared to controls. Hypercoagulability was common in both dogs with SARDS and controls, but dogs with SARDS were significantly more hypercoagulable on TEG. The role of hypercoagulability in the pathogenesis of SARDS remains to be determined.

Development of a High-Throughput Urosepsis Mouse Model.

Murine sepsis models are typically polymicrobial, and are associated with high mortality. We aimed to develop a high-throughput murine model that mimics a slow-paced, monomicrobial sepsis originating from the urinary tract. A total of 23 male C57Bl/6 mice underwent percutaneous insertion of a 4 mm catheter into the bladder using an ultrasound-guided method, previously developed by our group. The following day, Proteus mirabilis (PM) was introduced percutaneously in the bladder in three groups: g1-50 µL 1 × 108 CFU/mL solution (n = 10); g2-50 µL 1 × 107 CFU/mL solution (n = 10); and g3 (sham mice)-50 µL sterile saline (n = 3). On day 4, mice were sacrificed. The number of planktonic bacteria in urine, adherent to catheters, and adherent to/invaded into the bladder and spleen was assessed. Cell-free DNA, D-dimer, thrombin-antithrombin complex (TAT), and 32 pro-/anti-inflammatory cytokines/chemokines were quantified in the blood. All mice survived the 4 day postinterventional period. Mean weight loss was 11% in g1, 9% in g2, and 3% in the control mice. Mean urine CFU counts were highest in group 1. All catheters showed high catheter-adhered bacterial counts. Of the infected mice, 17/20 had CFU counts in the splenic tissue, indicating septicemia. Plasma levels of cell-free DNA, D-dimer, and the proinflammatory cytokines IFN-γ, IL-6, IP-10, MIG, and G-CSF were significantly elevated in infected mice versus controls. We present a reproducible, monomicrobial murine model of urosepsis that does not lead to rapid deterioration and death, and is useful for studying prolonged urosepsis.

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

Robust coagulation activation and coagulopathy in mice with experimental acetaminophen-induced liver failure

BackgroundPatients with acetaminophen (APAP)-induced acute liver failure (ALF) display both hyper- and hypocoagulable changes not necessarily recapitulated by standard hepatotoxic doses of APAP used in mice (eg, 300 mg/kg). ObjectivesWe sought to examine coagulation activation in vivo and plasma coagulation potential ex vivo in experimental settings of APAP-induced hepatotoxicity and repair (300-450 mg/kg) and APAP-induced ALF (600 mg/kg) in mice. ResultsAPAP-induced ALF was associated with increased plasma thrombin–antithrombin complexes, decreased plasma prothrombin, and a dramatic reduction in plasma fibrinogen compared with lower APAP doses. Hepatic fibrin(ogen) deposits increased independent of APAP dose, whereas plasma fibrin(ogen) degradation products markedly increased in mice with experimental ALF. Early pharmacologic anticoagulation (+2 hours after 600 mg/kg APAP) limited coagulation activation and reduced hepatic necrosis. The marked coagulation activation evident in mice with APAP-induced ALF was associated with a coagulopathy detectable ex vivo in plasma. Specifically, prolongation of the prothrombin time and inhibition of tissue factor–initiated clot formation were evident even after restoration of physiological fibrinogen concentrations. Plasma endogenous thrombin potential was similarly reduced at all APAP doses. Interestingly, in the presence of ample fibrinogen, ∼10 times more thrombin was required to clot plasma from mice with APAP-induced ALF compared with plasma from mice with simple hepatotoxicity. ConclusionThe results indicate that robust pathologic coagulation cascade activation in vivo and suppressed coagulation ex vivo are evident in mice with APAP-induced ALF. This unique experimental setting may fill an unmet need as a model to uncover mechanistic aspects of the complex coagulopathy of ALF.

Serpin family proteins as potential biomarkers and therapeutic drugs in stroke: A systematic review and meta-analysis on clinical/preclinical studies.

