Endogenous Thrombin Generation Research Articles

ENDOGENOUS THROMBIN GENERATION POTENTIAL IN PATIENTS WITH PULMONARY EMBOLISM AND ITS RELEVANCE TO THROMBOTIC AND INFLAMMATORY BIOMARKERS

SESSION TITLE: Pulmonary Vascular Disease Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Pulmonary embolism (PE) is a catastrophic outcome in patients with venous thrombosis. In the United States around 900,000 people (1 to 2 per 1,000) patients are affected each year with the mortality rate ranging from 10 – 30 %. Inflammation, hemostatic dysregulation and vascular complications contribute to the pathogenesis of PE. Endogenous thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) and the circulating biomarkers of inflammation, hemostatic activation and endothelial dysfunction in plasma samples obtained from the patients at the onset of PE. METHODS: Citrated blood samples from 78 patients with confirmed PE were collected at Loyola University Medical Center and Gottlieb Memorial Hospital under an IRB approved protocol. Normal human plasma (NHP) comprised of 25 male and 25 female samples were obtained from a commercial source (George King Biomedical, Overland park, KS.) Thrombin generation studies were carried out using a commercially available a kinetic system (Diagnostica Stago, Paris, France). Thrombin generation parameters such as peak thrombin, lag time and area under the curve (AUC) were compiled. Frozen samples were retrospectively analyzed for biomarkers, including D-Dimer, PAI-1, TNFa, IL6, IL1b, VEGF and TFPI using commercially available sandwich ELISA methods. Results were compiled as mean ± SD and analyzed for significance and correlation. Applicable statistical analysis was carried out by using GraphPad Prism software. RESULTS: On a cumulative basis, PE patients showed a wide variation in thrombin generation parameters. Peak thrombin levels (91.1 nM vs 160.0 nM) and AUC (554.7 nM*min vs 700.9 nM*min) were decreased in PE patients while lag time was increased (6.41 min vs 2.7 min). Biomarkers analysis showed elevated levels of D-Dimer (7521.3 ng/ml) in comparison to NHP (192.8 ng/ml), PAI (67.3 ng/ml) in comparison to NHP (10.5 ng/ml), TNF-a (3.2 pg/ml) in comparison to NHP (1.9 pg/ml), IL6 (99.9 pg/ml) in comparison to NHP (1.24 pg/ml), IL1b (1.6 pg/ml) in comparison to NHP (0.79 pg/ml) and TFPI (139.1ng/ml) in comparison to NHP (62.3 ng/ml) while and VEGF (31.24 pg/ml) levels were decreased in comparison to NHP (45.5 pg/ml. The biomarker levels did not show any correlation with peak thrombin levels with the exception of VEGF and TFPI. Samples with higher thrombin generation (>150 nM) showed marked elevation of all of the biomarkers studied. CONCLUSIONS: PE patients exhibit a wide variation in endogenous thrombin generation parameters. However, biomarkers of hemostatic activation, vascular dysfunction and inflammation were increased. CLINICAL IMPLICATIONS: Measurement of thrombin generation parameters along with the biomarkers of inflammation and endothelial dysfunctions may be useful in the overall management of PE patients. DISCLOSURES: No relevant relationships by Emily Bontekoe, source=Web Response No relevant relationships by Yevgeniy Brailovsky, source=Web Response No relevant relationships by Amir Darki, source=Web Response No relevant relationships by Jawed Fareed, source=Web Response No relevant relationships by ambar Farooqui, source=Web Response No relevant relationships by Debra Hoppensteadt, source=Web Response No relevant relationships by Omer Iqbal, source=Web Response No relevant relationships by Fakiha Siddiqui, source=Web Response

High-intensity interval training recuperates capacity of endogenous thrombin generation in heart failure patients with reduced ejection fraction

