Thrombin-antithrombin Complex Concentrations Research Articles

Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection

Inflammasome activation is a critical defense mechanism against bacterial infection. Previous studies suggest that inflammasome activation protects against Salmonella oral infection. Here we find inflammasome activation plays a critical role in the pathogenesis of Salmonella systemic infection. We show that in a systemic infection model by i.p. injection of Salmonella, deficiency of caspase-1 or gasdermin-D prolonged survival time, reduced plasma concentrations of the proinflammatory cytokines IL-1β, IL-6 and TNFα. These deficiencies also protected against coagulopathy during Salmonella infection as evidenced by diminished prolongation of prothrombin time and increase in plasma thrombin-antithrombin complex concentrations in the caspase-1 or gasdermin-D deficient mice. Activation of the NAIP/NLRC4 inflammasome by flagellin and/or the components of the SPI1 type 3 secretion system played a critical role in Salmonella-induced coagulopathy. In the absence of flagellin and SPI1, the Salmonella mutant strain still triggered coagulopathy through the caspase-11/NLRP3 pathway. Our results reveal a previously undisclosed role of the inflammasomes and pyroptosis in the pathogenesis of Salmonella systemic infection.

Andrographolide Suppresses Expressions of Coagulation and Fibrinolytic Inhibition-Related Factors in LPS-Induced Alveolar Epithelial Cell Type II via NF-κB Signal Pathway In Vitro

BackgroundAndrographolide (Andro) has been confirmed to ameliorate alveolar hypercoagulation and fibrinolysis inhibition via NF-κB pathway in acute respiratory distress syndrome (ARDS), but the specific target of Andro is unknown.PurposeOur aim is to explore the specific target of Andro through which the drug exerted its effects on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS.MethodsAECII was treated with different doses of Andro for 1 h, and then stimulated with LPS for 24 h. Expressions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and tissue factor pathway inhibitor (TFPI) were detected. Concentrations of thrombin-antithrombin complex (TAT), pro-collagen type III peptide (PIIIP), antithrombin III (ATIII) and activated protein C (APC) in cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). NF-κB signaling pathways activation was simultaneously determined. AECII with p65 down-/over-expression were used as control.ResultsAndro effectively inhibited TF and PAI-1 and promoted TFPI expressions on AECII induced by LPS stimulation. Andro also significantly suppressed the productions of TAT and PIIIP but promoted ATIII and APC secretions from the LPS-treated cell. Furthermore, Andro application obviously inhibited NF-κB signaling pathway activation provoked by LPS, as shown by decreased level of phosphorylation (p‑)-IKKβ/IKKβ, p-p65/p65 and p65 DNA binding activity. The effects of Andro on those factors were obviously strengthened by down- but were weakened by up-regulation of p65 gene in AECII cell.ConclusionsOur data demonstrates that targeting AECII is the mechanism by which Andro ameliorates alveolar hypercoagulaiton and fibrinolytic inhibition via NF-κB pathway in ARDS. Andro is worth to be clinically further studied in ARDS treatment.

Thrombin antithrombin complex concentration as an early predictor of deep vein thrombosis after total hip arthroplasty and total knee arthroplasty

AimEarly predictive markers of venous thromboembolism (VTE) after total hip arthroplasty (THA)/total knee arthroplasty (TKA) remain unclear. Our study identified early predictive markers for VTE after THA/TKA.MethodsA single-institution retrospective review study was conducted between May 2020 and April 2022 (n = 256). All patients underwent Doppler ultrasounds exam in preoperation and seventh day after surgery. Deep vein thrombosis (DVT) was defined by Doppler ultrasound of the lower extremities, which revealed thrombosis. Thrombin-antithrombin complex (TAT), thrombomodulin (TM), and plasmin-antiplasmin complex (PIC) concentration were tested from each patient’s preoperative and postoperative days 1, 4, 7, 14. These values were then accessed via receiver operating characteristic (ROC) curve analysis and further quantified the level of this risk by concentration.ResultsOn postoperative day 1 (pod-1), all patients’ TAT and PIC concentrations were significantly higher than those preoperatively (p < 0.05). The levels of TAT and PIC in patients in the DVT group on pod-1 were significantly higher than those in the non-DVT group (p < 0.05). At pod-1, the TAT concentration for DVT patients was 49.47 ng/mL compared to 20.70 ng/mL for non-DVT patients, PIC was 3.72μg/mL compared to 1.65μg/mL. ROC curve analysis demonstrated that a TAT concentration of 24.3 ng/mL had a sensitivity of 87.9% and a specificity of 69.1%.ConclusionTAT levels on pod-1 may predict DVT early after THA/TKA, which makes it possible for early intervention to decrease the incidence of DVT.

