A correlation of thrombin generation assay and clot waveform analysis in patients on warfarin.

OBJECTIVES: Thrombin generation assays and activated partial thromboplastin time (aPTT)-based clot waveform analysis (CWA), are some examples of global coagulation assays. Each modality evaluates different aspects of the clot forming process to globally define haemostasis with exclusive measurement parameters. Data on CWA are emerging, but its performance against other haemostatic assays is yet to be ascertained. This study evaluates the correlation between aPTT-based CWA and CAT parameters across a range of INR in warfarin-treated patients. PATIENTS/METHODS: A prospective study consisting of patients on warfarin anticoagulation with varying INR levels. CWA and CAT were performed for the study subjects. RESULTS: 54 samples were included covering an INR range from 1.33-6.89, with a mean of 4.31 +/- 1.13. For CAT parameters, endogenous thrombin potential (ETP) and peak thrombin were assessed. Both unadjusted and adjusted (adjusted for final plateau transmittance) aPTT-based CWA were evaluated for parameters min1 (maximum velocity), min2 (maximum acceleration) and max2 (maximum deceleration). Peak thrombin showed significant correlation with all CWA parameters (min1: r = 0.435, P<0.001; min2: r = 0.485, P<0.001; max2: r = 0.578, P<0.001; adjusted min1: r = 0.734, P<0.001, adjusted min2: r = 0.693, P<0.001; adjusted max2: r = 0.751, P<0.001). ETP correlated significantly with all CWA parameters except unadjusted min1 (min1: r = 0.235, P = 0.087; min2: r = 0.326, P = 0.016; max2: r = 0.437, P<0.001; adjusted min1: r = 0.610, P<0.001, adjusted min2: r = 0.563, P<0.001; adjusted max2: r = 0.642, P<0.001). CONCLUSION: We demonstrated a modest correlation between CAT and CWA parameters. Adjusted CWA improved this correlation. These findings provide additional understanding of CWA and it's role in the evaluation of global haemostatic function.

Protective role of serpina3c as a novel thrombin inhibitor against atherosclerosis in mice.

Abnormal vascular smooth muscle cell (VSMC) proliferation is a critical step in the development of atherosclerosis. Serpina3c is a serine protease inhibitor (serpin) that plays a key role in metabolic diseases. The present study aimed to investigate the role of serpina3c in atherosclerosis and regulation of VSMC proliferation and possible mechanisms. Serpina3c is down-regulated during high-fat diet (HFD)-induced atherosclerosis. An Apoe-/-/serpina3c-/--double-knockout mouse model was used to determine the role of serpina3c in atherosclerosis after HFD for 12 weeks. Compared with Apoe-/- mice, the Apoe-/-/serpina3c-/- mice developed more severe atherosclerosis, and the number of VSMCs and macrophages in aortic plaques was significantly increased. The present study revealed serpina3c as a novel thrombin inhibitor that suppressed thrombin activity. In circulating plasma, thrombin activity was high in the Apoe-/-/serpina3c-/- mice, compared with Apoe-/- mice. Immunofluorescence staining showed thrombin and serpina3c colocalization in the liver and aortic cusp. In addition, inhibition of thrombin by dabigatran in serpina3c-/- mice reduced neointima lesion formation due to partial carotid artery ligation. Moreover, an in vitro study confirmed that thrombin activity was also decreased by serpina3c protein, supernatant and cell lysate that overexpressed serpina3c. The results of experiments showed that serpina3c negatively regulated VSMC proliferation in culture. The possible mechanism may involve serpina3c inhibition of ERK1/2 and JNK signaling in thrombin/PAR-1 system-mediated VSMC proliferation. Our results highlight a protective role for serpina3c as a novel thrombin inhibitor in the development of atherosclerosis, with serpina3c conferring protection through the thrombin/PAR-1 system to negatively regulate VSMC proliferation through ERK1/2 and JNK signaling.

Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells.

BACKGROUND: Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. OBJECTIVE: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. METHODS: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. RESULTS: reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. CONCLUSION: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

A novel mechanism of ACE inhibition-associated enhanced platelet reactivity: disproof of the ARB-MI paradox?

