COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

Abstract Background Hospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined. Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability. Aim To assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis. Methods Blood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI; Final concentration 100μg/mL). Results 14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years; p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2; p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment. All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11; supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)]. Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL; p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL; p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min; p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1; p=0.3). Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins; p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1). Conclusion Plasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing. Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19. In the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). Figure 1 Figure 1. Disclosures Maguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding; Actelion: Research Funding; Leo Pharma: Research Funding; Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

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Thrombin-Generating Capacity of Microvesicles (MVs) in Patients with Immune Thrombocytopenia (ITP) before and during Treatment with Thrombopoietin-Receptor Agonists (TPO-RA)

Blood ◽

10.1182/blood-2018-99-116911 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2511-2511

Author(s):

Lamya Garabet ◽

Waleed Ghanima ◽

Marit Hellum ◽

Per Morten Sandset ◽

James B. Bussel ◽

...

Keyword(s):

Tissue Factor ◽

Research Funding ◽

Thrombin Generation ◽

Statistical Significance ◽

Small Sample ◽

Activity Assay ◽

Thrombotic Risk ◽

Increased Risk ◽

Significant Difference ◽

Before And After

Abstract Introduction: ITP is an acquired autoimmune disorder characterized by isolated thrombocytopenia and an increased risk of bleeding. Paradoxically, ITP is also associated with an increased risk of thrombosis, which may be exacerbated with TPO-RA-treatment. The underlying mechanism(s) involved in the development of thrombosis in ITP and especially in TPO-RA-treated ITP-patients remain poorly understood. MVs released from activated/apoptotic cells are procoagulant due to the presence of tissue factor (TF) and phospholipids such as phosphatidylserine (PS) on their membrane. MVs have been shown to be increased in ITP-patients but the prothrombotic role of these MVs before and after treatment with TPO-RA is unclear. We measured MV-associated thrombin generation, PS-dependent thrombin generation in plasma, TF-activity and PS-activity in plasma of ITP-patients vs controls and investigated the effect of TPO-RA on these measurements. Methods: In 15 controls and 11 ITP patients, before TPO-RA and 2 and 6 weeks after the initiation of TPO-RA, citrated plasma was prepared (2000gx20 min) and immediately frozen. After thawing of plasma, MVs were isolated by centrifugation (17,000gx30 min), the supernatant removed and the remaining MV-pellet washed twice. Isolated plasma-derived MVs were added to pooled normal plasma (PNP), and to obtain