Thrombin Generation Potential Research Articles

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19

BackgroundCOVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. ObjectivesFrom the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. MethodsWe utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. Results and ConclusionsCompared to controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. We conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

Association between venous thromboembolism-associated genetic variants, coagulation factor levels, and thrombin generation potential.

Recently three large meta-analyses of genome-wide association studies for venous thromboembolism (VTE) identified over 130 genetic variants. However, mechanisms by which newly identified and therefore underexplored VTE-associated genetic variants influence VTE remain unclear. To elucidate the mechanism, we investigated the association between 61 newly identified VTE-associated genetic variants and the levels of coagulation factor (F) VIII, FIX, FXI, and fibrinogen as well as thrombin generation parameters (lag time, peak, endogenous thrombin potential, time-to-peak, and velocity), which are well-known biological traits associated with VTE. This study was conducted on 5341 participants of the Netherlands Epidemiology of Obesity study. The associations between VTE-associated genetic variants and coagulation factor levels and thrombin generation parameters were examined using linear regression analyses, adjusted for age, sex, body mass index, oral contraceptive use, hormone replacement therapy, and menopausal status. Of 61 genetic variants, 33 were associated with one or more of the coagulation factor levels and thrombin generation parameters. Following multiple testing corrections, five genetic variants remained significant, of which MAP1A rs55707100 exhibited the most robust association with thrombin generation parameters and FXI levels (β=-5.33%, 95% confidence interval: -8.44, -2.22). Our findings shed light on the underlying mechanisms by which these genetic variants influence the risk of VTE.

First 24 Hour-Long Intensive Care Unit Testing of a Clinical-Scale Microfluidic Oxygenator in Swine: A Safety and Feasibility Study.

Microfluidic membrane oxygenators are designed to mimic branching vasculature of the native lung during extracorporeal lung support. To date, scaling of such devices to achieve clinically relevant blood flow and lung support has been a limitation. We evaluated a novel multilayer microfluidic blood oxygenator (BLOx) capable of supporting 750-800 ml/min blood flow versus a standard hollow fiber membrane oxygenator (HFMO) in vivo during veno-venous extracorporeal life support for 24 hours in anesthetized, mechanically ventilated uninjured swine (n = 3/group). The objective was to assess feasibility, safety, and biocompatibility. Circuits remained patent and operated with stable pressures throughout 24 hours. No group differences in vital signs or evidence of end-organ damage occurred. No change in plasma free hemoglobin and von Willebrand factor multimer size distribution were observed. Platelet count decreased in BLOx at 6 hours (37% dec, P = 0.03), but not in HFMO; however, thrombin generation potential was elevated in HFMO (596 ± 81 nM·min) versus BLOx (323 ± 39 nM·min) at 24 hours (P = 0.04). Other coagulation and inflammatory mediator results were unremarkable. BLOx required higher mechanical ventilator settings and showed lower gas transfer efficiency versus HFMO, but the stable device performance indicates that this technology is ready for further performance scaling and testing in lung injury models and during longer use conditions.

Hemostatic balance between pro- and anticoagulant is maintained during glucocorticoid treatment.

Glucocorticoids are associated with an increased risk of venous thrombosis. Glucocorticoid treatment increases coagulation factor and anticoagulant levels; however, its effect on hemostatic function remains unclear. This study aimed to investigate the changes in comprehensive coagulation profiles after glucocorticoid treatment in noninflammatory diseases to elucidate the direct contribution of glucocorticoids to hemostatic function. Patients diagnosed with primary immune thrombocytopenia requiring glucocorticoid treatment were prospectively enrolled in this study. Changes in coagulation factors and anticoagulants during glucocorticoid treatment and changes in thrombin generation potential were determined in the absence and presence of soluble thrombomodulin (sTM). Seven treatment cases (four for steroid pulse therapy and three for oral glucocorticoid therapy) in six patients with immune thrombocytopenia were examined. After glucocorticoid treatment, activated partial thromboplastin time significantly shortened, and activities of factor VIII, IX, XI, and XII significantly increased, except for von Willebrand factor antigen. Moreover, antithrombin and protein C (PC) activities significantly increased after glucocorticoid treatment. Two major parameters of thrombin generation potential, endogenous thrombin potential (ETP) and peak thrombin (Peak), significantly increased in the absence of sTM after glucocorticoid treatment. However, no significant increases in either parameter were observed in the presence of sTM. ETP-TM and Peak-TM ratios, which represent resistance to the anticoagulant effect of the PC pathway, significantly decreased after glucocorticoid treatment, suggesting that anticoagulant function via the PC pathway is elevated after glucocorticoid treatment. As glucocorticoids increase intrinsic coagulation factor and anticoagulant levels, hemostatic balance between pro- and anticoagulant functions is maintained.