PURPOSE: ACE inhibitors (ACEI) and angiotensin II receptor blockers (ARB) are important drugs in cardiovascular disease. However, little is known about which of these drug class is to be preferred. First analyses show that the blockade of the renin-angiotensin-aldosterone system (RAAS) influences platelet reactivity. Therefore, we evaluated the effects of ACEI and ARB on platelet reactivity and thrombin generation. METHODS: We conducted a time series analysis in 34 patients. We performed light transmission aggregometry (LTA) to evaluate platelet reactivity. Results are given as maximum of aggregation (MoA). Thrombin generation was measured as endogenous thrombin potential (ETP) via calibrated automated thrombogram. Flow cytometry was used to analyze protease-activated receptor (PAR)-1 expression. RESULTS: ACEI treatment significantly increased platelet reactivity already 4 h after initiation of treatment (prior vs. 4 h post ACEI: MoA 41.9 ± 16.2% vs. 55.2 ± 16.7%; p = 0.003). After switching from ACEI to ARB treatment, platelet reactivity decreased significantly (3 months after switching: MoA 34.7 ± 20.9%; p = 0.03). ACEI reduced endogenous thrombin potential significantly from before to 3 months after ACEI (ETP 1527 ± 437 nM × min vs. 1088 ± 631 nM × min; p = 0.025). Platelet thrombin receptor (PAR1) expression increased from 37.38 ± 10.97% before to 49.53 ± 6.04% after ACEI treatment (p = 0.036). CONCLUSION: ACEI enhanced platelet reactivity. This can be reversed by changing to ARB. The mechanism behind RAAS influencing platelet function seems to be associated with PAR-1 expression.

FVIII/VWF complex displays a greater pro-haemostatic activity than FVIII preparations devoid of VWF: Study in plasma and cell-based models.

INTRODUCTION: Plasma-derived FVIII/VWF complex was reported to be less sensitive to inhibitors than FVIII preparations devoid of VWF. AIM: To compare the efficacy of FVIII/VWF complex (Fanhdi) and five different VWF-free FVIII preparations in restoring thrombin generation and activation of thrombin-activatable fibrinolysis inhibitor (TAFI) in haemophilic plasma, with and without inhibitor, and in cell-based models. METHODS: Experiments were performed in haemophilic plasma supplemented with inhibitory IgG or in plasma samples obtained from haemophilia A patients without (n = 11) and with inhibitor (n = 12). Thrombin generation was evaluated by calibrated automated thrombography (CAT) under standard conditions, in the presence of activated protein C (APC) or thrombomodulin (TM), and in cell-based models including endothelial cells, either alone or in combination with platelets or tissue factor-expressing blood mononuclear cells. The kinetics of TAFI activation was determined by a two-stage functional assay in the absence and in the presence of APC. RESULTS: In haemophilic plasma without inhibitor, Fanhdi enhanced thrombin generation and TAFI activation as well as recombinant (2nd-4th generation) and plasma-derived FVIII preparations devoid of VWF. On the contrary, in plasma with inhibitor, Fanhdi displayed a greater ability to restore thrombin generation and TAFI activation under all tested conditions. Notably, in cell-based models including endothelial cells, Fanhdi proved more efficient than all other preparations in improving thrombin generation even in the absence of inhibitor. CONCLUSION: The greater pro-haemostatic activity of FVIII/VWF complex, either in haemophilic plasma with inhibitor or in the presence of endothelial cells, may offer therapeutic advantages.

Kaolin, used to trigger coagulation in thrombin generation test, increases sensitivity of the method in hemophilia patients.

: Thrombin generation test (TGT) is well established tool to research blood coagulation in plasma of hemophilia patients. Traditionally coagulation in this test is triggered by a tissue factor (TF), an extrinsic coagulation pathway activator. However, it is known that disorders of the intrinsic pathway are most important for coagulation in hemophilia. In this study, we hypothesized that triggering coagulation via the intrinsic pathway could increase a sensitivity of the TGT to monitor hemophilia treatment. The aim of this study was to compare thrombin generation in hemophilia A patients with inhibitors to factor VIII before and after infusion of bypassing agent [recombinant-activated factor VIIa (rVIIa)] using standard activation of coagulation by TF or by kaolin, an activator of coagulation by intrinsic pathway. Endogenous thrombin potential (ETP) in nine patients was measured. ETP before (ETP0) and 60 min after rVIIa infusion (ETP60) were compared. It was shown that ETP0 and ETP60 were significantly different when using any coagulation activator (paired Student's t test, P = 0.017 and 3.7 × 10 for clotting activation by TF and kaolin, respectively). The ratios of ETP60/ETP0 were 1.2 ±â€Š0.2 or 30.0 ±â€Š22.4 (mean ±â€ŠSD, n = 9) for coagulation activated by TF or kaolin, respectively, and were significantly different (paired Student's t test, P < 0.005). The TGT clearly distinguished between ETP0 and ETP60 in the case of any coagulation activator, but ETP increasing after rVIIa infusion was significantly higher when activated with kaolin. This provided increased sensitivity of this method for monitoring hemophilia therapy.

