Antithrombin Levels Research Articles

Genetic Risk Factors of Venous Thromboembolism in the East Algerian Population.

Many genetic risk factors have been identified for causing venous thromboembolism (VTE). Most of them affect the function of natural anticoagulant pathways, particularly the protein C system, although recent studies suggest a role of components of the hematopoietic pathway in the etiology of venous thrombosis. In this case-control study, we aimed to determine the frequency of prothrombin G20210A and factor V Leiden (FVL) G1691A polymorphisms and protein C, protein S, and antithrombin III deficiencies in the East Algerian population and to investigate whether these genetic factors are associated with VTE. On the other hand, our study tends to evaluate the status of JAK2V617F and calreticulin (CALR) mutations among these cases. The participants consisted of 121 cases with VTE and 146 healthy controls. Polymorphisms of FVL G1691A and prothrombin G20210A were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism. JAK2-V617F and calreticulin mutations were analyzed by quantitative PCR and PCR followed by capillary electrophoresis sequencing, respectively. Protein C, protein S, and antithrombin levels were determined and then hereditary deficiencies were identified. Of all cases and controls, none was a carrier of the antithrombin III deficiency, prothrombin gene G20210A, and CALR mutations. Only 1 case reported having a positive JAK2 mutation (mutant allele burden was 15%). The FVL mutation (GA/AA) was found in 14 (11.6%) cases and 2 (1.4%) controls and it was significantly different between both the groups ( P = .001). Deficiencies of protein S and protein C were detected in 17 (18.8%) cases. The univariate analysis resulted in a significant impact of FVL (odds ratio [OR] = 9.4, 95% confidence interval [CI] = 2.1-42.3; P = .003) and of protein S deficiency (OR = 16.9, 95% CI =2.1-132.8, P = .007) on the VTE status. Both factors stayed significant after adjustment for sex and age. The OR of the protein C deficiency was slightly elevated (OR = 6.4, 95% CI = 0.7-55.5), but it did not reach the level of statistical significance ( P = .091), and it was therefore not considered as a risk factor. In conclusion, coagulant factor V gene G1691A mutation and protein S deficiency constitute important genetic risk factors in patients with VTE in Eastern Algeria. The somatic mutation of JAK2 V617F and CALR mutations are less frequent causes of VTE, thus routine testing for these mutations is not recommended.

Effects of Fibrinogen Concentrate on Thrombin Generation, Thromboelastometry Parameters, and Laboratory Coagulation Testing in a 24-Hour Porcine Trauma Model.

Introduction:In a 24-hour porcine model of liver injury, we showed that fibrinogen supplementation does not downregulate endogenous fibrinogen synthesis. Here we report data from the same study showing the impact of fibrinogen on coagulation variables.Materials and Methods:Coagulopathy was induced in 20 German land race pigs by hemodilution and blunt liver injury. Animals randomly received fibrinogen concentrate (100 mg/kg) or saline. Coagulation parameters were assessed and thromboelastometry (ROTEM) was performed.Results:Fibrinogen concentrate significantly reduced the prolongations of EXTEM clotting time, EXTEM clot formation time, and prothrombin time induced by hemodilution and liver injury. A decrease in clot strength was also ameliorated. Endogenous thrombin potential was significantly higher in the fibrinogen group than in the control group, 20 minutes (353 ± 24 vs 289 ± 22 nmol/L·min; P < .05) and 100 minutes (315 ± 40 vs 263 ± 38 nmol/L·min; P < .05) after the start of infusion. However, no significant between-group differences were seen in other thrombin generation parameters or in d-dimer or thrombin–antithrombin levels. Fibrinogen–platelet binding was reduced following liver injury, with no significant differences between groups. No significant between-group differences were observed in any parameter at ∼12 and ∼24 hours.Conclusion:This study suggests that, in trauma, fibrinogen supplementation may shorten some measurements of the speed of coagulation initiation and produce a short-lived increase in endogenous thrombin potential, potentially through increased clotting substrate availability. Approximately 12 and 24 hours after starting fibrinogen concentrate/saline infusion, all parameters measured in this study were comparable in the 2 study groups.

Decrease in antithrombin III and prothrombin serum levels contribute to coagulation disorders during leptospirosis.

