scholarly journals Dynamic Hemostasis and Fibrinolysis Assays in Intensive Care COVID-19 Patients and Association with Thrombosis and Bleeding—A Systematic Review and a Cohort Study

Author(s):  
Christine Lodberg Hvas ◽  
Julie Brogaard Larsen ◽  
Kasper Adelborg ◽  
Steffen Christensen ◽  
Anne-Mette Hvas

AbstractPatients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19), the infectious pathology caused by severe acute respiratory syndrome coronavirus 2, have a high risk of thrombosis, though the precise mechanisms behind this remain unclarified. A systematic literature search in PubMed and EMBASE identified 18 prospective studies applying dynamic coagulation assays in ICU COVID-19 patients. Overall, these studies revealed normal or slightly reduced primary hemostasis, prolonged clot initiation, but increased clot firmness. Thrombin generation assay parameters generally were equivalent to the control groups or within reference range. Fibrinolysis assays showed increased clot resistance. Only six studies related their findings to clinical outcome. We also prospectively included 51 COVID-19 patients admitted to the ICU. Blood samples were examined on day 1, 3–4, and 7–8 with platelet function tests, rotational thromboelastometry (ROTEM), in vivo and ex vivo thrombin generation, and clot lysis assay. Data on thrombosis, bleeding, and mortality were recorded during 30 days. Primary hemostasis was comparable to healthy controls, but COVID-19 patients had longer ROTEM-clotting times and higher maximum clot firmness than healthy controls. Ex vivo thrombin generation was similar to that of healthy controls while in vivo thrombin generation markers, thrombin–antithrombin (TAT) complex, and prothrombin fragment 1 + 2 (F1 + 2) were higher in ICU COVID-19 patients than in healthy controls. Impaired fibrinolysis was present at all time points. TAT complex and F1 + 2 levels were significantly higher in patients developing thrombosis (n = 16) than in those without. In conclusion, only few previous studies employed dynamic hemostasis assays in COVID-19 ICU-patients and failed to reveal a clear association with development of thrombosis. In ICU COVID-19 patients, we confirmed normal platelet aggregation, while in vivo thrombin generation was increased and fibrinolysis decreased. Thrombosis may be driven by increased thrombin formation in vivo.

2016 ◽  
Vol 62 (5) ◽  
pp. 699-707 ◽  
Author(s):  
Armando Tripodi

Abstract BACKGROUND A gap exists between in vivo and ex vivo coagulation when investigated by use of the coagulation tests prothrombin time (PT) and activated partial thromboplastin time (APTT). The thrombin generation assay (TGA) has been developed to fill this gap. CONTENT TGA evaluates thrombin generation (resulting from the action of the procoagulant driver) and decay (resulting from the action of the anticoagulant driver), thus assessing the balance between the two. Coagulation of the test plasma (platelet poor or platelet rich) is activated by small amounts of tissue factor and phospholipids, and the reaction of thrombin generation is continuously monitored by means of a thrombin-specific fluorogenic substrate. Among the parameters derived from the thrombin-generation curve, the most important is the endogenous thrombin potential, defined as the net amount of thrombin that test plasmas can generate on the basis of the relative strength of the pro- and anticoagulant drivers. TGA is therefore the candidate assay to investigate hypo- or hypercoagulability. SUMMARY From my analysis of the literature, I draw the following conclusions. There is strong evidence that TGA is helpful to elucidate coagulation mechanisms in various clinical conditions that until recently were poorly understood (chronic liver disease; diabetes; inflammatory bowel disease, myeloproliferative neoplasms, nonalcoholic fatty liver disease). TGA is a promising laboratory tool for investigating hemorrhagic coagulopathies and monitoring replacement therapy in hemophiliacs, predicting the risk of recurrent venous thromboembolism after a first event, and monitoring patients on parenteral or oral anticoagulants. These applications require clinical trials in which TGA results are combined with specific clinical end points.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3631-3631
Author(s):  
Carsten Dan Ley ◽  
Thomas Lindebo Holm ◽  
Daniel Elenius ◽  
Heidi Lindgreen Holmberg ◽  
Jais Rose Bjelke ◽  
...  

