Expression and Release of Platelet Protein Disulfide (PDI) Isomerase Is Increased in Patients with Hemophilia a

Abstract Hemophilia A is an X-linked, recessive bleeding disorder caused by congenital factor VIII (FVIII) deficiency. Although the bleeding tendency largely depends on residual FVIII activity (FVIII:C), there is tremendous heterogeneity in bleeding frequency and severity among individuals with similar FVIII:C plasma levels. It is therefore likely that additional factors modulate thrombin generation and fibrin deposition in patients with hemophilia A. PDI is an abundant oxidoreductase with chaperone activity that is also present in human platelets and released upon activation. Preclinical studies indicate that extracellular PDI is critical to hemostasis, thrombosis and vascular inflammation. In particular, PDI has been implicated in monocyte/macrophage tissue factor activation, integrin regulation and platelet-associated thrombin generation. Furthermore, impaired PDI release has most recently been shown to contribute to the bleeding tendency of Hermansky-Pudlak syndrome, an inherited platelet function defect. To explore the role of platelet PDI in hemophilia A, we studied 24 patients (15 severely, 5 moderately and 4 mildly affected) in comparison to 12 age- and sex-matched controls. Expression of PDI antigen on resting platelets and platelets stimulated with either 20 µM ADP or 50 µM thrombin receptor activator peptide 6 (TRAP-6) was assessed by flow cytometry using a fluorescently labeled monoclonal antibody. Analysis of CD41 and CD62P (P-selectin) served as positive controls for constitutive platelet antigen expression and α-granule secretion, respectively. In addition, release of soluble PDI antigen into platelet supernatants was measured by ELISA. There was no significant difference in baseline CD41, CD62P and PDI antigen expression between patients and controls. Furthermore, ADP- and TRAP-6-induced CD62P expression was similar between the two groups (percent positive platelets in patients vs. controls: 28±14 vs. 32±15% and 80±12 vs. 83±9% for ADP- and TRAP-6-treated platelets, respectively). However, expression of PDI antigen on platelets stimulated with either ADP (3.3±2.1 vs. 1.5±1.2%, P<0.01) or TRAP-6 (3.4±1.7 vs. 2.1±1.3%, P<0.05) was significantly increased in patients compared to controls. While ADP-induced release of PDI antigen into platelet supernatants was similar between the two groups and not significantly different from baseline, stimulation with TRAP-6 resulted in significantly increased PDI antigen levels in platelet releasates from patients vs. controls (median, range): 1.5, 0.2-23.2 ng/mL vs. 0.4, 0.2-1.9 ng/mL (P<0.01). Importantly, in two patients with exceedingly high TRAP-6-induced PDI release over baseline (4.8 vs. 0.3 ng/mL and 23.2 vs. 2.8 ng/mL), findings were consistent when platelets were isolated and stimulated on a separate occasion (5.5 vs. 1.3 ng/mL and 10.2 vs. 0.2 ng/mL). Taken together, agonist-induced platelet PDI expression was significantly increased in patients with congenital hemophilia A. Furthermore, release of PDI antigen into supernatants of TRAP-6-activated platelets was significantly increased in patients compared to healthy controls. Up-regulation of platelet PDI may thus represent a compensatory mechanism under conditions of defective thrombin generation and fibrin deposition, and variations in platelet PDI expression and release could at least partially explain the heterogeneity in bleeding severity among patients with congenital hemophilia A and similar FVIII:C plasma levels. Disclosures Langer: Baxalta: Consultancy, Other: Travel support; Pfizer: Research Funding; CSL Behring: Consultancy, Other: Travel support, Research Funding. Voigtländer:CSL Behring: Other: Travel support. Holstein:CSL Behring: Consultancy, Other: Travel support, Research Funding.

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

Blood ◽

10.1182/blood-2018-99-118487 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4336-4336

Author(s):

Christina Berens ◽

Heiko Rühl ◽

Jens Müller ◽

Johannes Oldenburg ◽

Peter Brossart ◽

...