DNA Aptamers to Thrombin Exosite I. Structure-Function Relationships and Antithrombotic Effects.

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin receptors on platelet surface, thereby suppressing thrombin-stimulated formation of fibrin from fibrinogen and platelet aggregation. Earlier, we synthesized original antithrombin aptamer RE31 (5'-GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC-3') that contained (in addition to G-quadruplex) a hinge region connected to six pairs of complementary bases (duplex region). In this study, we compared properties of RE31 aptamer and its analogues containing varying number of bases in the duplex region and nucleotide insertions in the hinge region. Reduction in the number of nucleotides in the duplex region by 1 to 4 pairs (in comparison with RE31 aptamer) resulted in the decrease of the structural stability of aptamers (manifested as lower melting temperatures) and their ability to inhibit thrombin-stimulated fibrin formation in human blood plasma in tests of thrombin, prothrombin, and activated partial thromboplastin times. However, an increase in the number of bases by 1 to 2 pairs did not cause significant changes in the stability and antithrombin activity of the aptamers. Insertions into the hinge region of RE31 aptamer decreased its antithrombin activity. Investigation of RE31 antithrombotic properties demonstrated that RE31 (i) slowed down thrombin formation in human blood plasma (thrombin generation test), (ii) accelerated lysis of fibrin clot by tissue plasminogen activator in in vitro model, and (iii) suppressed arterial thrombosis in in vivo model. Based on the obtained data, RE31 aptamer can be considered as a potentially effective antithrombotic compound.

Concomitant assessment of rivaroxaban concentration and its impact on thrombin generation.

BACKGROUND: Reliable assays to measure direct oral anticoagulant (DOAC) levels and their activity in critical situations are needed. Drug levels alone are not representative of the effect of DOACs on an individual's coagulation. We developed a technique that provides direct assessment of the global effect of rivaroxaban on the individual's coagulation in addition to plasma concentrations. METHODS: DOAC concentrations were determined in fifty patients using rivaroxaban, with the new assay, Xross-CAT. The effect of rivaroxaban on coagulation (activity) was measured with thrombin generation (TG) in platelet poor plasma using 5 pM tissue factor on the same device. The levels were validated with the Biophen DiXal assay. The prothrombin time (PT) and dilute Russell viper venom time (dRVVT) were performed to estimate the effect on coagulation. RESULTS: The variability of Xross-CAT was below 12%. Xross-CAT correlates well with Biophen DiXaI (rs = 0.885). The bias, determined by Bland-Altman analysis, was 4.9% and the Passing-Bablok equation was y = 1.1x - 2.1. The correlation of plasma levels with TG was moderate (ETP rs = -0.548; Peak rs = -0.559), as for the PT (rs = 0.739) and the dRVVT (rs = 0.692). CONCLUSIONS: Xross-CAT shows a good correlation with Biophen DiXaI that was previously confirmed to accurately assess rivaroxaban levels. Bleeding and thrombotic complications are not necessarily associated with drug levels and could be influenced by concomitant risk factors. The main benefit of Xross-CAT is that it can be performed simultaneously with thrombin generation, providing an overview of the global anticoagulation status of a patient in relation to circulating DOAC levels.

Thrombin Generation in Chinese Pregnant Women and the Effect of Insulin Use on Thrombin Generation in Patients with GDM.

Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis. Calibrated automated thrombogram (CAT) is a test to monitor the thrombin generation (TG), a laboratory marker of thrombosis risk, and increases during normal pregnancy, but it is still unclear whether TG is related to the use of insulin in pregnant women with gestational diabetes mellitus (GDM). We performed thrombin generation by CAT on 135 normal pregnant women, including 43 in first trimester, 32 in second trimester, 60 in third trimester, respectively; 68 pregnant women with GDM were also enrolled, 19 patients with GDM using insulin to control blood glucose and 49 patients control their blood glucose through diet and exercise with noninsulin treatment. The overall CAT parameters were calculated using descriptive statistics method with mean ± standard deviation. Mean endogenous thrombin potential, peak thrombin generation, and StartTail time increased significantly with the pregnancy. There was no significant difference in TG test parameters except StartTail time(P = .003) in insulin-treated GDM group when compared to those without insulin in the GDM group. The normal ranges for CAT parameters in pregnant women were determined. Thrombin generation increased significantly in first trimester and remains stable in second and third trimester. The use of insulin in patient with GDM did not affect thrombin generation test. Our study helps to establish the reference range of thrombin generation in Chinese normal pregnant population and provide more basis to predict the risk of thrombus complicating during pregnancy.