Extracellular vesicles and coagulation in blood from healthy humans revisited

ABSTRACTBackground: In 2001, we studied the presence and coagulant properties of “microparticles” in the blood of healthy humans. Since then, multiple improvements in detection, isolation and functional characterization of the now called “extracellular vesicles” (EVs) have been made, and shortcomings were identified. Aim: To revisit the presence and function of EVs in blood from healthy humans. Methods: Blood was collected from 20 healthy donors. EV-containing plasma was prepared according to new guidelines, and plasma was diluted to prevent swarm detection. Single EVs were measured by flow cytometry with known sensitivity of fluorescence and light scatter. The haemostatic properties of EVs were measured by thrombin-, fibrin-, and plasmin generation. Plasma concentrations of thrombin-antithrombin complexes and prothrombin fragment 1 + 2 were measured to assess the coagulation status in vivo. Results: Compared to 2001, the total concentrations of detected EVs increased from 190- to 264-fold. In contrast to 2001, however, EVs are non-coagulant which we show can be attributed to improvements in blood collection and plasma preparation. No relation is present between the plasma concentrations of EVs and either TAT or F1 + 2. Finally, we show that EVs support plasmin generation. Discussion: Improvements in blood collection, plasma preparation and detection of EVs reveal that results from earlier studies have to be interpreted with care. Compared to 2001, higher concentrations of EVs are detected in blood of healthy humans which promote fibrinolysis rather than coagulation.

Role of tissue factor in the procoagulant and antibacterial effects of human adipose-derived mesenchymal stem cells during pneumosepsis in mice

BackgroundAdult mesenchymal stem cells (MSCs) improve the host response during experimental sepsis in animals. MSCs from various sources express a procoagulant activity that has been linked to the expression of tissue factor. This study sought to determine the role of tissue factor associated with adipose-derived MSCs (ASCs) in their procoagulant and antibacterial effects during pneumonia-derived sepsis.MethodsMice were infused intravenously with ASCs or vehicle after infection with the common human pathogen Klebsiella pneumoniae via the airways.ResultsInfusion of freshly cultured or cryopreserved ASCs induced the expression of many genes associated with tissue factor signaling and coagulation activation in the lungs. Freshly cultured and cryopreserved ASCs, as well as ASC lysates, exerted procoagulant activity in vitro as determined by a fibrin generation assay, which was almost completely inhibited by an anti-tissue factor antibody. Infusion of cryopreserved ASCs was associated with a rise in plasma thrombin-antithrombin complexes (indicative of coagulation activation) and formation of multiple thrombi in the lungs 4 h post-infusion. Preincubation of ASCs with anti-tissue factor antibody prior to infusion prevented the rise in plasma thrombin-antithrombin complex concentrations but did not influence thrombus formation in the lungs. ASCs reduced bacterial loads in the lungs and liver at 48 h after infection, which was not influenced by preincubation with anti-tissue factor antibody. At this late time point, microthrombi in the lungs were not detected anymore.ConclusionThese data indicate that ASC-associated tissue factor is responsible for systemic activation of coagulation after infusion of ASCs but not for the formation of microthrombi in the lungs or antibacterial effects.

