Differential Dose Dependent Effects of Recombinant FVIIa and Enhanced Activity Analogue (NN1731) on Thrombin Generation, Platelet Function and Clot Structure in Blood from Normal Volunteers and Patients with Factor VIII Deficiency with or without Factor VIII Inhibitor Antibodies.

Abstract Purpose: The onset of force production due to thrombin generation (TGT), the amount of force produced (PCF) and the elastic modulus (CEM) of clots have been shown to be dose dependent functions of increasing recombinant FVIIa (rFVIIa) concentrations in a variety of patient blood samples. We performed a dose dependent comparison of the effects of rFVIIa and NN1731, an enhanced activity analogue of rFVIIa, in blood from normal volunteers and from hemophilia patients with and without factor VIII inhibitors. Methods: Blood was obtained from six healthy controls, two patients with factor VIII deficiency, two patients with hemophilia and factor VIII inhibitors, and one patient with a spontaneous factor VIII inhibitor who were enrolled in an IRB approved study. The blood was spiked with increasing concentrations (0.32, 0.64, and 1.28 mcg/ml final) of either rFVIIa or NN1731. TGT, PCF and CEM were determined using the Hemodyne Hemostasis Analyzer. Results: rFVIIa (1.28 mcg/ml) shortened the TGT (from 7 to 4 mins), increased CEM (from 20 to 31 Kdynes/cm2) and increased PCF (from 5.6 to 6.6 Kdynes) in blood from healthy controls. The effects of NN1731 (1.28 mcg/ml) were similar for CEM and PCF parameters (31 Kdynes/cm2; and 6.8 Kdynes, respectively; however, the effects were slightly more pronounced for TGT (2 mins). Due to the prolongation of TGT, and the decrease in PCF and CEM in all patient samples, the effects of both rFVIIa and NN1731 were much more pronounced. In all cases, both agents shortened the TGT, and increased both the CEM and PCF. In all patients NN1731 was considerably more potent but the dose response to both agents showed significant patient dependent variability. An example of this variability is illustrated below in the kinetics of PCF development of two patients with hemophilia. At baseline both patients had profoundly prolonged TGT (>20 mins) and no force development. Patient P01 showed minimal response to increasing doses of rFVIIa and good response to NN1731. Patient P02 showed a nice response to both agents. Conclusions: This study suggests that recombinant analogue NN1731 of rFVIIa may be more potent than rFVIIa in correcting the thrombin generation, platelet function and clot structure deficiencies noted in patients with hemophilia with or without factor VIII inhibitors. Additionally, this study showed significant patient dependent variability at baseline and in response to both rFVIIa and NN1731. Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P01) with Severe Hemophilia A Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P01) with Severe Hemophilia A Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P02) with Severe Hemophilia A Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P02) with Severe Hemophilia A

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Tranexamic Acid Increases Clot Stability in Patients with Severe Hemophilia A.

Blood ◽

10.1182/blood.v108.11.1023.1023 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1023-1023

Author(s):

Anne-Mette Hvas ◽

Hanne Thykjaer Sorensen ◽

Lisbeth Norengaard Sorensen ◽

Kirsten Christiansen ◽

Jorgen Ingerslev ◽

...

Keyword(s):