Serpin is a superfamily of serine proteinase inhibitors. They have anticoagulative activities and immunoregulatory effects. The family has been widely studied in stroke patients and animal stroke models. However, results from clinical and preclinical studies are controversial. The systematic review and meta-analysis aimed to determine whether serpin activities are affected by stroke and whether members of the serpin family could be used in stroke treatment. Literature was systematically searched in six databases until September 5, 2022. In the included studies, 47 clinical studies (8276 subjects) reported concentrations of serpin proteins in stroke patients and healthy controls. In total, 41 preclinical studies (742 animals) reported neurological outcomes in animal models with serpin treatment and vehicle. Meta-analysis of clinical studies showed that both ischemic (IS) and hemorrhagic stroke patients had higher thrombin-antithrombin complex (TAT) levels and lower antithrombin (AT) levels which were persistent in the acute and subacute phase of IS. Meta-analysis of preclinical studies reported the efficacy of serpins in treating stroke. C1-INH and FUT175 reduced brain infarct size and improved sensorimotor and motor behavior in a dose- and time-dependent manner in the MCAO models. Our study confirmed the important roles serpin family proteins played in the onset, progression, and treatment of stroke. Among serpins, AT and TAT may be used as blood biomarkers in the early diagnosis of stroke. C1-INH and FUT175 could be potential medications for IS.

An impact of neutrophil extracellular traps to the prothrombotic state and tumor progression in gynecological cancer patients

Introduction. One of the leading causes in the mortality pattern of cancer patients is accounted for by thrombotic complications. Recent studies have shown that neutrophil extracellular traps (NETs) are involved in the activation of coagulation, contribute to the initiation and progression of thrombosis. In addition, NET-related effect on tumor progression and metastasis has been actively studied.Aim: to evaluate NET-related procoagulant activity in gynecological cancer patients.Materials and Methods. From April 2020 to October 2022, a prospective controlled interventional non-randomized study was conducted with 120 women. The main group included 87 patients aged 32 to 72 years with malignant neoplasms of the female genital organs and mammary glands who were hospitalized for elective surgical treatment or chemotherapy: uterine body cancer (subgroup 1; n = 18), ovarian cancer (subgroup 2; n = 26), cervical cancer – adenocarcinoma of the cervical canal (subgroup 3; n = 13), breast cancer (subgroup 4; n = 30). The control group consisted of 33 healthy women aged 32 to 68 years. In all women, plasma concentrations of citrullinated histone H3 (citH3), myeloperoxidase antigen (MPO:Ag), D-dimer, and thrombin–antithrombin (TAT) complexes were evaluated.Results. The magnitude of NETosis in cancer patients, assessed by level of citH3 (2.5 ± 0.7; 1.9 ± 0.8; 2.5 ± 0.7; 0.7 ± 0.5 ng/ml in four subgroups, respectively) and MPO:Ag (29.5 ± 13.1; 12.8 ± 3.7; 22.8 ± 8.7; 6.6 ± 2.5 ng/ml in four subgroups, respectively) was significantly higher compared to women in the control group (0.3 ± 0.1 ng/ml; p = 0.0001 and 2.5 ± 0.2 ng/ml; p = 0.0001). In parallel with increased NETosis markers in accordance with the disease stage, there was an increase in the concentration of hemostasis activation markers – D-dimer (1.7 ± 0.6; 2.0 ± 0.7; 1.4 ± 0.5; 1.5 ± 0.7 µg/ml in four subgroups, respectively) and TAT complexes (729.8 ± 43.9; 794.1 ± 164.8; 636.2 ± 149.5; 699.6 ± 165.7 pg/ml in four subgroups, respectively) exceeding their level in the control group (respectively, 0.4 ± 0.1 μg/ml; p = 0.0001 and 362.3 ± 0.1 pg/ml; p = 0.0001). The maximum values of parameters occurred at later stages according to the Classification of Malignant Tumours (tumor, nodus, metastasis, TNM). A significant correlation between TAT level and the concentrations of citH3 (r = 0.586; р = 0.04) and MPO:Ag was revealed (r = 0.631; р = 0.04).Conclusion. Tumor tissue creates milieu that stimulates NETs release, which, in turn, not only contribute to the creating a procoagulant state, but also might act as one of the factors that ensure tumor progression and metastasis. The development of targeted therapies acting on NETs has a potential to affect hemostasis in cancer patients and reduce rate of tumor growth and metastasis.