Blood Hemostatic Changes During an Ultraendurance Road Cycling Event in a Hot Environment

This study aims to examine blood hemostatic responses to completing a 164-km road cycling event in a hot environment. Thirty-seven subjects (28 men and 9 women; 51.8±9.5 [mean±SD] y) completed the ride in 6.6±1.1 hours. Anthropometrics (height, body mass [taken also during morning of the ride], percent body fat [%]) were collected the day before the ride. Blood samples were collected on the morning of the ride (PRE) and immediately after (IP) the subject completed the ride. Concentrations of platelet, platelet activation, coagulation, and fibrinolytic markers (platelet factor 4, β-thromboglobulin, von Willebrand factor antigen, thrombin-antithrombin complex, thrombomodulin, and D-Dimer) were measured. Associations between changes from PRE- to IP-ride were examined as a function of event completion time and subject characteristics (demographics and anthropometrics). All blood hemostatic markers increased significantly (P < .001) from PRE to IP. After controlling for PRE values, finishing time was negatively correlated with platelet factor 4 (r = 0.40; P = .017), while percent body fat (%BF) was negatively correlated with thrombin-antithrombin complex (r = -0.35; P = .038) and to thrombomodulin (r = -0.36; P = .036). In addition, male subjects had greater concentrations of thrombin-antithrombin complex (d = 0.63; P < .05) and natural logarithm thrombomodulin (d = 6.42; P < .05) than female subjects. Completing the 164-km road cycling event in hot conditions resulted in increased concentrations of platelet, platelet activation, coagulation, and fibrinolytic markers in both men and women. Although platelet activation and coagulation occurred, the fibrinolytic system markers also increased, which appears to balance blood hemostasis and may prevent clot formation during exercise in a hot environment.

Hemostyptic property of chitosan: Opportunities and pitfalls.

Chitosan is used in a wide field of applications and therapies and has been reported to be an effective hemostyptic. The objective of this study was to provide further information about the use of chitosan as a hemostyptic agent also taking into focus its hemocompatible effects. Human whole blood (n=5) was anticoagulated with heparin, treated with different chitosan concentrations (0, 2.5, 5, 7.5, 10, 12.5, 25 mg/mL) and incubated at 37°C for 30 minutes. Before and after incubation different parameters for coagulation and hemocompatibility were evaluated. Blood treated with high chitosan concentrations showed enhanced coagulation, which we evaluated with activated clotting time, activated partial thromboplastin time and concentration of thrombin-antithrombin complexes. Furthermore, we observed an activation of blood platelets, complement cascade and granulocytes in the groups treated with chitosan. Our data indicate that chitosan activates human blood coagulation and hence has good properties as a hemostyptic agent. However, inflammatory parameters were upregulated after direct contact with human blood indicating that systemic administration of chitosans should not be performed whereas the topical use of chitosan as a hemostypticum should not present any hazard with regard to adverse inflammatory reactions at the site of application.

Early Changes in the Antithrombin and Thrombin-Antithrombin Complex in Patients With Paroxysmal Atrial Fibrillation.

BackgroundData on coagulation changes in paroxysmal atrial fibrillation (PAF) are scarce. The aim of this study was to examine plasma antithrombin (AT) levels and activity as well as thrombin-antithrombin (TAT) complex levels in the early hours of the clinical manifestation of PAF.MethodsFifty-one patients (26 men and 25 women; mean age 59.84 ± 1.60 years) were consecutively selected with PAF duration < 24 hours, and 52 controls (26 men and 26 women; mean age 59.50 ± 1.46 years) matched the patients in terms of gender, age and comorbidities. Plasma levels and activity of AT and levels of the covalent TAT complex were studied once in each study participant.ResultsAT plasma levels in PAF patients were statistically significantly lower compared to controls (164.69 ± 10.51 vs. 276.21 ± 8.29 μg/mL, P < 0.001). Plasma activity of the anticoagulant was also significantly lower in PAF (71.33±4.87 vs. 110.72±3.09%, P < 0.001). TAT complex concentration in plasma was higher in the patient group (5.32 ± 0.23 vs. 3.20 ± 0.14 μg/L, P < 0.001).ConclusionWe can say that PAF is associated with significantly reduced AT levels and activity and increased levels of TAT complex during the first 24 hours after its manifestation. These changes indicate a reduced activity of AT anticoagulant system, which is a probable prerequisite for the established enhanced coagulation (high TAT complex levels).

SEPCR Levels in Chronic Myeloproliferative Diseases and Their Association with Thromboembolic Events: A Case-Control Study.