Reversal of Factor Xa Inhibitors by Andexanet Alfa May Increase Thrombogenesis Compared to Pretreatment Values.

Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed to neutralize FXa inhibitors. We studied the reversal effect of AA for these inhibitors in various anticoagulant and thrombin generation (TG) assays. Individual aliquots of normal human plasma containing 1 µg/mL of apixaban, betrixaban, edoxaban, and rivaroxaban, were supplemented with saline or AA at a concentration of 100 µg/mL. Clotting profiles include prothrombinase-induced clotting time, activated partial thromboplastin time, and prothrombin time. Factor Xa activity was measured using an amidolytic method. Thrombin generation was measured using a calibrated automated thrombogram. Differential neutralization of all 4 anticoagulants was noted in the activated clotting time and other clotting tests. The FXa activity reversal profile varied with an observed decrease in apixaban (22%), betrixaban (56%), edoxaban (28%), and rivaroxaban (49%). Andexanet alfa also led to an increased TG in comparison to saline. The peak thrombin was higher (40%), area under the curve (AUC) increased (15%), whereas the lag time (LT) decreased (17%). Andexanet alfa added at 100 µg/mL to various FXa supplemented systems resulted in reversal of the inhibitory effects, restoring the TG profile; AUC, LT, and peak thrombin levels were comparable to those of unsupplemented samples. Andexanet alfa is capable of reversing anti-Xa activity of different oral FXa inhibitors but overshoots thrombogenesis in both the saline and FXa inhibitor supplemented systems. The degree of neutralization of Xa inhibitor is specific to each agent.

Interruption of platelets and thrombin function as a new approach against liver fibrosis induced experimentally in rats.

AIM: Liver fibrosis is a serious health problem which is a critical cause of morbidity and mortality worldwide. It is the main complication of untreated chronic inflammatory liver diseases which can progress to liver cirrhosis, hepatocellular carcinoma, and finally death. Coagulation cascade plays a mechanistic role in the pathogenesis of different chronic inflammatory disease including atherosclerosis, stroke, and tissue fibrosis. The current study was designed to investigate the effect of inhibition of coagulation cascade on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. MATERIAL AND METHODS: The study was conducted in rats. Rats were treated with CCl4 subcutaneously for 6 consecutive weeks to determine the onset of coagulation system activation in relation to development of fibrosis. To investigate the effects of coagulation system inhibition in CCl4-induced liver fibrosis, the anticoagulants drugs dabigatran and clopidogrel were administrated orally concurrently with CCl4 treatment. KEY FINDINGS: The results of our study revealed that during the first week, there were significant elevations of fibrin, tissue factor expressions, and prothrombin time (PT) coupled with neutropenia without significant changes in liver fibrosis markers such as TGF-ß, α-SMA and collagen deposition. Starting from the second week, tissue injury markers including the oxidative, inflammatory and fibrosis markers as well as histopathological changes became evident progressively. Intriguingly, dabigatran and clopidogrel significantly normalized the biochemical and pathological changes. SIGNIFICANCE: In conclusion, activation of coagulation cascade is a triggering stimulus in the initiation of CCl4-induced liver fibrosis and the anticoagulant drugs may exert promising anti-fibrotic effect.

Nonhemostatic Activities of Factor Xa: Are There Pleiotropic Effects of Anti-FXa Direct Oral Anticoagulants?

Factor Xa (FXa) is the key serine protease of the coagulation cascade as it is the point of convergence of the intrinsic and extrinsic pathways, leading to the formation of thrombin. Factor Xa is an established target of anticoagulation therapy, due to its central role in coagulation. Over the past years, several direct oral anticoagulants (DOACs) targeting FXa have been developed. Rivaroxaban, apixaban, and edoxaban are used in clinical practice for prevention and treatment of thrombotic diseases. Increasing evidence suggests that FXa exerts nonhemostatic cellular effects that are mediated mainly through protease-activated receptors-1 and -2 and are involved in pathophysiological conditions, such as atherosclerosis, inflammation, and fibrosis. Direct inhibition of FXa by DOACs could be beneficial in these conditions. This is a narrative review that focuses on the cellular effects of FXa in various cell types and conditions, as well as on the possible pleiotropic effects of FXa-targeting DOACs.

Contradictory to its effects on thrombin, C1-inhibitor reduces plasmin generation in the presence of thrombomodulin.

