Calibrated Automated Thrombogram Assay Research Articles

Effects of dabigatran, rivaroxaban, and apixaban on fibrin network permeability, thrombin generation, and fibrinolysis

Introduction There are important pharmacological differences between direct oral anticoagulants (DOAC) and a deeper knowledge of how they influence different aspects of hemostasis in patients on treatment is desirable. Materials and methods Blood samples from patients on dabigatran (n = 23), rivaroxaban (n = 26), or apixaban (n = 20) were analyzed with a fibrin network permeability assay, a turbidimetric clotting and lysis assay, the calibrated automated thrombogram (CAT), plasma levels of thrombin-antithrombin complex (TAT) and D-dimer, as well as DOAC concentrations, PT-INR and aPTT. As a comparison, we also analyzed samples from 27 patients on treatment with warfarin. Results Patients on dabigatran had a more permeable fibrin network, longer lag time (CAT and turbidimetric assay), and lower levels of D-dimer in plasma, compared with patients on rivaroxaban- and apixaban treatment, and a more permeable fibrin network than patients on warfarin. Clot lysis time was slightly longer in patients on dabigatran than in patients on rivaroxaban. Warfarin patients formed a more permeable fibrin network than patients on apixaban, had longer lag time than patients on rivaroxaban (CAT assay), and lower peak thrombin and ETP compared to patients on treatment with both FXa-inhibitors. Conclusions Results from this study indicate dabigatran treatment is a more potent anticoagulant than apixaban and rivaroxaban. However, as these results are not supported by clinical data, they are probably more related to the assays used and highlight the difficulty of measuring and comparing the effect of anticoagulants.

Measuring Thrombin Generation in Platelet Rich Plasma with the ST Genesia

Background: Thrombin generation (TG) is nowadays used more and more for diagnosing and monitoring of patients. The ST Genesia is a fully automated device for measuring TG in platelet poor plasma (PPP). As the samples are not being inverted prior to the measurement once loaded in the device, it was not known whether it was possible to measure TG in platelet rich plasma (PRP). Platelets can sediment in time possibly affecting TG results. The goal of this study was to evaluate the use of the ST Genesia for measuring TG in PRP in time and to investigate whether the assay is sensitive to platelet antagonists. Methods: Blood was taken from 6 healthy donors and PRP was immediately prepared by centrifuging the blood once for 15 minutes at 220g. The PRP was divided into 14 tubes: 11 tubes for measuring TG in time; i.e. after 0, 15, 30, 45, 60, 75, 90, 105, 120, 180, 240 minutes after the samples were loaded on board; 1 tube for platelet count measurement, 1 tube for TG with the Calibrated Automated Thrombogram (CAT) assay and 1 tube for freezing the PRP. The PRP reagent of the CAT assay was used for triggering TG that contains a low tissue factor concentration. Tukey's multiple comparisons test was used for statistical analysis. Results: The platelet count remained stable in time even without inverting the vial prior to the measurement up to 3 hours. After 4 hours, platelet count was significantly lower (p<0.01). TG in PRP remained stable in time up to 3 hours, after that lagtime and time-to-peak were extended by an average of 48% and 16%, respectively (p-values <0.0001 and 0.0121, respectively), while ETP and peak height remained stable (with low CV's of <6.0% and <7.3%, respectively). The ST Genesia proved to be sensitive to platelet antagonists aspirin (100µM) and Cangrelor (500nM) as peak height decreased with 22% and 24%, respectively. TG measured with the CAT and the ST Genesia was comparable. Measuring TG in PRP after being frozen at -80°C resulted TG kinetics similar to TG in PPP, with a shorter lagtime (-67%) and time-to-peak (-64%), with an increased peak height (+82%), and velocity index (+383%), and an unaffected ETP. Conclusion: The ST Genesia can be used for the reproducible measurement of TG in PRP up to two hours after loading the samples in the device.

Application of the thrombin generation assay in patients with antiphospholipid syndrome: A systematic review of the literature.