People with haemophilia A (HA) lack functional factor VIII (FVIII) and typically receive FVIII replacement therapy to prevent or treat bleeds. However, this requires frequent i.v. access, and efficacy is impaired in inhibitor patients. Mim8 is in development as a subcutaneous prophylactic treatment option for people with HA and HA with inhibitors. Like the recently approved emicizumab (Hemlibra®), Mim8 is a FVIII-mimicking human bispecific antibody bridging FIXa and FX. Mim8 is highly specific towards human FIXa (hFIXa) and human FX (hFX), preventing pre-clinical testing in standard rodent haemophilia models. Pharmacologic characterisation can be conducted in vitro and ex vivo utilizing human components. In vivo studies are feasible in primates due to high sequence homology between human and monkey FIX and FX, however, haemophilic mice are used for the most well-established and widely recognized bleeding models. Our aim was to establish a method to evaluate acute effects of Mim8 using in vivo bleeding models in HA mice, and to compare the potency and efficacy of Mim8 to a sequence-identical-analogue (SIA) of emicizumab. A protocol for dosing HA mice with hFIX and hFX was optimized based on in vitro Thrombin Generation Assay (TGA) in HA mouse plasma spiked with a range of hFIX and hFX concentrations. The thrombin levels required to stop bleeding in the in vivo Tail Vein Transection (TVT) model were known from previous studies. In mouse plasma with a clinically efficacious concentration of 300-350 nM of emicizumab SIA (Mahlangu J et al, N Engl J Med. 2018 Aug 30;379(9)), we found that roughly twice the normal human levels of hFIX and hFX were needed to achieve sufficient thrombin generation for the TVT model. To maintain concentrations at or above this level throughout the bleeding experiments, in vivo doses were set at 1.5mg/kg and 0.9mg/kg, respectively. Based on the in vitro optimization, the haemostatic effect of Mim8 was evaluated by three different methods: 1. Tail Vein Transection (TVT), a venous in vivo bleeding model sensitive to clinical doses of FVIII, and 2. Tail Clip (TC), an arteriovenous in vivo bleeding model with lower sensitivity to FVIII, presumably due to the more severe nature of the bleed, and 3. Ex vivo TGA on plasma from Mim8-dosed mice Briefly, mice were anaesthetized with isoflurane and dosed with hFIX, hFX and test compound. Thereafter, they were subjected to either the TVT bleeding model, the TC bleeding model, or cardiac puncture for plasma collection and ex vivo TGA. All mice were euthanized without awakening from anaesthesia. Both compounds were efficacious in vitro in TGA (Figure A). Potency of Mim8 was significantly greater compared to emicizumab SIA; the efficacy of approximately 40 nM Mim8 corresponded to 300 nM emicizumab SIA. At the highest concentrations (>1000 nM), Mim8 efficacy tapered off, but remained superior to 300 nM emicizumab SIA. In TVT in HA mice, bleeding was reduced in a dose-dependent manner with an ED50 of 0.05 mg/kg for Mim8 or 0.7 mg/kg for emicizumab SIA. Statistically significant reduction of blood loss was observed at doses of or above 0.1 mg/kg Mim8 and 10mg/kg emicizumab SIA, corresponding to measured plasma concentrations above 10 nM for Mim8 and 300nM for emicizumab SIA. In the more severe TC model, blood loss was significantly reduced after treatment with 10 mg/kg of Mim8, whereas the tested doses of emicizumab SIA were not efficacious (Figure B). Mice treated with 4.6 and 10 mg/kg of Mim8, corresponding to a plasma concentration of up to approx. 1000nM, bled significantly less than mice treated with emicizumab SIA. In agreement with the in vitro TGA results, the 22 mg/kg dose (plasma concentration >2200 nM) appeared less efficacious; association of FIXa and FX to different Mim8 molecules is the likely cause. The increased potency of Mim8 was confirmed in TGA ex vivo. In conclusion, we developed a method for evaluating the FVIII-mimetic compounds Mim8 and emicizumab SIA, which require human FIX and FX, in a murine system. This method may be applicable for testing of other FIXa-FX bridging compounds lacking rodent cross-reactivity. In Thrombin Generation Assay and the Tail Vein Transection model, Mim8 showed significantly increased potency compared to emicizumab-SIA, and the observed potency gain corresponded to in vitro findings in the human system. Furthermore, Mim8 could stop a severe bleed in the tail clip model, which was not possible with the tested doses of emicizumab SIA. Figure Disclosures Ley: Novo Nordisk A/S: Employment, Equity Ownership. Holm:Leo Pharma A/S: Employment, Equity Ownership; Novo Nordisk A/S: Employment, Equity Ownership. Elenius:Leo Pharma A/S: Employment, Equity Ownership; Novo Nordisk A/S: Equity Ownership, Other: Previous employment. Holmberg:Novo Nordisk A/S: Employment, Equity Ownership. Bjelke:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Loftager:Novo Nordisk A/S: Employment, Equity Ownership. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties. Kjellev:Novo Nordisk A/S: Employment, Equity Ownership.


