Calibrated Automated Thrombography Research Articles

Low molecular weight heparin decreases pro-coagulant activity in clinical MSC products

Background aimsMesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT). MethodsClinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load. ResultsPresence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher. DiscussionAll MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE.

Thrombosis in Young Women on Ocps Likely Is Caused By an Unmasking Effect of Estrogens on Underlying Thrombophilias

Background: Estrogen-containing oral contraceptives (OCPs) increase the risk for thrombosis 3-4 fold in otherwise healthy young women, although the absolute thrombosis rate in young OCP users is only 0.05% per year (Trenor CC, Pediatrics. 2011;127: 347). In our prospective cohort database 70% of lower extremity venous thromboembolism in adolescent girls were associated with OCP use, usually of recent onset (<6months). OCPs are associated with acquired resistance to activated protein C (APC), an important thrombin regulatory pathway, with an increase in fibrinolysis (Douxfils J, Contraception, 2020;102:396). A causative role for estrogens in thrombus formation in young women is not completely understood. Aims: This study evaluated thrombin generation, thrombin reduction by APC (activated protein C resistance, APCR), fibrinolysis and thrombophilia in healthy young males, healthy young females who were using or not using estrogen hormone therapy (i.e. oral contraceptive pills/OCPs) and young females with thrombosis who were using or not using OCPs at the time of thrombus presentation. The goal of this study was to measure the effects of OCPs on laboratory assays of coagulation and fibrinolysis to reveal potential mechanisms in increased thrombosis risk. Methods: This was an analysis of a consented, prospective cohort study (Thrombo-PICS). Healthy controls included men and women with no personal or family history of bleeding or clotting who were using or not using OCPs and on no other relevant medications. Study groups included females who presented with thrombosis with no OCP use, or OCP use for less than six months versus six or more months. All participants with a history of thrombosis were studied three or more months following the onset of thrombosis and no longer on OCPs. Laboratory assays performed included calibrated automated thrombography (CAT) with and without thrombomodulin (CAT TM) to determine thrombin generation (endogenous thrombin potential, ETP) and thrombin reduction with APC (% ETP reduction, APCR) respectively, and the euglobulin clot lysis time (ELT) to model fibrinolysis. Factors known to cause APCR and to be associated with thrombosis were also assayed, including factor V Leiden (FVL), the prothrombin gene mutation (PTM), protein C, protein S, lipoprotein(a), and the lupus anticoagulant (LA). Overall results were presented using descriptive statistics. Chi-Squared tests and paired t-tests were used to make paired comparisons. Parametric and non-parametric paired comparisons were used to evaluate group differences in ETP, % ETP reduction, ELT and presence thrombophilic traits relative to estrogen use and history of thrombosis. Results: Table 1 and Figure 1 display results. All groups of females with a history of thrombosis had decreased mean %ETP reduction compared to healthy females not on OCPs (p=0.02), with the lowest values occurring in females with thrombosis following less than six months of OCP use. There was also a lower prevalence of abnormal CAT TM values amongst healthy controls not on OCPs as compared to thrombosis patients not on OCPs (19 vs 51%, p=0.005).Healthy females on OCPs showed an increased ETP (p=0.002) and a marked reduction in ELT (p<0.001) as compared to all other groups. Females with a history of thrombosis, whether associated with OCPs or not, were similar to healthy females not using OCPs in regard to ETP and ELT. Furthermore, females who developed thrombosis on OCPs were more likely to have a thrombophilic trait than healthy controls on OCPs (48 vs 4%, p<0.001). Healthy men were found to have a greater %ETP reduction than healthy women (p=0.04), while having a lower ETP than healthy women (p=0.007). There were no differences in ELTs between healthy male and female controls (p=0.39). Discussion: OCPs were associated with increased thrombin generation, increased APCR and accelerated fibrinolysis in healthy female controls. The lower ELT in healthy controls using OCPs suggests that increased fibrinolysis may partially balance acquired APCR from estrogen use. We were unable to test fibrinolysis in females on OCPs prior to the onset of thrombosis. Females with thrombosis onset within six months of starting OCPs showed the highest prevalence of thrombophilic traits known to cause APCR. These data suggest that the APCR effect of OCPs potentially augments the prothrombotic effect of underlying thrombophilias in the promotion of clinical thrombosis.

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood.

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.