Involvement of monocyte-derived extracellular vesicle-associated tissue factor activity in convallatoxin-induced hypercoagulability.

Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. The findings of the present study have further clarified the procoagulant properties of CNT.

Pretreatment with Eupatilin Attenuates Inflammation and Coagulation in Sepsis by Suppressing JAK2/STAT3 Signaling Pathway.

Sepsis is an aggressive and life-threatening organ dysfunction induced by infection. Excessive inflammation and coagulation contribute to the negative outcomes for sepsis, resulting in high morbidity and mortality. In this study, we explored whether Eupatilin could alleviate lung injury, reduce inflammation and coagulation during sepsis. We constructed an in vitro sepsis model by stimulating RAW264.7 cells with 1 μg/mL lipopolysaccharide (LPS) for 6 hours. The cells were divided into control group, LPS group, LPS+ Eupatilin (Eup) group, and Eup group to detect their cell activity and inflammatory cytokines and coagulation factor levels. Cells in LPS+Eup and Eup group were pretreated with Eupatilin (10μM) for 2 hours. In vivo, mice were divided into sham operation group, cecal ligation and puncture (CLP) group and Eup group. Mice in the CLP and Eup groups were pretreated with Eupatilin (10mg/kg) for 2 hours by gavage. Lung tissue and plasma were collected and inflammatory cytokines, coagulation factors and signaling were measured. In vitro, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and tissue factor (TF) expression in LPS-stimulated RAW264.7 cells was downregulated by Eupatilin (10μM). Furthermore, Eupatilin inhibited phosphorylation of the JAK2/STAT3 signaling pathway and suppressed p-STAT3 nuclear translocation. In vivo, Eupatilin increased the survival rate of the mice. In septic mice, plasma concentrations of TNF-α, IL-1β and IL-6, as well as TF, plasminogen activator inhibitor 1 (PAI-1), D-dimer, thrombin-antithrombin complex (TAT) and fibrinogen were improved by Eupatilin. Moreover, Eupatilin alleviated lung injury by improving the expression of inflammatory cytokines and TF, fibrin deposition and macrophage infiltration in lung tissue. Our results revealed that Eupatilin may modulate inflammation and coagulation indicators as well as improve lung injury in sepsis via the JAK2/STAT3 signaling pathway.

THE EFFECTS OF DNASE I AND LOW-MOLECULAR-WEIGHT HEPARIN IN A MURINE MODEL OF POLYMICROBIAL ABDOMINAL SEPSIS.

Introduction: Cell-free DNA (CFDNA) has emerged as a prognostic biomarker in patients with sepsis. Circulating CFDNA is hypothesized to be associated with histones in the form of nucleosomes. In vitro, DNA activates coagulation and inhibits fibrinolysis, whereas histones activate platelets and are cytotoxic to endothelial cells. Previous studies have targeted CFDNA or histones in animal models of sepsis using DNase I or heparins, respectively, which has reduced inflammatory and thrombosis markers, thereby improving survival. In this study, we explored the possibility that the combination of DNase I and a low-molecular weight heparin (LMWH) may be a better therapeutic approach than monotherapy in a murine model of abdominal sepsis. Methods: C57Bl/6 mice (8-12 weeks old, both sexes) were subjected to either cecal ligation and puncture or sham surgery. Mice were given antibiotics, fluids, and either saline, DNase I (intraperitoneally, 20 mg/kg/8 h), LMWH (dalteparin, subcutaneously 500 IU/kg/12 h), or a combination of both (n = 12-31). Mice were monitored over 72 h for survival. Organs and blood were harvested for analysis. Levels of LMWH, CFDNA, IL-6, citrullinated histone-H3, thrombin-antithrombin complexes, and protein C were measured in plasma. Results: Administration of either DNase I (81.8%) or LMWH (83.3%, prophylactic range of 0.12 ± 0.07 IU/mL achieved) improved the survival of septic mice compared with saline- (38.7%) and combination-treated mice (48.8%, P < 0.05). Combination-treated mice also showed a small but insignificant improvement in survival compared with saline-treated cecal ligation and puncture mice. Monotherapies may be improving survival by reducing blood bacterial loads, citrullinated histone-H3, and thrombin-antithrombin complexes, and improving protein C levels. Conclusions: Compared with saline- and combination-treated mice, administration of monotherapies to septic mice improved survival. These findings suggest that there may be a negative drug-drug interaction between DNase I and LMWH when DNase I is administered intraperitoneally in a murine model of polymicrobial abdominal sepsis.