Pathogenic bacteria of the genus Leptospira are the causative agent of leptospirosis, an emergent infectious disease that affects humans and animals worldwide. Severe forms of the disease in humans include jaundice, multiple organ failure and intense haemorrhage. Up to now, mechanisms associated with the haemorrhage foci are poorly understood. We report in this work that, despite the low levels of antithrombin III in convalescent human serum samples, virulent, culture-attenuated and saprophyte strains of Leptospira are unable to bind and/or degrade this thrombin inhibitor, suggesting an indirect mechanism of pathogenesis. Lower levels of prothrombin were found in serum samples at the onset and convalescent phase of the disease when compared to normal human sera. The concomitant decreased levels of antithrombin III and prothrombin suggest a process of stimulated coagulation, which is corroborated by the increase of prothrombin fragment F1+2 in the serum samples. Data obtained with hamsters experimentally infected with virulent Leptospira interrogans serovars Kennewicki and Canicola strongly point out that haemorrhage is correlated with decreased levels of thrombin inhibitors and prothrombin. Activated coagulation might lead to an overconsumption of coagulation factors ultimately leading to bleeding and organ failure.

The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin.

Objective:Sepsis syndrome is usually accompanied by activation of blood coagulation mechanisms. Earlier studies found deficiencies of the 3 main natural anticoagulants, antithrombin, protein C, and protein S. However, none of these inhibitors block tissue factor, the prime trigger of coagulation during sepsis that is controlled specifically by the tissue factor pathway inhibitor (TFPI). The aim of this study was to characterize the fluctuations in the levels of natural anticoagulants, particularly TFPI, in the course of sepsis and to find out their association with the anticoagulant action of the low-molecular-weight heparin enoxaparin.Materials and Methods:We studied 51 consecutive patients with sepsis. Blood samples were collected from patients at baseline (0 h) and at 4, 12, and 24 h after enoxaparin administration. The following assays were undertaken using commercial kits: activated partial thromboplastin time, prothrombin time, thrombin time, total and free TFPI, protein C and protein S, antithrombin, fibrinogen, and anti-factor Xa.Results:Before enoxaparin administration, there was significant prolongation of the prothrombin time and activated partial thromboplastin time, and this remained the case in the 3 subsequent samples. There was marked reduction in the levels of antithrombin, protein C, and total and free protein S to below control values throughout the study. In contrast, plasma levels of both total and free TFPI were markedly elevated and increased after enoxaparin therapy. Anti-factor Xa levels were within the therapeutic range throughout. There was no difference in TFPI levels between those patients who died and those who survived.Conclusion:Sepsis triggered marked release of TFPI from endothelial cells. This persisted and was increased further following the administration of enoxaparin. In contrast, there was marked consumption of the natural coagulation inhibitors antithrombin, protein C, and protein S. These results go some way towards explaining why the therapeutic use of recombinant TFPI fails to correct sepsis-associated coagulopathy.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

Identification of Regulatory Mutations in SERPINC1 Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency.

Antithrombin is a crucial anticoagulant serpin whose even moderate deficiency significantly increases the risk of thrombosis. Most cases with antithrombin deficiency carried genetic defects affecting exons or flanking regions of SERPINC1.We aimed to identify regulatory mutations inSERPINC1 through sequencing the promoter, intron 1 and 2 of this gene in 23 patients with antithrombin deficiency but without known genetic defects. Three cases with moderate antithrombin deficiency (63–78%) carried potential regulatory mutations. One located 200 bp before the initiation ATG and two in intron 1. These mutations disrupted two out of five potential vitamin D receptor elements (VDRE) identified in SERPINC1 with different software. One genetic defect, c.42-1060_-1057dupTTGA, was a new low prevalent polymorphism (MAF: 0.01) with functional consequences on plasma antithrombin levels. The relevance of the vitamin D pathway on the regulation of SERPINC1 was confirmed in a cell model. Incubation of HepG2 with paricalcitol, a vitamin D analog, increased dose-dependently the levels of SERPINC1transcripts and antithrombin released to the conditioned medium. This study shows further evidence of the transcriptional regulation of SERPINC1 by vitamin D and first describes the functional and pathological relevance of mutations affecting VDRE of this gene. Our study opens new perspectives in the search of new genetic defects involved in antithrombin deficiency and the risk of thrombosis as well as in the design of new antithrombotic treatments.

Antithrombin for the prevention of intraventricular hemorrhage in very preterm infants.