External validation of the MidiCAT variant of thrombography: Comparison with calibrated automated thrombography and study of the centrifugation scheme

To perform Calibrated Automated Thrombography (CAT), the use of reduced plasma volumes (referred to as "MidiCAT") makes it possible to more efficiently use limited volumes of valuable biobanked plasma samples and decreases expenses for reagents. It is, however, unclear whether the MidiCAT procedure is suitable when thrombin generation (TG) is studied in the presence of added thrombomodulin (TG-TM). Moreover, a simplified centrifugation scheme would facilitate biobanking, if appropriate, for more sensitive coagulation studies. We aimed to compare the results of "MidiCAT" (halved plasma and reagent volumes) with those from regular CAT, in the absence or presence of TM, as well as to study the impact of a single-centrifugation scheme for plasma preparation before freezing. Plasma samples were prepared from the citrated blood from 20 Geneva hospital diverse patients without gross coagulation abnormalities with a single- or double-centrifugation scheme. Samples were kept frozen at -80°C and thawed just before the TG assay in duplicate under two conditions: 1 pM tissue factor (TF) or 5 pM TF + TM. (1) We externally validated "MidiCAT" and also extended the validation to TG-TM. Whatever the method (CAT or MidiCAT), intra-assay (assessed with duplicates) CV was below 6% (1 pM TF) or below 10% (5 pM TF + TM) for ETP. Agreement between the MidiCAT and CAT results was satisfactory; the p coefficients were above 0.95 for ETP and above 0.90 for most other parameters; biases for ETP were +10.0% (1 pM FT) and +13.5% (5 pM + TM). (2) The centrifugation scheme markedly affected the results obtained in the presence of TM, whereas the bias and limit of agreement (difference plots) were low for the no TM condition. The bias in the presence of TM was obvious, more marked with plasma samples sensitive to TM when double centrifuged: the lower the ETP-TM, the greater the relative difference between the ETP-TM of plasma samples prepared with just single centrifugation and the reference plasma samples. Thus, a single-centrifugation procedure, as is often used for plasma biobanking, is suitable for TG study only if it is not performed in the presence of TM.

Thromboelastography and thrombin generation assessments for pediatric severe hemophilia A patients are highly variable and not predictive of clinical phenotypes

BackgroundSevere hemophilia A (SHA) patients vary in severity of bleeding, arthropathy, and requirements for replacement factor VIII (FVIII). Baseline hemostatic activity assays using calibrated automated thrombography (CAT) and thromboelastography (TEG) may offer insights into the physiological basis of clinical heterogeneity. ObjectivesUse CAT and TEG to measure baseline hemostatic activity in a cohort of 30 pediatric SHA patients with available clinical data. Determine effect of contact activation inhibition with corn trypsin inhibitor (CTI). Assess heterogeneity among patients for baseline hemostatic activity and examine correlations between assay results and clinical parameters including FVIII dosing regimen, von Willebrand factor level, and Pettersson arthropathy score. MethodsSHA blood after FVIII washout was subjected to TEG, and platelet‐rich (PRP) and platelet‐poor plasma was used for CAT assays. Varying concentrations of tissue factor (TF) were used. Statistical analysis examined relationships between assay results, and clinical parameters. ResultsCTI treatment was required to obtain TEG and CAT results representative of baseline hemostatic activity. Weak activity was observed in assays with low TF concentrations (0.5–2 pM), and most but not all samples approached normal activity levels at high TF concentrations (10–20 pM). A significant positive correlation was observed between results of TEG and CAT‐PRP assays. Correlations were not detected between hemostatic assay results and clinical parameters. ConclusionsIn vitro hemostatic assay results of samples containing platelets showed concordance. Assay results were not predictive of FVIII requirements or correlated with other clinical parameters. SHA patient heterogeneity is influenced by factors other than baseline hemostatic activity.

Thrombin generation by calibrated automated thrombography in goat plasma: Optimization of an assay

The goat model of atrial fibrillation (AF) allows investigation of the effect of AF on coagulation. However, assays for goat plasma are not available from commercial sources. Calibrated automated thrombography (CAT) provides a global view of the coagulation profile by assessing in vitro thrombin generation (TG). We describe the customization of the CAT assay in goat platelet‐poor plasma (PPP) and in factor Xa (FXa)‐inhibitor‐anticoagulated PPP. TG was initiated in the presence of phospholipids and either (a) PPP reagent, reagent low, or reagent high; (b) goat brain protein extraction (GBP); or (c) Russell's viper venom‐factor X activator (RVV‐X). Contact activation was assessed by adding corn trypsin inhibitor. Different concentrations of prothrombin complex concentrate (PCC) were used to determine the sensitivity of both the GBP and RVV‐X method. To obtain FXa‐inhibitor anticoagulated plasma, rivaroxaban was added to plasma. TG settings with human reagents were not suitable for goat plasma. TG triggered with GBP increased peak height and ETP values. Similarly, the RVV‐X method produced comparable TG curves and was more sensitive to PCC titration. Finally, both methods were able to detect the decrease in clotting potential induced by FXa inhibition. This is the first study that reports the customization of the CAT assay for goats. The GBP and RVV‐X methods were comparable in triggering TG in goat plasma. The RVV‐X method seemed to better discriminate changes in TG curves due to increases in clotting potential as well as to FXa inhibition by rivaroxaban in goat plasma.