C1-inhibitor (C1INH) was shown to enhance thrombin generation (TG) in the presence of thrombomodulin (TM) by reducing production of activated protein C. Because C1INH is known to inhibit fibrinolytic system proteases, the objective of this study was to evaluate the effect of moderate (3 IU/ml) and high (16 IU/ml) C1INH concentrations on TG and plasmin generation (PG) in the presence of TM. These concentrations were evaluated based on expected maximum plasma levels following C1INH replacement therapy and recently suggested supraphysiologic dosing. TG and PG were investigated in platelet poor plasmas obtained from 21 healthy donors. An assay designed to monitor the continuous generation of the 7-amino-4-methylcoumarin fluorescence from substrates specific to thrombin or plasmin was used to evaluate the impact of C1INH activity. To characterize the C1INH effects on TG and PG, the thrombin and plasmin concentration peaks and production rates were calculated. TM addition to donor plasma shifted the concentration dependence of C1INH on TG parameters from reduction to enhancement. Conversely, PG parameters were significantly reduced by 16 IU/ml in both the presence and absence of TM. Moderate C1INH concentration (3 IU/ml) reduced TG and PG in the absence of TM but did not significantly affect these parameters in the presence of TM. Finally, 3 IU/ml of C1INH reduced PG more so than TG in the absence of TM. The presented results suggest a mechanism by which C1INH could potentiate thrombosis by inhibition of fibrinolysis.

Food-derived antithrombotic peptides: Preparation, identification, and interactions with thrombin.

Thromboembolism and its sequelae have been the leading causes of morbidity and mortality throughout the world. Food-derived antithrombotic peptides, as potential ingredients in health-promoting functional foods targeting thrombus, have attracted increasing attention because of their high biological activities, low toxicity, and ease of metabolism in the human body. This review presents the conventional workflow of preparation, isolation and identification of antithrombotic peptides from various kinds of food materials. More importantly, to analyze the antithrombotic effects and mechanism of antithrombotic peptides, methods for interaction of anticoagulant peptides and thrombin, the main participant in thrombosis, were analyzed from biochemistry, solution chemistry and crystal chemistry. The present study is intended to highlight the recent advances in research of food-derived antithrombotic peptide as a novel vehicle in the field of food science and nutrition. Future outlooks are highlighted with the aim to suggest a research line to be followed in further studies with the introduced research approach.

The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells1.

PURPOSE: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. METHODS: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. RESULTS: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. CONCLUSION: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

A pilot study documenting increased thrombin generation following abrupt withdrawal of heparin therapy in healthy dogs.

OBJECTIVE: To document if a transient hypercoagulable state occurs in healthy dogs following abrupt cessation of unfractionated heparin (UFH) therapy. DESIGN: Prospective experimental pilot study. SETTING: University research facility. ANIMALS: Seven adult random-source male dogs. INTERVENTION: Thromboelastography (TEG) and thrombin-antithrombin (TAT) complex formation were used to assess coagulation status in healthy dogs. Seven adult research dogs received 200-300 IU/kg subcutaneous UFH every 8 hours for 4 days. A final IV bolus of 100 IU/kg was given on day 4 and the peak measured heparin concentration 1 hour later is defined as the start of heparin withdrawal (time 0). Citrated whole blood samples were collected at baseline (prior to heparin administration) and 3, 6, 12, 30, and 48 hours after UFH withdrawal. At all time points, a kaolin-activated TEG was performed and citrated plasma for measurement of TAT concentration was collected for batch analysis. Fibrinogen concentration, PCV, total plasma proteins, and platelet count were measured at baseline and 48 hours after heparin withdrawal. MEASUREMENTS AND MAIN RESULTS: Compared to baseline, TAT was increased 12 hours after heparin withdrawal and returned to baseline by 30 hours. TEG clot formation time (K) was decreased 30 and 48 hours after heparin withdrawal. CONCLUSION: TAT results suggest that a transient increase in thrombin generation developed 12 hours after withdrawal of UFH therapy. Though clot kinetics were rapid compared to baseline beginning 30 hours after heparin withdrawal, a return to baseline was not documented. Future studies are warranted to determine the clinical relevance of these results and to evaluate the effect of UFH withdrawal in critically ill animals.

Alternative treatment options for pediatric hemophilia B patients with high-responding inhibitors: A thrombin generation-guided study.

Little is known about the challenging treatment of pediatric patients with hemophilia B and inhibitors due to disease rarity. We describe three patients diagnosed in childhood and followed up to 9 years. All three had allergic reactions to Factor IX, but two were later safely treated for bleeding episodes with activated prothrombin complex concentrates (APCC = FEIBA). The third was given only recombinant activated Factor VIIa. Based on ex vivo thrombin generation analysis, a new alternative treatment of combined bypassing agents was administered for bleeding episodes and several minor surgical procedures with no treatment-associated adverse events or thrombosis.