Thrombin Generation Parameters Research Articles

A Snapshot of Coagulopathy After Cardiopulmonary Bypass.

Cardiac surgery involving cardiopulmonary bypass (CPB) is often associated with important blood loss, allogeneic blood product usage, morbidity, and mortality. Coagulopathy during CPB is complex, and the current lack of uniformity for triggers and hemostatic agents has led to a wide variability in bleeding treatment. The aim of this review is to provide a simplified picture of the data available on patients' coagulation status at the end of CPB in order to provide relevant information for the development of tailored transfusion algorithms. A nonsystematic literature review was carried out to identify changes in coagulation parameters during CPB. Both prothrombin time and activated partial thromboplastin time increased during CPB, by a median of 33.3% and 17.9%, respectively. However, there was marked variability across the published studies, indicating these tests may be unreliable for guiding hemostatic therapy. Some thrombin generation (TG) parameters were affected, as indicated by a median increase in TG lag time of 55.0%, a decrease in TG peak of 17.5%, and only a slight decrease in endogenous thrombin potential of 7%. The most affected parameters were fibrinogen levels and platelet count/function. Both plasma fibrinogen concentration and FIBTEM maximum clot firmness decreased during CPB (median change of 36.4% and 33.3%, respectively) as did platelet count (44.5%) and platelet component (34.2%). This review provides initial information regarding changes in coagulation parameters during CPB but highlights the variability in the reported results. Further studies are warranted to guide physicians on the parameters most appropriate to guide hemostatic therapy.

Correlative analysis of nanoparticle tracking, flow cytometric and functional measurements for circulating microvesicles in normal subjects

IntroductionCirculating microvesicles (MV) can be analysed using a number of different techniques. The aim of this study was to evaluate the correlation between functional procoagulant based assays including thrombin generation, factor Xa activation test (XaCT), and phosphatidylserine factor Xa-activity by ELISA with optical MV enumeration by flow cytometry and nanoparticle tracking analysis. MethodsCitrated blood samples were collected from 60 healthy volunteer blood donors after informed consent. Platelet free plasma was prepared using a standardized published protocol. MV subsets were enumerated by flow cytometry (BDFACS Canto) after staining with specific antibodies for platelets (CD41), endothelial cells (CD105), red cells (CD235) monocytes (CD14), tissue factor (CD142) and for phosphatidylserine expression by binding to annexin V. A standardized protocol using counting beads was employed. Nanotracking analysis was performed on both scatter and fluorescent settings after MV staining with quantum dot stain, Qdot 655. Procoagulant function was assessed by the XaCT assay on an automated coagulation analyser and by thrombin generation assay measuring endogenous thrombin potential (ETP), lagtime, peak (PEAK) and time to peak (ttPEAK) using a Calibrated Automated Thrombogram (CAT). The statistical analysis was carried out with Statistica 12 software using non-parametric tests (Spearman rank order correlations, with significance set at p<0.05). ResultsIn normal healthy subjects, thrombin generation parameters correlated with levels of MV measured by flow cytometry. ETP, lagtime, ttPEAK and PEAK correlated with MV expressing phosphatidylserine (rs, Spearman rank order correlation was 0.29, 0.40, 0.31 and 0.34 respectively, p<0.05), and MV expressing tissue factor (rs was 0.29, 0.40, 0.31 and 0.34 respectively, p<0.05), whilst red cell derived MV correlated with lagtime, ttPEAK and PEAK (rs, was 0.35,0.30 and 0.3, respectively, p<0.05). Lagtime and ttPEAK negatively correlated with the clot based XaCT test (rs, was −0.34 and −0.30 respectively, p<0.05) and positively correlated with the ELISA MP-activity assay (rs=0.42 for both, p<0.05). In addition, endothelial MV levels weakly correlated with white cell counts (rs = 0.27, p<0.05). ConclusionsThrombin generation and flow cytometry for phosphatidylserine or tissue factor expressing MV correlate well as markers for procoagulant activity. A combination of optical or non-optical enumeration as well as functional methods may be required for a complete profiling of circulating MV.