Tranexamic Acid ◽

Factor Viii ◽

Blood Sample ◽

Whole Blood ◽

Hemophilia A ◽

Ex Vivo ◽

Maximum Velocity ◽

Clot Formation ◽

Severe Hemophilia ◽

Clot Stability

Abstract Patients with severe Hemophilia A have a compromised clot formation. In addition, it is suggested that severe Hemophilia A is associated with reduced clot stability. Hence, during the recent years antifibrinolytics have been increasingly used as part of the prophylactic haemostatic treatment e.g. during surgical procedures. However, so far only limited evidence supports the systematic use of antifibrinolytics in these patients. In the present laboratory and clinical study we examined whether reduced clot stability in severe Hemophilia A could be improved by treatment with the antifibrinolytic drug tranexamic acid in combination with recombinant factor VIII (rFVIII). Eight patients with verified severe Hemophilia A (factor VIII:C <0.01 IU/mL) were included. None of the patients had previous or present inhibitor against factor VIII (Bethesda titre < 0.6 IU/mL), and all patients were abstinent to factor VIII substitution for at least 72 hours prior to blood sampling. A baseline blood sample was obtained. Subsequently, the patients received an i.v. bolus injection of rFVIII aiming at increasing the functional level of factor VIII to around 50%. Ten minutes later a second blood sample was obtained followed by i.v. injection of tranexamic acid (10 mg per kg). After another 10 minutes the third blood sample was obtained. In order to provide documentation on the haemostatic potential of tranexamic acid we adopted a thrombelastographic model of continuous whole blood coagulation using ROTEM Coagulation Analyzers (Pentapharm®, Munich, Germany). The whole blood clot formation was evaluated using activation with recombinant human tissue factor (TF - Innovin®, Dade Behring, final dilution 1:17000). The clot stability was evaluated using reaction mixture containing TF (final dilution 1:17000) and recombinant single chain tissue plasminogen activator (t-PA - American Diagnostics, final concentration t-PA 2 nM). Using the software DyCoDerivAn™ GOLD (AvordusoL, Risskov, Denmark) we obtained dynamic parameters of clot initiation (CT, s) and clot propagation such as the maximum velocity of clot formation (MaxVel, mm × 100/s) and the time until maximum velocity of clot propagation (t, MaxVel, s). Whole blood clot stability was analysed by evaluation of the maximum clot formation (MCF, mm × 100) and by the area under the elasticity curve (AUEC, mm × 100/s). Initially, ex vivo experiments were performed with buffer to record a baseline profile. Subsequently, rFVIII (final plasma concentration 0.5 IU/mL) and tranexamic acid (final plasma concentration 0.2 mg/mL) were added. Following the ex vivo experiments, analyses were performed 10 minutes after the patients had been administered rFVIII and tranexamic acid, respectively. We found almost total accordance between ex vivo and in vivo results both in the TF assay and the TF + t-PA assay. As expected, rFVIII increased clot formation indicated by a significantly shorter CT, higher MaxVel and shorter t, MaxVel. Tranexamic acid induced no further measurable improvements of the clot formation profile. Analyses of the clot stability using the TF + t-PA assay showed that rFVIII increased MCF three fold, whereas adding tranexamic acid revealed a four fold increase. The AUEC increased 5 fold after rFVIII and 24 fold after addition of tranexamic acid. In conclusion the study documents that treatment with tranexamic acid in combination with rFVIII significantly improves clot stability in patients with Hemophilia A.

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In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽

10.1182/blood-2020-139712 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

Dougald Monroe ◽

Mirella Ezban ◽

Maureane Hoffman

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Plasma Levels ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor Ix ◽

Factor X ◽

Dose Dependent

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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Hemodialysis in a patient with severe hemophilia A and factor VIII inhibitor

Hemodialysis International ◽

10.1111/hdi.12429 ◽

2016 ◽

Vol 20 (4) ◽

pp. E11-E13 ◽

Cited By ~ 2

Author(s):

Natarajan Gopalakrishnan ◽

Thiruvengadam Usha ◽

Balasubramaniyan Thopalan ◽

Jeyachandran Dhanapriya ◽

Thanigachalam Dineshkumar ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Factor Viii Inhibitor ◽

Severe Hemophilia ◽

Viii Inhibitor

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Significance of Factor VIII Inhibitors by ELISA in Patients with Hemophilia A.