Bioactive Lipid Mediators Predict Platelet Function in Transfusion Recipients: A Phase 1 Randomized Clinical Trial in Healthy Humans

Background: Platelets are stored at room temperature for 5-7 days (RSP) and are transfused to patients who are bleeding or at risk of bleeding. Due to frequent and severe shortages, the FDA recently approved cold-stored platelets in plasma (CSP) for up to 14 days despite a lack of post-transfusion data. It is also unclear which donors are best suited to provide platelets for either RSP or CSP and which in vitro markers accurately predict post-transfusion RSP or CSP platelet function. Objective: To evaluate the post-transfusion function and predictors of post-transfusion function of platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). Methods: We conducted a randomized cross-over study in ten healthy humans. Subjects on ASA were randomized to receive either autologous RSP or CSP first. We obtained blood from recipients before and after transfusion for platelet function testing and samples from the storage bag before and after storage for lipid metabolite analysis. The first round was followed by a second round with transfusion of the alternative product and the same testing sequence. Results: RSP reversed platelet inhibition significantly better in αIIbβ3 integrin activation-dependent assays. In contrast, CSP led to significantly more thrombin generation in recipients. Overall, RSP contained more pro-inflammatory lipid mediators while CSP had more anti-inflammatory lipid mediators. In particular, LPC species including LPC-O species (formerly known as Lyso-platelet activating factor [LPAF]), LPE, and LPI were significantly more abundant in RSP than in CSP, while LPS was more abundant in CSP than in RSP. Similarly, the relative increase of LPC, LPE, and LPI, and linoleic acid-dependent oxylipins, such as diHOMEs and HODEs were higher in RSP recipients than in CSP recipients. Linoleic acid-dependent oxylipins correlated positively with platelet aggregation in units and recipients of RSP and CSP. Polyunsaturated fatty acids (PUFAs) showed a negative correlation with platelet aggregation in both RSP and CSP units and recipients. Most importantly, LPC-O (LPAF) species showed a strong negative correlation with endogenous thrombin generation potential in CSP units after collection (pre-storage), post-storage, and in transfusion recipients. Conclusion: Results from our trial provide the first efficacy data of autologous and extended-stored CSP in plasma. We report the first correlational data between unit and recipient lipid mediators and post-transfusion platelet function. Taken together, we show that the main mechanism of post-transfusion CSP function appears to be based on procoagulant properties. In the future, testing platelet donors for LPC-O (LPAF) species could identify donors whose platelets are better suited for cold storage than room-temperature storage.

Blood Coagulation and Platelet Function Are Impaired By the ARP2 p.R258G Variant in Mice