Abstract 531: In Patients with Uncontrolled Arterial Hypertension Platelets and Coagulation Factor XI are Responsible of Modifications of Thrombin Generation

Background: Hypertensive crisis is an extreme phenotype of increased blood pressure that can lead to organ failure and thrombotic complications. We recently found in experimental hypertension an angiotensin II driven factor XI (FXI)-thrombin amplification loop leading to vascular injury. Objective: We wanted to evaluate, in patients with uncontrolled hypertension (HT), the role of platelet and factor XI in thrombin generation (TG). Patients and methods: Prospectively, 52 patients with uncontrolled HT (16 grade I and 36 grade II/III) were examined at the outpatient clinic and emergency room of the University Medical Center Mainz, and 16 controls. Calibrated automated thrombography (CAT) was used to measure TG in platelet rich plasma (PRP, adjusted to 150.109/L of platelets), platelet free plasma (PFP), with platelets from controls and uncontrolled HT patients resupsended in a pool of healthy controls and in the presence or absence of an antibody blocking FXI activation pathway. Results: We found an increased systolic blood pressure in HT patients, compared to control patients (179±1.9 mmHg for grade II/III patients, 139±1.2 mmHg for grade I patients and 123±2.5 mmHg for controls); age, BMI as well as weight were not different. TG in PRP showed a positive and significant correlation between blood pressure and the other TG parameters as Endogenous thrombin potential (ETP), peak TG and velocity of TG. ETP was significantly increased in grade II/III patients compared to the control group. TG parameters in HT patients and controls were completely blunted by the inhibition of FXI pathway by blocking its apple 3 domain. This result indicates an involvement of the FXI thrombin loop in thrombin generation in PRP of uncontrolled HT patients. Without platelets TG showed no difference between HT patients and controls. Platelets resuspended from HT or controls restored the differences in ETP, peak and velocity between the 2 groups indicating that increased TG in HT patient mostly depends on platelet overreactivity. Conclusion: These results point out the important role of platelet overreactivity as well as FXI-dependent thrombin activation pathway in hypertension. Monitoring the prothrombotic state of platelets might add to risk stratification of patients with HT.

PO-55 - Individual variation in hemostatic alterations caused by tyrosine kinase inhibitors – a way to improve personalized cancer therapy?

During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients. To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis. We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations. At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients, see Figure1. Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

Alterations in coagulation following major liver resection.

The international normalised ratio is frequently raised in patients who have undergone major liver resection, and is assumed to represent a potential bleeding risk. However, these patients have an increased risk of venous thromboembolic events, despite conventional coagulation tests indicating hypocoagulability. This prospective, observational study of patients undergoing major hepatic resection analysed the serial changes in coagulation in the early postoperative period. Thrombin generation parameters and viscoelastic tests of coagulation (thromboelastometry) remained within normal ranges throughout the study period. Levels of the procoagulant factors II, V, VII and X initially fell, but V and X returned to or exceeded normal range by postoperative day five. Levels of factor VIII and Von Willebrand factor were significantly elevated from postoperative day one (p < 0.01). Levels of the anticoagulants, protein C and antithrombin remained significantly depressed on postoperative day five (p = 0.01). Overall, the imbalance between pro- and anticoagulant factors suggested a prothrombotic environment in the early postoperative period.

Rare coagulation disorders: an emerging issue.

Rare coagulation disorders: an emerging issue.

Paired comparison of methylene blue- and amotosalen-treated plasma and cryoprecipitate.