ObjectiveConsumptive coagulopathy is associated with increased mortality in patients with heart failure (HF). Physical activity influences the risk of major vascular thrombotic events. This study investigates how high-intensity interval training (HIIT) affects the capacity of endogenous thrombin generation (TG) by modulating circulatory procoagulant microparticles (MPs) in HF patients. MethodsThirty-eight HF patients with reduced ejection fraction (HFrEF) and 38 age- and gender-matched normal counterparts (NC) were recruited into this study. The HFrEF group performed HIIT (3-min intervals at 40% and 80%VO2peak) on a bicycle ergometer for 30 min/day, 3 days/week for 12 weeks, whereas the NC group did not receive any form of intervention. Plasma TG kinetics, procoagulant MPs, coagulation-related factors, and oxidative stress/proinflammatory status were analyzed. ResultsThe HFrEF group exhibited (i) less endogenous thrombin potential (ETP) and TG rate, (ii) lower concentration/activity of tissue factor (TF) and counts of TF-rich MPs derived from blood cells, and (iii) higher vascular endothelial shedding and plasma myeloperoxidase and interleukin-6 concentrations, compared to the NC group did. However, HIIT elevated TG rate and TF concentration/activity in plasma, as well as, TF-rich MP counts derived from blood cells in patients with HFrEF. Moreover, the exercise regimen also decreased vascular endothelial shedding and plasma myeloperoxidase and interleukin-6 concentrations in HFrEF patients. ConclusionHFrEF reduces the capacity of endogenous TG in plasma, which is associated with decreased (or consumed) circulatory procoagulant MP levels. However, HIIT alleviates HFrEF-declined endogenous TG capacity and vascular endothelial damage through recuperating TF-related coagulation activity and suppressing oxidative stress/proinflammatory status.

Thrombin generation and fibrin clot structure after vitamin D supplementation.

ObjectiveVitamin D deficiency is associated with increased risks of arterial and venous cardiovascular events. Hypothetically, supplementation with vitamin D may lead to a less prothrombotic phenotype, as measured by global coagulation assays and fibrin clot structure.MethodsIn this prospective cohort study, we enrolled adult outpatients attending the Primary Care Division of the Geneva University Hospitals with a severe vitamin D deficiency (25-hydroxyvitamin-D3 (25-OHD) <25 nmol/L), excluding obese patients or with a recent acute medical event. We evaluated changes in coagulation times, thrombin generation assay, clot formation and clot lysis time, 25-OHD and parathormone before and 1–3 months after cholecalciferol oral supplementation with one-time 300,000 IU then 800 IU daily. Paired t-tests with a two-sided alpha of 0.05 compared absolute mean differences.ResultsThe 48 participants had a mean age of 43.8 ± 13.8 years. After supplementation, 25-OHD levels increased from 17.9 ± 4.6 nmol/L to 62.5 ± 20.7 nmol/L 6.4 ± 3.0 weeks after inclusion. Endogenous thrombin potential and thrombin generation peak values both decreased significantly (−95.4 nM × min (95%CI −127.9 to −62.8), P < 0.001; −15.1 nM (−23.3 to −6.8), P < 0.001). The maximum absorbance by turbidimetry decreased significantly (P = 0.001) after supplementation. There was no change in clot lysis time, coagulation times or plasminogen activator inhibitor-1 and homocysteine levels.ConclusionsIn severe vitamin D deficiency, a high-dose cholecalciferol supplementation was associated with a reduction in thrombin generation and an average decreased number of fibrin protofibrils per fibers and fibrin fiber size measured by turbidimetry. This suggests that severe vitamin D deficiency may be associated with a potentially reversible prothrombotic profile.

High-Intensity Interval Training Recuperates Capacity of Endogenous Thrombin Generation in Patients With Heart Failure

High-Intensity Interval Training Recuperates Capacity of Endogenous Thrombin Generation in Patients With Heart Failure

Magnetic Accumulation of SPIONs under Arterial Flow Conditions: Effect of Serum and Red Blood Cells.

Magnetic drug targeting utilizes an external magnetic field to target superparamagnetic iron oxide nanoparticles (SPIONs) and their cargo to the diseased vasculature regions. In the arteries, the flow conditions affect the behavior of magnetic particles and the efficacy of their accumulation. In order to estimate the magnetic capture of SPIONs in more physiological-like settings, we previously established an ex vivo model based on human umbilical cord arteries. The artery model was employed in our present studies in order to analyze the effects of the blood components on the efficacy of magnetic targeting, utilizing 2 types of SPIONs with different physicochemical characteristics. In the presence of freshly isolated human plasma or whole blood, a strong increase in iron content measured by AES was observed for both particle types along the artery wall, in parallel with clotting activation due to endogenous thrombin generation in plasma. Subsequent studies therefore utilized SPION suspensions in serum and washed red blood cells (RBCs) at hematocrit 50%. Interestingly, in contrast to cell culture medium suspensions, magnetic accumulation of circulating SPION-3 under the external magnet was achieved in the presence of RBCs. Taken together, our data shows that the presence of blood components affects, but does not prevent, the magnetic accumulation of circulating SPIONs.