The Effect of Platelets on Thrombin Generation in Cord Blood and Peripheral Neonatal Blood in the Premature Infant; The Event Study

IntroductionInfants born very preterm (<32 weeks) are at increased risk of haemorrhage, particularly intraventricular haemorrhage which can result in short and long term morbidity. Routine clinical laboratory tests such as the prothrombin time and activated partial thromboplastin time are frequently abnormal in this patient group but do not appear to correlate with clinical outcomes. Moreover, while reduced levels of circulating coagulation factors and platelet hypo-reactivity have been reported, plasma thrombin generation (TG), has been reported to be similar or even enhanced in very preterm infants when compared to term infants.Extracellular vesicles, comprising of lipid-bound nanoparticles released from cells (including platelets), may potentially play a role in modulating neonatal haemostasis. We aimed to characterize phospholipid (PL)-dependent plasma thrombin generation in platelet-rich (PRP) and platelet-poor plasma (PPP) in premature infants in both umbilical cord blood & peripheral neonatal blood using calibrated automated thrombography (CAT).MethodsIn this prospective observational study, PRP and PPP was prepared by centrifugation from citrated umbilical cord blood and peripheral neonatal blood collected from premature infants (24 - 31 weeks) and healthy term controls (>37 weeks). No samples were collected from heparinised lines. Parameters of plasma thrombin generation in PRP were assessed using CAT, with thrombin generation stimulated by tissue factor only (TF; final concentration 1pM). CAT was also repeated in PPP using only TF and no exogenous PL (rendering the assay dependent upon the endogenous PL content of plasma).ResultsIn the analysis of umbilical cord blood PRP, plasma thrombin generation was accelerated in the preterm infant group with a significantly shorter time to peak TG observed. The other parameters of TG were similar in both groups (Table 1).In the subset of infants from whom peripheral blood samples were available, there was further evidence of enhanced plasma TG in preterm PRP relative to term infants (Table 2). In this subgroup, the lag time and time to peak thrombin were significantly shorter in the preterm group and peak thrombin was significantly higher.TG was also assessed in both PPP and PRP prepared from umbilical cord blood samples in a subgroup of infants (n=10 term, n=6 preterm) using 1pM TF only, rendering the assay dependent on the phospholipid content of plasma. No difference was observed in any CAT parameters, suggesting that neonatal PPP phospholipid content (potentially from circulating extracellular vesicles) is sufficient to support thrombin generation in the absence of exogenous phospholipid.ConclusionThese preliminary data suggest that neonatal PRP, measured in both umbilical cord blood and peripheral neonatal blood, has similar thrombin generation or may even be hypercoagulable, compared with healthy term controls. Moreover, neonatal plasma phospholipid appears to support thrombin generation in the absence of exogenous phospholipid. This ongoing prospective study aims to further characterize the platelet-dependency of neonatal thrombin generation and evaluate the potential role of extracellular vesicles in neonatal haemostasis. [Display omitted] DisclosuresMaguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Daiichi-Sankyo: Research Funding; Bayer Pharma: Research Funding.

Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

IntroductionHematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment.We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. MethodsThis is a single-centre, case-control, non-interventionist, prospective study.During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed:Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway.Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software.TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA).The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. ResultsThose patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests.Table 1 shows the results obtained.Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI.Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI.Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. ConclusionsUBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient.Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes.TFPI inhibition might emerge as a good therapy for some of these patients.Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field.This work was supported by Novo Nordisk Pharma S.A.Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LTlagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. [Display omitted] DisclosuresAlvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Sphingosine-1-phosphate receptors modulate platelet activation and thrombin formation