Cryoprecipitate is used in the treatment of patients with acquired hypofibrinogenaemia. Studies have not directly compared cryoprecipitate produced following pathogen inactivation (PI) of fresh-frozen plasma (FFP) using different systems. The effects of methylene blue (MB) and amotosalen (AS) PI systems on the quality of FFP and cryoprecipitate were investigated in a paired study. Seven group A and 7 group O pools of plasma were prepared and split into individual units and rapidly frozen to produce FFP. Units of FFP were thawed and either PI treated with MB or amotosalen, or left untreated (control). Samples of FFP along with the corresponding cryoprecipitate were tested for a range of coagulation factors, thrombin generation (TGT) and rotational thromboelastometry (ROTEM). AS-FFP showed a smaller decrease following treatment for most coagulation factors analysed than MB-FFP, except fibrinogen (antigen) and factor VII, partly due to lower volume losses. There was no significant difference between treatment methods for fibrinogen content of cryoprecipitate with treated units meeting current UK specification, or TGT and ROTEM parameters studied. MB-cryo contained a significantly higher content of FVIII and lower content of FXIII when compared to AS-cryo, with no difference in fibrinogen activity.

Computational simulation and comparison of prothrombin complex concentrate dosing schemes for warfarin reversal in cardiac surgery

Prothrombin complex concentrate (PCC) is increasingly used for acute warfarin reversal. We hypothesized that computational modeling of thrombin generation (TG) could be used to optimize the timing and dose of PCC during hemodilution induced by cardiopulmonary bypass (CPB). Thrombin generation patterns were modeled in anticoagulated patients (n=59) using a published computational model. Four dosing schemes were evaluated including single full dose (median, 41.2IU/kg) of PCC before or after CPB, ½-dose before and after CPB, or 1/3-dose before CPB plus 2/3-dose after CPB. Hemodilution was modeled as 40 or 60% dilution of factors from baseline. The lag time (s) of TG, and peak thrombin level (nM) were evaluated. Prolonged lag time, and reduced peak TG were due to low vitamin K-dependent (VKD) factors, and pre-CPB PCC dose-dependently restored TG to near-normal or normal range. After 40% dilution, TG parameters were similar among 4 regimens at the end of therapy. The recovery of VKD factors was less when PCC was given before CPBafter 60 % dilution, but TG parameters were considered hemostatically effective (>200nM) with any regimen. Withholding the full dose of PCC until post-CPB resulted in severely depressed TG peak (median, 47nM) after 60 % dilution, and some supra-normal TG peaks after treatment. Pre-CPB administration of full or divided doses of PCC prevents extremely low TG peak during surgery, and maintains hemostatic TG peaks in both 40 and 60% hemodilution models. Although PCC's hemostatic activity appears to be highest using the full dose after CPB, hypercoagulability may develop in some cases.

Comparability of “Enoxamed” a Tunisian Generic Enoxaparin with the Originator Product: Non-clinical and Clinical Studies

Enoxaparin is a complex, biologically derived low-molecular-weight heparin. The manufacturing process for biologics is complex which makes difficult to obtain exact replicas of the reference biologic. This is why there is a critical need to ensure that generics of biologic medicines are safe and effective. This study was carried out to assess the comparability of a Tunisian generic enoxaparin (Enoxamed®, Unimed Laboratory, Tunisia) with the originator product (Lovenox®; Sanofi US, Bridgewater, New Jersey) through non-clinical in vitro study in healthy volunteers and clinical studies (phases III and IV) in patients with acute coronary syndrome (ACS). For non-clinical study, blood from healthy volunteers was used to compare the effect of both formulations using anti-Xa activity and the thrombin generation test (TGT). All parameters of TGT were analyzed. For clinical studies, ACS patients were randomly assigned to receive Enoxamed® (27 in phase III and 120 in phase IV) or Lovenox® (23 in phase III and 118 in phase IV) and anti-Xa activity was measured 4 h thereafter. In healthy volunteers, the two products inhibited thrombin generation in a concentration-dependent manner. According to the profiles obtained from the TGT, Enoxamed® had similar potency as Lovenox®. In ACS patients, anti-Xa activity was found no difference between Enoxamed® and Lovenox®. No difference in major cardiovascular events was observed at 30 days after initial admission. Our finding combining anti-Xa activity and thrombin generation parameters would support the possibility to translate this biological efficacy to the clinical setting. The generic enoxaparin Enoxamed® showed comparability and then the main regulatory criteria of bioequivalence with the originator product.