Preterm birth remains the major risk factor for the development of intraventricular hemorrhage, an injury that occurs in 25% of very low birth weight infants. Intraventricular hemorrhage is thought to be venous in origin and intrinsic thromboses in the germinal matrix are likely to play a triggering role. Antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. Very low birth weight newborn infants have low levels of antithrombin and the risk of developing intraventricular hemorrhage is increased by the presence of hypercoagulability in the first hours of life. The administration of anticoagulants such as antithrombin may offset the increased risk of developing intraventricular hemorrhage. Anticoagulants may also reduce the risk of developing parenchymal venous infarct, a condition known to complicate intraventricular hemorrhage. To assess whether the prophylactic administration of antithrombin (started within the first 24 hours after birth) reduces the incidence of germinal matrix-intraventricular hemorrhage in very preterm neonates when compared to placebo, no treatment, or heparin. We searched the Cochrane Central Register of Controlled Trials (The Cochrane Library 2015), MEDLINE (1996 to 22 November 2015), EMBASE (1980 to 22 November 2015), and CINAHL (1982 to 22 November 2015). No language restrictions were applied. We searched the abstracts of the major congresses in the field (Perinatal Society of Australia and New Zealand and Pediatric Academic Societies) from 2000 to 2015. Randomised controlled trials, quasi-randomised controlled trials and cluster trials comparing the administration of early, i.e. within the first 24 hours of life, antithrombin in very preterm infants (gestational age < 32 weeks, any birth weight). For each of the included trials, two authors independently extracted data (e.g. number of participants, birth weight, gestational age, antithrombin formulation (plasma-derived or recombinant), mode of administration, and duration of therapy, etc.) and assessed the risk of bias (e.g. adequacy of randomization, blinding, completeness of follow-up). The primary outcomes considered in this review are intraventricular hemorrhage and severe intraventricular hemorrhage. Two randomized controlled trials, for a total of 182 infants, met the inclusion criteria of this review. Both trials compared antithrombin with placebo. We found no significant differences in the rates of intraventricular hemorrhage (typical RR 1.30, CI 95% 0.87 to 1.93, typical RD 0.09, 95% CI -0.05 to 0.23; 2 studies, 175 infants; I² = 18% for RR and I² = 42% for RD) and severe intraventricular hemorrhage (typical RR 1.04, CI 95% 0.55 to 1.94; typical RD 0.01, 95% CI -0.11 to 0.12; 2 studies, 175 infants; I² = 0% for RR and I² = 0% for RD). Among secondary outcomes, we found no significant differences in terms of neonatal mortality (typical RR 2.00, CI 95% 0.62 to 6.45; typical RD 0.04, 95% CI -0.03 to 0.12; 2 studies, 182 infants; I² = 46% for RR and I² = 61% for RD) and in the other specified outcomes, such as bronchopulmonary dysplasia. The quality of the evidence supporting these findings is limited due to the imprecision of the estimates. The administration of antithrombin seems not to reduce the incidence and severity of intraventricular hemorrhage in very preterm infants. Limited evidence is available on other clinically relevant outcomes. Given the imprecision of the estimate, the results of this systematic review are consistent with either a benefit or a detrimental effect of antithrombin and do not provide a definitive answer to the review question.

Management of systemic unfractionated heparin anticoagulation during therapeutic plasma exchange.

Therapeutic plasma exchange (TPE) may remove medications from the patient's plasma. Data is limited on the effect of TPE on unfractionated heparin (UFH). A retrospective review was performed of patients receiving TPE and continuous IV infusion UFH from 1/1/2008 to 6/30/2010. TPE with plasma or 5% albumin for replacement fluid and pre and post anti-Xa levels within approximately six hours were analyzed. Three patients had 15 TPE with plasma replacement. Anti-Xa levels decreased 47% (mean, -0.25 IU/mL) for two TPE when UFH was not changed, 78% (-0.35 IU/mL) for one TPE when the UFH rate was decreased 25%; and 61% (mean -0.72 IU/mL) for two single volume TPE and 87% (-0.65 IU/mL) for one 1.5 plasma volume TPE when UFH was stopped. During nine TPE, the UFH rate was increased by 65% resulting in a mean increase in the anti-Xa level (mean 0.06 IU/mL, 30%). One patient had five single plasma volume TPE with 5% albumin. Anti-Xa levels decreased when the UFH was not changed (-0.06 IU/mL, 38%) and increased when UFH was increased by 30% (0.19 IU/mL, 61%) and 69% (mean 0.04 IU/mL, 15% in three TPE). The PTT increased with all albumin procedures, with more marked increases observed when the UFH rate was increased, while the antithrombin level decreased (mean 65%). Heparin was removed from the patient's plasma during TPE. Adjustment of the dose during TPE may be necessary to maintain therapeutic drug levels. Methods for monitoring UFH therapy may not agree. J. Clin. Apheresis 31:507-515, 2016. © 2016 Wiley Periodicals, Inc.