Abstract Background The bioactive lipid sphingosine-1-phosphate (S1P) is known as a modulator of various cell functions, including cell proliferation, migration, adhesion and survival. Hence, S1P plays a crucial role in both inflammatory and hematopoietic regulations. It is also known that S1P affects key processes of hemostasis, as platelets store and upon activation release large amounts of the phospholipid. S1P acts through binding to its five G-coupled receptors (S1PR1–5). Accumulating evidence suggests that S1P is involved in mechanisms of atherogenesis linking platelet activation and thrombus formation to inflammatory responses. However, the precise role of S1P and its receptors during platelet activation and hemostasis are still under debate. Purpose Until today, the effects of S1P in hemostasis are poorly understood. The aim of this study is to investigate the functions of specific agonists and antagonists for each of the S1PRs 1–5 on platelet aggregation and thrombin generation. Methods Blood was obtained from healthy volunteers after written informed consent. For both platelet aggregation studies using light transmission aggregometry (LTA) and thrombin generation measurements quantified by calibrated automated thrombography (CAT), platelet-rich-plasma (PRP) was used. PRP was generated by centrifugation from whole blood. PRP was incubated with either 10 μM S1PR-agonists or -antagonists for 15 minutes. Subsequently, platelets were activated with adenosine diphosphate (ADP) or collagen to evaluate potential synergistic or antagonistic effects. Results Platelet aggregation weakens after 2–3 hours. As described in the literature, this is presumably due to activation effects and partial release of platelet granules. Determined by LTA, we found that pre-incubation with the S1PR1-agonist CYM5442 maintains and even elevates ADP- but not collagen-induced platelet aggregation for up to 1.5 h. This effect was reversed by co-incubation with the S1PR1-antagonist Ex26. We only observed this findings for S1PR1. No effect on platelet aggregation was detected when PRP was incubated with S1PR1–5-agonists or -antagonists (10 μM) alone. In comparison, none of the S1PR-agonists or -antagonists did directly affect thrombin formation in PRP measured by CAT. However, co-incubation with a S1PR4-agonist and ADP (5 μM) shortened the lag time to thrombin formation significantly. Furthermore, co-incubation with collagen (5 μg/mL) and agonists for S1PR1, 3 or 4 did reduce the time-to-peak (ttpeak) of thrombin formation. Conclusion Activation of S1PR1 maintains the ADP-induced platelet aggregation. In addition, co-activation of S1PR1, 3 or 4 and collagen enhances and accelerates thrombin generation. Consequently, S1P and its receptors appear to play an important role in different processes of hemostasis. Thus, S1PRs may be promising future therapeutic targets for diseases involving elevated platelet activation such as coronary heart disease or diabetes. Funding Acknowledgement Type of funding sources: None.

Measuring the Anticoagulation Effects of Exosite I Thrombin Inhibitors: Utility and Limitations of Clotting Assays, Thromboelastography, and Calibrated Automated Thrombography

The ability to anticoagulate patients at elevated risk for thrombosis without inducing a bleeding diathesis remains an unmet need of patient care both in a hospital setting and at home. Traditional anticoagulants including warfarin and heparin effectively inhibit clotting, but incur a bleeding risk and require continued monitoring after a stable profile has been established. Current generation direct oral anticoagulants (DOACs) including dabigatran, rivaroxaban, and apixaban overcome many of the liabilities of warfarin and heparin (e.g. lack of target specificity and need for monitoring), but are still susceptible to bleeding diathesis. Thus there remains a need for novel anticoagulants that offer an improved therapeutic index. The recombinant human antibody JNJ-64179375 (JNJ-9375) is a novel thrombin inhibitor that binds to the exosite I region with high affinity and selectivity. In contrast to DOACs that inhibit the protease active site, JNJ-9375 inhibits the interaction of thrombin with its substrates including fibrinogen and PAR-1 while retaining active site and exosite II mediated interactions. This targeted and distinct mechanism of action which modulates the actions of thrombin offers the potential for efficacious anticoagulation with the potential for reduced risk of bleeding compared to active site-specific DOACs.To assess the ability to measure exosite I-specific thrombin inhibitors effects in blood, we compared JNJ- 9375 to the active site ligand dabigatran in clotting assays, thromboelastography (TEG), and calibrated automated thrombography (CAT) assays. We focused on clinically relevant concentrations of these inhibitors based on the efficacious doses reported in the RE-LY study for dabigatran (NCT00262600) and a Phase 1 study for JNJ-9375 (NCT02949206). In conventional clotting assays (prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT)) dabigatran and JNJ-9375 each prolonged clotting times in a dose-dependent manner. TT assays were the most sensitive to dabigatran and JNJ-9375, with 80 nM dabigatran prolonging the clotting time by 5-fold and 100 nM JNJ-9375 prolonging the clot time 4-fold over vehicle control. PT and aPTT assays were less sensitive than the TT assay, requiring ≥ 300 nM dabigatran and ≥ 1 µM JNJ- 9375 to double the PT or aPTT clotting times. In TEG assays both JNJ-9375 and dabigatran prolonged the reaction time (R), with 200 nM JNJ-9375 prolonging R by 1.3- to 1.5-fold and 100 nM dabigatran prolonging R from 1.6- to 1.8-fold over buffer control. In CAT assays initiated with 5 pM tissue factor (PPP Reagent, Stago), dabigatran inhibited peak thrombin and Endogenous thrombin potential (ETP) with an approximate IC50 of 1 µM. In contrast, titration of JNJ-9375 up to 1 µM increased both the Peak thrombin and ETP ≥ 20% above buffer control. This increase in peak thrombin and ETP with JNJ-9375 was also observed with other exosite I-specific ligands such as hirugen, suggesting a general effect of exosite I binding on the primary endpoints of the CAT assay. This insensitivity of the CAT assay to exosite I ligands likely reflects the inability of these molecules to inhibit proteolysis of the peptidyl substrate z-Gly-Gly-Arg-AMC, which directly binds to the active site and is used as the readout of thrombin activity.In summary, of the assays tested the TT clotting assay offers the greatest sensitivity to detect multiple classes of direct thrombin inhibitors in plasma, while TEG can detect direct thrombin inhibitors in whole blood. In contrast, the CAT assay is insensitive to exosite I ligands such as JNJ-9375. These results highlight the importance of the relationship between the endpoint of a given coagulation assay and the mechanism of action of the anticoagulant being tested. DisclosuresBunce:Janssen Research & Development: Employment. Huang Devine:Janssen Research & Development: Employment. Chintala:Janssen Research & Development: Employment.