Influence of protein Z plasma level on thrombin generation assay

Introduction: Protein Z (PZ) is a vitamin K-dependent factor, involved as a cofactor of PZ-dependent protease inhibitor (ZPI) that inhibits the activated factor X on phospholipid surface. A severe PZ deficiency could be associated with ischemic arterial diseases. The aim of this study was to evaluate the influence of PZ plasma levels on thrombin generation (TG) and to detect a hypercoagulable state in patients with PZ deficiency.Patients and Methods: Young patients with personal history of arterial thrombosis and PZ deficiency were included. PZ concentrations were assessed using ELISA method. TG assay was performed using PPP containing 5 pM or 1 pM tissue factor and 4 μM phospholipids with and without thrombomodulin. Then, we focussed on the influence of successive overloads with purified PZ in two patients with PZ deficiency (Pat1PZdef and Pat2PZdef) and industrial PZ deficiency (IndPZdef).Results: First, in 13 patients with PZ deficiency, none influence of PZ level was reported on TG assay parameters. Second, TG profiles performed on Pat1PZdef, Pat2PZdef and IndPZdef were close to the reference TG profile. In presence of TM, there was no difference in TG parameters according to PZ plasma levels. After each overload, real PZ plasma levels were underestimated; differences in standards of purified PZ between the two manufacturers might explain such results.Conclusion: The absence of influence of PZ concentration on TG assay is reliable to the absence of well-demonstrated clinical consequences of PZ deficiency in arterial thrombosis. Clinical consequences of protein Z deficiency warranted further studies.

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Upregulation of Microparticles, Tissue Factor, Adhesion Molecules, Nitric Oxide and Adiponectin in End Stage Renal Disease

Introduction: End stage renal disease (ESRD) represents the final stage of chronic kidney disease characterized by kidney failure (GFR <15 mL/min/1.73 m2). To understand the pathophysiology of hemostatic dysregulation in ESRD, we measured the circulating levels of microparticles (MP), tissue factor (TF), adhesion molecules, such as p-selectin (P-Sel), soluble ICAM (s-ICAM), nitric oxide (NO) and adiponectin (AD).Methods: Citrated blood plasma samples were collected from 119 ESRD patients undergoing maintenance hemodialysis to profile various thrombotic inflammatory biomarkers. 100 normal plasma samples were collected from healthy individuals and were used as controls. MP levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France). NO were measured using a kit from R&D systems (Minneapolis, Minnesota) and ELISA based methods for TF, P-Sel, s-ICAM and adiponectin were obtained from R&D systems. Chromogenic substrate methods were used to measure the heparin levels and thrombin generation.Results: MP levels were elevated in the ESRD group (28.1nm + 6.1nm vs. the control 8.9nm +1.3nm). Tissue factor levels were found to be increased in the ESRD group (20.4±6.1pg/ml) vs the control (11.9±2.8pg/ml). The nitric oxide level was markedly higher in the ESRD group (32±17uM) vs the controls (7±3uM). The p-selectin levels were also elevated in the ESRD group (46±20ng/ml) vs the control (31±3ng/ml). The soluble ICAM levels were higher in the ESRD group (250±112ng/ml) vs the control (180±19ng/ml). Interestingly, the adiponectin levels were also increased in the ESRD group (19.2±9.3ug/ml) vs the control (11.2±4.1ug/ml). Measured variations in thrombin generation parameters were noted. Detectable levels of heparin (.05 - 0.20 U/ml) were present in the ESRD group.Summary/Conclusions: These studies suggest that MP, TF, NO, p-selectin and s-ICAM levels are increased in the ESRD patient. It is of interest to note that despite that a significant number of ESRD patients were diabetic; the AD levels were increased in this group. These results also suggest that while ESRD represents a pro-inflammatory/hypercoagulable state, the repeated administration of heparin and other drugs may contribute to the regulation of the hemostatic process and inflammatory balance in these patients. DisclosuresNo relevant conflicts of interest to declare.

