Thrombin generation for monitoring hemostatic therapy in hemophilia A: a narrative review

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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The in Vitro Effect on Thrombin Generation of Adding rFVIII or rFIX to Hemophilia a or b Plasma in the Absence or Presence of Concizumab

Blood ◽

10.1182/blood-2020-140272 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Marianne Kjalke ◽

Søren Andersen

Keyword(s):

Drug Interaction ◽

Thrombin Generation ◽

Hemophilia A ◽

Plasma Samples ◽

Publicly Traded ◽

Plasma Pool ◽

Generation Capacity ◽

Drug Drug Interaction ◽

Vitro Effect

Introduction: Lack of factor VIII/IX (FVIII/FIX) in hemophilia A/B (HA/HB), respectively, results in reduced thrombin generation, leading to recurrent/spontaneous bleeds. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody, currently under clinical investigation for subcutaneous prophylaxis of HA/HB patients with/without inhibitors. Breakthrough bleeds occurring in HA/HB patients while on concizumab prophylaxis may be treated with FVIII/FIX. We aimed to compare the in vitro effect of recombinant FVIII (rFVIII) and FIX (rFIX) in HA and HB plasma, respectively, in the absence or presence of concizumab. Methods: rFVIII/rFIX was added to HA/HB pooled plasma at 0.25, 0.5 or 1 IU/mL (corresponding to post-administration plasma concentrations of 12.5, 25 and 50 IU/kg rFVIII and 12.5−25, 25−50 and 50−100 IU/kg rFIX) in the absence or presence of concizumab (1,500, 4,500 or 15,000 ng/mL). In a separate experiment, 33 plasma samples from eight HA patients, who were on concizumab prophylaxis as part of the phase 2 explorer5 trial (NCT03196297), were spiked with 0.5, 1 and 1.5 IU/mL rFVIII. Pre-dose samples (before concizumab prophylaxis) from seven of these patients were also included. Thrombin generation was measured after initiation with 1 pM tissue factor (PPP-Low, Thrombinoscope). Statistical analysis of the effects conferred by each (combination of) drug(s) was performed by ANOVA analyses. Results: A significant (p<0.001) and concentration-dependent increase in thrombin peak was observed when HA plasma pool samples were spiked with rFVIII, both in the absence and presence of concizumab. Likewise, concizumab increased the thrombin peak both in the absence and in presence of rFVIII. Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL only slightly increased the thrombin peak further, demonstrating that a close-to-maximal effect on thrombin peak was achieved at 1,500 ng/mL concizumab. The effects of concizumab and rFVIII were mainly additive with an up to 20% additional effect caused by drug-drug interaction. The addition of rFVIII to explorer5 patient plasma samples resulted in a significant and concentration-dependent increase in thrombin peak. The effects observed for rFVIII and concizumab were exclusively additive. The thrombin peak obtained with 1.0 IU/mL rFVIII before concizumab administration was lower than with 0.5 IU/mL rFVIII in the presence of concizumab. This suggests that a 2-fold reduced rFVIII dose may be sufficient to achieve the same plasma thrombin generation capacity as with the standard rFVIII dose in the absence of concizumab. The addition of rFIX to a HB plasma pool increased the thrombin peak significantly (p<0.001) and in a concentration-dependent manner both in the absence and presence of concizumab (1,500 ng/mL). Likewise, concizumab increased the thrombin peak at all rFIX concentrations (p<0.001). Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL had no or limited further effect. The effects of concizumab and rFIX were mainly additive with an up to 10% effect conferred by negative drug-drug interaction for 1 IU/mL rFIX combined with concizumab >1,500 ng/mL and 0.5 IU/mL rFIX combined with 15,000 ng/mL concizumab, i.e., a 10% smaller effect of rFIX was observed in the presence of concizumab than in its absence. The thrombin peak obtained upon adding 1.0 IU/mL rFIX to plasma without concizumab was similar to the thrombin peak in the presence of concizumab and 0.5 IU/mL rFIX. This suggests that in the presence of concizumab, a 2-fold reduced dose of rFIX would be sufficient to obtain the same plasma thrombin generation capacity as with 1.0 IU/mL rFIX in the absence of concizumab. Conclusion: rFVIII/rFIX increased the thrombin peak in HA and HB plasma, respectively, both in the absence and presence of concizumab. The combined effects of rFVIII/rFIX with concizumab were mainly additive with an up to 20% additional effect caused by drug-drug interaction with rFVIII and a 10% reduction with rFIX. No signs of exaggerated thrombin generation were observed by combining concizumab with rFVIII/rFIX. Therefore, the data support rFVIII/rFIX use for bleed treatment in patients on concizumab prophylaxis. As rFVIII/rFIX and concizumab have additive effects in terms of thrombin generation capacity, data suggest that clinical effectiveness could be achieved with rFVIII/rFIX doses in the lower range recommended for such products. Disclosures Kjalke: Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company. Andersen:Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company.