The antiphospholipid syndrome (APS) is classified by the presence of antiphospholipid antibodies (aPL) and thrombotic and/or adverse obstetric outcomes. The diagnosis and risk assessment of APS is challenging. This systematic review investigated if the thrombin generation (TG) assay could be helpful for APS diagnosis and risk assessment. A systemic review was performed by searching two databases (MEDLINE and Embase) until March 31, 2022, using a search strategy with two concepts: APS and TG, and related keywords. Two reviewers independently screened the articles based on predefined inclusion and exclusion criteria. Data extraction and quality assessment with the Newcastle-Ottawa Scale (NOS) were performed independently. Synthesis Without Meta-analysis guidelines were followed for data synthesis reporting. Fourteen studies with 677 APS and 1,349 control subjects were included with variable quality according to the NOS. Twelve studies measured TG via the calibrated automated thrombogram (CAT) method using a fluorogenic substrate, whereas two used a chromogenic substrate-based TG assay. One study compared the CAT assay to the fully-automated ST Genesia® (Stago, France). Two studies initiated TG using platelet-rich plasma, whereas the rest of the studies used platelet-poor plasma. Resistance to activated protein C (aPC) was examined in ten studies. They reported a significant increase in aPC-resistance in APS patients compared to healthy controls, aPL-carriers, and thrombotic controls. Based on two studies, the prevalence of aPC-resistance was higher in APS patients compared to healthy controls and thrombotic controls with odds ratios of 5.9 and 6.8-12.8, respectively (p < 0.05). In contrast, no significant difference in aPC-resistance was found between APS patients and autoimmune disease controls. Furthermore, 7/14 studies reported TG-parameters including peak height, endogenous thrombin potential, lag time, and time to peak, but these outcomes were highly variable between studies. Furthermore, TG methodology between studies differed greatly, impacting the comparability of the studies. aPC-resistance measured with TG was increased in APS patients compared to healthy and thrombotic controls, but the diagnostic and prognostic value is unclear compared to current diagnostic strategies. Studies of other TG-parameters were heterogeneous and more research is needed to identify their potential added value in APS diagnosis. https://www.PROSPERO/, identifier: CRD42022308363.

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces hypertension and vascular inflammation

Abstract Background Arterial hypertension is one of the most important modifiable risk factors for all-cause morbidity and mortality worldwide. Angiotensin II (Ang II) plays a pathogenic role in the development of hypertension, vascular dysfunction, inflammation and tissue damage. In the context of hypertension, heme-oxygenase 1 (HO-1) gene expression is upregulated as an antioxidant defense system in response to AngII through its action on heme catabolism, which generates carbon monoxide (CO), ferritin and biliverdin, which is reduced to bilirubin by biliverdin reductase A (BLVRA). Previous studies have shown the important role of HO-1 in the maturation and migration of immune cells. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion damage. However, the role of myeloid cell specific HO-1 in the detrimental effects of AngII induced vascular dysfunction has not yet been explored. Objectives To investigate the potential vascular protection of myeloid cell specific overexpression of HO-1 in AngII-induced arterial hypertension. Methods Hypertension was induced in 8–13 weeks old male mice with selective over-expression of HO-1 (HO-1indLysMcre) in myelomonocytic cells versus LysMCre/wt by AngII infusion (1mg/kg/d). Blood pressure was recorded by tail-cuff. Bilirubin levels were quantified in plasma by High Performance Liquid Chromatography (HPLC). The quantification of adherent and rolling leukocytes in carotid arteries was detected by intravital video microscopy (IVM). Endogenuos Thrombin potential (ETP) was measured in platelet rich plasma (PRP) and platelets poor plasma (PPP) by Calibrated Automated Thrombogram (CAT) assay. Endothelium-dependent vasodilation was assessed in isolated aortic rings by concentration-relaxation curves in response to acetylcholine (ACh). Results AngII-infused HO-1indLysMcre had decreased blood pressure values and improved endothelial function as compared to LysMcre controls. IVM revealed reduced leukocyte rolling and adhesion to the vascular endothelium in AngII infused HO-1indLysMcre mice compared to controls, paralleled by reduced expression of NOX-2 mediated oxidative stress and vascular inflammation in AngII-induced arterial hypertension by decreasing VCAM-1, CCR2 and MCP-1 expression. mRNA analysis revealed an increased expression of BLVRA in liver, spleen, heart and aortic tissues in response to AngII, which was higher in HO-1indLysMcre mice than in controls. By CAT assay we registered a decrease ETP in PPP and PRP in HO-1ind mice infused with AngII compatible with less abundance of inflammatory platelets, potentially regulated by BLVRA activity. This was supported by increased in bilirubin levels in response to AngII, which was higher in HO-1indLysMcre mice than in controls. Conclusion Myeloid cell specific overexpression of HO-1 confers anti-inflammatory protection to the vasculature in AngII induced hypertension. This effect is, at least in part, mediated by BLVRA. Funding Acknowledgement Type of funding sources: None.

Assessment of thrombin generation in horses using a calibrated automated thrombogram.