Blood ◽

10.1182/blood.v112.11.3398.3398 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3398-3398

Author(s):

Jamie R Brewer ◽

Sandra Harris ◽

David Green ◽

Anaadriana Zakarija

Keyword(s):

Factor Viii ◽

Functional Status ◽

Hemophilia A ◽

Statistical Significance ◽

Bleeding Event ◽

Clinical Information ◽

Prior History ◽

Factor Viii Inhibitor ◽

Severe Hemophilia ◽

Viii Inhibitor

Abstract Background: The Bethesda assay traditionally has been used to detect Factor VIII inhibitors in patients with Hemophilia A, but recent evidence suggests that it is not sensitive to all inhibitors, particularly non-inhibitory or low-titer antibodies. Methods: Patients with Hemophilia A without prior history of inhibitor were recruited. Study questionnaire collected demographic and clinical information, bleeding history and factor usage over the preceding 6 months. Functional status was assessed by the Hemophilia Activities List (HAL). Factor VIII inhibitor was assessed by both the Bethesda assay and Factor VIII inhibitor ELISA (GTI Diagnostics). T-test was performed to assess statistical significance. Results: Data is available for 26 patients, 19 severe, 2 moderate and 7 mild. All subjects had a negative Bethesda assay, but 10 (39%) had a detectable inhibitor by ELISA. 9/10 inhibitor patients had severe hemophilia, while one had mild hemophilia. In severe hemophiliacs, there were no differences in age, HIV status, CD4 count, Hepatitis C positivity or viral load between those with and those without inhibitors. Inhibitors were more frequent in those using plasma-derived concentrates 4/5 (80%), than in those using recombinant products 6/14 (43%), p=0.15. There was no difference in bleeding frequency or functional status in patients with or without inhibitors, although those with inhibitors had more frequent infusions.(Table). In patients on prophylaxis, those with inhibitors had a higher bleeding frequency compared to those without an inhibitor, (p =0.2). 11 patients were not on prophylaxis and had a higher bleeding frequency (p = 0.02) than patients on prophylaxis irrespective of inhibitor presence. However those with inhibitors required more factor doses per bleed compared to those without an inhibitor (4.4 vs. 1.5, p=0.16) even though the mean factor dose was the same (25.3 units/kg vs 25.2 units/kg). Conclusions: The Factor VIII ELISA assay detected inhibitors in 39 % of Hemophilia A patients who had undetectable inhibitors by standard Bethesda assay. This data suggests that these inhibitors may be clinically relevant, given that inhibitor patients who are not on prophylaxis require more doses of factor per bleeding event. Further study is necessary to determine mechanism and clinical significance of these Factor VIII inhibitors. Table. Characteristics of severe hemophilia patients with and without ELISA Factor VIII inhibitor All severe (n=19) Inhibitor (n=9) No inhibitor (n=10) Age 43.4 40.8 45.7 Plasma-derived factor 5 (26.3%) 4 (44.4%) 1 (10%) Total bleeds/6 months 8.6 8.1 9.0 Muscle bleeds/6 months 1.3 0.8 1.7 Joint bleeds/6 months 7.3 7.3 7.3 Factor infusions/6 months 50.9 57.2 45.2 On prophylaxis 8 (42%) 4 (44.4%) 4 (40%) Total bleeds/6 months 4.3 5.8 2.8 Not on prophylaxis 11 (58%) 5 (55.6%) 6 (60%) Total bleeds/6 months 11.7 10 13.2 Factor doses/bleed 2.9 4.4 1.5

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Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽

10.1182/blood.v128.22.3798.3798 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3798-3798

Author(s):

Lilley Leong ◽

Irina N. Chernysh ◽

Yifan Xu ◽

Cornell Mallari ◽

Billy Wong ◽

...