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In Hemophilia Α Plasma Treated with Emicizumab, Factor IXa in Activated Prothrombin Complex Concentrates Is the Dominant Contributor to Enhanced Thrombin Generation

Blood ◽

10.1182/blood-2020-139586 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-17

Author(s):

Dougald Monroe ◽

Mirella Ezban ◽

Maureane Hoffman

Keyword(s):

Thrombin Generation ◽

Hemophilia A ◽

State Level ◽

Peak Level ◽

Coagulation Factors ◽

Major Effect ◽

Factor Ix ◽

Factor X ◽

Factor Ixa ◽

Activated Prothrombin Complex Concentrates

Background. Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa and factor X has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to treating this bleeding in hemophilia A patients with inhibitors is to give an activated prothrombin complex concentrate (APCC; FEIBA) (disfavored in NHF MASAC #255). APCC contains a number of coaguation factors including prothrombin, factor X (FX), and factor IX (FIX). APCC also contains activated factor X (FXa) and factor IX (FIXa). Previous work has shown that when APCCs are added to hemophilia A plasma containing emicizumab there is a significant increase in thrombin generation [J Thromb Haemost 2018;16:1580-1591]. The goal of this work was to study thrombin generation in hemophilia A plasma with emicizumab and to examine the role of the zymogen and activated components of an APCC in the increased thrombin generation. Methods. In hemophilia A plasma, thrombin generation assays were done using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). The components of APCC were studied at concentrations that should mimic the levels seen in the plasma of a patient given a dose of 50 U/kg: prothrombin 1800 nM; FX 130 nM; FIX 90 nM; and FIXa 0.4 nM. Results. When initiated with low TF, hemophilia A plasma alone had essentially no thrombin generation. As expected, adding emicizumab enhanced thrombin generation. The addition of zymogen coagulation factors, prothrombin, FIX, and FX, separately or together gave a small increase in thrombin generation. However, addition of FIXa to emicizumab gave a large increase in peak thrombin. In hemophilia A plasma with emicizumab and FIXa, addition of prothrombin further increased thrombin generation and specifically increased the peak level of thrombin. Further addition of FX or FIX, separately or together, only minimally increased thrombin generation. Discussion. The strong contribution of factor IXa to the effects of APCCs on thrombin generation in hemophilia A plasma depends on the presence of emicizumab. In the absence of emicizumab, a study of the individual components of APCC showed that a combination of FXa and prothrombin at levels found in APCCs had the major effect on thrombin generation [Haemophilia. 2016;22:615-24]. That study found that FIXa did not increase thrombin generation. However, in the presence of emicizumab, despite the weak solution phase affinity of the bifunctional antibody for FIXa, small amounts of FIXa were the most significant contributor to thrombin generation. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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Potentiation of Thrombin Generation in Hemophilia A Plasma by Coagulation Factor VIII and Characterization of Antibody-Specific Inhibition

PLoS ONE ◽

10.1371/journal.pone.0048172 ◽

2012 ◽

Vol 7 (10) ◽

pp. e48172 ◽

Cited By ~ 14

Author(s):

Bhavya S. Doshi ◽

Bagirath Gangadharan ◽

Christopher B. Doering ◽

Shannon L. Meeks

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Hemophilia A ◽

Coagulation Factor ◽

Coagulation Factor Viii ◽

Specific Inhibition

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Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2007.02861.x ◽

2008 ◽

Vol 6 (3) ◽

pp. 486-493 ◽

Cited By ~ 57

Author(s):

M. TROSSAËRT ◽

V. REGNAULT ◽

M. SIGAUD ◽

P. BOISSEAU ◽

E. FRESSINAUD ◽

...

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Hemophilia A ◽

Thrombin Generation Assay

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Thrombin generation and bleeding in cardiac surgery: a clinical narrative review

Canadian Journal of Anesthesia/Journal canadien d anesthésie ◽

10.1007/s12630-020-01609-4 ◽

2020 ◽

Vol 67 (6) ◽

pp. 746-753 ◽

Cited By ~ 1

Author(s):

John Fitzgerald ◽

Robert McMonnies ◽

Aidan Sharkey ◽

Peter L. Gross ◽

Keyvan Karkouti

Keyword(s):

Cardiac Surgery ◽

Thrombin Generation ◽

Narrative Review ◽

Clinical Narrative

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Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Blood ◽

10.1182/blood.v95.4.1330.004k28_1330_1335 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1330-1335 ◽

Cited By ~ 98

Author(s):

Cornelis van 't Veer ◽

Neal J. Golden ◽

Kenneth G. Mann

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Plasma Concentrations ◽

Factor Xa ◽

Factor Vii ◽

Lag Phase ◽

Single Chain

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.

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Hemophilia A in Cardiac Operations: A Model of Reduced Thrombin Generation

The Annals of Thoracic Surgery ◽

10.1016/j.athoracsur.2010.10.077 ◽

2011 ◽

Vol 91 (5) ◽

pp. 1606-1608 ◽

Cited By ~ 9

Author(s):

Susanne Lison ◽

Michael Spannagl ◽

Wulf Dietrich

Keyword(s):

Thrombin Generation ◽

Hemophilia A ◽

Cardiac Operations

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Factor VIII Efficacy and Tissue Factor Levels in Hemophilia A Patients Undergoing Total Knee Replacement.