The amount of thrombin generated reflects the endogenous thrombin potential (ETP), which depends on the balance of pro- and anticoagulant factors. The calibrated automated thrombogram (CAT) allows for the direct measurement of thrombin generation during the clotting process. (1) To describe the results of the CAT assay in horses, (2) to establish intra-assay and intra- and interindividual variation of thrombin generation in healthy horses, and (3) to compare in vitro low-molecular-weight heparin (LMWH) sensitivity between healthy and sick horses. The hypothesis for the last objective is that inhibition of thrombin generation in sick horses requires higher heparin concentrations. The plasma of 10 healthy mixed breed horses was used for the determination of normal thrombin generation parameters (lag time, time to peak, peak thrombin concentration, and ETP). Five of the healthy horses were compared with five horses with systemic inflammatory response syndrome (SIRS). In vitro heparin sensitivity was determined using LMWH. The intra-assay variation was small (<5%) for all parameters. Relatively large intra- and interindividual variation were observed in healthy horses. Four of the five sick horses with SIRS had a thrombogram compatible with a hypercoagulable state. The in vitro heparin sensitivity test suggested decreased sensitivity to LMWH in hypercoagulable states. The CAT assay could detect coagulopathy in horses. In vivo experiments are needed to confirm that it can be used to monitor responses to LMWH therapy.

Potential value of the calibrated automated thrombogram in patients after a cerebral venous sinus thrombosis; an exploratory study

BackgroundCerebral venous sinus thrombosis (CVST) is a relatively rare, but potentially lethal condition. In approximately 15% of the patients, the cause of CVST remains unclear. Conventional clotting tests such as prothrombin time and activated partial thromboplastin time are not sensitive enough to detect prothrombotic conditions nor mild haemostatic abnormalities. The calibrated automated thrombogram (CAT) is a physiological function test that might be able to detect minor aberrations in haemostasis. Therefore, we aimed to detect the presence of a prothrombotic state in patients who endured idiopathic CVST with the CAT assay.MethodsFive adult patients with an idiopathic, radiologically proven CVST that had been admitted during the past 3 years were included in this study. The control group consisted of five age/gender matched healthy volunteers. Exclusion criteria were known haematological disorders, malignancy (current/past) or hormonal and anticoagulant therapy recipients. We obtained venous blood samples from all participants following cessation of anticoagulation. Using the CAT assay, we determined lag time, normalized endogenous thrombin potential (ETP), ETP reduction and normalized peak height. In addition, prothrombin concentrations were determined.ResultsWe found no significant differences in lag time (4.7 min [4.5–4.9] vs 5.3 min [3.7–5.7], p = 0.691), normalized ETP (142% [124–148] vs 124% [88–138], p = 0.222), ETP reduction (29% [26–35] vs 28% [24–58], p > 0.999), and normalized peak height (155% [153–175] vs 137 [94–154], p = 0.056) between patients and their age/gender matched controls. In addition, prothrombin concentrations did not significantly differ between patients and controls (120% [105–132] vs 127% [87–139], p > 0.999).ConclusionReasons for absent overt hypercoagulability within this study population may be the small patient sample, long time since the event (e.g. 3 years) and avoidance of acquired risk factors like oral contraception. Given the fact that CVST is a serious condition with a more than negligible risk of venous thrombosis event recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST.

Evaluation of Global Coagulation Assays in Normal and Thrombocytopenic Populations