Contraction (retraction) of blood clots in patients with ischemic stroke

To study a possible pathogenetic role of the blood clot contraction and its disturbances in the acute stage of ischemic stroke (IS). Using a new instrumental technique to study the dynamics of clot contraction in vitro, the authors have determined quantitative parameters of clot contraction (the extent and rate of contraction, duration of the lag-period) in the blood of 85 patients with acute IS. The contractile activity of blood clots was substantially reduced compared to the blood of healthy subjects. Correlations between hemostatic and contractile parameters suggest that the reduced clot contraction in stroke is due to the lower platelet count and impaired platelet functionality, higher levels of fibrinogen and antithrombin III as well as higher hematocrit and hemoglobin contents, leukocytosis, and changes in the biochemical blood composition. The results show that the reduced ability of clots may be a novel pathogenic mechanism that aggravates the course and outcomes of IS.

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).

Effects of Preoperative Glycyrrhizin Infusion for the Prevention of Venous Thrombosis on the Tissue Expression of Antithrombin in a Rat Model.

Using a thrombus model prepared by ligation of the inferior vena cava (IVC), the influences of the glycoside, glycyrrhizin, on plasma antithrombin levels and antithrombin mRNA expression levels in the liver and IVC with the inhibition of venous thrombosis were investigated. The rat IVC was exposed and ligated for 24 h immediately after the intravenous administration of 300 mg/kg glycyrrhizin. Among antithrombotic drugs, the Xa inhibitor, fondaparinux sodium, was used as a control drug. The mean thrombus weight was significantly smaller in the glycyrrhizin-treated group (18.3 mg) than in the saline-treated group (34.3 mg). In contrast, the inhibition of thrombosis was not observed in the fondaparinux-treated group. Antithrombin mRNA expression levels in the liver were significantly higher in the ligated groups than in the baseline control group. The mean plasma antithrombin level was significantly lower in the glycyrrhizin group (96.6%) than in the saline group (114.4%), but was not significantly different from that in the baseline control group (102.4%). The pretreatment with glycyrrhizin inhibited venous thrombosis, and antithrombin mRNA expression levels in the liver and IVC as well as plasma antithrombin levels were significantly lower than those in the saline group.

Restoration of Procoagulant Factor Levels after Cardiac Surgery Causes Thrombin Generation to Rise to Thrombotic Levels

IntroductionCardiac surgery with cardiopulmonary bypass (CPB) causes hemodilution and coagulation factor consumption. The administration of unfractionated heparin accelerates thrombin inactivation and leads to elevated consumption of antithrombin (AT). The heparin-mediated release of tissue factor pathway inhibitor (TFPI) from the vessel wall reduces prothrombin activation. CPB surgery thus affects both pro- and anticoagulant processes, which are reflected in changes in a patient's thrombin generation potential.In this study we investigated how cardiac surgery changes thrombin generation (TG), prothrombin conversion and thrombin inactivation. In addition, we studied the consequences of these changes in pro- and anticoagulant processes for transfusion management using in silico experimentation.MethodsThirty patients undergoing cardiac surgery with CPB were included and samples were collected before and after surgery and protamine administration. AT and prothrombin levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve.The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The effect of PCC administration in combination with AT was modeled as an increase of prothrombin conversion and antithrombin levels. The effect of in silico transfusion of PCC on coagulation was quantified by TG parameters.ResultsCPB surgery causes a reduction of plasma coagulation factor levels: AT (47%, p<0.001), prothrombin (63%, p<0.001), and decreases TG peak height by 71% (p<0.001). After surgery prothrombin conversion is significantly impaired (402 ± 218 vs 701 ± 181 nM; p<0.001), which is partly compensated for by lower thrombin inactivation (63%, p<0.001), resulting in a reduction of TG.We modeled the effect of the transfusion of prothrombin complex concentrate with or without added antithrombin on TG. If the prothrombin conversion capacity was increased to pre-surgery levels in the absence of antithrombin, TG peak height increases to 454 ± 72 nM (figure A). A slight increase of prothrombin conversion (up to 83% of the pre-surgery level) brings TG peak height into the normal range (386±61 nM), but the ETP remains elevated (3278 ± 1141 nM). The optimal way to restore thrombin generation is the simultaneous infusion of PCC and antithrombin. Our in silico experiments indicate that this restores both the peak height (362 ± 69 nM) and the ETP (1601 ± 369 nM∙min) to the pre-surgery level (figure B).ConclusionsCardiac surgery with CPB causes the hemostatic balance to shift toward bleeding due to a reduction of prothrombin conversion. This is only partially compensated by a reduction of thrombin inactivation, resulting in reduced TG after surgery.Post-surgery bleeding can be (and often is) remedied by the transfusion of PCC. However, full restoration of the prothrombin conversion capacity results in a TG profile that predicts a prothrombotic state, as an ETP value in the highest quartile is associated with a 5 times elevated thrombosis risk according to Winckers et al. Equilibrated restoration of the hemostatic capacity asks for the simultaneous supplementation of both pro- and anticoagulant factors, i.e. PCC containing AT or fresh frozen plasma. [Display omitted] DisclosuresKremers:Synapse bv: Employment. Bloemen:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Al Dieri:Synapse bv: Employment. Hemker:Synapse bv: Employment.