Laboratory Assays to Address the Problem of Heparin-Protamine Balance Following Neonatal Open Heart Surgery

Introduction: Neonates are especially susceptible to bleeding during and after cardiopulmonary bypass (CPB) surgery seemingly by virtue of a proportionately larger foreign surface exposure area for blood, significant hemodilution, and a coagulation system that differs from older patients. During CPB, heparin is administered to prevent thrombin formation. After CPB, it is reversed with protamine sulfate. Although administered to reverse anticoagulation, high levels of protamine sulfate paradoxically inhibit blood coagulation. This study evaluated the effect of heparin anticoagulation and protamine sulfate reversal on the accuracy of diagnostic coagulation tests and bleeding outcomes in a neonatal population undergoing CPB for congenital heart defects in the first 30 days of life.Methods: Blood samples were obtained at baseline (Draw 1), on CPB (Draw 2), following protamine reversal (Draw 3) and upon admission to the cardiac intensive care unit (Draw 4). Thrombin time (±protamine), FXa activity, activated partial thromboplastin time (APTT), Rotational Thromboelastometry (ROTEM) and Calibrated Automated Thrombography (CAT) were performed. Excessive bleeding was defined by meeting one or more of the following criteria: ≥ 84 mL/kg total chest tube output (CTO) in the first 24 hours, ≥ 7 mL/kg/hr CTO for ≥2 consecutive hours in the first 12 hours, or re-exploration in the first 24 hours for bleeding or tamponade.Results: This prospective observational study enrolled 44 neonates. Of these, 16 (36%) had excessive bleeding. The thrombin time assay was sensitive to heparin but was only minimally impacted by protamine sulfate, unless heparin was in the sample. Therefore, it was selected as the “gold-standard” assay for measurement of heparin. Relative heparin levels, determined by rate of fibrin formation, inversely correlated with thrombin generation measured using CAT, particularly in comparison to the thrombin generation rate in Draw 4 samples (exponential fit R2= 0.333). Higher plasma heparin concentration was present in 85% of the patients at Draw 4 than at Draw 3, indicating postoperative heparin rebound. This was important because, although the plasma heparin concentration in bleeders varied greatly, those patients with the two highest heparin concentrations at Draw 4 had excessive bleeding. The FXa activity assay was altered by protamine and by heparin. Therefore, samples with elevated FXa activity but normal thrombin time likely had high protamine. There were 4 such samples and 2 were bleeders. Additional coagulation assays were compared to the thrombin time assay to assess their performance for detection of heparin. ROTEM and APTT assays also correlated weakly (R2= 0.0531 with P value 0.1527 and R2 = 0.1549 with P value 0.0099, respectively) with the thrombin time assay at Draw 4. However, no correlation was present at Draw 3.Conclusions: Bleeding remains a common complication in neonates undergoing CPB for repair of heart defects. While there are many possible causes for the bleeding, excess heparin or protamine may contribute to its severity in a subset of cases. The simple to perform and inexpensive thrombin time test readily and accurately detects excess heparin. Increased use of this test may be beneficial, particularly for detection of heparin rebound that is observed in approximately 85% of patients. Excess protamine may also contribute to bleeding in these patients, and can be assessed using a combination of thrombin time and factor Xa activity assays. DisclosuresMast:Novo Nordisk: Research Funding.

Elevated anti-human factor Xa activity in rabbit and rodent plasma: Implications for preclinical assessment of human factor X in animal models of hemostasis

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma.

Coagulation Changes during Central Hypovolemia across Seasons.