IntroductionQuantitative platelet count is not always a true reflection of an individual's bleeding risk with thrombotic complications previously reported in thrombocytopenic patients. A better laboratory assessment of bleeding risk in these patients is required. Global coagulation assays such as thromboelastography (TEG), thrombin generation with calibrated automated thrombogram (CAT) and fibrin generation assessment with overall haemostatic potential (OHP) assay may provide more complete assessment of an individual's coagulation profile. We aim to evaluate how global coagulation assays differ between thrombocytopenic patients and normal controls.MethodsBlood samples were collected from thrombocytopenic patients (platelet count <150x109/L). The samples were processed for baseline investigations and global coagulation assays. Citrated whole blood was analysed for thromboelastography using the TEG® 5000 analyser and additional citrated blood samples were double spun at -2500G to obtain platelet-poor plasma (PPP) and stored at -80°C within 2 hours of sample collection. The stored PPP were then used for later testing with CAT and OHP, using standard manufacturer guidelines. The results were compared to our previously collected healthy normal control population (n=90).ResultsFifty-eight samples (30 males, 28 females) with mean age of 57.5 years were collected. This included 34 patients with chemotherapy or malignancy-related thrombocytopenia and 24 participants with immune thrombocytopenia or other secondary causes of thrombocytopenia. In comparison to the normal control population (average platelet count 245 x 109/L), thrombocytopenic participants (average platelet count 79 x 109/L, p<0.001) had reduced clot lysis (LY30, 0.0% vs 0.6%; p<0.001) without significant difference in other TEG® parameters including comparable clot strength (maximum amplitude, 59.3 vs 57.8 mm; p=0.38). Thrombin generation using CAT showed reduced endogenous thrombin potential (1252.2 vs 1353.0 nM.min; p=0.040) while fibrin generation showed no difference in the OHP parameters (p>0.05).On sub-analysis, participants with marked thrombocytopenia (platelet count 0 - 50 x 109/L; n=13) had significantly more hypocoagulable TEG® parameters including prolonged clot formation time (K-time, 3.4 vs 2.3 min; p=0.002) as well as reduced α-angle (41.2° vs 57.9°; p=0.001), clot strength (47.2 vs 57.8 nM.min; p=0.002) and clot lysis (0.0% vs 0.6%; p<0.001). Interestingly, the CAT parameters generally did not meet significance, possibly due to small numbers in this subgroup, apart from prolonged lag time (4.0 vs 3.1 min; p=0.018) although there was a trend towards lower endogenous thrombin potential (1184.0 vs 1353.0; p=0.056). Conversely, participants with platelet count 101 - 150 x 109/L appeared more hypercoagulable on TEG® with shorter clot formation time (1.7 vs 2.3 min; p<0.001) and increased clot strength (66.4 vs 57.8 mm; p<0.001) compared to normal controls. CAT parameters were not significantly different in this subgroup. Fibrin generation was relatively preserved in both sub-groups as well. When comparing the 2 groups of thrombocytopenic patients (ITP vs chemotherapy/malignancy) patients, there was no difference in all the parameters of the three global coagulation assays apart from reduced clot lysis in the latter group (p=0.018)ConclusionGlobal coagulation assays, particularly thromboelastography is useful in measuring the differences in coagulation in thrombocytopenic patients. Marked thrombocytopenia (<50 x 109/L) showed hypocoagulable parameters, which validate current clinical practice of managing this population as high bleeding risk. In contrast, thrombocytopenic patients with platelet counts between 101 - 150 x 109/L were found to be hypercoagulable, providing a possible explanation of overcompensation, which may lead to some thrombocytopenic patients experiencing thrombotic events. Clot lysis was consistently markedly reduced in thrombocytopenic population, suggesting again, possible compensatory mechanisms to reduce bleeding risk. CAT and OHP assay using platelet-poor plasma, on the other hand, does not appear to be sensitive in differentiating the thrombocytopenic and normal healthy populations. Larger prospective studies will be undertaken to confirm these findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Exploring the utility of a novel point‐of‐care whole blood thrombin generation assay following trauma: A pilot study

IntroductionPlasma thrombin generation kinetics as measured by the calibrated automated thrombogram (CAT) assay is a predictor of symptomatic venous thromboembolism after trauma. We hypothesized that data from a new prototype assay for measurement of thrombin generation kinetics in fresh whole blood (near patient testing of thrombin generation), will correlate with the standard CAT assay in the same patients, making it a potential tool in the future care of trauma patients. MethodsPatients were enrolled from June 2018 to February 2020. Within 12 hours of injury, blood samples were collected simultaneously for both assays. Variables compared and correlated between assays were lag time, peak height, time to peak, and endogenous thrombin potential. Data are presented as median with interquartile range (IQR). Spearman and Pearson correlations were estimated and tested between both assays; a P value of <0.05 was considered to be significant. ResultsA total of 64 trauma patients had samples analyzed: injury severity score = 17 (IQR), 10‐26], hospital length of stay = 7.5 (IQR), 2‐18) days, age = 52 (IQR, 35‐63) years, 71.9% male, and 42.2% of patients received a transfusion within 24 hours of injury. Thrombin generation parameters between plasma and whole blood were compared and found that all parameters of the two assays correlate in trauma patients. ConclusionIn this pilot study, we have found that a novel point‐of‐care whole blood thrombin generation assay yields results with modest but statistically significant correlations to those of a standard plasma thrombin generation assay. This finding supports studying this device in a larger, adequately powered study.

Validation of the Role of Thrombin Generation Potential by a Fully Automated System in the Identification of Breast Cancer Patients at High Risk of Disease Recurrence.