Blood ◽

10.1182/blood.v104.11.4029.4029 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4029-4029

Author(s):

Wolfgang Wegert ◽

Manuela Krause ◽

Inge Scharrer ◽

Ulla Stumpf ◽

Andreas Kurth ◽

...

Keyword(s):

Total Knee Replacement ◽

Plasma Levels ◽

Thrombin Generation ◽

Hemophilia A ◽

Lag Time ◽

Major Surgery ◽

Plasma Samples ◽

Time To Peak ◽

Thrombin Generation Assay ◽

Total Knee

Abstract The changes of tissue factor (TF) blood levels in patients undergoing major surgery has been reported presenting controversial data. Whether this TF is hemostatically active or if it interacts with other coagulation factors, e.g. FVIII, is still unclear, making thrombotic risk and complications assessment for even more difficult. We analyzed plasma samples from four male patients aged 27–55 with severe hemophilia A without inhibitory antibodies, undergoing total knee replacement, which all gave informed consent. Initial FVIII doses before intervention was 75–80 U/kg. Treatment following intervention was targeted at 100 % FVIII serum levels. None received heparin. No bleeding events occurred during the observation period. The samples were taken at these timepoints (TP): 1. before preoperative FVIII substitution, 2. at the time of first incision (intervention start), 3. at circulation arrest release + 90 s after prosthesis implantation, 4. final suture (intervention end), 5. 24 h and 6. 48 h after intervention to assay procedurally induced TF production. Coagulation analyses were carried out using a fluorometric thrombin generation assay (TGA) in platelet rich plasma (PRP), RoTEG (rotation thrombelastography) in whole blood and a TF ELISA for the plasma samples’ TF levels. Both clotting function tests were started using TF diluted 1:100.000 and calcium chloride 16,7 mM (final conc.). TGA parameters were ETP, PEAK (maximum thrombin generation velocity), TIME TO PEAK, LAG TIME. TGA parameters directly related to thrombin activity (ETP; PEAK) showed no change during the intervention, but a sharp decrease 24 h later with a partial recovery 48 h later. TGA time marks (TIME TO PEAK, LAG TIME) changed in an inverse way, except for the difference from LAG TIME and TIME TO PEAK, which shortened continously after circulation arrest removal. RoTEG was characterized using 4 parameters: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF) and clot formation rate (CFR). After preoperative FVIII substitution, CT decreased by 10 % and CFT by 50 % in 48 h. MCF stayed unchanged during the intervention and the following 24 h, but increased by 20 % at 48 h. CFR increased by 10 % during intervention, and by 20 % from 24 to 48 h. TF ELISA showed preoperative TF plasma levels from 11 to 271 pg/ml. After release of circulation arrest (TP 3) TF concentration increased sharply (4 times the initial value), which was not detectable in the samples taken at TPs 2 and 4. TF levels further increased at TPs 5 and 6 to 170 % and 317 % resp. Altogether, TF plasma levels elevated after major surgery seem to correspond to a potential risk factor for postoperative thrombosis, especially when elevation is induced after intervention. However, functional coagulation assays do not change uniformly, as the thrombin generation assay reflects no marked changes under intervention, but in the period after(24–48 h). Changes in the RoTEG whole blood clotting assay are not dramatic but indicate a thrombophilic shift in coagulation balance also pronouned at 24–48h, too. These results demonstrate that increased coagulability after orthopedic surgery detected using functional clotting assays correlates with increased TF levels, but further studies must be performed to prove this relation in healthy individuals.

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Thromboelastography in Children with Coagulation Factor Deficiencies.

Blood ◽

10.1182/blood.v106.11.2139.2139 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2139-2139 ◽

Cited By ~ 1

Author(s):

Meera B. Chitlur ◽

Indira Warrier ◽

Madhvi Rajpurkar ◽

Wendy Hollon ◽

Lolita Llanto ◽

...

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Hemophilia A ◽

Coagulation Factor ◽

Response To Treatment ◽

Factor Vii ◽

P Value ◽

Factor V ◽

Mean Values ◽

Significant Difference

Abstract The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients. Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects. Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following: Table 1 TEG Parameters (mean values) SHA (n=29) Mod.H (n=4) SHA+I (n=4) OFD (n=6) Control(n=19) MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. Amplitude MTG(mm*100/sec) 8.7 9.6 1.3 9 17 TMG(min) 27.5 16.6 62.7 17.5 8.9 R(min) 22 14 56 15 7 K(min) 7 4 41 4 2 Max.Amplitude, MA (mm) 59 56 12 58 62 The rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): Figure Figure Table 2 TEG Parameters (Mean values) MTG < 9 (n=16) MTG > 9 (n=13) p value TMA: Time to MA; MTG(mm*100/sec) 5.5 12.6 <0.001 TMG (min) 33 20 0.009 R(min) 26 16 0.004 K(min) 9 3.4 0.03 MA(mm) 56.1 62.3 0.01 TMA(min) 60 38 0.006 13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9. Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

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