Keyword(s):

Plasma Levels ◽

Research Funding ◽

Thrombin Generation ◽

Myeloproliferative Neoplasms ◽

Final Concentration ◽

Factor X ◽

Prothrombinase Complex ◽

Significant Difference ◽

Thrombin Formation

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

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Assessment of Bleeding Phenotype in Hemophilia Α By a Novel Point-of-Care Global Assay

Blood ◽

10.1182/blood-2019-131252 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4662-4662

Author(s):

Debnath Maji ◽

Michael A Suster ◽

Divyaswathi Citla Sridhar ◽

Maria Alejandra Pereda ◽

Janet Martin ◽

...

Keyword(s):

Whole Blood ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Point Of Care ◽

Blood Samples ◽

Healthy Group ◽

Significant Difference ◽

Bleeding Score ◽

Bleeding History

Introduction: Patients with Hemophilia A have considerable phenotypic heterogeneity with respect to clinical severity based on their baseline factor levels. As clinical bleeding risk is helpful to individualize factor replacement therapy in hemophilia patients, previous studies have utilized direct and indirect methods of thrombin generation to classify individual bleeding phenotypes, however, with variable results. An easy to use, point-of-care, global assay to assess bleed phenotype, can be a useful tool in the clinical setting to determine intensity of prophylaxis therapy for patients with hemophilia. We have previously introduced a novel, point-of-care (POC), dielectric microsensor, ClotChip, and demonstrated its sensitivity to factor replacement in patients with severe hemophilia A. We aim to further test the ability of ClotChip in assessment of a bleeding phenotype, as described by a bleeding score, in patients with hemophilia A. Methods: After IRB approval, 28 patients with hemophilia A of varying severity and well-characterized bleeding history, were enrolled in this study at the time of trough factor levels. The bleeding history was extracted from patient charts and included number of bleeds (joint and soft-tissue), annual factor usage in terms of units/kg, and number of target joints. These parameters were used to generate a bleeding score (range: 0 - 24), and patients were divided in to 2 categories with scores between 0 - 12 (n=14) and > 12 (n=14). Healthy volunteers (n=17) were accrued as controls. Whole blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate. Samples were then tested with the ClotChip within 2 hours of collection. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) and features a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. Our previous studies have shown that the ClotChip readout is sensitive to the global coagulation process and the time to reach a peak in permittivity (Tpeak) is a sensitive parameter to assess coagulation factor defects. Thrombin generation assay (TGA) using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. TGA was not available for 4 hemophilia and 2 control samples. Endogenous thrombin potential (ETP) parameter of TGA was used in this study to assess thrombin generation. Data are reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to test for statistical significance between groups with P < 0.05. Spearman's correlation test was used to derive correlation statistics. Results: ClotChip exhibited a mean Tpeak of 2186s ± 1560s for hemophilia patients in the group with higher bleeding scores (i.e. score >12), a mean Tpeak of 931s ± 496s for the group with lower bleeding scores (i.e. score <12) and a mean Tpeak of 441s ± 74s for the healthy group (Figure 1A). A significant difference in Tpeak was found between the group with higher bleeding scores compared to the group with lower bleeding scores (P = 0.002) as well as between higher bleeding scores and the healthy group (P < 0.0001). However, no significant difference in the TGA ETP parameter was detected between the groups with higher bleeding scores (mean ETP: 470 ± 814) and lower bleeding scores (mean ETP: 471 ± 897) (Figure 1B). ETP exhibited a statistical difference between the healthy group (mean ETP: 3462 ± 575) and both hemophilia groups (P < 0.0001). We also carried out studies to investigate the correlative power of the ClotChip Tpeak parameter to the TGA ETP parameter when including additional blood samples that were collected at various times during a hemophilia patient's prophylaxis regimen. The ClotChip Tpeak parameter exhibited strong negative correlation to the TGA ETP parameter (Spearman's rs= -0.73, P < 0.0001). Conclusions: Our studies suggest that a novel dielectric microsensor (ClotChip) could be useful in assessing bleeding phenotype in hemophilia A patients, allowing rapid assessment of hemostasis using a miniscule amount of whole blood (<10 µL) at the POC. Further studies are needed to determine if ClotChip data can be used to individualize prophylactic factor replacement regimens in hemophilia A patients. Disclosures Maji: XaTek, Inc: Patents & Royalties: 9,995,701. Suster:XaTek, Inc: Consultancy, Patents & Royalties: 9,995,701. Mohseni:XaTek, Inc: Consultancy, Patents & Royalties. Ahuja:XaTexk Inc.: Consultancy, Patents & Royalties, Research Funding; Rainbow Children's Foundation: Research Funding; Bayer: Consultancy; Biovertiv Sanofi: Consultancy; Genentech: Consultancy.