Objective: Venous, arterial, and microcirculatory events are frequently encountered in the clinical course of essential thrombocytosis and polycythemia vera. We aimed to investigate the levels of soluble endothelial protein C receptor (sEPCR) in myeloproliferative diseases to see whether there was a difference between the patients with and without history of thromboembolism. Materials and Methods: The study included patients with polycythemia vera (n=12), patients with essential thrombocytosis (n=13), and controls (n=29). In all groups, we measured proteins C and S, antithrombin and sEPCR levels, and plasma concentrations of thrombin-antithrombin complex, prothrombin fragments 1+2, and D-dimer. Results: Comparing the patients with and without history of thromboembolic attack, statistically significant differences were not observed in terms of sEPCR, D-dimer, thrombin-antithrombin complex, prothrombin fragments 1+2, and hematocrit levels (p=0.318, 0.722, 0.743, 0.324, and 0.065, respectively). Conclusion: Significant increase in the parameters that reflect activation of coagulation, such as sEPCR, thrombin-antithrombin complex, prothrombin fragments 1+2, and D-dimer, reflects the presence of a basal condition that leads to a tendency toward thrombosis development in ET and PV when compared to healthy controls.

Thrombin-Activatable Fibrinolysis Inhibitor Is Activated in an Instant Blood-Mediated Inflammatory Reaction After Intraportal Islet Transplant

Activated thrombin-activatable fibrinolysis inhibitor is a coagulation factor in some thrombotic diseases. However, available data on whether thrombin-activatable fibrinolysis inhibitor is activated in islet transplant are limited. In this study, changes of plasma-activated thrombin-activatable fibrinolysis inhibitor levels in instant blood-mediated inflammatory reaction after islet transplant were assessed. Plasma concentrations of thrombin-antithrombin complex, D-dimer, C-peptide, and activated thrombin-activatable fibrinolysis inhibitor were assessed at 0 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours after an intraportal islet transplant using rats via an enzyme-linked immunosorbent assay, or solid-phase, 2-site chemiluminescent immunometric assay. We recovered the liver at 1 hour after the transplant for histologic examination. Thrombin-antithrombin complex, C-peptide, and activated thrombin-activatable fibrinolysis inhibitor levels increased immediately after we stopped islet infusion, and their peak levels occurred at 1 hour after islet infusion. D-dimer levels increased continually after islet infusion was stopped, and peaked 24 hours after infusion. Histologic examination of the liver 1 hour after islet infusion revealed frequent portal venous thrombi, with entrapped islets. The entrapped islets showed a disrupted morphology. Activated thrombin-activatable fibrinolysis inhibitor was generated and peaked 1 hour after islet transplant according with activating coagulation, indicating that thrombin-activatable fibrinolysis inhibitor is activated and accumulated at levels in instant blood-mediated inflammatory reaction was sufficient to affect fibrinolysis.

Expression of the PAR-1 protein on the surface of platelets in patients with chronic peripheral arterial insufficiency - preliminary report.

The activation of pro-coagulation mechanisms associated with the vascular wall's immune and inflammatory responses wall to injury plays a crucial role in the mechanisms of the induction and progression of atherosclerosis. The aim of this study was to determine the role of protease activated receptors (PAR-1) expressed on the surface of blood platelets in the pathogenesis of chronic peripheral arterial obliterative disease (PAOD) in patients with obliterative atherosclerosis (n = 24) and diabetic macroangiopathy (n = 16), as well as in the controls (n = 12). In addition to the expression of PAR-1, serum/plasma concentrations of thrombin-antithrombin complex (TAT), the von Willebrand factor (vWF), the platelet-derived growth factor, monocyte chemotactic protein, the soluble form of the platelet endothelial cell adhesion molecule, thrombin activatable fibrinolysis inhibitor and interleukin 6 (IL-6) were determined. Compared to the controls, PAOD patients were characterized by significantly higher levels of PAR-1 expression, vWF, TAT and IL-6. Individuals with diabetic macroangiopathy did not differ significantly from individuals with obliterative atherosclerosis in terms of PAR-1 expression. Upon activation with thrombin receptor antagonist peptide (TRAP), the levels of PAR-1 were comparable in all analyzed groups. In patients with diabetic macroangiopathy, a significant association was observed between the expression of PAR-1 on the surface of the platelet and the serum TAT concentration, as well as between TAT and serum IL-6 concentration. Enhanced expression of PAR-1 on the thrombocyte surface in chronic PAOD patients occurs equally in cases of diabetic macroangiopathy and in individuals free from this endocrine pathology.