Background The measurement of thrombin generation (TG) potential by the calibrated automated thrombogram (CAT) assay provides a strong contribution in identifying patients at high risk of early disease recurrence (E-DR). However, CAT assay still needs standardization and clinical validation. Objective In this study, we aimed to validate the role of TG for E-DR prediction by means of the fully automated ST Genesia system. Methods A prospective cohort of 522 patients from the HYPERCAN study with newly diagnosed resected high-risk breast cancer was included. Fifty-two healthy women acted as controls. Plasma samples were tested for protein C, free-protein S, and TG by ST Genesia by using the STG-ThromboScreen reagent with and without thrombomodulin (TM). Results In the absence of TM, patients showed significantly higher peak and ETP compared with controls. In the presence of TM, significantly lower inhibition of ETP and Peak were observed in patients compared with controls. E-DR occurred in 28 patients; these patients had significantly higher peak and endogenous thrombin potential (ETP) in the absence of TM compared with disease-free patients. Multivariable analysis identified mastectomy, luminal B HER2-neg, triple negative subtypes, and ETP as independent risk factors for E-DR. These variables were combined to generate a risk assessment score, able to stratify patients in three-risk categories. The E-DR rates were 0, 4.7, and 13.5% in the low-, intermediate-, and high-risk categories (hazard ratio = 8.7; p < 0.05, low vs. high risk). Conclusion Our data validate the ETP parameter with a fully automated standardized system and confirm its significant contribution in identifying high-risk early breast cancer at risk for E-DR during chemotherapy.

Effects of rivaroxaban and dabigatran on global hemostasis in patients with atrial fibrillation.

: The study was aimed to evaluate the effects of two standard doses of rivaroxaban and dabigatran on global hemostatic assays in patients with atrial fibrillation. The study included 52 patients treated with rivaroxaban (15/20 mg), 50 on dabigatran (110/150 mg) and 20 healthy individuals. Platelet-poor plasma was used for determination of three global hemostatic assays, namely endogenous thrombin potential (ETP), calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP). Rivaroxaban and dabigatran reduced ETP (P < 0.01) although OHP (P < 0.05) was diminished only by dabigatran. Strong correlations were noticed between ETP parameters and the plasma concentrations of rivaroxaban (ETP, r = -0.51; c-max, r = -0.85; t-lag, r = 0.83; t-max, r = 0.66) as well as with plasma concentration of dabigatran (ETP, r = -0.75; c-max, r = -0.74; t-lag, r = 0.73; t-max, r = 0.52). Analysis of dabigatran concentrations under 50 ng/ml showed that ETP parameter has area under the concentration-time curve-receiver operating characteristic value of 0.879 (95% confidence interval 0.776-0.980). Dabigatran treatment paradoxically increased area under the concentration-time curve and peak values although rivaroxaban decreased peak values (P < 0.01). However, significant correlation between CAT parameters and plasma concentration of both direct oral anticoagulants was not observed. We confirmed that the CAT assay is inappropriate for estimation of dabigatran effects and is not fully sensitive as regards rivaroxaban. The ETP assay can potentially be the appropriate method for estimation of global hemostatic capacity as regards both direct oral anticoagulants. The role of OHP needs to be confirmed in additional studies. ETP parameter of chromogenic assay has promising potential in exclusion of high plasma concentrations of dabigatran.

Hyper-coagulability in obese patients accurately identified by combinations of global coagulation assay parameters

BackgroundRoutine laboratory methods are insensitive to hyper-coagulation, which may be detected by global hemostasis tests. Calibrated Automated Thrombogram (CAT) is a gold standard method to measure thrombin generation and coagulation potential. Thrombodynamics (TD) is a new global assay that monitors the spatio-temporal propagation of blood coagulation, separating initiation from amplification/propagation phases of coagulation and visualizing fibrin clot formation. AimWe investigated whether CAT and/or TD can identify hyper- and hypo-coagulable states in patients with well-characterized phenotypes and which parameters could be used as potential predictors of thrombotic risk. MethodsBlood was collected from: (1) forty healthy volunteers; (2) twelve obese patients scheduled for bariatric surgery (BMI ≥ 35 kg/m2); (3) nine patients under therapy with vitamin K-antagonists (median INR 2.7); (4) eight patients treated with low molecular weight heparins (anti-Xa activity between 0.5 and 0.7 U anti-Xa/ml); (5) ten patients with hemophilia A or B. Tissue factor induced thrombin generation was measured with CAT. Propagation of thrombin generation and clot growth from a tissue factor coated surface were monitored with TD. ResultsThrombin generation and fibrin clot formation parameters were significantly higher in obese patients compared to healthy volunteers and anticoagulated or hemophilic patients. ROC analysis of combined CAT or CAT/TD parameters (integrating thrombin generation and fibrin clot formation) demonstrated an excellent accuracy in detecting hyper-coagulability. ConclusionCombinations of CAT assay parameters and of parameters of thrombin generation burst with final fibrin clot properties allow recognizing accurately hyper-coagulable plasma and may represent predictive markers for thrombotic events.