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Which One, Generic Vs Original Glivec Is More Effective in Patients with Chronic Myeloid Leukemia?

Blood ◽

10.1182/blood-2019-127622 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4156-4156

Author(s):

Ekin Kircali ◽

Guldane Cengiz Seval ◽

Sinem Civriz Bozdag ◽

Selami Kocak Toprak ◽

Meltem Kurt Yuksel ◽

...

Keyword(s):

Treatment Failure ◽

Research Funding ◽

Chronic Phase ◽

Response Rates ◽

Molecular Response ◽

Optimal Response ◽

Generic Group ◽

Significant Difference ◽

Generic Groups ◽

Support Research

Introduction:Generic imatinib formulations are increasingly being used as more affordable alternatives worldwide and a few studies have evaluated the safety and efficacy of these formulations prospectively. We have retrospectively analyzed our CML cohort in terms of first line treatment of Glivec versus generic imatinib. This study aims to evaluate the safety and efficacy of generic imatinib products in chronic phase chronic myeloid leukemia as first line treatment. Methods:We have retrospectively analyzed our CML cohort from January 2000 to December 2018 treated with either Glivec or one of generic imatinib formulations. All of our patients (with 1 exception) were initiated imatinib in chronic phase in less than 56 days from diagnosis. All of our patients were followed in accordance with European Leukemia Net (ELN) 2013 recommendations and national hematology association CML guidelines and response definitions were applied according to ELN 2013 criteria. Event free survival (EFS) was defined as the time between treatment initiation and either loss of hematological response, progression to accelerated phase (AP) or blastic phase (BP), or death from any cause. Progression free survival was defined as the time between treatment initiation and transformation to AP, BP or death while on imatinib. For statistical analyses SPSS version 21.0 was used. All p values < 0.05 were considered statistically significant. Results:A total of 192 patients were analyzed comparing 102 (53.1 %) patients on Glivec with 90 patients on (476.9 %) generic formulations. 99 (51.6 %) were female patients. The median age of our population was median 46 years (14-88 years) for Glivec and median 51 years (19-79 years) for generic group (p=0.01). Risk stratifications according to Sokal, Hasford and ELTS scores were run for both Glivec and generic formulation groups. Most of the patients had low risk according to Sokal (137, 71.4%) and Hasford (116, 60.4 %) but intermediate risk according to ELTS (113, 58.9 %) scoring systems. There was no statistically significant difference in the gender distribution, Sokal, Hasford, ELTS scores and ECOG between the two groups. The median time to initiate imatinib treatment was 23.5 (1- 156) days for Glivec group and 13 (1- 51) days generic group (p< 0.05). But the late onset of the treatment was not associated with treatment failure or death. The median follow up was 119.8 (3.7- 250.5) months for Glivec group and 43.6 (2- 150) months for generic groups, respectively (p< 0.05). This difference might be explained by the fact that Glivec has been on the market for about two decades. Similar rates of grade> 2 hematological and non- hematological toxicity were seen in Glivec (4.9 %) and generic groups (3.3 %), respectively (p> 0.05). The rates of treatment failure at 3 months were significantly higher in generic formulation (6.7 %) group than Glivec (2.9 %) group (p< 0.05). Also, the rates of treatment failure at 6 months were significantly higher in generic formulation (3.3 %) group than Glivec (0.9 %) group (p< 0.05). Optimal molecular response rate at 3 months was 76.5 % (n=78) for Glivec and 32.2 % (n=29) for generic groups (p< 0.001). Also, optimal molecular response rate at 6 months was 69.6 % (n=71) for Glivec and 45.6 % (n=41) for generic groups (p= 0.01). Median EFS was found significantly higher for Glivec group compared to generic group (168 mos (95% CI: 159-177 mos) vs 74.6 mos (95% CI: 56-93); p<0.001) (Figure). Conclusion: We found that complete hematological response rates at 3 and 6 months were similar in both groups, but in early phase of treatment the optimal response rates of Glivec group was statistical significantly higher than generic group. Generic group presented with a lower rate of optimal response at 3 months but 13.4 % improvement in optimal response rates was observed at six months. No significant difference in safety concerns was observed between the groups. We recommend that these results from single center should be clarified in a prospective, randomized study including larger population. Figure Disclosures Özcan: AbbVie: Other: Travel support, Research Funding; MSD: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Other: Travel support; BMS: Other: Travel support; Jazz: Other: Travel support; Sanofi: Other: Travel support; Abdi Ibrahim: Other: Travel support; Janssen: Other: Travel support, Research Funding; Bayer: Research Funding; Celgene Corporation: Research Funding, Travel support; Takeda: Honoraria, Other: Travel support, Research Funding; Archigen: Research Funding; Roche: Other: Travel support, Research Funding. Beksac:Celgene: Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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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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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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Increased Thrombin Generation Capacity in Septic Patients with DIC.