Introduction: Venous thromboembolism (VTE) displays significant heritability, yet the genes explaining this heritability are not completely characterized. We previously identified that tissue factor pathway inhibitor heterozygosity ( Tfpi+/−) on the homozygous F5L background ( F5L/L) produces a lethal perinatal thrombotic phenotype. Using a sensitized ENU mutagenesis screen, we discovered an autosomal dominant missense mutation in the Actr2 gene (p.R258G, Actr2G) that suppresses thrombosis and restores survival to F5L/LTfpi+/− mice. Inheritance of the Actr2Gmutation is associated with nearly complete suppression of the lethal F5L/LTfpi+/- phenotype. Actr2 codes for the ARP2 protein, an essential component of the ARP2/3 complex, which mediates actin cytoskeletal rearrangements during platelet activation. We hypothesized that the Actr2G variant affects blood coagulation and platelet function to reduce thrombosis. Methods: To investigate potential effects of the Actr2G mutation on the coagulation cascade, prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma-dependent thrombin generation assays (TGA) were used to evaluate clot formation. A mouse tail clip assay was used to measure bleeding time and quantify blood loss. Finally, platelets were assessed for functional defects using platelet spreading and aggregometry assays. Results: Actr2 +/Gplasma had significantly prolonged PT and aPTT compared to wild type controls (N>17, p<0.03). There was also a significant reduction in Actr2+/G thrombin peak height compared to wildtypes (N=10; p=0.03). Despite the delayed time to clot formation, Actr2+/G mice did not exhibit a bleeding phenotype and showed no significant differences in blood loss from tail clip assays. Activated Actr2+/G platelets exhibited reduced surface area and adhesion (N=5; p<0.02), as well as significant defects in collagen induced whole blood aggregation (N=10; p<0.02). Discussion: The results of the PT, aPTT and TGA indicate reduced thrombin generation potential, suggesting a defect in the common pathway of coagulation. This hypocoaguability may be due to a decrease in circulating procoagulant factors, or an increase in endogenous thrombin inhibitors. Both the significantly decreased aggregation and defective platelet spreading in the Actr2+/G mice indicate that Actr2+/G platelets are less responsive to agonists, a protective mechanism that contributes to overall thrombosis suppression. Conclusion: In summary, our studies demonstrate the pleiotropic effects that the Actr2G mutation exerts in suppressing thrombosis. The combination of altered plasma coagulation and platelet reactivity likely contribute to suppression of the F5L/LTfpi+/− phenotype. Defects in these actin-mediated processes precipitated by Actr2+/G are the likely root of the F5L/LTfpi+/- phenotype rescue, demonstrating the power of this gene in maintaining hemostatic balance. However, the combination of these thrombosis suppressing phenotypes is not significant enough to prolong the bleeding time in mice with the Actr2G mutation. Thus, our results suggest that targeting actin proteins such as ARP2 for novel antithrombotic drug therapies may have clinical applicability with limited effects on bleeding.

Factor XI Inhibition With Heparin Reduces Clot Formation in Simulated Pediatric Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) supplies circulatory support and gas exchange to critically ill patients. Despite the use of systemic anticoagulation, blood exposure to ECMO surfaces causes thromboembolism complications. Inhibition of biomaterial surface-mediated activation of coagulation factor XI (FXI) may prevent device-associated thrombosis. Blood was collected from healthy volunteers (n = 13) following the U.S. Army Institute of Surgical Research standard operating procedure for testing in an ex vivo ECMO circuit. A roller-pump circuit circulated either 0.5 U/ml of unfractionated heparin alone or in combination with the anti-FXI immunoglobulin G (IgG) (AB023) for 6 hours or until clot formation caused device failure. Coagulation factor activity, platelet counts, time to thrombin generation, peak thrombin, and endogenous thrombin potential were quantified. AB023 in addition to heparin sustained circuit patency in all tested circuits (5/5) after 6 hours, while 60% of circuits treated with heparin alone occluded (3/8), log-rank p < 0.03. AB023 significantly prolonged the time to clot formation as compared to heparin alone (15.5 vs . 3.3 minutes; p < 0.01) at the 3-hour time point. AB023 plus heparin significantly reduced peak thrombin compared to heparin alone (123 vs . 217 nM; p < 0.01). Inhibition of contact pathway activation of FXI may be an effective adjunct to anticoagulation in extracorporeal life support.