2015 ◽  
Vol 113 (04) ◽  
pp. 851-861 ◽  
Author(s):  
Michal Ząbczyk ◽  
Margareta Blombäck ◽  
Jacek Majewski ◽  
Grzegorz Karkowski ◽  
Hakan N. Wallen ◽  
...  

SummaryAtrial fibrillation (AF) is a prothrombotic condition, involving increased thrombin generation and fibrinogen concentrations. Vitamin K antagonists (VKAs) prevent arterial thromboembolism if optimal anticoagulation is achieved by individualised drug doses, assessed by determining the Prothrombin time-related International Normalized Ratio (Pt-INR). There is evidence that formation of tight-laced fibrin networks is pathogenic in prothrombotic diseases. This study was performed among AF patients, to test whether long-term treatment with VKAs affects the structure of fibrin networks, and whether the effect is altered by employing different coagulation triggers: exogenous thrombin (1 IU/ml), 10 pM tissue factor (TF) or a commercial Pt-INR reagent (containing 400-fold more TF). In the thrombin-based method, fibrin network porosity (scanning electron microscopy) and liquid permeability (flow measurements) correlated inversely to fibrinogen concentrations, while positive correlations to the degree of anticoagulation were shown with the Pt-INR reagent. In the method with 10 pM TF, the two above relationships were detected, though the influence of Pt-INR was more profound than that of fibrinogen concentrations. Moreover, greater shortening of clot lysis time (CLT) arose from more permeable clots. As a coagulation trigger, 10 pM TF vs exogenous thrombin or the Pt-INR reagent is more informative in reflecting the in vivo process from thrombin generation to fibrin formation. Since fibrin network permeability rose in parallel to elevations of INR and shortening of CLT in AF patients, antithrombotic effects on prevention of thrombotic complications may be achieved from impairment of thrombin generation, resulting in formation of permeable clots susceptible to fibrinolysis.


2018 ◽  
Vol 114 (8) ◽  
pp. 1178-1188 ◽  
Author(s):  
Daniel S Gaul ◽  
Julien Weber ◽  
Lambertus J van Tits ◽  
Susanna Sluka ◽  
Lisa Pasterk ◽  
...  

AbstractAimsSirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).Methods and resultsUsing a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).ConclusionsSirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.


2019 ◽  
Vol 7 (2) ◽  
pp. 291-292
Author(s):  
Evi Kalodiki ◽  
Fredrik Wexels ◽  
Ola Dahl ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
...  

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1457-1457
Author(s):  
Daniel Lechner ◽  
Marietta Kollars ◽  
Sabine Eichinger ◽  
Paul Alexander Kyrle ◽  
Ansgar Weltermann

Abstract Background: Cisplatin-based chemotherapy is a risk factor of venous thromboembolism in cancer patients. The underlying pathogenesis remains unclear. We hypothesized an apoptotic effect of cisplatin on endothelial cells (EC) inducing a release of small membrane vesicles, so-called microparticles (MP) which are known to cause hemostasis activation. Objectives: To quantify the release of MP from EC following administration of cisplatin and to investigate MP-associated procoagulant mechanisms. Methods: Two EC lines (HUVEC, HMVEC-L) were exposed to cisplatin (1, 2.5, 5, 10, and 20 μM) for up to 120 h. Cell viability was assessed by quantification of mitochondrial dehydrogenase activity, counts and procoagulant activity of MP were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen levels were determined by ELISA. Results: EC viability decreased in a dose- and time-dependent manner and was accompanied by an increasing release of MP into culture media (maximum: HUVEC + 544%; HMVEC-L + 1738%). In parallel, procoagulant activity of media increased by up to 150% (HUVEC) and 493% (HMVEC-L), respectively. The procoagulant activity was almost abolished by annexin V but was not suppressed by a monoclonal TF-antibody. TF antigen levels on MP were persistently low even at high cisplatin concentrations. Conclusion: At pharmacologically relevant concentrations, cisplatin induced a marked release of procoagulant MP from EC. Negatively charged phospholipids but not TF on MP were decisive for total thrombin generation. Further studies are warranted to investigate the cisplatin-induced release of EC-derived MP in vivo.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2180-2180
Author(s):  
Sari Jalkanen ◽  
Satu Mustjoki ◽  
Kimmo Porkka ◽  
Jukka Vakkila