Could Hemorrhagic and Thrombotic Risk Due to Peg-Asparaginase be Identified By Coagulative Tests Using Fibtem and Antithrombin Levels? A Single Centre Study

Introduction. Hypofibrinogenemia and its correlation with hemorrhagic risk is usually detected by serum fibrinogen level with Clauss test. Recently the role FIBTEM test of rotational thromboelastometry (ROTEM) has been evaluated for prevention and management of acute bleeding in intesive care and surgical settings. We applied it for monitoring fibrinogen deficiency for the first time in children affected by acute lymphoblastic leukemia (ALL) treated with Peg-asparaginase (PEG-Asp). Antithrombin levels (AT) were measured as parameter for thrombotic risk. This drug impairs protein synthesis and causes severe coagulative deficiencies, with both hemorrhagic and thrombotic complications. Guidelines for the management of coagulation abnormalities being missing, we tried to identify parameters for management and prevention.Methods. Eightytwo children (age 1-17 years, mean 6 year) affected by ALL treated with AIEOP BFM ALL 2009 protocol at our Institution were studied. Platelet count and coagulation tests (protrombin time PT/INR, activated partial tromboplastin time aPTT, AT, serum fibrinogen level (SF) at Clauss test and fibrinogen function at MCF) were performed before and after each PEG-Asp administration. We previously found linear correlation with SF levels only for MCF > 9 mm. We then tried to identify clinically significant deficiency of SF. Severe hypofibrinogenemia was considered when Clauss test showed SF levels < 1g/L. MCF limit for severe fuctional hypofibrinogenemia was considered <= 5mm according to our experience in congenital and acquired hypofibrinogemia (cardiac surgery, liver transplant and intensive care settings). Severe AT deficiency was considered at <50%.Results. Severe hypofibrinogenemia was found in 240 Clauss test (24.3%), but only in 44 (4.5%) of MCF at FIBTEM. Only 28% of SF at Clauss test < 1 g/L had an MCF value <= 4mm. Thirty-five percent of children presented MCF <=4mm with a prolonged alteration in 5.3% of them, mostly observed during Induction phase, when steroid therapy was concomitant. Prolonged administration of Peg-Asp (10 doses in 20 weeks) did not cause a prologed hypofribrinogenemia. Fibrinogen concentrates was supplemented only at MCF >= 2mm or at 3-4 mm in association with INR > 2.8. Fresh frozen plasma administration was avoided. No significant bleeding was observed. Severe AT deficiency was found in 287 measurements (23%) and in 57% of patients. AT deficiency was more frequent according to the number of PEG-Asp doses, in particular the percentage of affected patients increased in the different phases of chemotherapy (Table 1), being observed in all patients receiving prologed PEG-Asp therapy. AT was suggested at level of < 50%, but it was performed only in 54% of cases. Three thrombotic events were registered (3.6%) all in presence of AT levels <50%.Conclusions. The frequency of fibrinogen functional deficiency as detected by MCF level at FIBTEM was inferior than hypofibrinogenemia at Clauss test. The MCF level chosen in our study to identify a parameter for hemorrhagic risk was helpful to select patients for coagulant therapy with fibrinogen concentrates. AT deficiency was more frequent than fibrinogen deficiency and was related to the numer of PEG-Asp doses. These tests can help in the management of patients with coagulative disturbancies in order to prevent seriuos adverse events.Table 1Frequency of MCF and AT deficiencies according to number of PEG-Asp dosesN° of doses12410MCF <=4mm25%42%43%40%AT < 50%19%65%86%100%N° of patients6759710 DisclosuresNo relevant conflicts of interest to declare.