Thrombin lag time is increased in children with mild asthma

BackgroundInflammation and coagulation are closely linked events. Thrombin is the key enzyme in coagulation system and also has roles in inflammation. ObjectiveThe aim of our study was to evaluate thrombin generation in children with mild asthma. MethodsForty-two children with mild asthma and 49 healthy children were included in the study. All patients performed spirometry. Thrombin generation tests (TGT) were performed with a calibrated automated thrombogram (CAT) in children without asthma exacerbation during the last six months. During CAT assay thrombogram curves were obtained. The area under the curve showed endogenous thrombin potentials and indicated the total amount of endogenous thrombin generated; the peak height showed the highest thrombin value, thrombin lag time and time to thrombin peak were measured. ResultsThrombin lag time was significantly longer in children with asthma (3.98±1.2min) compared to those in the control group (3.29±0.6min) (p<0.01). Children with asthma also had longer thrombin tail time compared to the control group (19.5±8.9min vs. 16.7±2.9min, p=0.02). Thrombin peak was inversely correlated with FEF 25–75 (r=−0.41, p<0.01). Thrombin lag time was inversely correlated with FEF 25–75 (r=−0.39, p<0.01). ConclusionInflammation in mild asthma seems to disturb coagulation but this disturbance may not be so strong as to increase thrombin levels and may only affect the initiation phase of thrombin generation.

In vitro effects of dalteparin on thrombin generation in canine plasma.

The calibrated automated thrombogram (CAT) is a functional thrombin generation (TG) assay that may provide a new approach for monitoring anticoagulant therapy in dogs. The effects of dalteparin on TG variables in dogs are unknown. Objectives were to establish normal TG variable ranges in dogs and measure the in vitro TG variables in canine pooled platelet-poor plasma (PPP) spiked with different dalteparin concentrations. In the first experiment, plasma samples from 25 adult healthy Beagle dogs and 11 client-owned healthy dogs of multiple breeds was measured individually for obtaining normal TG values. In the second experiment, separate pools of the remaining PPP from 24 of the 25 previous adult Beagles and from 45 different client-owned dogs were spiked with dalteparin at 9 concentrations with increasing anti-factor Xa (anti-FXa) activity. Activated partial thromboplastin time, tissue factor-induced TG, and anti-FXa activity were measured for each concentration. Concentration-response relationships were determined with ADAPT v.5, using various nonlinear regression models for stimulatory or inhibitory effects. Thrombin generation ranges of client-owned dogs and Beagles were equivalent only for time-to-peak (P < .05). In vitro dalteparin resulted in a concentration-dependent decrease in endogenous thrombin potential (ETP) in pooled PPP. The estimated dalteparin concentration that produced half the maximal inhibition of baseline ETP (IC50 ) was 0.289 U/mL. Thrombin generation and anti-FXa activity were more sensitive than APTT to detect the effects of dalteparin. The CAT assay can measure the effects of dalteparin in canine plasma, resulting in significant dose-dependent decreases in ETP, prompting further in vivo investigation.

Effect of dalteparin administration on thrombin generation kinetics in healthy dogs.

Dalteparin is used to prevent thrombotic complications in dogs. Measurement of anti-factor Xa (anti-FXa) activity is currently used for monitoring therapy, but remains a nonfunctional test. The calibrated automated thrombogram (CAT) could be a suitable approach for functional monitoring. We hypothesized that the CAT will detect decreased endogenous thrombin potential (ETP) in healthy dogs receiving dalteparin. Twenty-four healthy adult Beagles were randomly allocated to 4 equal groups. A single subcutaneous (SC) dose of 50 U/kg, 100 U/kg, or 150 U/kg of dalteparin was given. Platelet-poor plasma (PPP) was collected over a 24-hour period and evaluated by thrombin generation (TG) via CAT, anti-FXa activity, and APTT. Analysis was performed with a repeated-measures general linear mixed model, and the treated groups were compared to a placebo group. Time, dose, and time-dose interaction significantly affected ETP (P < .0001 for all effects), peak (P < .0001 for all effects), rate index (P < .0006 for all effects), and anti-FXa activity (P < .0001 for all effects). No significant time trend was detected in the control group. Dogs receiving the 100 U/kg dalteparin SC injection showed the most homogeneous response of ETP inhibition among treated groups. The % inhibition of ETP from baseline increased nonlinearly as a function of anti-FXa activity (r2 = .8186). The CAT assay can be employed to measure the effects of dalteparin at different doses in healthy dogs, showing sensitivity to time- and dose-dependent changes in ETP and other TG variables. Further investigation of the CAT as a tool for monitoring low molecular weight heparin therapy in dogs is warranted.