OC 17.1 The Association between Venous Thrombosis-Associated Genetic Variants and Coagulation Factor Levels and Thrombin Generation Potential

OC 17.1 The Association between Venous Thrombosis-Associated Genetic Variants and Coagulation Factor Levels and Thrombin Generation Potential

PB0072 Thrombin Generation Potential and the Risk of Severe COVID-19 Infection

PB0072 Thrombin Generation Potential and the Risk of Severe COVID-19 Infection

PB0196 Concizumab Enhances Thrombin Generation Potential in Plasma from Patients with FV-Amsterdam

PB0196 Concizumab Enhances Thrombin Generation Potential in Plasma from Patients with FV-Amsterdam

Women with severe postpartum hemorrhage have a decreased endogenous thrombin potential before delivery

BackgroundSevere postpartum hemorrhage (PPH), defined as a blood loss ≥1000 mL, is associated with maternal morbidity and mortality. ObjectivesWe aimed at characterizing coagulation properties of predelivery plasmas from pregnant women with thrombin generation assay and hemostatic biomarkers (plasminogen activator inhibitor-1, tissue factor [TF], and thrombomodulin). MethodsA nested case-control study was conducted within the “Study of Biological Determinants of Bleeding Postpartum,” a French prospective cohort study, in order to compare women with severe PPH (cases) and controls matched for age, body mass index, term, and mode of delivery. Plasma was collected at entry in the delivery room, and blood loss was measured objectively. The predelivery endogenous thrombin generation potential (ETP) was measured in plasma using calibrated automated thrombinography and low TF concentration. Hemostatic biomarkers were measured using ELISA kits. ResultsA total of 142 women (71 cases and 71 controls) were investigated. There was no difference in the median lag phase, thrombin peak, and time to peak between cases and controls. However, median predelivery ETP was lower in cases than in controls (2170 vs 2408 nM.min, P < .0001), independently of mode of delivery and PPH etiology. Median plasminogen activator inhibitor-1 and TF levels were higher in cases compared with controls (107.4 vs 68.1 ng/mL, P = .0003; 34.4 vs 27.4 pg/mL, P = .007), whereas thrombomodulin levels did not differ between the 2 groups. ConclusionAmong thrombin generation assay parameters, predelivery ETP levels may have a predictive value for severe PPH.

Synergistic inhibitory effects of clopidogrel and rivaroxaban on platelet function and platelet-dependent thrombin generation in cats.

Dual antithrombotic treatment (DAT) with clopidogrel and rivaroxaban sometimes is prescribed to cats with hypertrophic cardiomyopathy at risk of thromboembolism. To date, no studies have evaluated their combined effects on platelet function. Evaluate the safety of DAT in healthy cats and compare, ex vivo, platelet-dependent thrombin generation and agonist-induced platelet activation and aggregation in cats treated with clopidogrel, rivaroxaban, or DAT. We hypothesized that DAT would safely modulate agonist-induced platelet activation and aggregation more effectively than single agent treatment. Nine apparently healthy 1-year-old cats selected from a research colony. Unblinded, nonrandomized ex vivo cross-over study. All cats received 7 days of rivaroxaban (0.6 ± 0.1 mg/kg PO), clopidogrel (4.7 ± 0.8 mg/kg PO), or DAT with defined washout periods between treatments. Before and after each treatment, adenosine diphosphate (ADP)- and thrombin-induced platelet P-selectin expression was evaluated using flow cytometry to assess platelet activation. Platelet-dependent thrombin generation was measured by fluorescence assay. Platelet aggregation was assessed using whole blood impedance platelet aggregometry. No cats exhibited adverse effects. Of the 3 treatments, only DAT significantly decreased the number of activated platelets (P = .002), modulated platelet activation in response to thrombin (P = .01), dampened thrombin generation potential (P = .01), and delayed maximum reaction velocity (P = .004) in thrombin generation. Like clopidogrel, DAT inhibited ADP-mediated platelet aggregation. However, rivaroxaban alone resulted in increased aggregation and activation in response to ADP. Treatment combining clopidogrel and rivaroxaban (DAT) safely decreases platelet activation, platelet response to agonists, and thrombin generation in feline platelets more effectively than monotherapy with either clopidogrel or rivaroxaban.