SARS-CoV-2 infection results in upregulation of Plasminogen Activator Inhibitor-1 and Neuroserpin in the lungs, and an increase in fibrinolysis inhibitors associated with disease severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in coagulation activation although it is usually not associated with consumption coagulopathy. D-dimers are also commonly elevated despite systemic hypofibrinolysis. To understand these unusual features of coronavirus disease 2019 (COVID-19) coagulopathy, 64 adult patients with SARS-CoV-2 infection (36 moderate and 28 severe) and 16 controls were studied. We evaluated the repertoire of plasma protease inhibitors (Serpins, Kunitz, Kazal, Cystatin-like) targeting the fibrinolytic system: Plasminogen Activator Inhibitor-1 (PAI-1), Tissue Plasminogen Activator/Plasminogen Activator Inhibitor-1 complex (t-PA/PAI-1), α-2-Antiplasmin, Plasmin-α2-Antiplasmin Complex, Thrombin-activatable Fibrinolysis Inhibitor (TAFI)/TAFIa, Protease Nexin-1 (PN-1), and Neuroserpin (the main t-PA inhibitor of the central nervous system). Inhibitors of the common (Antithrombin, Thrombin-antithrombin complex, Protein Z [PZ]/PZ inhibitor, Heparin Cofactor II, and α2-Macroglobulin), Protein C ([PC], Protein C inhibitor, and Protein S), contact (Kallistatin, Protease Nexin-2/Amyloid Beta Precursor Protein, and α-1-Antitrypsin), and complement (C1-Inhibitor) pathways, in addition to Factor XIII, Histidine-rich glycoprotein (HRG) and Vaspin were also investigated by enzyme-linked immunosorbent assay. The association of these markers with disease severity was evaluated by logistic regression. Pulmonary expression of PAI-1 and Neuroserpin in the lungs from eight post-mortem cases was assessed by immunohistochemistry. Results show that six patients (10%) developed thrombotic events, and mortality was 11%. There was no significant reduction in plasma anticoagulants, in keeping with a compensated state. However, an increase in fibrinolysis inhibitors (PAI-1, Neuroserpin, PN-1, PAP, and t-PA/PAI-1) was consistently observed, while HRG was reduced. Furthermore, these markers were associated with moderate and/or severe disease. Notably, immunostains demonstrated overexpression of PAI-1 in epithelial cells, macrophages, and endothelial cells of fatal COVID-19, while Neuroserpin was found in intraalveolar macrophages only. These results imply that the lungs in SARS-CoV-2 infection provide anti-fibrinolytic activity resulting in a shift toward a local and systemic hypofibrinolytic state predisposing to (immuno)thrombosis, often in a background of compensated disseminated intravascular coagulation.

Effect of plasma thrombin-antithrombin complex on ischemic stroke: a systematic review and meta-analysis