Blood ◽

10.1182/blood.v108.11.4054.4054 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4054-4054

Author(s):

Kazuo Kawasugi ◽

Ryousuke Shirasaki ◽

MIzuho Noguti ◽

Haruko Tashiro ◽

Moritaka Gotoh ◽

...

Keyword(s):

Thrombin Generation ◽

Bleeding Tendency ◽

Fibrin Deposition ◽

Activity Peak ◽

Thrombin Activity ◽

Healthy Female ◽

Generation Capacity ◽

Laboratory Changes ◽

Clinical Conditions ◽

Normal Controls

Abstract Variety clinical conditions may cause systemic activation of coagulation, ranging from insignificant laboratory changes to severe disseminated intravascular coagulation (DIC). DIC consists of widespread systemic activation of coagulation, resulting in diffuse fibrin deposition in small and midsize vessels. However, little is known about thrombin generation capacity in patients with DIC. To investigate the thrombin generation capacity, we measured thrombin generation in septic patients with DIC (n=20) and acute promyelocytic leukemia (APL)-induced DIC (n=5). Thrombin generation was determined by the Throbogram-Thrombinoscope assay (Thermo Electron Corporation, Netherlands). The analyzed TG parameters ware the peak of thrombin activity (Peak) and endogenous thrombin potential (ETP). The thrombin antithrombin complexes (TAT) levels were higher in both DIC patients as reported by others. In the septic patients with DIC, we found significant elevations in peak of thrombin activity and ETP as compared with normal controls (determined in 17 healthy males and 14 healthy female). However, the peak of thrombin activity and ETP levels were severely decreased by 60% n the APL patients with DIC. There was slightly correlation between the ETP and TAT levels in septic patients with DIC. Also, there was correlation between the ETP and bleeding tendency in APL patients with DIC. These results suggest that assess of thrombin generation capacity may be helpful in the making the diagnosis in septic patients with DIC. It appears that assess of ETP may contribute to evaluate bleeding tendency in APL patients with DIC. Sepsis with DIC APL with DIC Controls (n=31) *(P<0.05) significantly different controls Peak (nM) 325.3 ± 55.5* 88.2 ± 47.2* 281.2 ± 40.9 ETP (nM.min) 1 ± 326.1* 652.1 ± 274.5* 1469.2 ± 227.3 TAT (μg/ml) 28.4 ± 17.5* 22.5 ± 15.3* <3

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Kinetics of Thrombin Generation in Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.3535.3535 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3535-3535 ◽

Cited By ~ 1

Author(s):

Anna D. Petropoulou ◽

Grigoris T. Gerotziafas ◽

Kostas Zervas ◽

A. Mpanti ◽

Michel Meyer Samama ◽

...