Thrombin Generation in Children after Acute Venous Thromboembolism

Introduction: Venous thromboembolism (VTE) is a major and growing problem in children. The annual rate of VTE has increased by 70% in the U.S hospitalized children in the past decade. Accompanying this increase in VTE is the attendant increase in chronic, post-thrombotic sequeale of VTE. Strategies to identify children at high risk of developing poor outcomes after initial management of VTE are unclear. We hypothesized that 'global coagulation assessment' may identify children likely to suffer from poor thrombosis outcomes following an initial event. In this pilot study, we therefore, evaluated thrombin generation (TG) after an acute episode of provoked VTE in children. Methods: Baseline patient, thrombus characteristics and thrombophilia testing were prospectively collected for 50 consecutive children with acute, provoked VTE after informed consent. Subjects with cancer, protein C, protein S and antithrombin deficiency, persistent antiphospholipid positivity and inflammatory disorders were excluded. Thrombin generation, using corn trypsin inhibited treated plasma by calibrated automated thrombogram was measured in the presence and absence of thrombomodulin (TM), 4 weeks after completion of anticoagulation therapy, and compared to 50 healthy controls. All cases were treated according to the 9th edition of American College of Chest Physicians Clinical Practice Guidelines. Results: The mean age of VTE cases was 14 years (range: 6 weeks to 18 years). Two thirds (33/50) of VTE cases consisted of deep venous thrombosis of the limbs and/or pulmonary embolism, and nearly 20% (10/50) were cerebral sinovenous thrombosis. Thrombin generation of subjects showed significantly higher endogenous thrombin potential (ETP) compared to healthy controls (1150 ± 281 vs. 763 ± 274 nM.min; p &lt;0.001). There were no significant differences in the other TG parameters (lag time and thrombin peak) between VTE cases and healthy controls. Inhibition by TM was attenuated in patients compared to controls (22% vs. 45.5% p&lt;0.001). A significant negative correlation was found between those with elevated FVIII levels (&gt;150%) at end of therapy and TM induced reduction of ETP (r=0.297 vs. 0.171; p&lt;0.01). In conclusion, a novel finding in children with provoked VTE was persistent elevation of TG after successful completion of anticoagulation therapy. A role for FVIII as an important cause of hypercoagulability after anticoagulation therapy is reflected by the reduced inhibitory effect of TM at high FVIII levels. Further studies are ongoing to determine the utility of TG as a biomarker for predicting poor outcomes after VTE in children. Disclosures Journeycake: CSL, Baxalta, NovoNordisk: Consultancy; ATHN: Research Funding; Biogen: Speakers Bureau; ATHN: Membership on an entity's Board of Directors or advisory committees.

Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG.Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography.Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket.We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma.Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresBos:Bayer Hemophilia Awards: Research Funding.

Comparison of platelet‐derived and plasma factor VIII efficacy using a novel native whole blood thrombin generation assay

We have recently developed a successful gene therapy approach for hemophilia A in which factor VIII (FVIII) expression is targeted to platelets by the αIIb promoter. Levels of platelet-expressed FVIII (2bF8) achieved by gene therapy may vary between individuals due to differences in ex vivo transduction and gene expression efficiency. Accurate assays to evaluate 2bF8 efficacy are desirable. To compare the hemostatic efficacy of 2bF8 with replacement therapy over a wide therapeutic dose range. Efficacy of 2bF8 was assessed using a new transgenic mouse model expressing high 2bF8 levels (LV18(tg) ). Blood from LV18(tg) mice or FVIII(null) mice infused with recombinant FVIII was mixed with FVIII(null) blood at different ratios ex vivo to achieve several concentrations of 2bF8 or plasma FVIII. Samples were evaluated with a novel native whole blood thrombin generation assay that uses recalcified whole blood without the addition of tissue factor to initiate coagulation. FVIII dose dependency was observed in all five thrombin generation parameters. While the total amount of thrombin generated was similar, 2bF8 significantly accelerated thrombin generation compared with plasma FVIII. Remarkably, a 10-fold lower dose of 2bF8 than plasma FVIII (0.2% vs. 2%) significantly shortened the onset and peak of thrombin generation compared with FVIII(null) blood. Using a new transgenic mouse model, we showed that the novel native whole blood thrombin generation assay established here can be used to monitor platelet targeted FVIII gene therapy. The higher therapeutic efficacy of 2bF8 compared with factor replacement therapy seemed to be due to acceleration of thrombin generation.