Buffy coat-derived platelets cryopreserved using a new method: Results from in vitro studies

Cryopreservation for the long-term storage of platelets (PLTs) is a useful method to overcome the limits of platelet shortage. This is an in vitro prospective study to evaluate the count, viability, and function of buffy coat-derived pooled platelet concentrates (BC-PLTs), treated with dimethyl sulphoxide (DMSO) and cryopreserved (CRY BC-PLTs) at −80 °C with a modified Valeri method. PLTs were stored in 6% DMSO with a patented kit. Overall, 49 BC-PLTs from 245 healthy volunteer donors were prepared, cryopreserved, and analysed before and after 3, 6, and 9 months of storage. In flow cytometry, a statistically significant reduction in CD 42b (92.7 ± 4.29% at T0 vs. 23.6 ± 27.5% at T3, 16.38 ± 12.54% at T6, and 17.3 ± 9.6% at T9) and PAC-1 (1.9 ± 1.34% at T0 vs. 0.62 ± 0.4% at T3, 0.63 ± 0.83% at T6, and 0.49 ± 0.48% at T9) was observed after storage. CRY BC-PLTs showed a good and stable endogenous thrombin generation potential (nM min): 529.25 ± 98.64 at T0 vs. 533.04 ± 103.15 at T9 months. CRY BC-PLTs showed a good viability in vitro, according to currently accepted criteria for cryopreserved PLTs.

Continuous thrombin generation in whole blood: New applications for assessing activators and inhibitors of coagulation

Hemostatic tests have been utilized to clarify the blood coagulation potential. The novel thrombin generation (TG) assay of this study provides explicit information and is the most physiologically-relevant hemostatic test ex vivo. We describe how this assay allows for TG under a number of relevant circumstances. First, whole blood (WB) from healthy individuals was analyzed ± 5 pM tissue factor (TF) and ± contact pathway inhibition. Without an exogenous initiator TG was decreased and delayed, but addition of 5 pM TF shortened the lag phase and increased peak thrombin. Additional experiments included fresh WB from a trauma patient analyzed for endogenous activity and TG from healthy donors subjected to TG antagonists which prolonged the lag phase whereas TG agonists consistently shortened the lag phase in a dose dependent manner. Lastly, platelet-poor plasma was reconstituted with packed red blood cells and TG was monitored in the presence and absence of both TF as an activator and PCPS as a phospholipid surface. Our data illustrate the potential that this continuous TG assay has in the evaluation of disorders relevant to blood coagulation and in the monitoring of treatments administered in response to these disorders.

Platelet tissue factor activity and membrane cholesterol are increased in hypercholesterolemia and normalized by rosuvastatin, but not by atorvastatin

Background and aimsHigh plasma LDL-cholesterol (LDL-C) and platelet responses have major pathogenic roles in atherothrombosis. Thus, statins and anti-platelet drugs constitute mainstays in cardiovascular prevention/treatment. However, the role of platelet tissue factor-dependent procoagulant activity (TF-PCA) has remained unexplored in hypercholesterolemia. We aimed to study platelet TF-PCA and its relationship with membrane cholesterol in vitro and in 45 hypercholesterolemic patients (HC-patients) (LDL-C >3.37 mmol/L, 130 mg/dL) and 37 control subjects (LDL-C <3.37 mmol/L). The effect of 1-month administration of 80 mg/day atorvastatin (n = 21) and 20 mg/day rosuvastatin (n = 24) was compared. MethodsPlatelet TF-PCA was induced by GPIbα activation with VWF-ristocetin. ResultsCholesterol-enriched platelets in vitro had augmented aggregation/secretion and platelet FXa generation (1.65-fold increase, p = 0.01). HC-patients had 1.5-, 2.3- and 2.5-fold increases in platelet cholesterol, TF protein and activity, respectively; their platelets had neither hyper-aggregation nor endogenous thrombin generation (ETP). Rosuvastatin, but not atorvastatin, normalized platelet cholesterol, TF protein and FXa generation. It also increased slightly the plasma HDL-C levels, which correlated negatively with TF-PCA. ConclusionsPlatelets from HC-patients were not hyper-responsive to low concentrations of classical agonists and had normal PRP-ETP, before and after statin administration. However, washed platelets from HC-patients had increased membrane cholesterol, TF protein and TF-PCA. The platelet TF-dependent PCA was specifically expressed after VWF-induced GPIbα activation. Rosuvastatin, but not atorvastatin treatment, normalized the membrane cholesterol, TF protein and TF-PCA in HC-patients, possibly unveiling a new pleiotropic effect of rosuvastatin. Modulation of platelet TF-PCA may become a novel target to prevent/treat atherothrombosis without increasing bleeding risks.