In Vitro Assessment of Altered Thrombin-Fibrinogen Interaction As a Mechanism for Acute Traumatic Coagulopathy

IntroductionThe acute traumatic coagulopathy (ATC) which develops within 30 min following severe trauma with tissue damage and shock is defined by an increased prothrombin time (PT) and international normalized ratio (INR). While reduced thrombin might be expected in conjunction with elevated PT, recent clinical studies reveal paradoxically elevated thrombin generation potential in patients with ATC. We therefore hypothesized that the quantity of thrombin and the timing of thrombin-fibrinogen interactions both have an impact on clot quality; the exuberant production of thrombin found in trauma results in improper clot formation.MethodsIn vitro studies were conducted in human blood products and simplified synthetic plasma (consisting of purified human coagulation factors in HEPES buffered saline). Turbidimetry was used to observe fibrin crosslinking, while thromboelastography (TEG) was used to quantify clot formation parameters. Quantitation of fibrin(ogen) degradation products (FDPs) was conducted with the STA-R Evolution coagulation analyzer and by ELISA. A fluorogenic substrate was used to observe thrombin generation.ResultsIncreasing the amount of prothrombin or thrombin (0-1400nM) in prothrombin-immunodepleted citrated plasma resulted in reduced clot times. The same dose response was examined in a buffered mixture of fibrinogen (300 mg/dl), FXIII (31.25nM), Ca2+ (2mM), and FXa (170nM-only used with prothrombin samples). However, while increasing prothrombin increased clot strength in both FII-deficient plasma and in the synthetic plasma, direct addition of thrombin decreased clot strength and by 3-fold at 1000nM versus 100nM (Figure 1; *p<0.05; **p<0.01; ***p<0.001 between groups at given concentration); fibrin density was similarly reduced in turbidimetric assays. In TEG, the thrombin dose response did not affect whole blood or platelet-rich plasma, but in platelet-poor plasma the same clot strength inhibition trend was observed. Thrombin generation from the combination of prothrombin (0-1000 nM), FXa (170 nM), and Ca2+ (2 mm) was found to be saturated above an initial prothrombin concentration of 500nM.An examination of FDPs from plasmin-degraded fibrinogen ± thrombin ± FXIII showed that FDPs from both crosslinked and uncrosslinked fibrin had 4-fold higher amounts of fibrin monomer and D-dimer than those produced from plasmin digestion of pure fibrinogen. When FDPs from thrombin +fibrinogen ± FXIII were supplemented back into fresh fibrinogen (0-50% of the final mixture) and allowed to clot again, there was a concentration-dependent decrease in fibrin formation rate (control: 0.15 OD/min; 50% FDP: 0.07 OD/min; p<0.001) which was not observed in samples treated with non-thrombin exposed FDPs. The use of these FDPs allowed for simulations of trauma patient plasma to be constructed using concentrations of plasma proteins associated with both "normal" trauma and the hypocoagulable state which manifests in ATC. When combining relevant levels of thrombin, fibrinogen, FDPs, and antithrombin III as are found in trauma patients that trend toward either good or bad outcomes, differences in TEG tracings can be observed illustrating the validity of these in vitro systems (Figure 2).ConclusionsThe dose responses of prothrombin versus thrombin reveal the significance of the timing of these reactions on proper clot formation. The conversion of prothrombin to thrombin mediated through FXa and FVa results in strong clots under normal circumstances. These results were reflected in both turbidimetry and TEG, indicating that fibrin crosslinking is being hindered by the presence of excess thrombin even in the presence of the antithrombin III. Additionally, FDPs from crosslinked substrates reduce new clot-making efficiency. Excess thrombin and significant increases in FDPs (which can result from fibrinolytic feedback loops) both contribute to an in vitro phenotype that may represent an underlying factor in the development of ATC. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rebalanced Thrombin Generation in Liver Cirrhosis Calls for Tailored Transfusion Protocols