Keyword(s):

Factor Viii ◽

Whole Blood ◽

Neutralizing Antibodies ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Clot Formation ◽

Factor Vii ◽

Wild Type ◽

Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

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Thrombomodulin-modified thrombin generation after in vivo recombinant factor VIII treatment in severe hemophilia A

Haematologica ◽

10.3324/haematol.13039 ◽

2008 ◽

Vol 93 (9) ◽

pp. 1351-1357 ◽

Cited By ~ 12

Author(s):

A. W.J.H. Dielis ◽

W. M.R. Balliel ◽

R. van Oerle ◽

W. T. Hermens ◽

H. M.H. Spronk ◽

...

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Hemophilia A ◽

Recombinant Factor Viii ◽

Severe Hemophilia

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A Native Whole Blood Thrombin Generation Assay Allows Discrimination of Whole Blood Samples with FVIII Levels below 1%

Blood ◽

10.1182/blood.v124.21.4215.4215 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4215-4215

Author(s):

Christina K Baumgartner ◽

Jonathan C Roberts ◽

Paula M Jacobi ◽

Sandra L Haberichter ◽

Qizhen Shi ◽

...

Keyword(s):

Tissue Factor ◽

Whole Blood ◽

Thrombin Generation ◽

Hemophilia A ◽

Blood Samples ◽

Time Points ◽

Fviii Activity ◽

Post Infusion ◽

Whole Blood Samples ◽

Dose Dependent

Abstract Monitoring the correction of abnormal bleeding tendencies during the treatment of patients with hemostatic disorders is essential to evaluate success of therapy. While single clotting factor assays provide valuable information, global coagulation assays are desirable to better understand the overall hemostatic condition of patients. In Hemophilia A, severity of the clotting defect is traditionally evaluated by determining FVIII activity using chromogenic or clotting assays. Evaluation of thrombin generation in plasma samples for the assessment of bleeding tendencies in hemophilic patients has been suggested. Discriminating between samples with FVIII levels below 1%, however, has been challenging using FVIII activity and thrombin generation assays. We previously reported a native whole blood thrombin generation assay (nWB-TGA) that uses recalcification of whole blood samples without the addition of tissue factor to initiate clotting. We have shown that this assay is sensitive to varying levels of FVIII in vitroand to platelet targeted FVIII gene therapy in a murine model of Hemophilia A. The objective of the present study was to determine if the nWB-TGA can be used to monitor Hemophilia A patients during FVIII therapy and if this assay allows discrimination of whole blood samples with FVIII levels below 1%. Using the nWB-TGA we evaluated thrombin generation in a severe hemophilia A patient carrying an intron 22 inversion. Numerous data points were obtained from 15 different FVIII infusions, each targeting a FVIII level of 50%. Samples collected at least 72 hours (hrs) post infusion (>6 half-lives, calculated FVIII levels <1%) significantly differed from healthy control samples in all thrombin generation parameters. Compared with healthy controls (6.9 ± 0.6 min; mean ± SEM) the hemophilic patient had a lag time (LT) of 24.8 ± 3.4 min. Peak time in healthy controls and the patient was 10.1 ± 1 min and 35 ± 5 min, peak thrombin was 528 ± 78 nM and 124 ± 20 nM, endogenous thrombin potential (ETP) was 1949 ± 117 nM and 1201 ± 50 nM, and thrombin generation rate was 196 ± 58 nM/min and 21 ± 6 nM/min, respectively. While previous studies on thrombin generation in plasma samples mainly reported on differences in peak thrombin and ETP, spiking of hemophilic blood with increasing concentration of recombinant FVIII in vitro revealed that the LT was FVIII dose dependent in our assay. When hemophilic blood was reconstituted with FVIII to a 2%, 5% and 50% level, the LT was 22.5 ± 1.6 min, 16.1 ± 1.7 min and 8.8 ± 0.6 min, respectively. All other thrombin generation parameters were FVIII dose dependent as well. A FVIII dependent LT was also apparent in vivo, when we monitored the patient after FVIII infusions. LT was 6.4 ± 0.2 min at 15 min, 8.5 ± 0.4 min at 24 hrs, and 13.8 ± 0.5 min at 48 hrs post FVIII treatment. We identified the lack of tissue factor as being key to a FVIII dose dependent LT. At all post infusion time points the LT was approximately 5 min when tissue factor was added to the assay. To our surprise, looking at specific time points equal to or greater than 72 hrs post infusion enabled us to discriminate these samples based on the LT (72 hrs: LT= 13.0 ± 0.6 min, 84 hrs: LT= 19.5 ± 0.8 min, 96 hrs: 36.0 ± 4.4 min). The ETP, commonly used as a variable parameter in previous thrombin generation reports, however, was not different among 72, 84 and 96 hrs post FVIII infusion samples. Strikingly, FVIII activity determined by chromogenic and one stage clotting assay was below detection limit (1% FVIII:C) in samples obtained 72 hrs post infusion or later. Thus, the patient in our study displayed considerable thrombin generation determined by the nWB-TGA at post FVIII infusion time points when FVIII levels were considered undetectable with currently available technology. Our data suggest that the different LT observed in 72, 84 and 94 hrs post infusion samples is possibly related to differences in residual FVIII levels below 1%. In conclusion, the nWB-TGA provides a useful tool to monitor efficacy of FVIII replacement therapy and might assist in tailoring individual FVIII treatment regimens. This close to physiological whole blood assay allows distinguishing blood samples with FVIII levels below 1% in vivo, and might help to explain the heterogeneity in bleeding phenotypes observed in severe hemophilia A patients. This assay may also be useful in assessing therapeutic benefit of “long acting” FVIII or FIX products. Disclosures No relevant conflicts of interest to declare.