Abstract Abstract 2180 Poster Board II-157 Introduction. Aberrant phosphorylation of the BCR-ABL1 tyrosine kinase (TK) is characteristic of chronic myeloid leukemia (CML). This oncoprotein interacts directly with intracellular signaling proteins, alters the responsiveness of cytokine receptors and regulates secretion of autocrine cytokines. Targeted inhibition of BCR-ABL1 with TK inhibitor (TKI) imatinib mesylate (IM) is the current standard treatment of CML. For overcoming IM resistance or intolerance, 2nd generation TKIs (nilotinib, dasatinib) with broader kinase inhibition profile have been approved for clinical use. Although in vitro results suggest that TKIs are immunosuppressive, no increases in opportunistic infections or secondary malignancies have been observed to date. In contrast, in some TKI-treated patients immunoactivation in the form of chronic lymphocytosis linked to excellent therapy responses has recently been shown. Dynamic monitoring of aberrant cytokine signaling pathways would aid in understanding and predicting the development of TKI-resistance or adverse/off-target effects. The aim of this study was to analyze the responsiveness of leukocytes to cytokine stimuli in CML patients at diagnosis and during TKI therapy using single-cell profiling of phosphoprotein networks by multiparameter flow cytometry. Patients and methods. The study consisted of 4 healthy controls, 6 CML patients at diagnosis, 6 IM patients and 5 dasatinib patients. Stimuli included GM-CSF, IL-2+IL-10+IFNα and IL-4+IL-6+IFNγ and they were added immeadately to freshly drawn whole blood ex vivo. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD4+ CD25neg T helper cells (Th), CD4neg lymphocytes and CD4+CD25+ T cells including regulatory T-cells (Treg). Analysis was performed with heatmap function of Cytobank software (http://cytobank.stanford.edu/public/). Results. Unstimulated phosphoprotein levels reflecting the activation state of leukocytes in vivo did not differ between healthy controls and CML patients at diagnosis or during dasatinib therapy. Strikingly, in IM patients, baseline levels of pSTAT3 were relatively high indicating in vivo occurring activation of leukocytes in this patient group. We next studied ex vivo responsiveness of immune effector cells with cytokines and found clear differences between healthy controls and CML patients. At CML diagnosis. GM-CSF/pERK1+pSTAT5a, IFNa/pSTAT1,and IL-4/pSTAT6 (stimulus/readout) as well as pSTAT3 responses with all stimuli were suppressed in monocytes. In granulocytes, GM-CSF/pSTAT1 levels were diminished. In Th and Treg lymphocytes, IL-6/pSTAT3 responses were markedly pronounced, while IL-10/pSTAT3 responses were not affected when compared to healthy controls. Such difference was not observed in CD4neg lymphocytes. During TKI therapy. Most patients (9/11) were in cytogenetic remission at the time of analysis. The unresponsiveness of myeloid cells at diagnosis was restored by IM or dasatinib therapy in most, but not all patients. Similarly, in Th and Treg lymphocytes TKI-therapy normalized the enhanced IL-6/pSTAT3 responses that were evident at diagnosis. However, in Th and Treg cells pSTAT3 responses provoked by IL-10 were particularly prominent. Interestingly, one dasatinib patient with aberrant constant blood NK-lymphocytosis and monocytosis had uniquely strong IFNg/pSTAT1 and IL-4/pSTAT6 responses in monocytes. Furthermore, one patient who have stayed in persistent remission after IM discontinuation had exceptionally high pSTAT3 responses with all of stimuli used. Similar kind of signaling profile was unseen with the other patients and could reflect immunoactivation related to leukemia control. Conclusions. Dynamic single-cell profiling of signaling networks is feasible in CML patients and can be used to study mechanisms of aberrant immune reactivity in TKI-treated patients. The method could be particularly suitable for assessing candidate patients for TKI discontinuation. Although in vitro results suggest immunosuppressive effects of TKIs on lymphocytes, leukocytes ex vivo from patients were able to respond similarly to cytokine stimuli as in healthy controls. Disclosures: Mustjoki: BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3350-3350
Author(s):  
Wei Zhang ◽  
Suying Dang ◽  
Thomas Wisniewski