measureable thrombin generation, antibodies against tissue factor pathway inhibitor were also added to the PNP. The ability of MVs to generate thrombin was measured by the calibrated automated thrombogram method and the thrombin generation parameters lag time (LT), peak, endogenous thrombin potential (ETP), time to peak (ttPeak) and velocity index (VI) were calculated by the Thrombinoscope software. To estimate the contribution of procoagulant PS, PS-activity in plasma (PS equivalents) was measured with the Zymuphen MP-activity assay. In addition, thrombin generation was measured directly in plasma where only TF (1pM), but not PS, had been added (PS-dependent thrombin generation). TF-activity in plasma was measured with the Zymuphen MP-TF-activity assay. Friedman test with Dunn's multiple comparisons was used to compare measurements in ITP-patients before and after TPO-RA-treatment. Kruskal-Wallis test was used to compare measurements in ITP-patients and controls. Results: Median age of ITP-patients and controls: 53 and 50 years. Eight (73%) were on romiplostim and three (27%) were on eltrombopag. Median values (IQR) for all measurements before, 2 weeks and 6 weeks on TPO-RA-treatment and in controls are shown in the table. ITP-patients before treatment with TPO-RAs vs controls: No significant difference was found in MV-associated thrombin generation, PS-activity or TF-activity in plasma. There was a trend towards a higher peak, VI and ETP and lower ttPeak in PS-dependent thrombin generation in plasma of ITP-patients vs controls; lack of statistical significance may be due to the small sample size. TPO-RA-treated ITP-patients (2 and 6 weeks) vs controls: MV-associated thrombin generation: significant changes after 2 weeks (shorter LT/ttPeak (p=0.03/p=0.03), higher peak/VI (p=0.04/p=0.04)). PS-dependent thrombin generation in plasma: significant changes after 2 weeks (shorter ttPeak (p=0.007), higher peak/VI (p=0.003/p=0.001)), and after 6 weeks (shorter ttPeak (p=0.002), higher peak/ETP/VI (p=0.004/p=0.02/p=0.002)). PS-activity in plasma: significant changes after 2 and 6 weeks (p=0.006/p=0.02). TF-activity in plasma: no significant changes. ITP-patients before vs after TPO-RA-treatment (2 and 6 weeks): Only MV-associated thrombin generation was significantly increased after 2 weeks (higher peak/VI (p=0.03/p=0.04)). Conclusions: Compared with controls, treatment with TPO-RAs increases PS-dependent thrombin generation and PS-activity in plasma that is partly accompanied by an increase in MV-associated thrombin generation, but not with an increase in TF-activity in plasma. Similarly, we find a trend of increase in PS-dependent thrombin generation in ITP vs controls. This suggests that PS-positive MVs, most likely released from activated/apoptotic platelets, may contribute to the pre-existing increased thrombotic risk present in at least some of the patients with ITP, and may be used as a potential marker to estimate the risk of future thromboembolic events in TPO-RA-treated ITP-patients. Disclosures Ghanima: Roche, Amgen, Novartis, Bayer, BMS: Other: Personal Fees, Research Funding; GlaxoSmithKline and Pfizer: Other: Personal Fees. Bussel:Prophylix: Consultancy, Research Funding; Protalex: Consultancy; Uptodate: Honoraria; Novartis: Consultancy, Research Funding; Amgen Inc.: Consultancy, Research Funding; Momenta: Consultancy; Rigel: Consultancy, Research Funding.

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Tissue Factor Pathway Inhibitor Mediates Attenuation of Tissue Factor-Stimulated Thrombin Generation in Early Onset Preeclampsia

Blood ◽

10.1182/blood.v128.22.1431.1431 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1431-1431

Author(s):

Karl Egan ◽

Hugh O'Connor ◽

Barry Kevane ◽

Fergal Malone ◽

Amani Al Zadjali ◽

...

Keyword(s):

Tissue Factor ◽

Early Onset ◽

Research Funding ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Inflammatory Disorder ◽

Contact Activation ◽

Thrombotic Risk ◽

Tissue Factor Pathway ◽

Early Onset Preeclampsia

Abstract Introduction Pregnancy increases the risk of venous thromboembolism (VTE) in women. Interestingly, preeclampsia, an extremely pro-inflammatory disorder specific to pregnancy is associated with a lower than expected increase in thrombotic risk compared to other proinflammatory disorders. The mechanism underling this lower thrombotic risk is unknown. The aim of this study was to investigate the coagulation balance, a major determinant of VTE risk, in early onset preeclampsia (EOP) patients. EOP is the most inflammatory form of preeclampsia, characterised by the development of hypertension and proteinuria prior to 34 weeks gestation. Methods Platelet-poor plasma was collected from patients with early onset preeclampsia (EOP n=26), matched pregnant (n=20) and non pregnant controls (n=16). Calibrated automated thrombography, an assay of thrombin generation and TFPI assays were performed. Data are expressed as mean ± standard deviation. Results During the study period, 15,299 women delivered and 40 patients developed EOP, of whom 26 were successfully recruited with consent. For comparison, 16 non pregnant controls and 20 pregnant controls were also recruited. When artefactual contact activation was inhibited by using corn trypsin inhibitor as anticoagulant, EOP patients were characterised by a decrease in the rate and extent of Tissue Factor (TF) thrombin generation compared to pregnant controls. There was a prolongation of the time to peak thrombin generation (16 ± 4 minutes vs. 13 ± 2 minutes, p < 0.05), a decrease in the velocity index (25 ± 17nM/minute vs. 41 ± 27nM/minute, p < 0.05), and a decrease in peak thrombin generation (150 ± 80 nM IIa vs. 210 ± 70 nM IIa, p < 0.05 ) in EOP compared to pregnant controls. This reduction in the rate and extent of thrombin generation was most amplified in patients with severe EOP (multi-organ involvement) compared with moderate EOP. This lower overall procoagulant state seen was further emphasised by the increase in sensitivity to the anticoagulant activity of exogenously added activated protein C and thrombomodulin observed in EOP. Again, the increased sensitivity to APC and thrombomodulin was most apparent in severe EOP cases. Previous studies have shown that preeclampsia is characterised by a increase in plasma TFPI activity. As such, we investigated whether increases in plasma TFPI activity explained the reduction in TF-dependent thrombin generation. Consistent with previous studies, plasma tissue factor pathway inhibitor (TFPI) levels and plasma TFPI activity significantly increased in EOP, most notably in severe EOP cases. There was a significant inverse correlation between total TFPI levels and peak thrombin generation (r2 = -0.64, p < 0.05) and TFPI activity and peak thrombin generation (r2= -0.52, p < 0.05). The inhibition of TFPI with a polyclonal anti-TFPI antibody abolished the attenuation in thrombin generation seen in severe EOP. Conclusion In conclusion, TF-dependent thrombin generation is reduced in patients with early onset preeclampsia due to increases in plasma TFPI activity. These findings may partially explain the lower thrombosis risk observed in patients with early onset preeclampsia relative to comparable systemic proinflammatory conditions. These data also have future potential in helping clinicians to manage competing bleeding and thrombotic risks in these very high-risk patients. Disclosures Maguire: Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

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Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Blood ◽

10.1182/blood-2021-146803 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3207-3207

Author(s):

Patrick Van Dreden ◽

Joseph Gligorov ◽

Evangelos Terpos ◽

Mathieu Jamelot ◽

Michele Sabbah ◽

...

Keyword(s):

Endothelial Cell ◽

Tissue Factor ◽

Platelet Activation ◽

Research Funding ◽

Thrombin Generation ◽

Cell Activation ◽

Control Group ◽

Clotting Time ◽

Endothelial Cell Activation ◽

Active Cancer

Abstract Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

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Factor Xa Inhibitors Can Be Differentiated in Xa, IIa and Microparticle Generation and Other Whole Blood Based Assays.

Blood ◽

10.1182/blood.v110.11.931.931 ◽

2007 ◽

Vol 110 (11) ◽

pp. 931-931 ◽

Cited By ~ 1

Author(s):

Omer Iqbal ◽

Cafer Adiguzel ◽

Debra Hoppensteadt ◽

Josephine Cunannan ◽

Jawed Fareed

Keyword(s):

Tissue Factor ◽

Whole Blood ◽

Thrombin Generation ◽

Factor Xa ◽

Final Concentration ◽

Factor Xa Inhibitors ◽

Saline Control ◽

Functional Assay ◽

Control Value ◽

Anticoagulant Agents

Abstract Currently used oral anticoagulants such as Vitamin K antagonists have drawbacks, which reportedly limit their safety and efficacy. Oral Factor Xa and IIa inhibitors are claimed to overcome these limitations. Factor Xa inhibitors provide more complete suppression of thrombin generation than Factor IIa inhibitors. Four Factor Xa anticoagulant agents, both direct and indirect, namely A,B,C and D with Ki values 6–200 nM, were studied in various assays at equigravimetric final concentration of 10 ug/ml to determine their relative potencies. Apparent differences in their biochemical profiles were noted in thrombin generation, Factor Xa generation and microparticle generation inhibition assays. Furthermore, the anticoagulant potential of these Xa inhibitors was studied in celite activated clotting time (ACT) and modified celite ACT system using different concentrations of tissue factor. Microparticle generation was performed using agent- supplemented whole blood that was incubated for three minutes with varying concentrations of tissue factor. Following three minutes the reaction was stopped using EDTA stop solution. The functional assay for the determination of microparticle procoagulant activity in the plasma was performed using Zymuphen MP-Activity assay kit from Hyphen BioMed (Neuville-sur-Oise, France). While three of the Xa inhibitors studied showed microparticle generation inhibition levels of 31.37 nM, one agent provided a level of 17.87 nM compared to a saline control value of 39.28nM. Supplementation studies were also carried out in whole blood PT, APTT and Heptest assays in the concentration range of 0 to 10 ug/ml. The whole blood prothrombin time assay showed a maximum of 208.8, 61.3, 77.8 and 15.9 seconds at a final concentration of 10 ug/ml for the respective anticoagulant agents when compared to a normal value of 12.3 seconds. Similarly the whole blood APTT assay showed a maximum of 267.9, 161.8, >300 and 71.9 seconds respectively when compared to a control value of 46.8 seconds. The whole blood Heptest assay showed a varying maximum anticoagulant effect from >300, 42.7, >300 and >300 seconds respectively when compared to a control value of 13.8 seconds. In each of these assays Factor Xa inhibitors showed concentration-dependent effects and had varying potencies. In TF-mediated platelet activation assays the different Xa agents produced varying effects which were not proportionate to their Ki values. Differentiation of Factor Xa inhibitors have a clinical impact in dosage selection, dosage adjustment and their monitoring when given in large dosages. Synthetic factor Xa agents exhibit Ki values of 20–200 nM, while pentasaccharide -AT complex has a Ki value of 60 nM. However, these results do not translate into proportionate anticoagulant effects to their Ki values. Large-scale clinical studies are necessary to validate these preliminary results.

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Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Blood ◽

10.1182/blood-2021-151203 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4235-4235

Author(s):

Paula Acuña ◽

Elena Monzón Manzano ◽

Elena G Arias-Salgado ◽

María Teresa Alvarez Román ◽

Mónica Martín ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Function ◽

Research Funding ◽

Thrombin Generation ◽

Bleeding Tendency ◽

Contact Activation ◽

Normal Range ◽

Coagulation Tests ◽

In Vitro Treatment

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

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Platelet-Dependent Thrombin Generation Induced By ADP or Activated Factor X Does Not Contribute to Increased In Vivo Thrombin Formation in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-118487 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4336-4336

Author(s):

Christina Berens ◽

Heiko Rühl ◽

Jens Müller ◽

Johannes Oldenburg ◽

Peter Brossart ◽

...

Keyword(s):

Plasma Levels ◽

Research Funding ◽

Thrombin Generation ◽

Myeloproliferative Neoplasms ◽

Final Concentration ◽

Factor X ◽

Prothrombinase Complex ◽

Significant Difference ◽

Thrombin Formation

Abstract Introduction: Myeloproliferative Neoplasms (MPN), including the clinical entities Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by an increased thrombotic risk, the pathomechanisms of which are not well-understood. It has been suggested that an increased sensitivity of platelets to adenosin diphosphate (ADP) contributes to the hypercoagulable state in PV and ET through increased thrombin generation. In the present study we analyzed plasma levels of thrombin and platelet-dependent thrombin generation in MPN patients with an additional focus on prothrombin activation by the prothrombinase complex. Methods: A total of 33 blood samples were obtained from patients with MPN (PV, n=18; ET, n=5; PMF, n=10) and from 33 healthy blood donors that served as controls. In vitro thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was assessed using the Calibrated Automated Thrombogram (CAT) assay. To induce thrombin generation either ADP (1 µmol/L final concentration) or activated factor X (FXa, 10 ng/mL final concentration) were applied. To further characterize the MPN-associated hypercoagulable state in vivo, plasma levels of free thrombin were measured using an oligonucleotide-based enzyme capture assay (OECA). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally. Results: In PRP of MPN patients a slightly higher ADP-induced peak thrombin concentration (Cpeak) was observed than in the healthy controls, with 106 (79-130) vs. 84 (65-110) nmol/L (median and interquartile range, p=.026). There was no statistically significant difference in the ADP-induced endogenous thrombin potential (ETP) in MPN patients (1445, 1194-1643 nmol/L·min) compared with the controls (1417, 1258-1814 nmol/L·min). There was no statistically significant difference in the FXa-induced Cpeak and ETP between MPN patients and controls, with 106 (79-127) vs. 97 (82-128) nmol/L, and 1424 (1165-1560) vs. 1641 (1193-1841) nmol/L·min, respectively. With 0.68 (<0.46-1.20) pmol/L, plasma levels of free thrombin were significantly higher (p=.025) in MPN patients than in the control group, in which median thrombin levels were below the limit of detection. Plasma levels of F1+2 and TAT were also higher in the MPN group than in healthy controls, with 0.31 (0.17-0.50) vs. 0.18 (0.13-0.25) nmol/L (p=.002) and 4.36 (2.53-6.76) vs. 2.36 (<2.00-2.68) ng/mL (p=.003), respectively. Conclusion: Increased plasma levels of thrombin, F1+2, and TAT indicate enhanced in vivo thrombin formation in MPN patients. Despite a slightly increased ADP sensitivity of MPN-platelets, the total amount of thrombin generated in PRP from MPN patients is not increased. This makes it unlikely that a 'hyperreactivity' of MPN platelets, resulting in increased activities of the prothrombinase complex on the platelet surface, contributes to the increased thrombin formation in MPN patients. Disclosures Berens: Shire: Research Funding; Biotest: Research Funding; Pfizer: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Rühl:Shire: Research Funding; Swedish Orphan Biovitrum: Consultancy, Research Funding; Grifols: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding.

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Tissue Factor Bearing Microparticles in the Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v118.21.5174.5174 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5174-5174

Author(s):

Brady L Stein ◽

Brandon McMahon ◽

Ivy Weiss ◽

James Marvin ◽

Hau C. Kwaan

Keyword(s):

Sample Size ◽

Tissue Factor ◽

Thrombin Generation ◽

Myeloproliferative Neoplasms ◽

Flow Cytometric Analysis ◽

Jak2 V617f ◽

Lag Phase ◽

Thrombotic Risk ◽

Larger Sample

Abstract Abstract 5174 Background: Thrombosis is a well recognized complication in the myeloproliferative neoplasms (MPN), essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). The mechanism for thrombosis is not well-established, nor are there relevant biomarkers to predict risk and/or recurrence. Circulating cellular microparticles (MP) containing procoagulant tissue factor (TF) have been shown to correlate with thrombotic risk in many forms of cancer and cardiovascular diseases. To investigate the role of MP in the MPN, we studied 16 patients (ET=5; PV=6; PMF=2; post-ETMF=1, and MPN NOS=2) and compared results to 15 healthy subjects. Methods: Citrated blood samples were collected from the 16 MPN patients and 15 controls. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 50 μL of PPP was added to 200 μL PBS (without Mg/Ca) and centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of buffer for labeling with TF, CD41a (platelets), CD14 (monocytes), CD66b (neutrophils), and CD33 (myeloid lineage). Following incubation, PBS was added to the suspension to a volume of 1ml for flow cytometric analysis (LSR Fortessa, FlowJo software). Electronic triggering was done on side-scatter, and acquisition regions were defined based on sizing beads (0.3 to 1.0 micron) along with annexin A5 positivity. Using MP sediment, TF activity was measured using chromogenic assays (Actichrome TF ELISA, American Diagnostica,) and thrombin generation (TGT) was assayed (Technothrombin TGA, diaPharma), with results expressed as lag phase, velocity-index, peak thrombin, and area under the curve (AUC). The Wilcoxon-Rank Sum test was used to compare group differences (MPN vs. control) in median values. Results: Among the MPN patients, 7(44%) were male, and the median age was 60 years. 11 (69%) were JAK2 V617F positive, and 3 (19%) had a prior history of thrombosis (2 hepatic vein thromboses, 1 myocardial infarction). At the time of collection, 14 (93%) were on aspirin, 1 (6%) was on Coumadin, and 5 (31%) were on Hydroxyurea. The median total MP number was increased in MPN patients vs. controls (243580 vs. 83120; p=0.0057). The median percentage of TF-bearing MP's was also significantly greater in MPN patients compared to controls (35.5% vs. 12%; p=0.0003). When comparing MPN patients to controls, these TF-bearing MP were derived from CD14 (31.5% vs. 2%; p<0.001) and CD41a (24% vs. 7%; p=0.0157), respectively, reflecting monocyte and platelet origins of the MPs. The TF-bearing MPs in MPN patients (N=10) were functionally active compared to controls (N=10) (median TF activity: 1.7 pM vs. 0.03 pM; p=0.0022). Thrombin generation assays were performed in 13 MPN patients and 9 controls, and were comparable: mean lag phase (14.4 vs. 10.15 minutes; p=0.26); mean velocity index (21.13 vs. 14.78; p=0.31); mean peak thrombin generation (111.66 nM vs. 120.41 nM; p=0.75); and mean AUC (3218.77 vs. 3807.41 p=0.37). Conclusion: Compared to controls, samples of JAK2 V617F-positive and negative MPN patients revealed a higher median total number of microparticles. Further, the proportion of TF-bearing MPs was higher in MPN patients, and of monocyte and platelet origin, suggesting their possible role in thrombotic complications. Though the MPs in MPN patients appear functional, based on higher TF activity, functional assays with thrombin generation testing failed to reveal a difference between MPN patients and controls. A lack of difference in TGT may suggest the presence of one or more inhibitors present in the MPN; the nature of this inhibitor is under investigation. Future studies, with a larger sample size and prospective follow-up are indicated to determine the role of MPs in predicting incident or recurrent thrombosis in the MPN. In addition, with a larger sample size, differences by MPN disease class and JAK2 V617F status will be uncovered. Disclosures: No relevant conflicts of interest to declare.

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Characterization of a Novel Human Factor VIIa Chimera with Increased Tissue Factor-Independent Activity for Emergency Hemostasis

Blood ◽

10.1182/blood-2019-124428 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3623-3623

Author(s):

Ammon Fager ◽

Maureane Hoffman ◽

Dougald Monroe

Keyword(s):

Tissue Factor ◽

Research Funding ◽

Protein C ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Hek293 Cells ◽

Cell Protein ◽

Acute Bleeding ◽

Hemostatic Efficacy

While prophylactic treatment with emicizumab has shown remarkable efficacy in patients with hemophilia A, the treatment options for traumatic, perioperative, and breakthrough bleeding in hemophilia A or B patients with inhibitors remain extremely limited. Recombinant Factor VIIa (rFVIIa) is routinely used to promote hemostasis in hemophilia patients with inhibitors and is recommended as first line therapy for acute bleeding, especially for patients on emicizumab. In addition, rFVIIa has extensive off-label use for hemostasis in cardiovascular surgery, trauma, and intracranial hemorrhage. The hemostatic efficacy of rFVIIa depends on its ability to bind activated platelets and promote thrombin generation by activating Factor X (FX) in a tissue factor (TF)-independent manner. However, the use of rFVIIa requires frequent high doses at significant cost, and is limited by an inconsistent response. Therefore, there is a critical need for new strategies to treat acute bleeding in hemophilia patients with inhibitors and others requiring emergency hemostasis. We have previously shown that human platelets express endothelial cell protein C receptor which contributes to the platelet binding and activity of rFVIIa. Based on this work, we designed a novel FVIIa chimera (PC-FVIIa) with the potential for increased hemostatic efficacy and an enhanced safety profile compared to rFVIIa. The purpose of the current study was to characterize the in vitro activity of this chimera. A cDNA construct encoding the Gla and EGF1 domains of human Protein C along with the EGF2 and catalytic domains of human FVIIa was synthesized and cloned into HEK293 cells. Stable transfectants were selected and PC-FVIIa was purified from the media. Protein electrophoresis of eluates confirmed bands consistent with the expected molecular weight. Similar to rFVIIa, we found that autoactivation of PC-FVIIa readily occurs in the presence of calcium and phospholipid (15% PS/41% PC/44% PE). Autoactivation is rapidly accelerated by the addition of Factor Xa (FXa) and significantly impaired in the absence of either calcium or phospholipid. There was no significant difference between activated PC-FVIIa and rFVIIa in their ability to cleave a synthetic FVIIa substrate. As the Gla and EGF1 domains of rFVIIa are primarily responsible for binding TF, we hypothesized that PC-FVIIa would have little to no affinity for TF. Indeed, we found that the interaction between PC-FVIIa and TF is too weak to be measured in an assay designed to detect weak TF binding. We therefore determined the TF-independent activity of PC-FVIIa using FXa and thrombin generation assays. For some experiments, phospholipid vesicles were incubated with rFVIIa or PC-FVIIa. Plasma levels of FX were added and FXa generation was assessed using a chromogenic substrate. In these assays, the rate of FX activation by PC-FVIIa was significantly higher than that of rFVIIa (Figure 1). To determine the ability of PC-FVIIa to promote thrombin generation, we performed a modified calibrated automated thrombography assay in the absence of TF. Hemophilia A plasma was incubated with PC-FVIIa or rFVIIa in the presence of phospholipid. Subsequent thrombin generation was assessed by monitoring cleavage of a fluorogenic substrate. No appreciable thrombin generation was seen in plasma alone. Adding rFVIIa resulted in a shorter lag time than PC-FVIIa. However, PC-FVIIa led to significantly higher peak thrombin concentration and endogenous thrombin potential compared to rFVIIa. Finally, we used a prothrombinase detection system to determine the activity of PC-FVIIa on the surface of platelets activated with thrombin plus a collagen receptor agonist to generate highly procoagulant platelets. Once again, the rate of thrombin generation was significantly higher with PC-FVIIa as compared to rFVIIa, consistent with a higher rate of FX activation on the platelet surface. Taken together, these data suggest that the PC-FVIIa chimera has the potential for increased hemostatic efficacy compared to rFVIIa. Additional studies will characterize the in vivo activity of PC-FVIIa. However, the lack of affinity for TF represents a potential advantage for PC-FVIIa since long-term exposure to high levels of rFVIIa can lead to thrombosis in TF-rich tissues. As such, PC-FVIIa warrants further study as a potential therapeutic agent with unique characteristics compared to rFVIIa. Disclosures Fager: Otello Medical Inc.: Research Funding. Hoffman:Novo Nordisk A/S: Honoraria, Research Funding. Monroe:Novo Nordisk: Honoraria, Research Funding.

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Stability Of Thrombin Generation In Cirrhotic Patients

Blood ◽

10.1182/blood.v122.21.2361.2361 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2361-2361

Author(s):

Thomas Sinegre ◽

Armand Abergel ◽

Géraldine Lamblin ◽

Marc G Berger ◽

Aurélien Lebreton

Keyword(s):