Abstract 263: Prothrombotic State Exists In Long-COVID Even After Mild Acute Infection

Long-term consequences of COVID-19 (long-COVID) are becoming a significant burden to health care. Many people remain symptomatic for months or years after recovery from COVID-19, including some who had only mild symptoms during acute infection. Sustained increases in pro-thrombotic markers have been reported in patients who were initially hospitalized with acute COVID-19. However, whether patients with less severe acute infection develop a prothrombotic-state during long-COVID is unclear. Our objective was to measure prothrombotic factors in long-COVID patients with mild disease (non-hospitalization) during acute infection. We recruited 47 consecutive adult patients with long-COVID symptoms seen in the Post-COVID clinic. All patients had a confirmed COVID-19 diagnosis by RT-PCR or antibody test during acute infection and had at least one of the symptoms of long-COVID per WHO criteria. Twenty asymptomatic healthy subjects not previously diagnosed with COVID-19 were recruited as controls. Markers of platelet activation and platelet-neutrophil aggregates (PNA) were quantified using whole blood flow cytometry. Serum levels of cell-free DNA (cfDNA) and citrullinated histones (H3Cit) and thrombin generation potential in plasma was measured. At the time of recruitment (6 weeks to 2 years after acute infection), the most common post-COVID symptoms included fatigue, chest pain, shortness of breath, headache, and dizziness. In whole blood, activation with TRAP caused increased surface expression of P-selectin without inducing activation of integrin αII b β 3 on platelets (P&lt;0.001 and P=0.5 respectively vs control). Concurrently, there was increased PNA in the long-COVID cohort (P&lt;0.01 vs. control). H3Cit and cfDNA were elevated in long-COVID (P&lt;0.01 and P&lt;0.05 respectively vs. control) and thrombin generation potential was increased, reflected by shorter lag-time, higher peak thrombin and increased velocity index (P&lt;0.01, P&lt;0.05 and P&lt;0.0001 respectively vs control). Within the long-COVID cohort, these markers were not different for age, sex, BMI or time since infection. We conclude that a prothrombotic state persists for months to years in long-COVID patients who exhibited mild disease at the time of initial infection.

High risk oral contraceptive hormones do not directly enhance endothelial cell procoagulant activity in vitro.

Oral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs. Characterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERβ and inflammatory processes. Human umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERβ (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively. Neither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus. The OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro.

Decreased protein C activity, lower ADAMTS13 antigen and free protein S levels accompanied by unchanged thrombin generation potential in hospitalized COVID-19 patients

IntroductionCOVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown. Patients and methodsIn 118 adult Caucasian severe but non-critically ill COVID-19 patients (median age 58 years; 73 % men) and 46 controls, we analyzed in vitro plasma thrombin generation profile (calibrated automated thrombogram [CAT assay]) and investigated thrombophilia-related factors, such as protein C and antithrombin activity, free protein S level, presence of antiphospholipid antibodies and factor V Leiden R506Q and prothrombin G20210A mutations. We also measured circulating von Willebrand factor (vWF) antigen and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) antigen and activity. In patients, blood samples were collected on admission to the hospital before starting any therapy, including heparin. Finally, we examined the relationship between observed alterations and disease follow-up, such as thromboembolic complications. ResultsCOVID-19 patients showed 17 % lower protein C activity, 22 % decreased free protein S levels, and a higher prevalence of positive results for IgM anticardiolipin antibodies. They also had 151 % increased vWF, and 27 % decreased ADAMTS13 antigens compared with controls (p < 0.001, all). On the contrary, thrombin generation potential was similar to controls. In the follow-up, pulmonary embolism (PE) occurred in thirteen (11 %) patients. They were characterized by a 55 % elevated D-dimer (p = 0.04) and 2.7-fold higher troponin I (p = 0.002) during hospitalization and 29 % shorter time to thrombin peak in CAT assay (p = 0.009) compared to patients without PE. ConclusionsIn COVID-19, we documented prothrombotic abnormalities of peripheral blood. PE was characterized by more dynamic thrombin generation growth in CAT assay performed on admittance to the hospital.