Background and objectiveThrombin-antithrombin complex (TAT) is a prethrombotic marker, and its application in ischemic stroke is still uncertain. The purpose of this systematic review and meta-analysis is to evaluate the relationship between plasma TAT and ischemic stroke base on the current evidence.MethodsA systematic literature search was conducted for searching the relative studies that investigated the association of TAT and ischemic stroke in PubMed, EMBASE, and Cochrane library databases. Mean difference and 95% confidence interval as the effect sizes were synthesized by random effects model in Review Manager (RevMan) Version 5.4. The heterogeneity was investigated using the chi-square test and the possible sources of heterogeneity were explored by sensitivity analysis and meta-regression. The publication bias was estimated by Egger’s tests.ResultsA total of 12 eligible studies were included involving 1431 stroke cases and 532 healthy controls, of which six studies were eventually included in the meta-analysis. Plasma TAT in patients with ischemic stroke was significantly higher than that in healthy controls (MD 5.31, 95% CI = 4.12–6.51, P < 0.0001, I2 = 97.8%). There is a difference of TAT level in the same period among cardioembolic, lacunar, and atherothrombotic stroke (all P < 0.0001), in which the cardioembolic stroke with the highest level. Meanwhile, it is significant of TAT levels among various phases of cardioembolic stroke and the acute phase are markedly elevated (MD 7.75, 95CI%, 6.07–9.43, P < 0.001). However, no difference was found in the atherothrombotic (P = 0.13) and lacunar stroke (P = 0.34). Besides, the higher TAT level is closely related to the poor prognosis of patients with ischemic stroke, including higher recurrence, mortality, unfavorable recovery (modified Rankin scale > 2), and poor revascularization.ConclusionsThis study suggested that plasma TAT levels are different in ischemic stroke subtypes, which are closely associated with the progression and might have an effect on the prognosis.Systematic review registrationPROSPERO CRD: 42021248787

Abstract WP178: Markers Of Coagulation And Hemostatic Activation Predict Occult Malignancy In Cryptogenic Stroke Patients

Introduction: One potential cause of embolic strokes of undetermined source (ESUS) is occult malignancy. The objective of the study was to evaluate the frequency of and potential predictors of occult malignancy among ESUS patients. Methods: Consecutive ESUS patients seen at a single institution from May 1, 2017 to March 1, 2021 were included in this analysis. Patients with occult malignancy were defined as those who had newly diagnosed malignancy within one year of index stroke. We evaluated demographics, blood biomarkers and imaging to identify predictors of occult malignancy diagnosis after ESUS. Results: Of 365 patients [Median age: 66 (IQR 53-75), female 60%), non-White 52%], 35 (9.6%) had a history of malignancy before the index stroke and 8 (2.5%) were diagnosed with occult malignancy within 12 months of index stroke. Occult malignancies included colorectal cancer (4), blood cancer (3), and bladder cancer. Stage 4 malignancy was found in 1 (13%) patient diagnosed with occult malignancy. Elevation in ≥2 markers of coagulation and hemostatic activation (MOCHA) (d-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex, fibrin monomer) was significantly more frequent in occult malignancy versus other patients (88% vs 42%, Fisher exact p=0.023, OR=9.6 95%CI 1.2-79, p=0.035). MOCHA elevation in ≥2 markers for the detection of occult cancer had sensitivity 87.5%, specificity 57.9%, PPV 4.7%, NPV 99.5%. Conclusions: Hematologic markers such as MOCHA may be potentially useful to identify patients more likely to have underlying malignancy after ESUS. MOCHA abnormalities in &lt;2 markers effectively rules out occult malignancy as the cause of ESUS.

C1 inhibitor deficiency enhances contact pathway–mediated activation of coagulation and venous thrombosis

AbstractC1 inhibitor (C1INH) is a multifunctional serine protease inhibitor that functions as a major negative regulator of several biological pathways, including the contact pathway of blood coagulation. In humans, congenital C1INH deficiency results in a rare episodic bradykinin-mediated swelling disorder called hereditary angioedema (HAE). Patients with C1INH deficiency–associated HAE (C1INH-HAE) have increased circulating markers of activation of coagulation. Furthermore, we recently reported that patients with C1INH-HAE had a moderate but significant increased risk of venous thromboembolism. To further investigate the impact of C1INH deficiency on activation of coagulation and thrombosis, we conducted studies using patient samples and mouse models. Plasmas from patients with C1INH-HAE had significantly increased contact pathway–mediated thrombin generation. C1INH-deficient mice, which have been used as a model of C1INH-HAE, had significantly increased baseline circulating levels of prothrombin fragment 1+2 and thrombin-antithrombin complexes. In addition, whole blood from C1INH-deficient mice supported significantly increased contact pathway–mediated thrombin generation. Importantly, C1INH-deficient mice exhibited significantly enhanced venous, but not arterial, thrombus formation. Furthermore, purified human C1INH normalized contact pathway–mediated thrombin generation and venous thrombosis in C1INH-deficient mice. These findings highlight a key role for endogenous C1INH as a negative regulator of contact pathway–mediated coagulation in humans and mice. Further, this work identifies endogenous C1INH as an important negative regulator of venous thrombus formation in mice, complementing the phenotype associated with C1INH-HAE.