Plasminogen Activator Inhibitor-1 Deficiency Augments Visceral Mesothelial Organization, Intrapleural Coagulation, and Lung Restriction in Mice with Carbon Black/Bleomycin–Induced Pleural Injury

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1(-/-) mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1(-/-) mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1(-/-) mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial-mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1(-/-) mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.

Serial haemostatic monitoring of dogs with multicentric lymphoma

Lymphoma is the most common haematopoietic malignancy in dogs and it has been associated with hypercoagulability and subsequent thromboembolism. The objectives of this study were to serially characterize the haemostatic status of dogs with multicentric lymphoma. Thromboelastography, thrombin-antithrombin complex concentration and routine haematology and coagulation panels were measured. Twenty-seven dogs were included in the study and 15 completed the study in remission. At presentation, 81% (22/27) of dogs with multicentric lymphoma had altered haemostatic profiles consistent with hypercoagulability. Laboratory evidence of hypercoagulability did not resolve during treatment or for up to 1 month following attainment of clinical remission. Accelerated rate of clot formation at the time of chemotherapeutic protocol completion was associated with decreased survival time. We concluded that dogs with multicentric lymphoma were frequently hypercoagulable from presentation through 4 weeks after the completion of chemotherapy. Increased angle and shortened K in dogs that have successfully completed their chemotherapeutic protocol may be associated with shorter survival times.

In vitro and in vivo evaluation of the effect of elevated factor VIII on the thrombogenic process

Factor VIII (FVIII), a procoagulant cofactor, plays a crucial role in the intrinsic coagulation cascade. A causal association between elevated FVIII levels and venous thrombosis incidence has been established; no such association has been confirmed with arterial thrombosis. The independent role of elevated FVIII levels in arteriolar thrombosis was evaluated in a mouse model to determine the thrombogenic potential of elevated levels of FVIII. The in vitro thrombogenic effect of elevated FVIII levels was examined using thrombin-antithrombin (TAT) complex generation and thromboelastography (TEG) assays. The thrombogenic potential of acute and extended elevation of circulating FVIII levels was assessed using ferric chloride induced injury of the cremaster arterioles. The rate of TAT complex formation, and the final concentration of TAT complexes, significantly increased as FVIII levels were elevated from 100% to 400% FVIII activity. TEG analysis of fibrin and clot formation showed that as FVIII levels were elevated, the time to initial fibrin formation decreased and the rate of fibrin formation increased. The acute elevation of circulating FVIII to 400% FVIII activity resulted in significantly decreased times to vessel occlusion. Prolonged elevation of FVIII activity did not significantly affect time to vessel occlusion. In conclusion, acute elevations in FVIII levels result in a non-linear thrombogenic effect, with non-significant increases in thrombogenic risk within the physiological range (FVIII levels up to 200%). Prolonged elevation of plasma FVIII did not further increase the thrombogenic potential of elevated FVIII levels.

Effects of ulinastatin on coagulation in high-risk patients undergoing off-pump coronary artery bypass graft surgery

BackgroundBoth systemic inflammatory reaction and regional myocardial ischemia/reperfusion injury may elicit hypercoagulability after off-pump coronary artery bypass grafting (OPCAB). We investigated the influence of ulinastatin, which suppresses the activity of polymorphonuclear leukocyte elastase and production of pro-inflammatory cytokines, on coagulation in patients with elevated high-sensitivity C-reactive protein (hsCRP) undergoing OPCAB.MethodsFifty patients whose preoperative hsCRP > 3.0 mg/L were randomly allocated into the ulinastatin (600,000 U) or control group. Serum concentrations of thrombin-antithrombin complex (TAT) and prothrombin fragment 1+2 (F1+2) were measured preoperatively, immediately after surgery, and at 24 h after surgery, respectively. Secondary endpoints included platelet factor (PF)-4, amount of blood loss, and transfusion requirement.ResultsAll baseline values of TAT, F1+2, and PF-4 were higher than the normal range in both groups. F1+2 was elevated in both groups at immediate, and at 24 h after surgery as compared to baseline value, without any significant intergroup differences. Remaining coagulation parameters, transfusion requirement and blood loss during operation and postoperative 24 h were not different between the two groups.ConclusionsIntraoperative administration of ulinastatin did not convey beneficial influence in terms of coagulation and blood loss in high-risk patients with elevated hsCRP undergoing multivessel OPCAB, who already exhibited hypercoagulability before surgery.

Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats

There are concerns that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. We have shown that low-dose administration of a direct thrombin inhibitor, melagatran, significantly worsens the coagulation status induced by tissue factor injection in rats. We compared the effect of inhibition of thrombin and factor Xa for their potential to aggravate tissue factor-induced coagulation in rats. Hypercoagulation was induced by the injection of 2.8 U/kg tissue factor after administration of melagatran, heparin and edoxaban in rats. Blood samples were collected 10min after tissue factor injection. Platelet numbers, thrombin–antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1mg/kg, i.v.) suppressed platelet consumption and thrombin–antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1mg/kg, i.v.) significantly enhanced platelet consumption and thrombin–antithrombin complex generation. In addition, although melagatran (3mg/kg, i.v.) improved coagulation status when tissue factor was given 5min after the drug administration, and 2, 4 and 8h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15–234ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190ng/ml or more) of the compound. In contrast, edoxaban and heparin did not show any exacerbation under these examination conditions. These results show that subtherapeutic concentrations of melagatran are associated with coagulation pathway activation, whereas factor Xa inhibition with edoxaban has a low risk of paradoxical hypercoagulation.

Arterial antithrombotic activity of rivaroxaban, an orally active factor Xa inhibitor, in a rat electrolytic carotid artery injury model of thrombosis

Rivaroxaban, an oral, direct factor Xa inhibitor, has been approved in several countries for thromboprophylaxis after elective hip or knee arthroplasty based on favorable benefit-risk profile and improved efficacy compared to enoxaparin in reducing the composite of symptomatic and asymptomatic deep vein thrombosis, nonfatal pulmonary embolism, and all-cause mortality. Given the potential therapeutic utility of factor Xa inhibition in arterial thrombosis, we evaluated the antithrombotic activity of rivaroxaban in a model of arterial thrombosis in anesthetized rats in which thrombotic occlusion was induced by electrolytic injury of the carotid artery. Rivaroxaban, 0.3, 1 or 3 mg/kg, enoxaparin, 10 mg/kg, or vehicle were infused intravenously to anesthetized rats and time to occlusion as well as coagulation parameters monitored following carotid electrolytic injury. Although the lowest dose of rivaroxaban (0.3 mg/kg) did not prolong occlusion time compared to vehicle, rivaroxaban at 1 or 3 mg/kg prevented occlusion in all vessels during the 30-min observation period (median occlusion time >30 min), which was greater than that following a single dose of enoxaparin infused at a dose of 10 mg/kg (median time to occlusion = 21.6 min). Rivaroxaban was also effective following oral dosing at 3 mg/kg. Rivaroxaban's antithrombotic activity was paralleled by dose-dependent increases in prothrombin time (PT) and activated clotting time (ACT) without significant changes in activated partial thromboplastin time. Rivaroxaban also markedly increased Russell's viper venom time (RVVT) and decreased thrombin-antithrombin complex concentrations at all doses. These findings support the potential utility of rivaroxaban in arterial thrombotic disorders such as acute coronary syndrome, stroke and peripheral arterial disease.