Abstract Abstract 3350 HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 1911-1918 ◽  
Author(s):  
Matt W. Goschnick ◽  
Lai-Man Lau ◽  
Janet L. Wee ◽  
Yong S. Liu ◽  
P. Mark Hogarth ◽  
...  

AbstractWe investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin αIIbβ3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin αIIbβ3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal “insideout” integrin αIIbβ3 signaling (fluorescein isothiocyanate [FITC]–fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin αIIbβ3 “outside-in” signaling events in platelets and is necessary for stability of arterial thrombi in vivo.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4233-4233
Author(s):  
Maria-Isabel Bravo ◽  
Aida Raventós ◽  
Alba Pérez ◽  
Elena G Arias-Salgado ◽  
María Teresa Alvarez Román ◽  
...  

Abstract Introduction: Hemophilia A (HA) patients under emicizumab prophylaxis treatment may require the concomitant use of procoagulant factors for breakthrough bleedings or immune tolerance induction. Thromboembolic events have been described with the concomitant use of emicizumab and activated prothrombin complex concentrate (aPCC), but not with recombinant activated factor VII (rFVIIa). Previous studies showed that the in vitro combination of emicizumab and plasma-derived Factor VIII/Von Willebrand Factor (pdFVIII/VWF) had a non-additive effect on thrombin generation (TG)(Bravo M-I, et al J Thromb Haemost. 2020;18:1934-39). The aim of this study was to evaluate the TG resulting from ex vivo combination of plasma samples from HA patients treated with emicizumab, with a pdFVIII/VWF concentrate (Fanhdi ®, Grifols). Methods: Twelve adult patients with severe HA without inhibitors on prophylaxis with emicizumab and nine healthy controls were included in the study. Blood samples were drawn in citrate plus corn trypsin inhibitor tubes. Then, platelet poor plasma (PPP) was collected for the TG assay, which measures the whole kinetics of TG. Thrombin peak (TP) and endogenous thrombin potential (ETP) were calculated using calibrated automated thrombogram (Thrombinoscope ™ software, Stago) after in vitro activation of coagulation by trigger solution, PPP Reagent LOW TM (4 μM phospholipids/1 pM tissue factor), fluorogenic substrate and CaCl 2 (FLUKAkit TM) reagents (Diagnostica Stago). Fluorescence was read in a Fluoroskan Ascent reader (Thermo) equipped with a 390/460 filter set. Samples were spiked with increasing concentrations of pdFVIII/VWF (10 to 400 IU/dL), rFVIIa (0.9 µg/mL) or aPCC (0.5 U/mL). Results: TG from healthy control samples was measured to establish TP and ETP normal ranges. TP and ETP results obtained from HA plasma with emicizumab were lower than in healthy controls. The addition of pdFVIII/VWF as of 25 IU/kg (prophylaxis dose in HA w/o inhibitors) to samples from HA patients concomitantly treated with emicizumab restored TP and ETP levels within healthy controls normal range (Table 1). Increasing ex vivo concentrations of pdFVIII/VWF maintained TP and ETP similar to healthy controls. The highest concentration of concomitant treatment with pdFVIII/VWF (200 IU/kg) and emicizumab did not result in excessive TP and, importantly, ETP levels were always within the normal range. The combination with the bypassing agent rFVIIa moderately increased TP and ETP values up to normal range. However, when HA plasma was spiked with aPCC in the presence of emicizumab, TP and ETP dramatically increased above normal range resulting in a synergistic procoagulant profile. Conclusions: The concomitant use of pdFVIII/VWF in patients with prophylaxis with emicizumab did not trigger a multiplying effect on TG. These results were aligned with previous in vitro data and suggested the low risk of overdose and thrombotic events of concomitant treatment emicizumab with the pdFVIII/VWF concentrate in HA patients. Figure 1 Figure 1. Disclosures Bravo: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Raventós: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Pérez: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Bayer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Costa: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Willis: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®.


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