Clinical study on the feasibility of new thrombus markers in predicting massive cerebral infarction.

This study investigated the diagnostic performance of the thrombin-antithrombin complex (TAT), plasmin-α2 plasmin inhibitor complex (PIC), tissue plasminogen activator-plasminogen activator inhibitor complex (t-PAIC), and thrombomodulin (TM) in the early identification of massive cerebral infarction. A total of 423 patients with cerebral infarction confirmed by imaging examination were divided into the massive cerebral infarction (MCI) group and the non-massive cerebral infarction (NMCI) group. TAT, PIC, t-PAIC, and TM were measured immediately after admission. The diagnostic performance was analyzed by the receiver characteristic operating curve (ROC). The median plasma concentrations of TAT, PIC, and t-PAIC in patients with MCI at early onset were 5.10 ng/ml, 1.11 μg/ml, and 8.80 ng/ml, respectively, which were higher than those in patients with NMCI (2.20 ng/ml, 0.59 μg/ml, and 7.35 ng/ml), and the difference was statistically significant (P < 0.001). TAT was shown to be an independent risk factor for the development of massive cerebral infarction by a multivariate logistic regression analysis (OR = 1.138). A ROC curve analysis showed that PIC had the best performance in identifying MCI at an early stage (AUC = 82.8%), with a sensitivity of 80.7% and a specificity of 76.2% when the PIC concentration was ≥0.8 μg/ml; TAT had the highest specificity in identifying MCI, with a specificity of 80.6% when the TAT concentration was ≥3.97 ng/ml. The detection of PIC, TAT, t-PAIC, and TM is a comprehensive assessment of vascular endothelial damage and activation of the coagulation and fibrinolytic systems and has diagnostic value for early identification of patients with MCI, which, together with its ease of detection, can be used as a plasma marker for early identification of large vessel occlusion.

Assessment of Fibrinolytic Activity in Gastric Cancer

Objectives: To explore the relationship between thrombin-antithrombin complex (TAT), plasmin—α plasmin inhibitor complex (PIC), thrombomodulin (TM), tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAIC) and Gastric cancer. Methods: The plasma levels of TAT, TM, t-PAIC and PIC in patients with gastric cancer (40 in the initial diagnosis group and 35 in the post-treatment group) and 40 healthy controls were measured by chemiluminescent enzyme immunoassay. Then analyze the differences between these indicators in different stages of gastric cancer patients and healthy controls. Results: 1) The plasma levels of TAT, TM, t-PAIC and PIC in the newly diagnosed gastric cancer group were higher than those in the control group and the post-treatment group (P 0.05). 2) The plasma levels of TAT, TM, t-PAIC and PIC were not significantly different among the effective treatment group, the ineffective treatment group and the healthy control group. Conclusion: The changes of TAT, TM, t-PAIC and PIC levels are closely related to the development of patients with gastric cancer, and their increase may indicate that patients have a high risk of thrombosis.

Prevalence of Asymptomatic COVID-19 Infection in Hemodialysis Patients and the Risk of Hypercoagulability: Should we Consider Routine Screening?

Coronavirus disease 2019 (COVID-19) has become a pandemic in late 2019. Its clinical presentation varies from asymptomatic infection to severe respiratory failure. Infection control strategies to minimize the risk of transmission of COVID-19 in end-stage renal disease (ESRD) patients receiving in-center hemodialysis (HD) have been implemented. Development of humoral response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in adult patients with ESRD receiving HD has not been sufficiently reported. A total of 179 asymptomatic HD patients undergoing regular HD were screened for COVID-19 infection. Infection with SARS-CoV-2 was confirmed through a real-time reverse transcription polymerase chain reaction assay of nasopharyngeal swab specimens. They were classified into positive and negative groups according to the results of PCR. Of the 179 asymptomatic patients, we found that 23 patients (12.8%) were positive for COVID-19. Their mean age was 45.61 ± 13.38 years. There was a significant difference between both groups regarding C-reactive protein, lymphocytes, and platelet counts (P < 0.001). Also, TAT (thrombin-antithrombin complex) and D-dimer levels were significantly increased among the positive group (11.47 ± 1.51 vs. 7.53 ± 1.64 mcq/L, P < 0.001; 1171.52 ± 267.6 vs. 542.76 ± 107.06 ng/mL, P < 0.001, respectively). Asymptomatic SARS-CoV-2 infection is detected in HD patients. They carry the risk of hypercoagulability complications. We need more strict infection control measures and proactive diagnosis to limit the spread of the infection and lethal thromboembolic complications.