Characterization of the thrombin generation profile in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystemic inflammatory autoimmune disorder. Thrombotic events occur at a higher incidence among SLE patients. The investigation of thrombin generation (TG) with calibrated automated thrombogram (CAT) test as a global hemostasis assay is applicable for the overall functional assessment of the hemostasis. The aim of this study was to characterize the hemostatic alterations observed in SLE by CAT assay. In this study, CAT parameters and basic coagulation parameters of SLE patients (n = 22) and healthy control subjects (n = 34) were compared. CAT area under the curve (i.e.,endogenous thrombin potential) was lower than normal in SLE (807 vs. 1,159 nM*min, respectively), whereas other CAT parameters (peak, lag time, time to peak, and velocity index) and the basic coagulation tests were within the normal range. The presence of anti-phospholipid antibodies and the applied therapy was not associated with hemostasis parameters in SLE. We concluded that the reported high risk of thrombosis is not related to TG potential.

Procoagulant activity in gynaecological cancer patients; the effect of surgery and chemotherapy

BackgroundGynaecological cancers are associated with high rates of venous thromboembolism (VTE). Studies on ambulatory cancer patients do not support thromboprophylaxis during chemotherapy. Approximately 6–7% of gynaecological cancer patients suffer a postoperative VTE despite Low Molecular Weight Heparin prophylaxis (LMWH). Large cancer studies have shown that Calibrated Automated Thrombogram (CAT) and Microparticles (MP) assays may be useful in predicting VTE but data on gynaecological cancer patients is scarce. ObjectiveOur objective was to identify whether the CAT assay and MP functional assays have potential as biomarkers predictive of VTE in gynaecological cancer patients. Patients and methodsGynaecological cancer patients were investigated before surgery (n=146) and at 5, 14 and 42days post-surgery (n=78). Fourteen additional patients were investigated before chemotherapy and after 3 and 6 cycles of therapy. Thrombin generation was measured before and after addition of thrombomodulin. ResultsPatients with clear cell cancer (CCC) of the ovary and patients with endometrial cancer had higher ETP and peak thrombin compared with patients with benign disease. Patients who developed VTE (n=8) following surgery had enhanced thrombin generation prior to surgery which persisted during the post-operative period despite LMWH prophylaxis. Both neoadjuvant and adjuvant chemotherapy showed increased thrombin generation following addition of thrombomodulin. There were no differences in MP levels during the study. ConclusionsCAT assay shows potential as a promising biomarker for the prediction of VTE in gynaecological cancer patients. The identification of high risk patients combined with individualised LMWH prophylaxis might reduce VTE in this high risk group.

The Application of Thrombin Generation Test Is Compromised By Antithrombin III Deficiency

Background: Antithrombin III (ATIII) is a stoichiometric inhibitor of factor Xa and thrombin and a major physiological regulator of coagulation. Reduction of ATIII levels, for example in patients with congenital ATIII deficiency, causes a rise in active thrombin which is associated with risk of venous and arterial thrombosis (1, 2). The calibrated automated thrombogram (CAT) assay is widely used to measure thrombin generation in human plasma. Therewith, the fluorogenic substrate Z-G-G-R-AMC is cleaved by the thrombin emerging over time in the plasma sample. Thus, an increase in fluorescence signal reflects the amount of thrombin provided that fluorogenic substrate is in excess over thrombin. To account for substrate consumption, plasma color, and 'inner filter' effect, the fluorescence signal is compared to alpha-2-macroglobulin thrombin complex, the thrombin calibrator. In this complex, thrombin is protected from its physiological inhibitors, but retains its ability to cleave small substrates (3).Aim: We assessed the technical feasibility of measuring the effect of ATIII reduction on thrombin generation in normal and FVIII-inhibited human plasma in the tissue factor (TF) triggered CAT assay.Methods: CAT was performed in ATIII deficient plasma with 1 pM of TF to trigger thrombin generation. The plasma was supplemented with 0.125 - 2.5 µM ATIII to achieve ATIII plasma levels of 5 - 100% of normal. The ATIII titration was also done in ATIII deficient plasma inhibited with a goat polyclonal anti-human FVIII antibody to simulate hemophilia A conditions. Raw data analysis focused on the velocity of fluorescence signal increase, which is usually automatically converted to nM of thrombin by the Thrombinoscope software.Results: Reduced ATIII levels resulted in an apparent concentration dependent increase in thrombin generation in both normal and FVIII inhibited human plasma. For example, a reduction from 2.5 to 1.25 µM ATIII resulted in an apparent increase of 45 - 55% in thrombin peak and ETP values. Most importantly, analysis of the raw data showed that the fluorescence signal reached a plateau after 30 min (normal plasma) and 50 min (FVIII-inhibited plasma) in samples with <1.25 µM ATIII. The plateau (at ~1700 FU) is due to complete depletion of fluorescent substrate. In comparison, samples with ³1.25 µM ATIII did not reach a plateau and show substrate conversion until the end of the assay. In summary, a plasma level of at least 1.25 µM ATIII (50% of normal) was required to obtain thrombin generation profiles which were not compromised by fluorogenic substrate depletion. Below 1.25 µM ATIII, it is not possible to distinguish between inhibition of thrombin and excessive consumption of fluorescence substrate, and therefore, data may be misinterpreted.Conclusion: Thrombin generation at low ATIII plasma levels is difficult to interpret as the physiological inhibition of the generated thrombin is reduced. Consequently, the thrombin substrate is consumed, resulting in substrate depletion. Therefore, we conclude that the CAT assay is technically not suitable to assess the effect of ATIII levels < 1.25 µM ATIII corresponding to 50% of normal plasma. DisclosuresScheiflinger:Baxalta Innovations GmbH: Employment.