Lower body negative pressure (LBNP) application simulates hemorrhage. We investigated how seasons affect coagulation values at rest and during LBNP. Healthy participants were tested in cold (November–April) and warm (May–October) months. Following a 30-min supine period, LBNP was started at −10 mmHg and increased by −10 mmHg every five minutes until a maximum of −40 mmHg. Recovery was for 10 min. Blood was collected at baseline, end of LBNP, and end of recovery. Hemostatic profiling included standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and thrombin formation markers. Seven men (25.0 ± 3.6 years, 79.7 ± 7.8 kg weight, 182.4 ± 3.3 cm height, and 23.8 ± 2.3 kg/m2 BMI) and six women (25.0 ± 2.4 years, 61.0 ± 8.4 kg weight, 167 ± 4.7 cm height, and 21.8 ± 2.4 kg/m2 BMI) participated. Baseline levels of prothrombin (FII), tissue factor (TF) and markers for thrombin generation F1+2 and the thrombin/antithrombin complex (TAT) were higher during summer. Factor VIII, prothrombin fragment 1+2 (F1+2), TAT and the coagulation time showed significant increases during LBNP in both seasons. Some calibrated automated thrombography variables (Calibrated automated thrombography (CAT): lag, time to peak (ttPeak), peak) shifted in a procoagulant direction during LBNP in summer. Red blood cell counts (RBC), hemoglobin and white blood cell counts (WBC) decreased during LBNP. LBNP application reduced prothrombin time in winter and activated partial thromboplastin time in summer. Greater levels of FII, TF, F1+2, and TAT—a more pronounced LBNP-induced procoagulative effect, especially in CAT parameters (lag time (LT), Peak, ttPeak, Velindex)—were seen in summer. These results could have substantial medical implications.

Hypercoagulability (thrombin generation) in patients with cirrhosis is detected with ST‐Genesia

BackgroundThrombin generation assays (TGAs) performed with calibrated automated thrombography (CAT) in the presence of thrombomodulin (TM) indicate plasma hypercoagulability in cirrhosis. ObjectiveTo evaluate, in the presence of TM, the new ST‐Genesia automated device developed for improving TGA vs the previously used CAT method, with plasma samples of patients with cirrhosis. Patients/MethodsPlatelet‐poor plasma samples were prepared from citrated blood samples of 52 healthy controls and 85 patients with cirrhosis (severity evaluated using the Child‐Pugh score [CP]). TGAs were performed using CAT with PPP‐Reagent and ST‐Genesia with the STG‐ThromboScreen reagent, in the presence of TM. Endogenous thrombin potential (ETP) was chosen as the main parameter. ResultsWhatever the method, ETP values were higher in patients than in healthy controls. All patients identified as hypercoagulable with ST‐Genesia and STG‐ThromboScreen were found hypercoagulable with CAT and PPP‐Reagent. Conversely, eight and ten patients in the CP‐A and CP‐B classes respectively were identified as hypercoagulable only with CAT. The use of ST‐Genesia with the STG‐ThromboScreen reagent with TM led to a bias, with higher ETP values for healthy controls and lower for patients compared with CAT. Crossover analysis (CAT with the STG‐ThromboScreen reagent) evidenced a substantial effect of the STG‐ThromboScreen reagent; the analyzer (including calibration and data analysis) plays a lesser role. ConclusionST‐Genesia evidences hypercoagulability in patients with cirrhosis when TG is studied in the presence of TM, but the results are not interchangeable with those obtained with CAT.

Delayed Thrombin Generation Is Associated with Minor Bleedings in Venous Thromboembolism Patients on Rivaroxaban: Usefulness of Calibrated Automated Thrombography.

Bleeding is the most feared and difficult to predict adverse event of anticoagulation. We sought to investigate whether calibrated automated thrombography (CAT) parameters are associated with minor bleeding (MB) in anticoagulated patients following venous thromboembolism (VTE). Enrolled were 132 patients on rivaroxaban, 145 on vitamin K antagonists (VKA) and 31 controls who stopped anticoagulation. Prior to the next dose of the anticoagulant, we measured CAT parameters, along with rivaroxaban concentration and INR. During a median follow-up of 10 months, we recorded minor and major bleedings. On rivaroxaban, 27 (20.5%) patients with MB had longer time to start thrombin generation, lower peak thrombin generation and lower endogenous thrombin potential compared with subjects without MB (all p < 0.001). All CAT parameters, except for peak thrombin generation (p = 0.049), were similar in VKA patients with (n = 25, 17.2%) vs. without MBs. By logistic regression, time to start thrombin generation (p = 0.007) and unprovoked VTE (p = 0.041) independently predicted MBs on rivaroxaban. Major bleedings were more frequent in patients with MBs (17.3% vs. 1.8%, p < 0.001). Abnormal CAT parameters characterize VTE patients prone to MBs on rivaroxaban, but not on VKA. Time to start thrombin generation measured about 24 h since the last rivaroxaban dose might help predict MBs.