Characterization of a prothrombotic phenotype using thrombin generation and thrombin activity in cirrhosis and portal hypertension

BackgroundPatients with advanced chronic liver disease (ACLD) may develop a prothrombotic phenotype that seems to be more pronounced with more severe liver dysfunction. An imbalance of endogenous pro- and anticoagulants is not fully captured by routine coagulation assays. MethodsIn a cohort of ACLD patients undergoing hepatic venous pressure gradient (HVPG) measurement, we assessed thrombin generation (TGA) using two commercially available assays (Technothrombin and Thrombinoscope) with and without addition of soluble thrombomodulin (TM), as well as thrombin activity, alongside a panel of coagulation parameters. ResultsThe cohort encompassed 37 patients (median age 55.3 years, mean HVPG 16 ± 5 mm Hg). In the TM-modified Thrombinoscope TGA, the endogenous thrombin generation potential (ETP) was significantly increased in Child-Pugh-Score (CPS) B/C patients (N = 23, 62 %) compared to CPS A patients (N = 14, 38 %) (ETP: 546 nM∗min (443–696) vs. 404 nM∗min (289–573), p = 0.028). Using the Technothrombin TGA without TM, patients with CPS B/C had decreased ETP compared to CPS A patients (ETP: 2792 ± 1336 nM∗min vs. 5040 ± 816 nM∗min, p < 0.001) and with addition of TM (final concentration: 5 nM; ETP: 2545 ± 1327 nM∗min vs. 4824 ± 929 nM∗min, p < 0.001). Thrombin activity levels were 0.6pM in median (0.2–1.6pM) and above the level of detectability (0.10pM) in 94.6 % of patients but were not correlated to severity of cirrhosis (CPS A 0.7pM vs CPS B/C 0.4pM, p = 0.377) nor to parameters of TGA. ConclusionThrombin plasma levels are elevated in liver disease patients without apparent correlation to TGA or severity of cirrhosis. TGAs can be modified with TM to enable protein C-dependent anticoagulation, but result in differences with regard to severity of liver disease.

Evaluation of the procoagulant state in chronic immune thrombocytopenia before and after eltrombopag treatment—a prospective cohort study

BackgroundThrombopoietin receptor agonists are frequently used in treating immune thrombocytopenia (ITP) owing to high response rates and good tolerability. ITP is associated with an increased risk of thrombosis. Whether treatment with eltrombopag further increases this risk is controversial. The mechanisms behind the thrombotic risk in ITP are unclear. ObjectivesTo assess platelet function and hypercoagulability in patients with ITP and the effect of eltrombopag thereon. MethodsThis prospective multicenter study assessed adult primary patients with ITP who were starting eltrombopag treatment. Platelet (re)activity and hypercoagulability were measured in whole blood or plasma before start and after 2 to 3 weeks of eltrombopag treatment and compared with those of controls. Change over time was assessed by mixed-effects models, and the results were corrected for multiple testing. ResultsWe included 16 patients and 33 controls. At baseline, patients with ITP exhibited lower expression of glycoprotein VI, more activated platelets, and lower reactivity toward agonists compared with controls. β-Thromboglobulin levels reduced and thrombin generation peak height increased compared with those of controls. In line with this finding, patients with ITP showed high factor VIII (median, 217%; IQR, 174%-272%) and von Willebrand factor levels (median, 167%; IQR, 109%-198%). Eltrombopag treatment increased thrombin generation potential: lag time decreased and peak height and endogeneous thrombin potential increased. The latter changes were not significant after correction for multiple testing. ConclusionPatients with ITP in this study were in a hypercoagulable state, with preactivated platelets, increased thrombin generation potential, and increased levels of factor VIII and von Willebrand factor. Eltrombopag treatment further increased plasma thrombin generation potential but no other hemostatic parameters.