Evaluation of the hemostatic potential at coronary artery bypass surgery during long-term aspirin therapy

To estimate thrombin generation test parameters in patients with coronary heart disease during coronary artery bypass surgery under extracorporeal circulation after transfusion of donor platelet concentrates during long-term therapy with acetylsalicylic acid (ASA). A total of 148 patients with coronary heart disease who had undergone elective primary coronary artery bypass surgery under extracorporeal circulation during preserved therapy with ASA (75-100 mg/day) were examined. According to donor platelet concentrate transfusion, all the patients were divided into 2 groups: 1) 76 patients undergoing donor platelet transfusion and 2) 72 without this procedure. A control group consisted of 20 apparently healthy individuals. At the pre-, intra-, and early postoperative stages, the investigators evaluated the following thrombin generation test parameters: lag time (min); peak thrombin concentration (nM/l); time to peak (min); the area under the thrombin generation curve (nM), and thrombin generation rate (nM/min). During long-term ASA therapy, the patients were found to have an activated endogenous thrombin potential in the pre- and intraoperative periods, as evidenced by the high peak concentration of thrombin and the increased rate of its generation. At the same time, the time of prothrombinase complex activation and that of thrombin generation were longer than those in the control group. In the early postoperative period, the patients who had not been transfused with platelet concentrates with a further increase in the temporal parameters, showed a decreased hemostatic potential, reaching the control level, whereas donor platelet transfusion stimulated endogenous thrombin generation: the time to initiate clotting and that to reach the peak were shorter; in this case, the thrombin generation rate and concentrations increased, but the preoperative level was not reached. No perioperative (hemorrhagic or thrombotic ischemic) events were noted in the examined groups. The hemostatic potential was preserved in patients receiving long-term therapy for ASA. Taking into account laboratory and clinical findings, platelet concentrate transfusions are unnecessary for preventive purposes. The appropriateness of donor platelet transfusion should be strictly individually approached with regard to the laboratory parameters of the thrombin generation test, by minimizing the risk of perioperative ischemic and hemorrhagic events in each specific patient.

Thrombin Generation and Bleeding Phenotype during Personalized Prophylaxis with Recombinant Human FVIII in Previously Treated Patients with Severe Hemophilia A

Background and objectivesIn apharmacokinetic (PK)-guided personalized prophylaxis study with human-cl rhFVIII(Nuwiq®), a factor VIII (FVIII) concentrate from a human cell line,58% of the previously treated adult patients (PTPs) with severe hemophilia A received 2 or fewer prophylactic infusions per weekwith a median dosing interval of3.5 days. Seventy-three percent (73%) of patients did not experience any bleeding and 83% had no spontaneous bleeding episodes during the 6-month personalized prophylaxis period. The objective of this analysis was to evaluate the relationship between endogenous thrombin generation (TG) as well as FVIII plasma concentrations with the bleeding phenotype.Study design and methodsThis prospective, open-label, multicenter study included 66 adult PTPs with severe hemophilia A. After the previously given FVIII concentrate was washed out, patients received human-cl rhFVIII(60 IU/kg) for PK evaluation. Individual PK data were analyzed to determine doses and injection intervals that would theoretically result in a trough FVIII plasma level of ≥1%. Individualized prophylaxis lasted 6 months. TG and FVIII:C plasma concentrations were measured before and during the PK assessment, and trough TG and FVIII:C levels were measured 2, 4 and 6 months after the start of personalized prophylaxis. For TG, blood was drawn in trisodium citrate tubes (0.106 M) containing 1.45 µM corn trypsin inhibitor, centrifuged twice to obtain platelet poor plasma, and stored frozen until central analysis. TG was initiated by adding tissue factor (1 pM), and endogenous thrombin potential (ETP) was measured using the calibrated, automated TG assay.FVIII:C was measured using one-stage (automated APTT, Trinity Biotech, Siemens BCX-XP) and chromogenic assays (Coatest SP FVIII Kit, Chromogenix, Siemes BCS-XP).ResultsData on both baseline ETP and bleeding rate during PK-guided individualized prophylaxis were available for 32 patients. Twenty-one patients did not experience any bleeding episode, and 25 patients had no spontaneous bleeding episodes. The mean baseline ETP did not differ between patients with and without any bleeding (373 ± 334 vs 367 ± 168 nmol*min), but was considerably lower in patients who had spontaneous bleeding episodes compared with those without spontaneous bleeding episodes (164 ± 66 vs 426 ± 231 nmol*min). Data on trough ETPs and FVIII:C during personalized prophylaxis were available for 34 patients.Patients with low TG potential tended to experience more frequent spontaneous bleeding episodes during this phase of the protocol (Figure 1), while low FVIII:C levels seemed to be less related to spontaneous bleeding episodes.ConclusionsLow TG capacity seems to be correlated with increased frequency of spontaneous bleeding episodes, irrespective of trough FVIII plasma levels. Further prospective studies should be carried out to evaluate the value of this global coagulation parameter in the personalization prophylactic treatment in patients with hemophilia A in comparison with trough FVIII coagulant activity. [Display omitted] DisclosuresKlamroth:Biogen Idec: Other: honoraria for advisory boards and speaker fees; Baxalta: Other: honoraria for advisory boards and speaker fees ; Bayer: Other: honoraria for advisory boards and speaker fees; CSL Behring: Other: honoraria for advisory boards and speaker fees; NovoNordisk: Other: honoraria for advisory boards and speaker fees; SOBI: Other: honoraria for advisory boards and speaker fees; pfizer: Other: honoraria for advisory boards and speaker fees; Octapharma: Other: honoraria for advisory boards and speaker fees; uniqure: Other: honoraria for advisory boards and speaker fees. Windyga:Biogen: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Aspen: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Nordisk: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Alexion: Other: Speaker’s honorarium; Sanofi: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Octapharma: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Baxalta, now part of Shire: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Investigator Clinical Studies, Patents & Royalties, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau. Bichler:Octapharma: Employment. Knaub:Octapharma: Employment. Negrier:NovoNordisk: Membership on an entity’s Board of Directors or advisory committees, Other: Travel support, Research Funding; LFB: Membership on an entity’s Board of Directors or advisory committees; Alnylam: Research Funding; Bayer: Membership on an entity’s Board of Directors or advisory committees, Other: Travel support; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity’s Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Research Funding; SOBI: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.