Interconnectedness of global hemostasis assay parameters in simultaneously evaluated thrombin generation, fibrin generation and clot lysis in normal plasma

BackgroundFluorogenic thrombin generation (TG) assays and turbidity-based fibrin generation (FG)- and fibrinolysis (FL)-resistance assays have been sought to assess bleeding and clotting disorders. Theoretically, TG, FG and FL tests should provide overlapping information because thrombin is responsible for FG and induces protection from FL. The relationships between TG, FG and FL parameters remain poorly investigated, partly because existing experimental systems do not permit simultaneous detection of both TG and FG in the same sample of plasma, and are instead tested in separate experiments. Objectives and methodsWe evaluated the potential benefits of a combined TG/FG/FL assay by testing responses of normal plasma to a wide range of tissue factor (TF) and tissue plasminogen activator (tPA) concentrations. Correlations between multiple parameters extracted from the TG and FG/FL curves were also compared. ResultsRate of FG correlated well with TG peak height at all TF concentrations, but correlations between TG and FL parameters depended on the TF concentration. Without thrombomodulin, all FG/FL parameters at high TF could be predicted from TG parameters and no FL protection was observed. With thrombomodulin and high TF, TF-dependent FL protection did not correlate with TF-dependent TG. The fluorogenic thrombin substrate did not interfere with optical density readings, and meaningful tPA concentrations did not interfere with TG readings. ConclusionsIn normal plasma, TG, FG and FL parameters may provide interchangeable information. Evaluation of FL-resistance may provide additional data under special assay conditions, but the value of this information should be studied under disease conditions.

In vitro comparison of the effect of two factor XI (FXI) concentrates on thrombin generation in major FXI deficiency

Bleeding risk in factor XI (FXI) deficiency following surgery may be reduced by treatment with either of two FXI concentrates, but indications for their use are unclear and treatment has been associated with thrombosis. To quantify and compare the effects of two different FXI concentrates on thrombin generation (TG) in major FXI deficiency (FXI:C < 15 IU dL(-1) ). Thrombin generation was measured in controls (n = 50), FXI-deficient individuals pre and post in vitro spiking with FXI concentrates (n = 10), and in ex vivo samples following treatment with FXI concentrate (n = 3). Thrombin generation was significantly impaired in FXI deficiency but improved following FXI replacement in vitro and in vivo. LFB Hemoleven(®) had greater effect on TG than BPL FXI concentrate in vitro (equivalent in vivo doses 10, 20 and 30 U kg(-1) ): higher endogenous thrombin potential (ETP) (P < 0.0001), peak height (P < 0.01) velocity (P < 0.0002) and shorter lag time and time to peak (both P < 0.003). Some measurements with LFB Hemoleven(®) exceeded the reference range. At lower dose (5 U kg(-1) ), BPL FXI concentrate normalized all TG parameters and LFB Hemoleven(®) normalized the ETP but exceeded the reference range with other parameters. Both FXI concentrates improve TG in vitro in major FXI deficiency but differ in dose response, and for both products, doses lower than previously recommended normalized TG in vitro. Comparison of in vitro spiked and ex vivo samples suggest that in vitro results could be used to estimate an expected in vivo response to FXI replacement.