Translational insights into mechanisms underlying residual venous obstruction and the role of factor XI, P-selectin and GPVI in recurrent venous thromboembolism

BackgroundResidual venous obstruction (RVO) after deep vein thrombosis (DVT) is considered a risk factor of recurrent venous thromboembolism (VTE), arterial events and post-thrombotic syndrome (PTS). We hypothesized thrombo-inflammatory markers might be associated with RVO and clinical outcomes. Materials and methodsIn a DVT cohort with routine RVO-assessment and 5-year follow-up, patients were invited for blood withdrawal after stopping anticoagulants. Thrombin generation potential, coagulation enzyme:inhibitor complexes, soluble platelet markers and clinical markers were measured in platelet-poor plasma. Associations were represented as odds ratio (OR) or hazard ratio (HR) per standard deviation. ResultsPatients with RVO (102/306, 33 %) had higher rates of PTS (24 vs. 12 %, p = 0.008), but similar rates of recurrence (16 vs. 15 %, p = 0.91) and arterial events (7 vs. 4 %, p = 0.26). RVO was associated with thrombin peak height (OR 1.40 [1.04–1.88]), endogenous thrombin potential (ETP, OR 1.35 [1.02–1.79]), and CRP (OR 1.74 [1.10–2.75]). Recurrent VTE was associated with ETP (HR 1.36 [1.03–1.81]), FXIa:C1-inhibitor (HR 1.34 [1.04–1.72]), thrombin:antithrombin (HR 1.36 [1.16–1.59]), soluble P-selectin (HR 2.30 [1.69–3.11]), soluble glycoprotein VI (sGPVI, HR 1.30 [1.01–1.69]), D-dimer (HR 1.56 [1.31–1.86]), and factor VIII (HR 1.44 [1.15–1.82]). Arterial events were associated with sGPVI (HR 1.80 [1.25–2.59]). PTS was not associated with any marker. ConclusionsOur findings indicate RVO was associated with thrombo-inflammation, but this did not predict clinical outcomes in this setting. Importantly, we found recurrent VTE was associated with ongoing coagulation and platelet activation in patients well beyond the acute phase of DVT. Furthermore, sGPVI indicated an increased risk of arterial events, highlighting the role of platelets in arterial thrombosis following DVT.

The Nupower Study: Nuwiq for Perioperative Management of Patients with Haemophilia a on Emicizumab Regular Prophylaxis

Background: Surgery and other invasive procedures have the potential for excessive, uncontrolled bleeding in patients with hemophilia A. Simoctocog alfa (Nuwiq®) has been shown to be highly efficacious at controlling bleeding during and after surgery in hemophilia A patients. Sufficient factor VIII (FVIII) must be available for the surgery and to maintain postoperative coverage. Emicizumab is indicated for the prophylaxis of bleeding in patients with hemophilia A, but additional hemostatic cover may be required to manage bleeding during surgery, particularly major surgeries. To date, there are limited data available on the use of FVIII to provide additional hemostatic cover for surgery in patients on emicizumab. Aims: To evaluate the overall haemostatic efficacy of simoctocog alfa during major surgery in hemophilia A patients on regular emicizumab prophylaxis. Methods: NuPOWER is a prospective, open-label, single-arm, multicenter study to be conducted at approximately 15 study centers worldwide. Previously treated male patients ≥12 years of age with severe hemophilia A (FVIII:C <1%) and no current or past FVIII inhibitors, who have been on regular emicizumab prophylaxis for at least 3 months and scheduled for major elective surgery are eligible for the study. Simoctocog alfa will be administered once within 3 hours prior to surgery and repeated as necessary every 8-24 hours to achieve a FVIII plasma level of 80-100 IU/dL until adequate wound healing has been achieved. Simoctocog alfa dosing will continue for at least another 7 days to maintain a FVIII plasma level of 30-60 IU/dL (Figure 1). The primary endpoint is the overall perioperative hemostatic efficacy ("success" or "failure") of simoctocog alfa. Hemostatic efficacy will be assessed at the end of surgery and at the end of the postoperative period (i.e. up to 24 hours after the last injection of simoctocog alfa), both using an objective 4-point ordinal scale, and adjudicated by the Independent Data Monitoring Committee using a pre-defined algorithm. Secondary endpoints include thrombin generation potential, and incidence of adverse events including thrombotic events and inhibitor formation. Target enrollment is 23 patients to document 26 major surgeries. Each subject can have multiple independent surgeries that will be counted as separate procedures. Study start is planned for Q4, 2022. Conclusions: Results from the NuPOWER study will provide valuable data on the use of simoctocog alfa in patients on emicizumab prophylaxis who need to undergo major surgery. The data will help healthcare professionals to develop evidence-based guidelines for the management of bleeding in hemophilia A patients on emicizumab during surgery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Elevated Thrombin Generation Biomarkers and Their Relevance with Decreased Thrombin Generation Potential in Patients with Pulmonary Embolism