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An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN

Blood ◽

10.1182/blood.v90.8.3067 ◽

1997 ◽

Vol 90 (8) ◽

pp. 3067-3072 ◽

Cited By ~ 60

Author(s):

Cornelis van ‘t Veer ◽

Neal J. Golden ◽

Michael Kalafatis ◽

Paolo Simioni ◽

Rogier M. Bertina ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor V ◽

Severe Hemophilia ◽

Factor Viii Activity ◽

Thrombin Formation

Abstract The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.

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Case Report: Development of Factor VIII Inhibitor in a Patient with an Uncommon de novo Mutation in the Factor VIII Gene

Acta Haematologica ◽

10.1159/000495559 ◽

2019 ◽

Vol 141 (3) ◽

pp. 129-134

Author(s):

Kirk D. Wyatt ◽

Lea M. Coon ◽

Dawn N. Rusk ◽

Vilmarie Rodriguez ◽

Deepti M. Warad

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

De Novo ◽

De Novo Mutation ◽

Factor Viii Inhibitor ◽

Severe Hemophilia ◽

Factor Viii Gene ◽

Patient Reported ◽

Viii Inhibitor ◽

Mixed Ethnicity

The development of factor VIII inhibitors remains a significant clinical challenge in the management of hemophilia A. We present a patient of mixed ethnicity with severe hemophilia A who was found to have a F8 gene hemizygous c.5815G>T mutation resulting in an Ala1939Ser substitution (Ala1920Ser in legacy nomenclature) and possible splice site change that has been reported in only 1 patient previously. He developed an inhibitor shortly after starting replacement recombinant factor VIII (Advate®; Baxalta, Bannockburn, IL, USA) and was successfully treated with immune tolerance therapy. Our report describes the second patient reported to have severe hemophilia due to this mutation and the only case of a factor VIII inhibitor associated with this mutation.