Keyword(s):

Multiple Myeloma ◽

Endothelial Cell ◽

Plasma Levels ◽

Thrombin Generation ◽

Cell Damage ◽

Control Group ◽

Endothelial Cell Damage ◽

Thrombotic Risk ◽

Soluble Thrombomodulin ◽

Significant Difference

Abstract Thalidomide has emerged as a promising treatment for multiple myeloma (MM). Thrombosis is the most serious complication of thalidomide therapy, essentially when it is combined with dexamethasone. The pathogenesis of thrombosis in MM patients (pts) treated with thalidomide is not clear and probably of multi factorial origin. We used the Thrombin Generation test (TGT) and measured the plasma levels of soluble thrombomodulin (sTM) to better clarify the MM-related and thalidomide-related thrombogenicity. TGT was performed in citrated frozen platelet poor plasma (PPP). Blood was obtained from 26 MM pts, Salmon and Durie stage II and III, 62.5 years old (42–77), 9 males and 17 females, 10 treated with thalidomide (100–200mg/d orally) and dexamethasone (40mg/d for 4 days) (TD group) and 16 receiving no treatment (MM group). 13 healthy volunteers formed the control group. Thrombin Generation (TG) was initiated by adding the PPP reagent (Thrombogram-Thrombinoscope®) and the triggering solution (CaCl2 and fluorogenic substrate). We analyzed the endogenous thrombin potential (ETP), the Cmax and the velocity index of TG. The plasma levels of sTM in PPP were measured by a specific ELISA (Diagnostica Stago, France). In the MM group we observed an increase of the ETP, though not significant compared to the controls. The Cmax was almost equal to the control group value, while the velocity index of TG was statistically lower in the MM group compared to controls. In the TD group, a statistically significant increase of ETP was observed as compared to the control group. The Cmax was higher, compared to controls, though not significantly, whereas the velocity index of TG was almost equal to the control group value. There was no significant difference in the TG parameters between MM and TD groups. sTM in the control group was 45±14ng/ml. Both groups of pts had significantly increased sTM plasma levels as compared to the control but the difference between the two groups did not reach significance. Results are shown in Table 1. In patients with MM coexists an increase of sTM, a marker of endothelial cell damage, together with an increased TG capacity. The addition of thalidomide treatment is associated with a slight but not significant increase of ETP and Cmax. The co-existence of endothelial cell damage with increased TG capacity could be associated to the increased thrombotic risk in MM patients treated with thalidomide. This hypothesis will be controlled in a prospective study. Table 1: Thrombogram parameters and sTM plasma levels of studied pts. Control MM group TD group * Results significantly different between the MM and TD groups and the control group (p<0.05 vs the control group) ETP (nM×min) 1399±297 1651±478 1747±448* Cmax (nM) 366±54 342±52 402±99 Velocity Index (nM/min) 198±45 160±19* 184±65 STM (ng/ml) 45±14 84±42* 73±30*

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Defective TAFI Activation in Hemophilia Worsens Joint Bleeding

Blood ◽

10.1182/blood.v128.22.3752.3752 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3752-3752

Author(s):

Tine L Wyseure ◽

Esther J Cooke ◽

Paul J Declerck ◽

Joost CM Meijers ◽

Annette von Drygalski ◽

...