Фактор фон Виллебранда и ADAMTS13 как предикторы тромбоза у онкогинекологических пациенток на фоне химиотерапии

The incidence of venous thrombosis in patients undergoing chemotherapy is high. Activation of vascular endothelium during chemotherapy is followed by elevated concentrations of von Willebrand factor (vWF), decreased concentrations of ADAMTS13, and increased thrombotic risk. Objective. To study the significance of vWF:Ag and ADAMTS13:Ag concentrations and the ratio of vWF:Ag to ADAMTS13:Ag (VWF:Ag/ADAMTS13:Ag) as risk factors for thrombosis in gynecologic cancer patients undergoing chemotherapy. Patients and methods. The prospective controlled interventional non-randomized trial included 222 patients aged 34 to 72 years who underwent elective surgical treatment (138 patients) and chemotherapy courses (84 patients), and 50 healthy women in the control group. Concentrations of vWF:Ag and ADAMTS13:Ag, as well as markers of hemostatic activation such as thrombin-antithrombin complex (TAT) and D-dimer were determined in the patients’ plasma before therapy (baseline levels), 14 days after surgical treatment, and 14 days after 2 courses of chemotherapy. Results. Concentrations of vWF:Ag were significantly higher and concentrations of ADAMTS13:Ag were significantly lower among cancer patients both before and after surgical treatment and after chemotherapy compared to healthy controls. There was a significant difference in the dynamics of indicators in patients after chemotherapy compared to patients after surgical treatment. Significant changes in the vWF:Ag/ADAMTS13:Ag ratio between the groups of patients after surgical treatment and on the background of chemotherapy were also confirmed. A correlation was found between an increase in vWF:Ag concentrations, vWF:Ag/ADAMTS13:Ag ratio and TAT concentrations, and thus the risk of thrombosis. The study was conducted against the background of using standard thromboprophylaxis regimens in all patients, considering the high risk of thrombosis in all patients included in the study, according to standard thrombosis risk assessment scales. Conclusion. Concentrations of vWF:Ag, ADAMTS13:Ag and the vWF:Ag/ADAMTS13:Ag ratio can be considered as predictors of thrombosis during chemotherapy in gynecologic cancer patients. Key words: von Willebrand factor, ADAMTS13, cancer, chemotherapy

Взаимодействие внеклеточных ловушек нейтрофилов и антифосфолипидных антител у онкологических больных