In vivo anticoagulant effect of ethyl pyruvate in endotoxemic rats

Introduction Disseminated intravascular coagulation (DIC), a disorder of the blood coagulation function, is a common complication of severe sepsis. Ethyl pyruvate (EP), a derivative of pyruvate, is known for its anti-inflammatory and antioxidant properties. Recently, EP showed anticoagulant effects in vitro. Therefore, the aim of study is to investigate the in vivo anticoagulant effect of EP on a severe DIC model induced by lipopolysaccharide (LPS) in anesthetized Wistar rats. Materials and Methods The animals were intravenously infused with LPS (30 mg/kg) over a period of 4 h. One hour after LPS initiation, rats were treated with EP in three dosages (20, 40 and 60 mg/kg/4 h, i.v.). Results The intermediate and high doses of EP (40 and 60 mg/kg) prevented circulatory failure, improved renal and hepatic function, and reduced the plasma levels of TNF-α and IL-6 after LPS administration. All used doses of EP significantly prevented prothrombin time prolongation, and reduced mRNA expression of tissue factor in lung tissue induced by LPS. The two higher doses of EP attenuated plasma concentrations of thrombin-antithrombin complex. The high dose of EP (60 mg/kg) significantly preserved platelet counts, and improved survival rate. However, EP did not reduce the elevation of plasma plasminogen activator inhibitor-1 during endotoxemia. Conclusion EP demonstrates the in vivo anticoagulant effect and improved organ functions in a severe DIC model in rats, which is likely associated with the inhibitory effect on tissue factor mRNA expression and cytokines release. Its effectiveness in preventing DIC is not mediated by increase in fibrinolysis.

Impact of Endogenous Protein C on Pulmonary Coagulation and Injury during Lethal H1N1 Influenza in Mice

Influenza accounts for 5-10% of community-acquired pneumonia cases, and is a major cause of mortality. Sterile and bacterial lung injury are associated with procoagulant and inflammatory derangements in the lungs and down-regulation of the protein C (PC) pathway has been correlated with disease severity and mortality in severe bacterial pneumonia and sepsis. In addition, during lethal influenza pneumonia, pulmonary and systemic coagulation are activated, which can be attenuated by the administration of recombinant activated (A) PC. We here determined the role of endogenous PC in lethal H1N1 influenza A infection. Male C57BL/6 mice pretreated with an inhibitory monoclonal antibody directed against murine PC or a control antibody were intranasally infected with a lethal dose of a mouse-adapted H1N1 influenza A strain. Mice were killed at 48 or 96 hours after infection, after which lungs and bronchoalveolar lavage fluid were harvested, or observed for up to 9 days. Anti-PC antibody treatment aggravated pulmonary activation of coagulation as compared with control antibody treatment, as reflected by increased lung concentrations of thrombin-antithrombin complexes and fibrin degradation products, as well as intravascular thrombus formation. Anti-PC antibody treatment aggravated lung histopathology, but lowered bronchoalveolar neutrophil influx and total protein levels, and delayed mortality. In conclusion, endogenous PC has strong effects on the host response to lethal influenza A infection, inhibiting pulmonary coagulopathy and inflammation on the one hand, but facilitating neutrophil influx and protein leak and accelerating mortality on the other hand.

Analysis of thrombin-antithrombin complex contents in plasma and hematoma fluid of hypertensive intracerebral hemorrhage patients after clot removal

Animal experiments indicate that the cerebral thrombin is associated with secondary brain damage after intracerebral hemorrhage (ICH). This study was aimed to investigate the concentrations of thrombin-antithrombin complex (TAT) in hematoma fluid and plasma of the patients with ICH after surgery and analyze the correlation between TAT complex levels and severity of ICH. Sixty patients with ICH were enrolled. Craniotomy for removal of intracranial blood clot was performed within 24h after ICH. Hematoma fluid and plasma were collected on postoperative days 1, 2, and 4. The plasma obtained from healthy subjects and cerebrospinal fluid from patients without cerebrovascular diseases served as controls, respectively. Enzyme-linked immunosorbent assay was used to determine the concentrations of TAT complex in the patients and controls. TAT complex concentrations in both postoperative plasma and hematoma fluid of patients with ICH were significantly higher than those of the controls (P<0.01). In patients with ICH, hematoma fluid had a higher TAT complex level than plasma (P<0.01). The preoperative hemorrhage volume and postoperative TAT complex levels in plasma and hematoma fluid correlated positively with National Institutes of Health stroke scale and negatively with Glasgow coma score (P<0.01). This study indicates that TAT complex levels of plasma and hematoma fluid correlate positively with the severity of ICH. Determination of the plasma TAT complex concentration is helpful for the evaluation of the severity of post-ICH brain injury.