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Evaluation of the Procoagulant Properties of Freshly Prepared Platelet Microparticles As Compared to Fresh and Stored Platelets

Blood ◽

10.1182/blood.v128.22.2635.2635 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2635-2635 ◽

Cited By ~ 1

Author(s):

Hannah L McRae ◽

Grace Conley ◽

Majed A. Refaai

Keyword(s):

Blood Sample ◽

Whole Blood ◽

Thrombin Generation ◽

Blood Bank ◽

Particle Analysis ◽

Normal Donor ◽

Activation Markers ◽

Whole Blood Sample ◽

Platelet Microparticles

Abstract Background: Platelets are a critical component of primary and secondary hemostasis and clinically important in bleeding and thrombocytopenic patients. However, they remain logistically problematic in blood banking due to their short shelf life, complicated pooling process, high wastage rate and cost, and high rate of transfusion reaction. In vitro studies have shown that stored platelets exhibit minimal (if any) platelet aggregation, and the clinical presumption is that they retrieve their function post-transfusion in vivo. In addition, stored platelets have been shown to have increased levels of platelet microparticles (PMP) that are directly correlated to the length of the storage period. Clinical findings have revealed that patients with thrombocytopenic disorders such as HIT, ITP, TTP, SCD, arterial thrombosis, and certain types of cancer are also associated with increased levels of PMP, which may contribute to bleeding prevention and subsequent thrombosis in these patients. We hypothesized that the same principle may be attributed to stored platelets, and that the retrieved platelet function after transfusion may be due to the procoagulant properties of PMP. Here we evaluated a newly developed, freshly prepared PMP product as a possible means for topical and/or IV application in bleeding and/or thrombocytopenic patients. Methods: Platelet rich plasma (PRP) of healthy individuals was concentrated via centrifugation at 400xg for 8 minutes and a complete blood count (CBC) was performed. Five-day old platelet units were obtained from the blood bank. Platelet counts for fresh and stored PRP were standardized at approximately 1.2x106/μL. PRP was then briefly sonicated for two-second intervals, repeated four times (VibraCell VC50 Probe Sonicator, Sonics and Materials Inc.). The sonicated platelets were then centrifuged at 18,000xg for one hour to obtain platelet microparticles (PMP), and 75% of the supernatant was removed. The pellet was re-suspended in the remaining 25% of the supernatant to obtain concentrated PMP. A thrombocytopenic model was developed by replacing PRP from a normal donor whole blood sample with autologous plasma for a final platelet count of approximately 15x103/μL. The prepared PMP was mixed 1:4 with the thrombocytopenic whole blood sample and evaluated for clot kinetics via thromboelastography (TEG) (TEG5000 Thromboelastograph¨ Hemostasis Analyzer, Haemonetics), thrombin generation assay (TGA) (Calibrated Automated Thrombogram¨, Stago), activated clotting time (ACT) (International Technidyne Corporation, Hemochron¨ Signature Elite Whole Blood Microcoagulation System), and whole blood aggregation (Chrono-Log Lumi-Aggregometer, Model 700). PMP size and concentration were also measured via NanoSight (NanoSight NS300, Malvern) and flow cytometry was used to identify and measure PMP activity (FACSCantoª II, BD Biosciences). All analyses were performed according to manufacturer protocols. The student t-test was performed in all statistical analysis. Results: TEG revealed increased coagulability of PMP as compared to baseline whole blood, thrombocytopenic whole blood, and fresh and stored PRP via decreases in R time (p<0.001), K time (p<0.001), and α angle (p=0.006) (Table 1). TGA showed increased thrombin generation of PMP compared to fresh and stored platelets through increases in the peak (p<0.001), endogenous thrombin potential (ETP) (p<0.001), and velocity index (p<0.001) (Table 2, Figure 1). Particle analysis by NanoSight revealed a PMP concentration of 4.72x108/mL (Figure 2), and flow cytometric analysis of PMP was positive for platelet activation markers CD62 and CD42a (Figure 3). Discussion: Our study confirmed the significant procoagulant properties of our newly developed PMP product as compared to blood bank stored platelets. This PMP product may have useful clinical implications in the future that may reduce complications associated with platelet transfusions. Further in vivo studies will be conducted for evaluation of the potential for topical and IV application of PMP in thrombocytopenic mice. Disclosures No relevant conflicts of interest to declare.

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