Alterations of the thrombin generation profile in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder resulting in the erosion of the cartilage and bone. Systemic involvement including the cardiovascular system with the risk of atherosclerosis may also occur. Calibrated automated thrombogram (CAT), a commercially available thrombin generation assay is suitable for the general assessment of the functionality of coagulation system. In this study we performed CAT assay in RA patients and in non-affected control subjects (matched for age, sex and comorbidities). Among the CAT parameters Velocity Index increased (from 60 to 83 nM/min), Lag Time and Time to Peak decreased (from 3.47 to 2.83 min and from 6.98 to 5.58 min respectively) in RA. On the other hand, Endogenous Thrombin Potential values decreased (from 1242 to 1108 nM min). The observed alterations were not associated with the applied therapy. These results indicate that the velocity of thrombin formation is increased, while the thrombin generating capability is reduced in RA.

174-POS

Objectives Venous thromboembolism (VTE) remains a leading cause of maternal mortality. An effective predictive test would be very clinically important. Measurements of Calibrated Automated Thrombogram (CAT) parameters in the presence and absence of thrombomodulin have been correlated with risk of recurrent VTE. D-Dimers and soluble P-selectin are markers of thrombosis. We aimed to evaluate the CAT assay, D-Dimers and P-selectin as potential predictive tools for thrombosis in pregnancy. Methods The SCOPE (Screening for Pregnancy Endpoints) study is a prospective, multicentre cohort study of nulliparous women with the primary aim of developing screening tests to predict pre-eclampsia, small for gestational age infants and spontaneous preterm birth. Twenty patients with documented VTE in pregnancy or postpartum and 61 controls were identified from the SCOPE cohort (total of 5000 pregnant women). The control group was matched for age, gestation, BMI and ethnicity. CAT assay with and without presence of thrombomodulin was performed on citrated plasma collected at 15 weeks gestation, prior to any thrombotic event. Normalised thrombomodulin sensitivity ratios (n-TMsr) were calculated. D-dimers and P-selectin were also measured by ELISA. Results There were no significant difference in ETP, peak thrombin, lagtime , time to peak, n-TMsr, D-dimer or P-selectin levels between patients who subsequently developed VTE compared with those who remained thrombosis free. Standardisation of CAT assay is needed to ensure it is a clinically effective predictive tool. Conclusions CAT assay, D-dimer and P-selectin levels are not useful as predictors of VTE in a low risk population of primigravida screened at 15 weeks. Disclosures S.K. Ismail: None. L. Norris: None. A. Khashan: None. N. Simpson: None. G. Dekker: None. J. Myers: None. L. McCowan: None. L. Kenny: None. J.R. Higgins: None.

Phospholipid‐dependent procoagulant activity is highly expressed by circulating microparticles in patients with essential thrombocythemia

This study evaluates the functional procoagulant features of plasma microparticle (MP) to explore the MP contribution to the hypercoagulable state of patients with essential thrombocythemia (ET). Platelet-free plasma samples were obtained from 73 ET patients (37 positive for the JAK2V617F mutation) and 72 control subjects. The calibrated automated thrombogram (CAT) was performed in plasma samples to determine thrombin generation of MP-associated tissue factor (TF) and procoagulant phospholipid (PPL) activity, and the STA Procoag PPL assay to measure MP-PPL activity only. Both thrombin generation and PPL procoagulant activities were found significantly elevated in ET patients compared to controls, and were associated to significantly higher levels of TF antigen and FVIIa/AT complex. Thrombin generation was significantly greater in JAK2-V617F positive compared to JAK2-V617F negative patients and normal subjects. Significant correlations were found between the PPL-assay and the different parameters of the CAT assay. No difference was seen between the thrombosis and no thrombosis group. Prospective studies are needed to test whether MP-associated thrombin generation and procoagulant activity may predict for thrombosis in these patients.