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.

Anti-TNFα agents curb platelet activation in patients with rheumatoid arthritis

BackgroundCardiovascular disease is important in rheumatoid arthritis (RA). Tissue factor (TF) is expressed upon platelet activation and initiates coagulation. Anti-tumour necrosis factor-α (TNFα) agents seem to decrease RA-associated cardiovascular events....

Changes in thrombin generation in children after cardiac surgery and ex-vivo response to blood products and haemostatic agents.

Impaired haemostasis has been reported in children with congenital heart disease undergoing cardiopulmonary bypass. As thrombin generation encompasses all phases of the coagulation process, this analysis might provide the best assessment of global haemostasis. A prospective study was undertaken to test the hypothesis that thrombin generation reveals an impaired haemostasis after paediatric cardiac surgery and that ex-vivo addition of platelet concentrate and haemostatic agents improves thrombin generation. The study comprised 29 children with congenital heart disease, who underwent corrective surgery including cardiopulmonary bypass. Thrombin generation was analysed both in platelet-poor plasma and platelet-rich plasma. Analysis of the thrombin generation showed a significantly prolonged lag time (Pplatelet-poorandplatelet-richplasma < 0.001), decreased peak thrombin generation (Pplatelet-poorplasma = 0.013; Pplatelet-richplasma < 0.001) as well as a decreased endogenous thrombin generation potential (Pplatelet-poorandplatelet-richplasma < 0.001) after cardiopulmonary bypass compared to baseline. Ex-vivo addition of platelet concentrate, fibrinogen concentrate and recombinant factor VIIa improved thrombin generation significantly (all P < 0.001). Changes were most pronounced after addition of platelet concentrate. The present study showed that thrombin generation was significantly reduced after cardiopulmonary bypass in children, both when analysed in platelet-poor and platelet-rich plasma. The impaired haemostasis was not only restored after ex-vivo addition of platelet concentrate but also rVIIa improved the haemostatic capacity.

Effect of Asparaginase and Dexamethasone on FVIIa-at Complex and F1.2 in Children with Acute Lymphoblastic Leukemia: Evidence of Hypercoagulable State