Thrombin generation and international normalized ratio in inherited thrombophilia patients receiving thromboprophylactic therapy

Thrombin generation assay (TGA) is useful as a global functional test for assessing bleeding or thrombotic risk and its modification with therapy. We investigated TGA to assess anticoagulation status compared with the international normalized ratio (INR) system in patients with primary thrombophilia receiving and not undergoing thromboprophylaxis. We studied 50 patients with at least one thrombotic event and a confirmed diagnosis of inherited thrombophilia. Thrombin generation was measured in platelet-poor plasma by calibrated automated thrombography (CAT). Patients in optimal anticoagulation (INR: 2.0-3.0) showed an endogenous thrombin potential (ETP) of 14-56% of normal and a peak of 18-55% of normal. A significant inverse relationship between INR and thrombin generation parameters (ETP, peak and velocity index) and a linear correlation for lag time was found in patients treated with vitamin-K antagonists (VKA). Receiver-operating characteristics (ROC) analysis showed that the optimal cutoff for ETP was 1600.2 nM · min (111.6% of normal, with a sensitivity of 96.6% and a specificity of 92.9%) and for the peak was 298.3 nM (112.1% of normal, with a sensitivity of 96.4% and a specificity of 100%). According to this analysis, ETP was able to identify patients with increased thrombotic and hemorrhagic risk, correlating with severe clinical complications. TGA showed excellent sensitivity and specificity for assessing anticoagulation status in patients with primary thrombophilia receiving VKA, with significant advantages with regard to INR. Clinical data strongly support ETP as a valuable indicator of thrombotic or hemorrhagic risk in patients receiving or not receiving thromboprophylaxis.

Vitamin K antagonists favourably modulate fibrin clot properties in patients with atrial fibrillation as early as after 3 days of treatment: Relation to coagulation factors and thrombin generation

IntroductionAtrial fibrillation (AF) increases the risk of thromboembolism that is reduced by vitamin K antagonists (VKAs). We sought to investigate changes in plasma fibrin clot phenotype at the onset of oral anticoagulation. Materials and methodsForty consecutive AF patients (aged 45–83 years, CHA2DS2-VASc score 3.0±1.5) who started therapy with warfarin or acenocoumarol were studied. Plasma fibrin clot permeability (Ks), clot lysis time (CLT), along with clotting factors (F), thrombin generation (TG) profiles and protein C (PC) levels were determined on days 3, 5, 7, 28 and 56±1 since the first dose. ResultsAF patients had 16% higher median of Ks and 15% lower median of CLT as early as on day 3 of VKA therapy compared with the baseline (both p<0.001), reaching the plateau values on day 7 and 5, respectively. Higher Ks values on days 1 and 3 were found in AF patients with further stable anticoagulation (both p<0.05). Moreover, FIX explained 32% of the total variability in Ks. Multivariate analysis adjusted for potential confounders including time as a predictor showed that vitamin K-dependent (VKD) factors, PC and TG parameters were the predictors of Ks (all p<0.0001), while only the lag phase of TG and thrombin peak predicted CLT (both p<0.05) in AF patients. Regression analysis of time-series showed however, that CLT was also predicted by VKD factors and PC (all p<0.05). ConclusionsPlasma fibrin clot properties in AF patients are favourably modified as early as after 3days of VKA administration, which might contribute to antithrombotic effectiveness.

Hypercoagulability in adolescent girls on oral contraceptives-global coagulation profile and estrogen receptor polymorphisms.

C after being on OCPs. Protein S levels decreased with OCPs. Sixty percent of participants showed evidence of hypercoagulability on TG testing on OCPs. Higher thrombin peak and endogenous thrombin potential (ETP) were seen on TG after OCPs. With thrombomodulin, ETP and thrombin peak did not decrease after OCPs, signifying 'thrombomodulin resistance'. We demonstrated that OCPs induce a state of "variable" hypercoagulability in adolescents, predominantly through the protein S pathway. Genetic and nongenetic factors may account for the variable increase in hypercoagulability. Further research is needed to understand this.