A mathematical model of coagulation under flow identifies factor V as a modifier of thrombin generation in hemophilia A

BackgroundThe variability in bleeding patterns among individuals with hemophilia A, who have similar factor VIII (FVIII) levels, is significant and the origins are unknown. ObjectiveTo use a previously validated mathematical model of flow‐mediated coagulation as a screening tool to identify parameters that are most likely to enhance thrombin generation in the context of FVIII deficiency. MethodsWe performed a global sensitivity analysis (GSA) on our mathematical model to identify potential modifiers of thrombin generation. Candidates from the GSA were confirmed by calibrated automated thrombography (CAT) and flow assays on collagen‐tissue factor (TF) surfaces at a shear rate of 100 per second. ResultsSimulations identified low‐normal factor V (FV) (50%) as the strongest modifier, with additional thrombin enhancement when combined with high‐normal prothrombin (150%). Low‐normal FV levels or partial FV inhibition (60% activity) augmented thrombin generation in FVIII‐inhibited or FVIII‐deficient plasma in CAT. Partial FV inhibition (60%) boosted fibrin deposition in flow assays performed with whole blood from individuals with mild and moderate FVIII deficiencies. These effects were amplified by high‐normal prothrombin levels in both experimental models. ConclusionsThese results show that low‐normal FV levels can enhance thrombin generation in hemophilia A. Further explorations with the mathematical model suggest a potential mechanism: lowering FV reduces competition between FV and FVIII for factor Xa (FXa) on activated platelet surfaces (APS), which enhances FVIII activation and rescues thrombin generation in FVIII‐deficient blood.

Anticoagulant activity of fucosylated chondroitin sulfate isolated from Cucumaria syracusana

Fucosylated chondroitin sulfate (FCScs) isolated from sea cucumber Cucumaria syracusana was characterized by Fourier Transform InfraRed spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR) spectroscopy and high performance size exclusion chromatograph, a multi-angle laser light scattering detector, a viscometer and a differential refractive index (dRI) detector (HPSEC-MALLS-dRI). The anticoagulant activities of FCScs were studied by the classical clotting time assays and the purified systems containing thrombin and antithrombin or heparin cofactor II. The effect on thrombin generation was investigated using calibrated automated thrombography (CAT). The results obtained showed that the FCS with high sulfate content 31 % and relatively low average molecular weight of 36.3 kDa was isolated from C. syracusana in amount of ∼ 35.6 mg/g dry body wall. Structural analysis of this polysaccharide revealed the presence backbone structure of chondroitin sulfate chain branched by two types of fucose 2,4-O-di and 3,4-O-disulfated residues in respective ratios of 57.5 and 42.5 %. The FCScs exhibited a high anticoagulant activity mediated essentially by heparin cofactor II (HCII) and to lesser extent by antithrombin (AT) with IC50 values of 0.05 μg/mL and 0.09 μg/mL, respectively. Furthermore, the results of CAT assay showed that the velocity index decreases 3-times at 50 μg/mL in comparison with normal plasma. The overall results showed high anticoagulant activity attributed to the high sulfate content and abundance of disulfated fucose branches of FCScs which made it a promising candidate of anticoagulation drug.

Platelet-derived- Extracellular Vesicles Promote Hemostasis and Prevent the Development of Hemorrhagic Shock

Every year more than 500,000 deaths are attributed to trauma worldwide and severe hemorrhage is present in most of them. Transfused platelets have been shown to improve survival in trauma patients, although its mechanism is only partially known. Platelet derived-extracellular vesicles (PEVs) are small vesicles released from platelets upon activation and/or mechanical stimulation and many of the benefits attributed to platelets could be mediated through PEVs. Based on the available literature, we hypothesized that transfusion of human PEVs would promote hemostasis, reduce blood loss and attenuate the progression to hemorrhagic shock following severe trauma. In this study, platelet units from four different donors were centrifuged to separate platelets and PEVs. The pellets were washed to obtain plasma-free platelets to use in the rodent model. The supernatant was subjected to tangential flow filtration for isolation and purification of PEVs. PEVs were assessed by total count and particle size distribution by Nanoparticle Tracking Analysis (NTA) and characterized for cells of origin and expression of EV specific-surface and cytosolic markers by flow cytometry. The coagulation profile from PEVs was assessed by calibrated automated thrombography (CAT) and thromboelastography (TEG). A rat model of uncontrolled hemorrhage was used to compare the therapeutic effects of 8.7 × 108 fresh platelets (FPLT group, n = 8), 7.8 × 109 PEVs (PEV group, n = 8) or Vehicle (Control, n = 16) following severe trauma. The obtained pool of PEVs from 4 donors had a mean size of 101 ± 47 nm and expressed the platelet-specific surface marker CD41 and the EV specific markers CD9, CD61, CD63, CD81 and HSP90. All PEV isolates demonstrated a dose-dependent increase in the rate and amount of thrombin generated and overall clot strength. In vivo experiments demonstrated a 24% reduction in abdominal blood loss following liver trauma in the PEVs group when compared with the control group (9.9 ± 0.4 vs. 7.5 ± 0.5 mL, p < 0.001>). The PEV group also exhibited improved outcomes in blood pressure, lactate level, base excess and plasma protein concentration compared to the Control group. Fresh platelets failed to improve these endpoints when compared to Controls. Altogether, these results indicate that human PEVs provide pro-hemostatic support following uncontrolled bleeding. As an additional therapeutic effect, PEVs improve the outcome following severe trauma by maintaining hemodynamic stability and attenuating the development of ischemia, base deficit, and cardiovascular shock.