Purpose:Concomitant administration of asparaginase (ASP) and steroids, the backbone of front line acute lymphoblastic leukemia (ALL) therapy, are shown to increase the risk of thromboembolism (TE). On Dana-Farber Cancer Institute (DFCI) ALL Consortium studies TE is frequent during ASP intensification phase (Consolidation II) where pulse steroids are administered for 5 days at commencement of 3-week multiagent chemotherapy cycles (Grace et al, 2011). These observations indicate that combination therapy with ASP and steroids leads to a hypercoagulable state. However, there are no in vivo or in vitro data to support these clinical observations. Hence we undertook a study to evaluate the impact of concomitant administration of ASP and steroids on markers of endogenous thrombin generation namely prothrombin activation fragment 1.2 (F1.2) and, activated coagulation factor VII (FVIIa) and antithrombin (AT) complex (FVIIa-AT).Hypothesis:ASP administered with steroids results in increased levels of endogenous thrombin generation when compared to baseline or with ASP only therapy.Methods:Children (>1 to ≤18 yrs. of age) treated according to DFCI ALL 05-001 therapy protocol who are in remission following consolidation I therapy, without prior TE or ASP allergy, were eligible. Blood samples collected at 3 time points: prior to the first post-induction dose of E. Coli ASP ("baseline"), prior to Week 2 therapy (effects of ASP and Dex therapy) and prior to subsequent Week 1 (effect of ASP only) of three week cycle of Consolidation II therapy, were analyzed for F1.2 and FVIIa-AT complex. F1.2 levels were estimated by enzyme-linked immunosorbent assay (ELISA). FVIIa-AT levels were analyzed using the Asserachrome FVIIa-AT ELISA kits (Diagnostica Stago). Baseline parameters [time point (TP) 1] were compared to parameters after ASP and Dex (TP2), and after ASP only (TP3) therapy, using paired t-tests. Pearson correlation was used to evaluate the relationship between FVIIa-AT complex and F1.2.Results:This exploratory study included 9 patients (7 boys; 2 with high-risk ALL). The average age was 4.7 yrs. (range 1 to 8 yrs.). All patients received weekly E. Coli ASP during Consolidation II phase. As shown in Table 1, compared to baseline both F1.2 and FVIIa-AT complex levels were significantly increased at TP2 (ASP and Dex) and TP3 (ASP alone). In all but one patient the FVIIa-AT levels were higher following Dex therapy (TP2) compared to ASP alone (TP3). However this difference was not statistically significant [mean difference 83.4 (SD152.1), 95% CI -33.5, 200.4; p=0.138]. Similarly F1.2 levels were higher at TP2 compared to TP3 in all but 2 patients, but the difference was not statistically significant [mean difference -5.9 (SD 38.2), 95% CI-35.2, 23.4; p=0.656]. Following ASP alone therapy F1.2 and FVIIa-AT were significantly correlated (Pearson correlation coefficient 0.716, p=0.046).Conclusions:E. Coli ASP and Dex therapy is associated with significantly increased levels of F1.2 and FVIIa-AT levels compared to baseline, indicating activation of the coagulation cascade with increased endogenous thrombin generation. In the majority of patients combination of ASP and Dex had increased levels of F1.2 and FVIIa-AT compared to ASP alone therapy. However as a group this comparison was statistically insignificant. This may explain the increased risk of TE with concomitant ASP and Dex therapy as in Consolidation II phase on DFCI ALL therapy protocol. Small sample size is a limitation of our study. Hence we recommend a larger study to confirm our findings.Table 1VariableTP1 Mean(SD)TP2 Mean (SD)Mean Difference TP1/TP2 (SD)[95% CI]P valueTP3 Mean (SD)Mean Difference TP1/TP3 (SD)[95% CI]P valueFVIIa-AT (pmol/L)77.4 (25.7)296.4 (132.3)-291.0 (133.9)[-322.0,-116.0]0.001213.0 (131.4)-135.6 (117.6)[-226.0, -45.1]0.009F1.2 (nmol/L)110.7 (45.9)153.0 (78.8)-42.3 (50.4) [-81.1,-3.6]0.036158.9 (80.4)-48.2 (61.3)[-95.3,-1.1]0.046 DisclosuresNo relevant conflicts of interest to declare.

Increased RhoA/Rho-Kinase Activity and Markers of Endothelial Dysfunction in Young Adult Subjects with Metabolic Syndrome.

Metabolic syndrome, a chronic condition associated with higher risk of cardiovascular diseases, is increasingly prevalent in young adults. Dyslipidemia, proinflammatory cytokines, endothelial dysfunction signs, and RhoA/Rho-kinase (ROCK) activation are considered risk factors of cardiovascular diseases. The occurrence of these factors in young patients with metabolic syndrome but without type 2 diabetes or hypertension has not been fully studied. The objective of this study was to evaluate young subjects with enlarged waist circumference and dyslipidemia but without type 2 diabetes or hypertension,for markers associated with a higher risk of cardiovascular diseases. Thirty-two male patients aged 31 ± 1.3 years diagnosed with metabolic syndrome according to the National Cholesterol Education Program Adult Treatment Panel III guide for enlarged waist circumference, elevated triglycerides, and low HDL levels, but with blood pressure and fasting glucose within normal ranges, were evaluated for RhoA/ROCK activity in leukocytes, serum fatty acid methyl esters profile, proinflammatory cytokines, and oxidative stress markers in addition to thrombin generation and biochemical analysis. Age- and gender-matched healthy subjects were equivalently evaluated. Patients showed higher RhoA/ROCK activity, elevated levels of interleukin-6, soluble CD40L, monocyte chemoattractant protein, and high-sensitivity C-reactive protein (P < 0.001) as well as parameters of endogenous thrombin generation potential (P < 0.05) compared with healthy subjects. Increased thiobarbituric acid reactive substances, advanced oxidation protein product, and insulin levels and low nitric oxide biodisponibility (P < 0.001) were also found in patients as compared with controls. Palmitic acid was one of the saturated fatty acids found to be significantly elevated in patients compared with control subjects (P = 0.0087). Increased markers of cardiovascular risk are already present in young adults with metabolic syndrome but without type 2 diabetes or hypertension.