Keyword(s):

Board Of Directors ◽

Thrombin Generation ◽

Hemophilia A ◽

Bleeding Tendency ◽

Protective Effects ◽

Acquired Hemophilia ◽

Advisory Committees ◽

Fviii Inhibitor ◽

Vascular Beds ◽

The Difference

Abstract Background: Joint bleeds are common in hemophilia A or B and cause hemophilic arthropathy. It is clinically well recognized that patients with acquired hemophilia generally do not suffer from joint bleeding, but the molecular mechanisms responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia are ill defined. FVIII deficiency causes defective thrombin generation, impaired coagulation, and increased fibrinolysis. The latter is caused by impaired activation of thrombin activatable fibrinolysis inhibitor (TAFI). Our previous plasma-based analyses showed that clotting and thrombin generation were readily inhibited by an anti-FVIII antibody, whereas a 10-fold higher antibody concentration was required to inhibit thrombin-mediated TAFI activation. We hypothesize that residual TAFI activation occurring in acquired hemophilia, but not in congenital hemophilia, protects against joint bleeding. Here, we determine whether TAFI activation prevents joint bleeding in a mouse model of acquired hemophilia. Methods and results: A transient (anti-FVIII) acquired hemophilia A model was set up to compare joint bleeding in wild type (WT) vs. TAFI-/- mice. Joint bleeding was induced by a subpatellar needle puncture in the right knee. This model caused considerable joint bleeding in FVIII-/- mice as evidenced by the decreased hematocrit (Hct) 2 days post injury (D2 Hct) (D2 Hct= 29 ± 11 % (n= 9) vs. baseline Hct (46 ± 2 %); p< 0.0001). A single injection of the FVIII inhibiting antibody (GMA-8015; 0.25 mg/kg) in WT mice caused acquired hemophilia for up to 72 hours as evident from increased tail bleeding similar to that observed in FVIII-/- mice. Consistent with clinical findings, only minimal joint bleeding was observed in inhibitor-treated WT mice (D2 Hct= 44 ± 4 % (n= 15) for BALB/c and 40 ± 4 % (n= 17) for C57Bl/6J). Significant joint bleeding (D2 Hct= 36 ± 9% (n= 12) for C57Bl/6J; p< 0.05) could be induced by a higher dose of inhibitor (1 mg/kg), however bleeding remained considerably less severe than that observed in FVIII-/-mice. In vitro, the FVIII inhibitor readily inhibited thrombin generation but was relatively ineffective in inhibiting TAFI activation. Therefore, we tested our hypothesis that continued TAFI activation prevented severe joint bleeding in the inhibitor-treated WT mice. Indeed, administration of the FVIII inhibitor (0.25 mg/kg) in TAFI-/-mice resulted in excessive joint bleeding (D2 Hct= 25 ± 8 %; n= 14; p< 0.0001). Similarly, joint bleeding in WT mice was increased significantly when the FVIII inhibitor was co-administered with an inhibitory antibody against TAFI (D2 Hct= 34 ± 7 %; n= 13; p< 0.01). In contrast, TAFI deficiency did not increase tail bleeding with or without FVIII inhibitor, as determined by acute blood loss, 24-hour mortality, and Hct of the survivors at 24 hours post tail resection. These data clearly demonstrate that different vascular beds empower different mechanisms to curb bleeding and suggest that the protective effects of TAFI are specifically relevant for the vascular beds of the synovial joint. Activated TAFI (TAFIa) conveys multiple functions, including anti-fibrinolytic effects and numerous anti-inflammatory activities. Interestingly, tranexamic acid (TXA), a Lys analogue and potent anti-fibrinolytic agent, added at 50 mg/ml to the drinking water, did not reduce joint bleeding in FVIII-/- mice or TAFI-/- mice with the FVIII inhibitor, whereas TXA did correct tail bleeding in these mice. This suggests that the protective effects of TAFI on joint bleeding were independent of its anti-fibrinolytic effects and may result from its anti-inflammatory activities. This is supported by histological analysis at day 7 showing increased stromal proliferation and inflammatory cell recruitment in the joints of TAFI-/-mice. Conclusions:TAFI activation is impaired in congenital hemophilia but not in acquired hemophilia. Abrogation of TAFIa activity, either genetically or pharmaceutically, increased joint bleeding in mice with acquired hemophilia, indicating that TAFI may be responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia. Protective effects of TAFI were vascular bed specific and independent of its anti-fibrinolytic effects, suggesting that one or more of TAFIa's other substrates promote hemophilic joint bleeding. Disclosures von Drygalski: Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau. Mosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.

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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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