Activation of NETosis processes has been detected in numerous autoimmune and non-autoimmune diseases with increasing concentrations of antiphospholipid antibodies (APA). Neutrophil extracellular traps (NETs) are an important activator of the coagulation cascade and an integral component of arterial and venous clots in cancer patients. Therefore, APA and NETs may be the links in the pathogenesis of thrombosis in cancer patients. Objective. To evaluate the severity of NETosis, hemostatic system activation and APA circulation (anti-β2-glycoprotein 1 (anti-β2-GPI)) in cancer patients. Design. A prospective controlled interventional non-randomized study. Patients and methods. This study included 144 patients who were divided into two groups: the study group – 111 patients aged 34–72 years with malignant breast and gynecological diseases of stages I–III (endometrial cancer, n = 25; ovarian cancer, n = 36; cervical cancer, n = 17; breast cancer, n = 33), and the control group – 33 healthy women. Prior to therapy, serum concentrations of NETosis markers – citrullinated histone H3 (citH3) and human myeloperoxidase antigen (MPO:Ag), markers of hemostatic activation (D-dimer and thrombin-antithrombin complex), and anti-β2-GPI antibodies were assessed. Results. Anti-β2-GPI antibodies were significantly more common in patients with ovarian cancer compared to controls. A significant correlation between the circulation of anti-β2-GPI and the concentration of MPO:Ag was also detected in these patients. All patients in the study group showed a significant increase in the markers of NETosis and hemostatic activation compared to controls. The revealed correlation between the concentrations of NETosis markers and anti-β2-GPI antibodies in ovarian cancer suggests a possible pathogenetic role of APA in the progression of ovarian cancer and NETs-mediated prothrombotic state against the background of APA circulation. It can be assumed that APA formed in cancer patients may be one of the triggers of NETosis. NETosis itself contributes to the development of thrombotic events and stimulates further synthesis of APA. Perhaps it is the activation of NETosis that explains the worse prognosis in cancer patients with APA circulation. Conclusion. The results of this study improve the understanding of interactions between the immune and hemostatic systems. Further research is needed to determine the role of APA in NETosis, as well as the role of NETosis and APA circulation in tumor progression, metastasis, and cancer-associated thrombosis. Key words: neutrophil extracellular traps, NETosis, antiphospholipid antibodies, cancer, neutrophils, anti-β2-GPI

Association of High Mobility Group Box-Protein 1 and Platelet Microparticles in Patients After Hematopoietic Stem Cell Transplantation.

Thrombotic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly impact transplant outcomes. We focused on high mobility group box-protein (HMGB)1, one causative agent of thrombotic lesions in allo-HSCT, and investigated its association with platelets. We statistically analyzed available data from 172 patients with hematopoietic malignancies receiving allo-HSCT. A significant enhancement of monocyte-chemotactant protein-1, HMGB1, and platelet-derived microparticle (PDMP) levels was observed at day 0 after transplantation as compared to pre-transplantation. Multivariate analysis of the association among HMGB1 and 16 factors on day 0 revealed a significant correlation of HMGB1 levels with thrombin-antithrombin complex, interleukin-6, and PDMPs. High mobility group box-protein 1-induced procoagulant platelet induction and PDMP generation were performed in vitro using healthy platelets. High mobility group box-protein 1-induced PDMP generation was suppressed by toll-like receptor inhibitors and recombinant thrombomodulin. These results suggest that HMGB1 contributes to platelet activation in patients after allo-HSCT and is associated with PDMP-related thrombotic complications.

Inflammation as an exacerbation marker and target for prophylaxis against Coronavirus Disease 2019-related thrombosis

Objectives: There are currently no appropriate markers and target for prophylaxis against COVID-19-related thrombosis, especially in the not-severe cases. We tested the hypothesis that inflammation is a suitable marker and target for prophylaxis against COVID-19-related thrombosis. Methods: Data of all 32 COVID-19 patients admitted to Saitama Medical Center between January 1 and March 30, 2021, were analyzed. Patients were divided into severe (requiring oxygen, n=12) and non-severe (no requirement for oxygen, n=20), and also those with high C-reactive protein (CRP) level (cutoff value: 30 mg/L, n=21) and low-CRP (n=11). We also compared the clinical and laboratory data of a 46-year-old post-liver transplant male patient, who was treated with a combination of immunosuppressants (methylprednisolone, fludrocortisone, cyclosporine, and everolimus) with those of other COVID-19 patients, using the Smirnoff-Grubbs and Box plots tests. Results: The levels of CRP, ferritin, lactate dehydrogenase, aspartate aminotransferase, and thrombin-antithrombin complex (TAT) were significantly higher in the high-severity group than the low-severity group; while other coagulation parameters were comparable. The time between onset of illness and blood levels of lactate dehydrogenase, fibrinogen, D-dimer, TAT, and plasmin alpha2-plasmin inhibitor complex (PIC) were significantly higher whereas lymphocyte count was significantly lower in the high-CRP group. Extremely low levels of TAT, PIC, and plasminogen activator inhibitor-1 (PAI-1) were recorded in the liver transplant patient treated with immunosuppressants. The TAT, PIC, and PAI-1 levels were deemed outliers. Conclusions: Inflammation is a potentially suitable marker and target for prophylaxis against COVID-19-related thrombosis.