Endogenous thrombin potential in polycystic ovary syndrome: the association to body mass index, insulin resistance, and inflammation

Objectives: The objective of this study is to investigate plasma endogenous thrombin generation in four different phenotypes of polycystic ovary syndrome (PCOS) defined by Body Mass Index (BMI) and insulin resistance (IR). PCOS is diagnosed according to the Rotterdam criteria.Design: Multicenter cross-sectional study.Setting: Two major University Hospitals in the Capital region of Denmark.Patients: Hundred forty-eight European women with PCOS were consecutively recruited during April 2010–February 2012. Clinical examination, blood sampling, and DEXA scan were performed.Main outcome measures: Endogenous thrombin potential (ETP).Results: PCOS women with phenotype BMI > 25 + IR have increased potential of thrombin generation. ETP is associated with total body fat mass, IR, and CRP.Conclusions: Obese and insulin resistant women with PCOS have elevated level of ETP corresponding to increased risk of CVD. ETP is related to well-known CVD risk factors in PCOS but not in general to the Rotterdam criteria.

Thrombin generation, ProC(®)Global, prothrombin time and activated partial thromboplastin time in thawed plasma stored for seven days and after methylene blue/light pathogen inactivation.

Methylene blue pathogen inactivation and storage of thawed plasma both lead to changes in the activity of several clotting factors. We investigated how this translates into a global loss of thrombin generation potential and alterations in the protein C pathway. Fifty apheresis plasma samples were thawed and each divided into three subunits. One subunit was stored for 7 days at 4 °C, one was stored for 7 days at 22 °C and one was stored at 4 °C after methylene blue/light treatment. Thrombin generation parameters, ProC(®)Global-NR, prothrombin time and activated partial thromboplastin time were assessed on days 0 and 7. The velocity of thrombin generation increased significantly after methylene blue treatment (increased thrombin generation rate; time to peak decreased) and decreased after storage (decreased thrombin generation rate and peak thrombin; increased lag time and time to peak). The endogenous thrombin generation potential remained stable after methylene blue treatment and storage at 4 °C. Methylene blue treatment and 7 days of storage at 4 °C activated the protein C pathway, whereas storage at room temperature and storage after methylene blue treatment decreased the functional capacity of the protein C pathway. Prothrombin time and activated partial thromboplastin time showed only modest alterations. The global clotting capacity of thawed plasma is maintained at 4 °C for 7 days and directly after methylene blue treatment of thawed plasma. Thrombin generation and ProC(®)Global are useful tools for investigating the impact of pathogen inactivation and storage on the clotting capacity of therapeutic plasma preparations.

Thrombin Generation and Venous Thromboembolism Among Multiple Myeloma Patients Receiving Lenalidomide and Prednisolone Maintenance Following Autologous Stem Cell Transplantation (ASCT)