IntroductionMost coagulation factors are produced by the liver, and therefore in liver cirrhosis both pro- and anticoagulant factor levels are reduced. Cirrhosis patients show no clear systemic bleeding or thrombotic phenotype and thrombin generation (TG) is (almost) unaffected, therefore it has previously been proposed that coagulation is rebalanced in cirrhosis because both pro- and anticoagulant processes are affected to a similar extent. Nevertheless, clinical parameters such as the prothrombin time indicate a bleeding risk in cirrhosis patients, and based on these tests patients often receive blood product transfusion during surgery.In this study we investigated thrombin generation and its main underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation) to provide empirical evidence for the hypothesis of rebalanced thrombin generation in liver cirrhosis. In addition, we used in silico experimentation to study the consequences of rebalanced TG for current transfusion practices.Methods25 cirrhosis patients and 25 healthy subjects were enrolled in our study. The group of cirrhosis patients consisted of 22 Child-Pugh A and 3 Child-Pugh B patients. Prothrombin, antithrombin (AT) and α2Macroglobulin (α2M) levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve, and thrombin-antithrombin and thrombin-α2Macroglobulin complex formation was determined.The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The increase of prothrombin conversion was simulated by substituting the prothrombin conversion curve of a cirrhosis patient by the average healthy subject prothrombin conversion curve, to normalize prothrombin conversion in patients. Thrombin generation curves were calculated based on this normalized prothrombin conversion curve and the original thrombin inactivation parameters of each cirrhosis patient.ResultsProthrombin and antithrombin are significantly decreased in cirrhosis patients (74 % and 70 %, p<0.001), whereas the α2M level is significantly increased (p<0.001). Thrombin generation in healthy subjects and patients is similar (figure), although prothrombin conversion (65 %, p<0.001) and thrombin inactivation (73 % p<0.001) are markedly reduced in cirrhosis patients. Acquired AT deficiency results in a reduction of thrombin inactivation by AT (64 %, p<0.001), but the substitution of AT by α2M leads to increased thrombin inactivation by α2M (220 %, p<0.001).The effect of prothrombin complex concentrate transfusion on thrombin generation in cirrhosis patients was determined by computational modeling (figure). In silico normalization of prothrombin conversion, as would be achieved by PCC transfusion, causes TG to rise to levels that have been shown to be highly prothrombotic (ETP 2878 ± 1323 nM∙min, p<0.001).ConclusionsDespite large differences in prothrombin conversion and thrombin inactivation, TG in liver cirrhosis patients remains within the normal range (rebalanced), in contrast to the prothrombin time that predicts a bleeding risk in these patients. The transfusion of PCC based on a prolonged prothrombin time would result in a substantial elevation of thrombin generation. This indicates that standard transfusion protocols need to be tailored to the specific needs of cirrhosis patients. [Display omitted] DisclosuresKremers:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Hemker:Synapse bv: Employment.

Baseline Thrombin Generation Markers and Functional Antithrombin Levels in Sepsis Associated Coagulopathies Are Predictive of the Severity of Pathogenesis

Introduction: Severe sepsis remains the most common cause of death in critically ill patients. Thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). Thrombin generation markers and functional AT levels provide useful prognostic information in the management of these patients. The purpose of this study was to compare the baseline values of such thrombin generation markers of prothrombin fragment (F1.2), thrombin antithrombin complex (TAT) and functional antithrombin levels in patients enrolled in the Phase 2B and a Phase III study to investigate the therapeutic effect of recombinant thrombomodulin (ART-123) in sepsis associated coagulopathy.Materials and Methods: Plasma samples from seven hundred and fifty eight patients enrolled in the ART-123 study, a phase 2b, international, multicenter, randomized controlled trial, and patients enrolled in an ongoing (n=200) phase III clinical trial were profiled for F1.2 and TAT using commercially available ELISA methods (Siemens, Deerfield, IL, USA). Functional AT activity was measured using a chromogenic substrate method. All of these samples included blood drawn prior to the first of ART-123 or placebo treatment.Results: The results are tabulated in the following tables.Phase 2b Study (n=750) Table 1ParameterMeanStd ErrorMinMaxMedianQ1Q3F1.2 (pmol/L)515.541.7109,239305182571.5TAT (ng/ml)26.74.50.31,213.87.54.215.1AT (% NHP)84.91.383.020672.882.3101.3Phase 3 Study (n=176) Table 2ParameterMeanStd ErrorMinMaxMedianQ1Q3F1.2 (pmol/L)604.259.418.88,618401.8207.6695TAT (ng/ml)28.94.21.86524.812.46.1227.6AT (% NHP)51.424.82.1122.650.332.567.3Discussion: The inclusion criteria for the phase 2b study was based on the revised ISTH criteria and the phase III study used an INR>1.4, 30K=<PLT<150K, shock, and/or respiratory organ dysfunction as criteria at baseline. The median values were TAT (7.5 ng/ml Phase 2b study; 12.4 ng/ml Phase III), F1.2 ( 305 pmol/L Phase 2b; 401.8 pmol/L Phase III) and AT ( 72.8 %, Phase 2b; 50.3% ,Phase III). There was a difference noted in the markers of thrombin generation between the two studies. The TAT and F1.2 were higher and the AT was lower in the Phase III study suggesting a higher generation of thrombin in this study. Therefore the strict criteria used in the Phase III study shows that this patient group had more activation of coagulation compared to the Phase 2b patients. Furthermore the modified criterion used for the recruitment of patients in the phase III study provides a defined project population to investigate the therapeutic effects of ART-123. These observations also underscore the importance of inclusion criterion for sepsis associated coagulopathy patients to validate the therapeutic effects of newer drugs such as ART-123. DisclosuresWilliams:Asahi Kasei Pharma America: Employment.