Rivaroxaban Effects Illustrate the Underestimated Importance of Activated Platelets in Thrombin Generation Assessed by Calibrated Automated Thrombography.

Background: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism. Methods: We compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban. Results: In the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve. Conclusions: AgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting.

Low Sensitivity to Thrombomodulin Correlates with Liver Stiffness in Advanced Chronic Liver Disease

Advanced chronic liver disease (ACLD) is characterized by changes in the coagulation system that embrace not only hypo-, but also hyper-coagulability. Global hemostatic tests such as thrombin generation (TG) assays are used to evaluate the hemostatic balance, in order to better assess bleeding and thrombotic risks. In addition, procoagulant state in ACLD patients has been demonstrated using modified TG assays with thrombomodulin (TM), a cofactor for protein C activation. Evaluation of the liver stiffness by transient elastography (Fibroscan) is a reliable method for the diagnosis of cirrhosis and disease staging. Increased liver stiffness is associated with clinically significant portal hypertension (PVT) in patients with ACLD. Here, we aimed to investigate whether TG parameters in patients with ACLD could be associated to liver stiffness. Of 147 enrolled patients with ACLD, we excluded 69 patients (5 patients received anticoagulation, 3 were on chemotherapy, 48 with other liver diseases and 13 had a history of orthotopic liver transplantation), leaving a study sample of 78 patients. Liver stiffness measurement (LSM) was performed by transient elastography in each patient. Cut-offs of 13 and 21 kPa were used to subgroup the studied ACLD patients sample, as a LSM of ≥13 kPa has been associated with the diagnosis of cirrhosis and ≥21 kPa with the development of a clinically significant PVT (Deillon et al., 2019). TG was evaluated by ST Genesia® Thrombin Generation System (STG) using Thromboscreen with/without TM (TS-TM/+TM) assay and the Calibrated Automated Thrombography (CAT) using PPP reagent low (Stago) with/without TM. TG was measured by STG in 50 patients with ACLD. ETP inhibition by TM correlated with LSM (r =-0.339, p=0.0184). The discriminant analysis (ANOVA with multiple comparison) among the study groups showed that ETP inhibition by TM was higher in ACLD patients with LSM &lt;13 kPa than in those with LSM ≥13 but &lt;21 kPa (p=0.003), indicating a lower sensitivity to TM in patients with liver stiffness values ≥13 but &lt;21 kPa. However, there was no difference in ETP inhibition by TM between the group with LSM ≥13 but &lt;21 kPa and the group with LSM ≥21 kPa (p=0.995). TG was measured by CAT in 56 patients with ACLD and ETP inhibition by TM was evaluated using a normalized ETP ratio in presence and in absence of TM. Similarly to STG data, ETP inhibition by TM measured by CAT correlated with LSM (r=0.4108, p=0.0018). The discriminant analysis among the study groups revealed that ETP inhibition by TM was higher in ACLD patients with LSM &lt;13 kPa than in those with ≥13 but &lt;21 kPa (p=0.0098) as well as with the group with LSM ≥21kPa (p=0.0022), indicating a lower sensitivity to TM in patients with liver stiffness values ≥13 kPa. However, there was no difference in ETP inhibition by TM between the group with LSM ≥13 but &lt;21 and the group with LSM ≥21 kPa (p=0.721). In addition, TG was measured by both by STG and CAT in 35 patients with ACLD in order to compare the two methods. ETP inhibition by TM measured by STG correlated with the normalized ETP inhibition determined by CAT (r = -0.61, p&lt;0.0001). Low sensitivity to TM measured in ST Genesia® and CAT, correlated with liver stiffness in patients with ACLD. Sensitivity to TM discriminated between ACLD groups with low (≤13 kPa) and high (≥13 kPa) LSM, confirming a procoagulant state in patients with ALCD. However, it did not differ between patients with ACLD with high LSM (≥13 and &lt;21 kPa) and patients prone to the decompensation defined by higher (≥21 kPa) LSM. Sensitivity to TM measurements determined by ST Genesia® correlated with those obtained with CAT. Further studies are needed to determine whether sensitivity to TM and LSM in patients with ACLD could help to identify patients at higher risk of portal vein thrombosis Disclosures No relevant conflicts of interest to declare.