BackgroundLenalidomide is effective in the treatment of multiple myeloma (MM) but is associated with an increased risk of venous thromboembolism (VTE). The mechanism underlying this increase risk remains unclear. As routine coagulation tests do not reflect true in vivo coagulation, global coagulation assays such as thrombin generation might be a better surrogate marker of thrombosis risk.AimTo sequentially monitor thrombin generation in myeloma patients receiving lenalidomide and alternate day prednisolone (RAP) maintenance following ASCT and correlating it with objectively confirmed VTE.MethodsThe LEOPARD study was a phase II, open label, single arm, multi-centre study of RAP (lenalidomide 10mg/continuous daily increasing to 15mg after 8 weeks and alternate day prednisolone 50mg) maintenance therapy commencing 6-8 weeks after a single MEL200 ASCT as part of first-line therapy for patients with MM. RAP was continued until toxicity or relapse/progression.Enrolled patients were systematically observed for symptomatic objectively confirmed VTE. Plasma samples were collected and frozen pre-ASCT, post-ASCT prior to commencing RAP maintenance and at six months post ASCT. Thrombin generation (TG) was measured using calibrated automated thrombography (CAT™) following a single thaw, blinded to the clinical outcome following completion of the study.Results28 of 60 patients enrolled in the LEOPARD had available all three predetermined plasma samples for analysis and remained on RAP at 6m post ASCT. Female=14, male=14 with a median age of 61 years (range, 41-71). Of the 60 patients, 44 and 6 patients received low-dose Aspirin and Enoxaparin as thromboprophylaxis post ASCT respectively. Ten patients received no thromboprophylaxis. Overall, 3 out of 60 patients (5%) enrolled had objectively confirmed VTE. Two episodes of symptomatic proximal deep vein thrombosis occurred at 9, and 14 months post ASCT; both patients were in complete remission and remained on RAP. One patient developed symptomatic pulmonary embolism at 14 months post ASCT and was in partial remission.TG as measured by endogenous thrombin generation (ETP) did not differ significantly between pre-ASCT, post-ASCT (pre RAP maintenance) and 6months post ASCT. The mean ETP was 1682nM pre ASCT, 1645nM post ASCT (pre RAP maintenance) and 1537nM 6 months post ASCT. Similarly, there was no significant difference between peak thrombin (PTG) at each of the time points with mean PTG of 376nM pre ASCT, 388nM post ASCT (pre RAP maintenance) and 367nM 6 months post ASCT. Moreover, no apparent difference in TG between the 3 patients who experienced a VTE and the remainder of the cohort was evident (for the 3 VTE patients mean ETP, 1396.8nM pre RAP maintenance and 1385.8 nM 6 months post ASCT; mean peak thrombin is 216.5 pre RAP maintenance and 221.7 6 months post ASCT).ConclusionThe risk for VTE was low in this cohort of MM patients receiving maintenance RAP following ASCT most of whom were receiving pharmacological thromboprohylaxis. This is supported by the findings of no significant difference in TG (both ETP and PTG) at pre-ASCT, post-ASCT (pre RAP maintenance) and 6 months post ASCT. A late accumulative risk for VTE due to RAP cannot be excluded with 3 late episodes of VTE observed. A larger study is needed to confirm these findings. DisclosuresSpencer:Celgene: Consultancy, Honoraria.

Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms.

Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from patients with sepsis. NETs released from phorbyl myristate-activated neutrophils enhance thrombin generation in platelet-poor plasma. This effect was DNA dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with coagulation factor XII- and coagulation factor XI-depleted plasma). In platelet-rich plasma treated with corn trypsin inhibitor, addition of phorbyl myristate-activated neutrophils increased thrombin generation and shortened the lag time in a toll-like receptor-2- and toll-like receptor-4-dependent mechanism. Addition of DNase further augmented thrombin generation, suggesting that dismantling of the NET scaffold increases histone-mediated, platelet-dependent thrombin generation. In platelet-poor plasma samples from patients with sepsis, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation. These studies examine the procoagulant activities of CFDNA and histones in the context of NETs. Our studies also implicate a role for the intrinsic pathway of coagulation in sepsis pathogenesis.

Direct thrombin inhibitors, but not the direct factor Xa inhibitor rivaroxaban, increase tissue factor‐induced hypercoagulability in vitro and in vivo

BackgroundIncreased hypercoagulability has been reported with low doses of direct thrombin inhibitors but not with direct factor Xa inhibitors.ObjectivesTo compare the effects of rivaroxaban with those of melagatran and dabigatran on thrombin generation (TG) and tissue factor-induced hypercoagulability and to explore the possible involvement of the thrombin–thrombomodulin/activated protein C system.MethodsIn normal human plasma and in protein C-deficient plasma, TG was investigated in vitro in the presence and absence of recombinant human soluble thrombomodulin (rhs-TM). TG was determined by calibrated automated thrombography and an ELISA for prothrombin fragments 1+2 (F1+2). In an in vivo rat model, hypercoagulability was induced by tissue factor; levels of thrombin–antithrombin (TAT) and fibrinogen and the platelet count were determined.ResultsRivaroxaban inhibited TG in a concentration-dependent manner. In the absence of rhs-TM, melagatran and dabigatran also inhibited TG concentration dependently. However, in the presence of rhs-TM, lower concentrations of melagatran (119–474 nmol L–1) and dabigatran (68–545 nmol L−1) enhanced endogenous thrombin potential, peak TG, and F1+2 formation in normal plasma but not in protein C-deficient plasma. In vivo, rivaroxaban dose-dependently inhibited TAT generation, whereas melagatran showed a paradoxical effect, with an increase in TAT and a small decrease in fibrinogen and platelet count at lower doses.ConclusionLow concentrations of the direct thrombin inhibitors melagatran and dabigatran enhanced TG and hypercoagulability, possibly via inhibition of the protein C system. In contrast, rivaroxaban reduced TG and hypercoagulability under all conditions studied, suggesting that it does not suppress this negative-feedback system.