A Pilot Study of Antithrombin Replacement Prior to Cardiopulmonary Bypass in Neonates.

Neonates have low levels of antithrombin. Inadequate anticoagulation during cardiopulmonary bypass (CPB) due to low antithrombin activity may result in a poor preservation of the coagulation system during bypass. We hypothesize that antithrombin replacement to neonates prior to CPB will preserve the hemostatic system and result in less postoperative bleeding. A randomized, double-blinded, placebo-controlled pilot study of antithrombin replacement to neonates prior to CPB was conducted. Preoperative antithrombin levels determined the dose of recombinant antithrombin or placebo to be given. Antithrombin levels were measured following the dosing of the antithrombin/placebo, after initiation of bypass, near the completion of bypass, and upon intensive care unit admission. Eight subjects were enrolled. No subject had safety concerns. Mediastinal exploration occurred in two antithrombin subjects and one placebo subject. Antithrombin activity levels were significantly higher in the treated group following drug administration; levels continued to be higher than preoperatively but not different from the placebo group at all other time points. Total heparin administration was less in the antithrombin group; measurements of blood loss were similar in both groups. A single dose of recombinant antithrombin did not maintain 100% activity levels throughout the entire operation. Although no safety concerns were identified in this pilot study, a larger trial is necessary to determine clinical efficacy.

Evaluation of Some Plasma Coagulation Factors in Women with Spontaneous Miscarriage.

It has been reported that 15-20% of parous female have experienced at least one miscarriage, while 3% of them have experienced two miscarriages. The goal of this study was to evaluate the plasma level of coagulation factors in women with a history of spontaneous abortions. In this case-control study, 82 women with a history of two or more abortions referred to the six private gynecologic clinics in Gorgan city without any structural abnormality were recruited during 2011-2012. Plasma levels of antithrombin III (ATIII) using colorimetric assay, protein C, protein S, factor V Leiden and lupus anticoagulant (LAC) using coagulation method were measured. The control group was women with a history of normal delivery and no abortions. Those under anti-coagulant therapy were excluded from the study. Data were entered into the computer using the Statistical Package for the Social Sciences (SPSS, SPSS Inc., Chicago, IL, USA) version 16 and analyzed by Chi-square, t test and non-parametric tests. At least one abnormality was reported in 35 cases (42.7%). Among them, protein C deficiency was the most prevalent (30.5%). ATIII was abnormal in 17.1% and lupus anti-coagulant was abnormal in 8.5%. Factor V Leiden was normal in all cases and protein S deficiency was only seen in one case. We suggest to perform these tests in regards to the thrombophilia in cases with spontaneous abortions in order to find an early cure for this treatable disorder.

Takayasu arteritis and antiphospholipid antibody syndrome in an elderly woman

Takayasu arteritis and antiphospholipid antibody syndrome in an elderly woman

Monitoring unfractionated heparin in children: a parallel-cohort randomized controlled trial comparing 2 dose protocols

AbstractMonitoring unfractionated heparin (UFH) is crucial to prevent over- or under-anticoagulation. However, the optimal parameters for monitoring UFH in children are not well established. The study objectives were to investigate (1) the relationship between UFH dose and its anticoagulant effect as assessed by anti-Xa, activated partial thromboplastin time (aPTT) and activated clotting time (ACT); (2) other factors influencing UFH effect; (3) the agreement between the assays; and (4) the association between UFH effect and clinical outcome. HEARTCAT was a parallel-cohort randomized controlled trial comparing high-dose (100 U/kg bolus followed by age-based continuous infusion in randomized children) vs low-dose UFH (50 U/kg bolus) during cardiac catheterization in children. Blood samples were drawn before and after UFH administration at 30, 60, and 90 minutes. Four-hundred and two samples of 149 patients were evaluable. Anti-Xa, aPTT, and ACT all showed good discrimination between UFH doses. Regression models demonstrated the following determinants of UFH effect: UFH dose, age, baseline antithrombin (for anti-Xa), and baseline levels of aPTT and ACT, respectively. UFH effects were lower in infants compared with older children, which was more pronounced at low-dose than at high-dose UFH. Agreement between the 3 assays was poor. Most aPTT values were above therapeutic range or beyond measuring limit and thus of limited value for UFH monitoring. No association of UFH dose or effect with clinical outcome could be observed. In conclusion, all assays reflected a significant UFH dose-effect relationship, however, with poor agreement between the respective tests. The age-dependency of UFH effect was confirmed. Notably, the influence of age on UFH effect was dose-dependent.