Background: Pulmonary embolism (PE) is a catastrophic complication in patients with venous thrombosis and is the third leading cardiovascular cause of death after myocardial infraction and stroke. Inflammation, hemostatic dysregulation, and generation of thrombotic mediators contribute to the pathogenesis of PE. Persistent thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) in PE patients and demonstrate its relationship with thrombin generation biomarkers, such as prothrombin fragment 1+2 (F1+2), thrombin anti-thrombin complex (TAT) and D-Dimer. Methods: Citrated blood samples from 201 patients with confirmed PE were collected under an IRB approved protocol. Fifty normal human plasma (NHP) samples were used as a reference. Thrombin generation studies were carried out by using fluorogenic substrate method (calibrated automated thrombogram, Diagnostica Stago; Paris, France). Thrombin generation parameters such as peak thrombin, lag time and endogenous thrombin potential (ETP) were compiled. Plasma samples were analyzed for such biomarkers as prothrombin fragment 1+2 (F1+2), thrombin anti-thrombin complex (TAT), and D-Dimer by using commercially available Sandwich ELISA methods. Results were compiled as mean ± SD and applicable statistical analysis was performed by using GraphPad Prism software. Result: PE patients exhibited a wide variation in thrombin generation parameters. ETP in PE patients decreased to 549.2±508.4 nM*min compared to NHP 701.8±214.6 nM*min, thrombin levels in PE patients also decreased to 83.7±80.1 nM compared to NHP 158.5±31.5 nM, while lag time increased 5.4±12.7 mins to 2.6±0.6 mins compared to NHP. Biomarker analysis showed elevated levels of F1+2 (972.3±1508.0 vs 294.2±105.0 pmol/L, 3.3-fold), TAT (34.9±54.3 vs 2.3±1.1ug/L, 15.3-fold), and D-Dimer (6720.0±4810.0 vs 182.6±205.8 ug/ml, 36.8-fold) compared to NHP. Endogenous thrombin potential levels demonstrate moderate and positive correlations with F1+2 (r = 0.22) while weak correlations were noted with TAT (r = 0.14) and D-Dimer (r = 0.05). F1+2 possess strong correlation with TAT (r = 0.55) and moderate for D-Dimer (r = 0.21). TAT also showed strong correlation with D-Dimer (r = 0.45). Thrombin generation parameters and biomarker levels based on PE severity has been shown in figure 1. Marked increase in thrombin generation biomarker levels were noted with increase PE severity. However, for the thrombin generation parameters, there is no differences were noted between the low grade and sub-massive PE, while the ETP and peak thrombin levels decreased, and lag time increased for the massive PE. Conclusion: These studies suggest that persistent thrombin generation in PE patients results in the consumption of prothrombin complex. However, such biomarkers of thrombin generation as F1+2, TAT and D-Dimer are formed, leading to higher circulating levels. Simultaneous measurement of thrombin generation biomarkers and thrombin generation parameters may be useful to determine the pathogenesis of thrombotic complications and optimization of treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal