Efficacy and Safety of Recombinant Factor IX-FIAV and Bypassing Agents in Thrombin Generation Analyses in Hemophilia A Patient Plasma: The FIVITAS Study

Factor (F)IX-FIAV, a FIX variant with four amino acid substitutions that functions independently of the cofactor VIIIa, has been previously shown to ameliorate the hemophilia A (HA) phenotype in vivo [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. Here we evaluated the efficacy of purified recombinant FIX-FIAV in severe, moderate, and mild hemophilia A patient plasma employing thrombin generation and intrinsic clotting activity (aPTT) analyses. The combination of FIX-FIAV with current hemophilia A therapeutics was used for preclinical safety assessment. Plasma was obtained from 21 HA patients, seven per HA phenotype, with a median age of 38 years [interquartile range (IQR) 27.5 - 49.5]. The plasma levels of FIX, FX, prothrombin, and antithrombin of all included patients were within the normal range. To determine the effect of FIX-FIAV on FXIa-triggered thrombin generation parameters (lag time, endogenous thrombin potential (ETP)), plasma was spiked with 100% (5 µg/ml in severe/mild) or 125% (6 µg/ml in moderate) FIX-FIAV prior to analysis. FIX-FIAV significantly shortened the lag time in all patient plasmas irrespective of disease severity (Figure 1) with an overall median of 4.4 min [IQR 3.6 - 6.9] in the absence of FIX-FIAV vs. 3.1 min [IQR 2.4 - 4.5] in the presence of FIX-FIAV (p<0.0001, Table 1). Similar observations were obtained following aPTT analyses: median clotting time of 115.5 sec [IQR 105 - 173.9] without vs. 97.7 sec [IQR 91.8 - 136.2] with 100% FIX-FIAV (p = 0.0039). Conversion of the thrombin generation lag time to FVIII-like activity using a FVIII calibration for each individual patient plasma revealed that FIX-FIAV mitigated the HA phenotype from severe to moderate, from moderate to mild, and from mild to normal (Table 1). Interestingly, following the addition of FIX-FIAV a minor but significant decrease in ETP was observed for non-severe HA patient plasma (p = 0.016 for mild and p = 0.016 for moderate), while FIX-FIAV increased the median ETP in severe HA plasma by 2.1-fold (p = 0.22) (Figure 2, Table 1). This may result from competition between the added FIX-FIAV and endogenous FIX for interaction with residual functional FVIII. In line with this, experiments performed in the presence of an anti-FVIII antibody that inhibits FVIII activity significantly enhanced the ETP in all patient plasmas (p = 0.016 for each individual severity, Figure 2) in addition to shortening the lag time (Figure 1). Next, we evaluated the combination of FIX-FIAV with bypassing agents (1 IU/mL aPCC or 1.5 mg/mL rFVIIa) in nine patient plasmas, three per phenotype. Addition of aPCC or rFVIIa to FIX-FIAV-spiked plasma did not significantly affect aPTT clotting times nor ETP values in comparison to adding aPCC or rFVIIa only, respectively. The median lag time shortened significantly, albeit modestly, for the combination of FIX-FIAV with aPCC in comparison to conditions with aPCC only: 5.2 min [IQR 3.0 - 6.4] vs. 5.5 min [IQR 3.6 - 7.7], p = 0.0078, respectively. Similar findings were obtained when combining FIX-FIAV with rFVIIa relative to rFVIIa only: median lag time 4.9 min [IQR 2.2 - 6.1] vs. 5.3 min [IQR 3.3 - 8.8], p = 0.0039. Hence, no substantial synergistic effect was observed when combining FIX-FIAV with bypassing agents aPCC or rFVIIa for HA. In contrast, combining approximate hemostatic levels (55 µg/ml) of emicizumab, a bispecific antibody mimicking FVIIIa, with FIX-FIAV resulted in a ~1.1-fold reduced lag time and ETP relative to emicizumab alone (p = 0.016 and p = 0.004 respectively). This is suggestive of a minor synergistic procoagulant effect, which is consistent with the FIX(a)-FX(a) bridging capacity of emicizumab. In conclusion, FIX-FIAV could serve as a potential treatment for hemophilia A as it mitigates the hemophilia A phenotype in patient plasma, also in the presence of an inhibitory anti-FVIII antibody. While further safety assessment is warranted, no severe procoagulant effects were observed for the combination of FIX-FIAV with conventional hemophilia A therapeutics. Figure 1 Figure 1. Disclosures Romano: Swedish Orphan Biovitrum B.V.: Other: Travel grant and aforementioned Research Funding in the form of the Young Investigator's Award 2020, Research Funding. Liu: uniQure Biopharma B.V.: Current Employment. McCreary: uniQure Biopharma B.V.: Ended employment in the past 24 months. Leebeek: Roche: Other: DSMB member of a study sponsored by Roche; uniQure Biopharma B.V.: Consultancy, Research Funding; Swedish Orphan Biovitrum B.V.: Other: Travel support, Research Funding; Biomarin: Consultancy; CSL Behring: Consultancy, Research Funding; Shire/Takeda: Consultancy, Research Funding. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

Iron-driven alterations on red blood cell-derived microvesicles amplify coagulation during hemolysis via the intrinsic tenase complex

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived pro-inflammatory and oxidatively reactive mediators (e.g. extracellular hemoglobin, heme and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring FXa and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

RBD targeted COVID vaccine and full length spike-protein vaccine (mutation and glycosylation role) relationship with procoagulant effect

Related COVID vaccine production many different strategies was followed by the producers. Observing some rare event of thrombosis after some COVID-19 vaccination, it is interesting to verify if the Target used for the manufacturing can be involved in a different procoagulant activity or not. Some vaccine are suspended in some country or under a deep new verify- investigation by the regulatory agency. (EU or USA). This fact it is relevant. The target SPIKE-PROTEIN FULL LENGTH modified or not or towards the RBD domain can be a relevant factor.

Tabernaemontana divaricata Stem and Latex Proteases as Haemostatic Agent with Temporally Spaced Intense Fibrinogenolytic and Mild Fibrinolytic Activity

Background: Proteases play a crucial role in the pharmacological properties of latex producing plants. Some of them exhibited intervention with fibrinogenolysis and/or fibrinolysis, two crucial wound healing events. Objective: To evaluate wound healing potential of crude and partially purified enzyme from Tabernaemontana divaricata (stem and latex). Materials and Methods: Proteolytic activity, clot inducing/dissolving potential, fibrinogen polymerization, recalcification time, blood clot lysis and Tricine-SDS PAGE for enzyme treated fibrinogen and human plasma clot were performed. Results: Latex PPE exhibited significant proteolytic activity (115.8 ± 0.3 U/ml) compared to that of the stem (28.78 ± 0.2 U/ml). Enzyme preparations exhibited temporally spaced clot inducing and subsequent dissolving properties favoring hemostatic effect, procoagulant effect being dominant and the first event. Significant reduction in fibrinogen absorbance at 540 nm with time, recalcification time and human fibrinogenolytic product analysis on Tricine PAGE substantiated procoagulant effect. Disappearance of Aα and Bβ fibrinopeptides by both stem and latex PPEs in the PAGE was observed. γ subunits were completely hydrolysed by latex PPE, however, it showed comparative resistance to stem PPE. Reduction in blood clot weight and fibrin subunit intensity supported thrombolytic property. Conclusion: The study provides evidence of the procoagulant and thrombolytic activity associated with T. divaricata proteases.

Study of the Effect of Fibrinogen, Factor XIII and Recombinant Activated Factor VII in a Model of Trauma-Induced Coagulopathy

Introduction: Trauma-induced coagulopathy (TIC) is a multifactorial condition secondary to severe trauma. In TIC, early fibrinogen (FI) replacement and low dose of recombinant activated factor VII (rFVIIa) may positively impact outcome. Factor XIII (FXIII), on the other hand, may stimulate in vitro clot formation and clot stability. We hypothesized that combination of FI, rFVIIa and FXIII might normalize clot formation more effectively than the isolated use of each concentrate in a model of TIC. Aim: Evaluation of the procoagulant effect of isolated or combined use of FI, rFVIIa and FXIII in a model of TIC. Methods: TIC in vitro model was obtained by dilution of whole blood from seven healthy controls with isotonic saline (NaCl 0.9%) (2:3 whole blood:saline ratio). FI, rFVIIa and FXIII were spiked in combination or alone until obtaining final levels of 2 g/L, 1 μg/mL and 100 IU/dL respectively. Procoagulant effects of the different concentrates or their mixtures were evaluated by Rotational Thromboelastometry (ROTEM®, Werfen) triggered using starTEM® (calcium chloride 0,2 M) and exTEM® reagent (source of tissue factor) diluted with saline up to 1:100.000 (final dilution) for a better evaluation of both the extrinsic and intrinsic pathways of coagulation. The values of clotting time (CT: time until 2 mm of amplitude, in seconds), amplitude (parameter proportional to the clot strength) at 5 minutes (A5, in mm) and clot formation time (CFT: time from CT to 20 mm of amplitude, in seconds) were evaluated. Statistical analysis of differences was performed by One-Way ANOVA test assuming no paring of data and using the Holm-Sidak's correction for multiple comparisons with a family-wise significance and confidence level of 0.01. Statistical significance was set at p< 0.05. Results/Discussion: Data are summarized in Table I and Figure 1. CT needed the combination of two of more concentrates to reach the normal range suggesting that the administration of FI alone in TIC may not be enough to restore the patients' hemostatic potential. In regard to the clot strength evaluated by A5, the addition of FXIII or rFVIIa alone or in combination did not improve the value of A5 that was only normalized by the addition of FI. This effect of FI was increased in the presence of FXIII or rFVIIa which indicated that normal levels of FI might be required for rFVIIa or FXIII to be effective emphasising the possible benefit of the combinatory therapy. Like observed in A5, the velocity of clot formation evaluated by the CFT was normalised only by the addition of FI. However, the combination of FI plus FXIII + rFVIIa had a stronger effect on CFT compared with the combination of FI + FXIII or FI + rFVIIa, indicating that the improvement of thrombin generation due to rFVIIa plus an increment of fibrin formation and net stabilization through the contribution of higher levels of FI and FXIII respectively, might provide a beneficial synergistic procoagulant effect in TIC. Conclusion: The use of FI in TIC may contribute to increase the patient's hemostatic potential but might not be enough. Combinatory therapies based on the administration of FI, rFVIIa and FXIII might be of better benefit in this setting. Ex-vivo studies using blood of patients with stablished TIC might bring new insights on the possible advantages of this combinatory therapy to design more effective protocols to treat this frequent and life-threatening acquired condition. Disclosures Canales: Sandoz: Honoraria; iQone: Honoraria; Janssen: Speakers Bureau; Janssen: Honoraria; Roche: Speakers Bureau; Karyopharm: Honoraria; Sandoz: Speakers Bureau; Novartis: Honoraria; Takeda: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Janssen: Speakers Bureau; Roche: Speakers Bureau; Sandoz: Speakers Bureau; Takeda: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Gilead: Honoraria. Butta:NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Pfizer: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Grifols: Research Funding. Alvarez Román:NovoNordisk,: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy; Grifols: Research Funding. Jiménez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria.

Thein Vitroprocoagulant Effects of Standard and Extended Half-Life Recombinant Factor IX Concentrates in Patients on Prophylaxis with Emicizumab

Introduction: Emicizumab is a humanized, monoclonal, bispecific antibody that binds factor (F) FX and FIXa allowing thrombin generation in the absence of FVIII, which is used for routine treatment of patients with Hemophilia A (HA) with and without inhibitors. Plasma level of FIX will be an important limiting factor for the formation of the FX-FIXa-emicizumab ternary complex in the absence of FVIII, suggesting the potential use of FIX concentrates to regulate the procoagulant function of emicizumab and therefore, to use it as an alternative treatment in certain circumstances to stop or prevent bleedings in patients on prophylaxis with emicizumab. At the present time there are several recombinant FIX concentrates with differences in their content of activated FIX (FIXa) and in their half-life (standard or extended) that may differ in their procoagulant effects when combined with emicizumab. Aim: The aim of this study was to evaluate if there were differences in thein vitroprocoagulant effects of two recombinant FIX concentrates, one with standard half-life (rFIX Nonacog Alfa, BeneFIX®, Pfizer) and the other with extended half-life (rFIX fused to rAlbumin, Albutrepenonacog Alfa, Idelvion®, CLS Behring) in samples from patients on prophylaxis with Emicizumab. Methods: This is a prospective and transversal pilot study that was approved by the Ethics Committee from La Paz University Hospital. Two patients with haemophilia A (HA) with inhibitors in prophylaxis with emicizumab were recruited and one haemophilia B (HB) patient was included as a control for the effects of FIX. Blood samples were collected in tubes with corn trypsin inhibitor (CTI, Haematologic Technologies, USA), to block thecontact phase and to only evaluate coagulation mediated by the extrinsic pathway. Levels of FIXa in concentrates of FIX were quantified using the Spectrozyme® FIXa chromogenic substrate (LOXO) and measuring the increase in OD at 405 nm. Rotational thromboelastometry (ROTEM) was performed using whole blood activated by a low concentration of tissue factor solution (final dilution 1:50,000 of EXTEM reagent) plus recalcification. Parameters evaluated in ROTEM were CT (cloting time), defined as time until detection of a clot firmness of 2 mm; and CFT (clot formation time), defined as time between detection of a clot firmness from 2 to 20 mm. Calibrated automated thrombogram (CAT)was performed using platelet free plasma (PFP) activated by low concentration of tissue factor plus phospholipids (PPP-Reagent LOW®, Stago). Parameters evaluated in CAT were: Peak, defined as maximum thrombin concentration reached, in nM; and ETP, defined as the total amount of thrombin generated over time, in nMxmin. Results: The presence of FIXa activity assayed by a chromogenic substrate was not detectable with 20 IU of Idelvion while BeneFIX® showed a specific concentration-dependent FIX activity that was blocked with the serine protease inhibitor EGR-chloromethylketone (Figure 1). ROTEM (Figure 2) and CAT (Figure 3) results showed that the addition of increased concentrations of both concentrates of rFIX produces an enhanced procoagulant effect of Emicizumab similar to the effect produced by the addition of the bypassing agent rFVIIa (NovoSeven®, NovoNordisk). These results also showed that it is necessary three-four times higher concentration (U/dl) of Idelvion® to get similar procoagulant effects that those obtained with BeneFIX®. The higher procoagulant effects of BeneFIX® were also observed in samples of a patient with severe HB. Conclusion: Global coagulation assays suggest that increasing endogenous FIX levels with two rFIX concentrates that have different FIXa content and half-life, produce an enhanced procoagulant effect of Emicizumab opening the idea of the use of these concentrates as an alternative treatment for bleedings in patients with inhibitors on prophylaxis with Emicizumab. Further studies need to be performed to evaluate the procoagulant activity of the concomitant use of different rFIX concentrates and Emicizumab, and to assess security of this therapeutic approach. This work was supported by grants from FIS-FONDOS FEDER (PI19/00631 and P19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Fernandez-Bello: Novartis:Speakers Bureau;Stago:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Current Employment, Research Funding, Speakers Bureau;Roche:Speakers Bureau;SOBI,:Research Funding;Pfizer:Speakers Bureau.García Barcenilla:Pfizer,:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Bayer:Speakers Bureau;Novartis:Speakers Bureau;NovoNordisk:Research Funding, Speakers Bureau.Alvarez Román:Roche:Speakers Bureau;Novartis:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Pfizer,:Research Funding, Speakers Bureau;Bayer:Consultancy;SOBI,:Consultancy, Research Funding, Speakers Bureau;Grifols:Research Funding;NovoNordisk,:Research Funding, Speakers Bureau.Martín:NovoNordisk:Speakers Bureau;SOBI:Research Funding;Pfizer:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Novartis:Speakers Bureau.Rivas Pollmar:Novartis:Speakers Bureau;Roche:Speakers Bureau;Pfizer:Speakers Bureau.Canales:Roche:Honoraria;Celgene:Honoraria;Roche:Honoraria;Janssen:Honoraria;Novartis:Honoraria;Sandoz:Speakers Bureau;Sandoz:Honoraria;Janssen:Speakers Bureau;iQone:Honoraria;Sandoz:Honoraria;Gilead:Honoraria;Takeda:Speakers Bureau;Novartis:Honoraria;Karyopharm:Honoraria;Janssen:Honoraria;Takeda:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau;Sandoz:Speakers Bureau;Roche:Speakers Bureau;Karyopharm:Honoraria.Butta:Grifols:Research Funding;Novartis:Speakers Bureau;ROCHE:Research Funding, Speakers Bureau;Pfizer:Speakers Bureau;SOBI:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Speakers Bureau.Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer:Honoraria;F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer:Consultancy;Grifols, Novo Nordisk, Takeda, Sobi, Pfizer:Research Funding.

Cerebral Infarction Secondary to Vascular Infiltration of Glioblastoma Confirmed in High-Resolution Vessel Wall MRI

Acute cerebral infarction secondary to glioblastoma is rarely reported, and its mechanism is still controversial. Vascular infiltration of malignant cell, direct mass effect and local procoagulant effect were considered as main pathophysiology. We report a case of glioblastoma-related cerebral infarction documented by high resolution vessel wall magnetic resonance imaging, which reveals concentric enhancement of arterial wall and intraluminal thrombus without direct compression. These findings suggest tumor cell infiltration of intracranial vessels is one of the important mechanisms of glioblastoma-related cerebral infarction.

Emicizumab, the factor VIII mimetic bi-specific monoclonal antibody and its measurement in plasma

ObjectivesEmicizumab, a monoclonal antibody mimicking the function of factor (F) VIII in the activation of FX by FIXa, is widely used for prophylaxis in hemophilia patients with or without inhibitors to FVIII. Although it is administered at fixed dose, its measurement could be occasionally required. In principle, the emicizumab procoagulant effect could be assessed by the one-stage assay (OSA) currently used to measure FVIII. However, the OSA for FVIII presents with limitations. Furthermore, owing to its potent FVIII-like activity, emicizumab interferes with the measurement of the inhibitor to FVIII, which is often needed in patients on emicizumab.MethodsWe prepared test samples by spiking a FVIII-deficient plasma with graded amounts of emicizumab. We modified the OSA for FVIII and tested plasma samples for emicizumab concentrations. Furthermore the chromogenic assay (CA) for FVIII with bovine reagents was used to assess for the FVIII inhibitor in patients on emicizumab.ResultsSlight modification of the OSA for FVIII (i.e., higher test plasma dilution and longer coagulometer acquisition time) made the regular OSA as a reliable laboratory tool to measure emicizumab concentration as shown by the identity of the regression (observed vs. expected) lines. Furthermore, the inhibitors to FVIII in patients on emicizumab, which were negative when measured by the regular Bethesda assay, were reliably measured by the CA assay employing bovine reagents.ConclusionsThe methods currently used to measure FVIII can be easily modified to make the general clinical laboratory able to assist clinicians when dealing with patients on emicizumab.