Protein C ◽

Thrombin Generation ◽

Activated Protein C ◽

Final Concentration ◽

Control Group ◽

Healthy Controls ◽

Thrombotic Risk ◽

Compensated Cirrhosis ◽

Cirrhotic Patients ◽

The Stability

Abstract Introduction Cirrhotic patients have a significant impairment of hemostasis. Most procoagulant and anticoagulant factors decrease but there are still exceptions such as factor VIII. Since many years, new approaches allowed to consider the cirrhotic patients as patients exposed to a thrombotic risk in many circumstances. In this concern, global tests as Thrombin Generation Assay (TGA) have an interest and many studies using thrombomodulin (TM) demonstrated a resistance to activated protein C (aPC) pathway. These assays evaluate the variations of both procoagulant factors and anticoagulant factors. However, the stability of thrombin generation in these patients is unknown. The aim of this study was to evaluate the stability of thrombin generation parameters (with and without TM and aPC) in cirrhotic patients compared to healthy volunteers. Patients and Methods 8 cirrhotic patients and 15 healthy controls were included in this study. For each patient, the follow-up period covers three months including three blood samples (D0, W6 and W12). Patients included in the study are cirrhotic (Prothrombin Time < 70% and / or liver dysmorphia and / or Fibroscan> 20 kPa and / or histology and / or association of portal hypertension and liver failure) of alcoholic origin. They are exclusively male, free of hepatocellular carcinoma and not anticoagulated. Thrombin Generation was performed in platelet-poor plasma in the presence / absence of TM (3.6 nmol/L) and in the presence/absence of aPC (1 nmol/L). The thrombin generation test was carried out according to the principle described by Hemker using Fluoroskan Ascent®, and the analysis software ThrombinoscopeTM. Reagent contains phospholipids (4 microM / L) and tissue factor (5 pM/L). TM is soluble thrombomodulin purified from rabbit lungs at final concentration 3.6 nmol/L. Human aPC was used at final concentration of 1 nmol / L (IC 50). The results are expressed as ratios: with and without TM and with and without PCa. Lag Time (LT), Endogenous Thrombin Potential (ETP), Peak Height (PH) and Time to Peak (TP) were analyzed. Statistical analysis compared the coefficient of variation (CV) between the control group and cirrhotic patients for each ratio (with and without TM with and without PCa) and that for each parameter by the Mann-Whitney test. Results Patients included in the study have chronic liver disease. Seven of them have compensated cirrhosis (CHILD A) and one cirrhotic patient has complicated cirrhosis (infection). The average age of cirrhotic is 50 (range 37-57). For both TM or aPC, all parameters of TGA are stable in cirrhotic patients compared with healthy controls. Regarding ETP, the most used parameter, CV of the ratio with and without TM is 12% (2-24) for the controls and 13% (2-22) for the cirrhotic patients while CV of the ratio with and without aPC is 11% (3-24) for the controls and 11% (6-21) for the cirrhotic patients. Similar matches were found for PH: 12% (3-25) for the controls versus 12% (1-22) for the cirrhotic patients with TM and 12% (1-20) for the controls versus 11% (5-19) for the cirrhotic patients. Similar results were found for LT and TP. Conclusions The Thrombin generation - performed with Thrombomodulin and activated Protein C and expressed with a ratio - in patient with cirrhosis is similarly stable than the thrombin generation in the control population. These observations can be used in the management and the design of further studies involving cirrhotic patients. Now, it is possible to follow the cirrhotic patients with TGA in order to detect the occurrence of a hypercoagulability state. However patients included in the study were mostly compensated cirrhosis. Further studies are needed to evaluate the stability of thrombin generation in the other subgroups of cirrhotic patients. Disclosures: No relevant conflicts of interest to declare.

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Evaluation of a Blood Coagulation Factor IX Variant That Functions Independently of Factor VIII As an Alternative Treatment for Hemophilia A

Blood ◽

10.1182/blood-2019-124120 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1110-1110

Author(s):

Viola J.F. Strijbis ◽

Ka Lei Cheung ◽

Pavlina Konstantinova ◽

Ying Poi Liu ◽

Sander J van Deventer ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Alternative Treatment ◽

Active Site ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Activation Mechanism ◽

Hek293 Cells ◽

Synergistic Enhancement

The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo. To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV. Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (<13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven. Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A. Disclosures Strijbis: uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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