Contact Activation Inhibition and Platelet Rich Plasma Are Required in Order to Differentiate Bleeders from Non-Bleeders in FXI Deficiency Using the Thrombin Generation Assay

Abstract Introduction: FXI deficiency is characterised by a variable bleeding tendency. No clear correlation exists between bleeding and either FXI:C level or genotype. Some patients with major FXI deficiency (FXI:C <15 IU/dL) do not exhibit excessive bleeding, even after surgery or trauma, while others with partial deficiency (FXI:C 20-60 IU/dL) report significant haemorrhagic symptoms. The variation in bleeding tendency is observed between individuals with the same FXI:C level and also between individuals within families who share the same F11gene mutation. The thrombin generation (TG) assay quantifies the overall haemostatic potential of an individual through the measurement of thrombin production. While reduced TG has been observed in some FXI deficient individuals, there are conflicting reports about the ability of the TG assay to distinguish between patients with a history of bleeding (bleeders) and those without (non- bleeders). Aims: To identify the optimal TG assay conditions which discriminate between normal controls and individuals with different levels of FXI deficiency. To investigate which conditions allow the TG assay to differentiate between bleeders and non-bleeders. Methods: 97 adults with FXI deficiency were studied together with 50 controls. Bleeding histories were taken from each individual and citrated blood samples, both with addition of corn trypsin inhibitor (+ CTI) and without (- CTI). Platelet rich (PRP) and platelet poor plasmas (PPP) were prepared. TG was performed using the CAT method with tissue factor (TF) concentrations of 5pM, 1pM and 0.5pM in the following samples: PPP + CTI, PPP - CTI, PRP + CTI, PRP - CTI. TG results were compared between controls and 3 patient groups: partial FXI deficiency (FXI:C 20-60 IU/dL) (n=78), major low level FXI deficiency (FXI:C 3-15 IU/dL) (n=10) and major very low level FXI deficiency (FXI:C ≤2IU/dL) (n=9) using unpaired t-test or Mann Whitney U tests. A subgroup of 74 patients was divided into bleeders and non-bleeders based solely on their experience of tonsillectomy and /or dental extraction prior to diagnosis of FXI deficiency. Those with excessive bleeding (requiring blood product transfusion or return to theatre/dentist for re-suturing or packing) were classified as bleeders (n=24) and those without, as non-bleeders (n=50). The optimal TG assay conditions to differentiate between bleeders and non-bleeders groups were identified with Receiver Operator Curves (ROC) using Area Under Curve (AUC) values. Results: TG performed in PPP with CTI was unable to differentiate between controls and all of the 3 patient groups. In all other sample types the best discrimination between controls and FXI deficient patients was seen at low TF (0.5pM) with a sequential reduction in peak height and ETP measurements observed as FXI:C levels decreased across the 3 patient groups (see Figure 1). When TG results were compared between the bleeder (n=24) and non-bleeder (n=50) groups, peak height and ETP measured in PRP with CTI were best able to differentiate between the two groups (peak height ROC AUC = 0.9362, P<0.0001) (ETP ROC AUC=0.9362, P<0.0001). PPP samples and those without CTI were unable to segregate bleeders from non-bleeders as effectively. Conclusions: In the presence of contact activation inhibition, FXI:C levels have minimal impact on thrombin generation in PPP but do influence thrombin generation in the presence of platelets. TG measured at low TF in PPP and PRP samples without CTI (with in vitro contact activation of FXI possible) was able to differentiate between controls and individuals with different levels of FXI deficiency but not between bleeders and non-bleeders.TG performed at low TF in PRP with CTI samples demonstrated the greatest differentiation between bleeders and non-bleeders in FXI deficiency providing further support for the clinical importance of platelet interaction with FXI in this disorder and suggesting that these test conditions may best mimic those in vivo. Acknowledgments: This work is supported by a Fellowship Project Award (Bayer Hemophilia Awards Program), an unrestricted grant (LFB Biotechnologies) and a Wycherley Fellowship grant. Figure 1A Figure 1A. ETP measurements in PPP and PRP samples and Peak height in PRP samples with and without CTI (TF 0.5pM) performed in a control group and 3 patient groups. Line and error bars represent mean and SD of each group. *P<0.05, **P<0.01, ***P< 0.001, ****P<0.0001, ns=not significant. Figure 1B Figure 1B. Figure 1C Figure 1C. Disclosures Pike: Bayer: Honoraria, Research Funding; LFB Biotechnologies: Honoraria, Research Funding. Bolton-Maggs:Bio-Products Laboratory (U.K.): Consultancy; LFB Biotechnologies: Consultancy.

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Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Blood ◽

10.1182/blood-2021-151203 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4235-4235

Author(s):

Paula Acuña ◽

Elena Monzón Manzano ◽

Elena G Arias-Salgado ◽

María Teresa Alvarez Román ◽

Mónica Martín ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Function ◽

Research Funding ◽

Thrombin Generation ◽

Bleeding Tendency ◽

Contact Activation ◽

Normal Range ◽

Coagulation Tests ◽

In Vitro Treatment

Abstract Introduction: Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

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Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram

Thrombosis and Haemostasis ◽

10.1160/th09-03-0180 ◽

2009 ◽

Vol 102 (11) ◽

pp. 936-944 ◽

Cited By ~ 64

Author(s):

Kellie Machlus ◽

Emily Colby ◽

Jogin Wu ◽

Gary Koch ◽

Nigel Key ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Relative Sensitivity ◽

Epidemiological Studies ◽

Peak Height ◽

Clotting Factor ◽

Time To Peak ◽

High Throughput Method ◽

Assay Conditions

SummaryElevated procoagulant levels have been correlated with increased thrombin generation in vitro and with increased venous thromboembolism (VTE) risk in epidemiological studies. hrombin generation tests are increasingly being employed as a high throughput method to provide a global measure of procoagulant activity in plasma samples. The objective of this study was to distinguish the effects of assay conditions [tissue factor (TF), thrombomodulin, platelets/lipids] and factor levels on thrombin generation parameters, and determine the conditions and parameters with the highest sensitivity and specificity for detecting elevated factor levels. Thrombin generation was measured using calibrated automated thrombography (CAT) in corn trypsin inhibitor (CTI)-treated platelet-free plasma (PFP) and plateletrich plasma (PRP). Statistical analysis was performed using logarithms of observed values with analysis of variance that accounted for experiment and treatment. he relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX,VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. For all conditions, inclusion of thrombomodulin prolonged the LT and decreased the peak and ETP; however, addition of thrombomodulin did not increase the ability of CAT to detect elevated levels of individual procoagulant factors. In conclusion, CAT conditions differentially affected the sensitivity of thrombin generation to elevated factor levels. Monitoring the peak height and/ or ETP following initiation of clotting in PFP with 1 pM TF was most likely to detect hypercoagulability due to increased procoagulant factor levels.

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The Relationship between Thrombin Generation Assay and FVIII Levels in Patients with Mild to Moderate Haemophilia (A)

Blood ◽

10.1182/blood-2018-99-111675 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2454-2454

Author(s):

Pu-Lin Luo ◽

Steven K. Austin ◽

Kiran Parmar ◽

Dan P Hart ◽

Michael Laffan

Keyword(s):

Research Funding ◽

Thrombin Generation ◽

Haemophilia A ◽

Contact Activation ◽

Extrinsic Pathway ◽

Two Stage ◽

One Stage ◽

Thrombin Generation Assay ◽

The Relationship

Abstract Introduction Haemophilia A (HA) phenotypes (mild, moderate and severe) are based on the baseline FVIII levels, however considerable variation in the bleeding phenotype exists between patients with similar FVIII level. Moreover, approximately 40% of patients with mild HA have large discrepancies between FVIII measured by one stage (FVIII:C1) and two stage methods (FVIII:Chr2) and it is unclear which method correlates best with in vivo FVIII function and bleeding phenotype. The Thrombin Generation assay (TGA), a global measure of haemostasis may be a better predictor of bleeding phenotype but pre-analytical factors such as contact activation can confound the results. Choice of initiating conditions may also be critical in determining sensitivity: recent studies have suggested that initiation with FIXa rather than tissue factor (TF) in detecting low levels of FVIII:C in severe HA, however its utility in mild to moderate HA patients has yet to be determined. The aim of this study is to establish the relationship between FVIII:C and TGA and the influence of contact factor activation in TF and FIXa triggered TGA in patients with mild to moderate HA. Methods This is a prospective cohort study. Patients aged >18 with known congenital HA and FVIII:C 0.01- 0.2 iu/ml were recruited from 3 Haemophilia Comprehensive Care Centres in London. Peripheral blood was drawn into citrate Vacutainer tubes containing 0.106M trisodium citrate (1:9 volume) and Vacutainer tubes preloaded with CTI (50µg/ml). Samples underwent double centrifugation (2500g) to obtain platelet free plasma. Thrombin generation assay, using a standard calibrated automated thrombogram was triggered with either TF (1pmol) or FIXa (5nM). Factor FVIII levels were assessed by one stage APTT based (FVIII:C1) and two stage chromogenic (FVIII:Chr2) methods. Mutation analysis was carried out in all patients. Results 40 patients were recruited in the study. Five patients (13%) had standard FVIII discrepancy (FVIII:C1/FVIII:Chr2>1.5) with 4 different FVIII mutations located on the inter-domain surface of the A2 domain (p.Tyr683Ser, p.Arg550Cys, p.Gly498Arg, p.MET681.Le). One patient had reverse FVIII discrepancy. In TF triggered TGA, the presence of CTI resulted in significant reduction in mean ETP (nmol .min)(455. vs 278, p<0.01, 95% CI 104-243), mean Peak thrombin (nM) (37.81 vs 16.54, t(6.6) p<0.01 95%CI 14.7-27.3), and mean Velindex (nM/min) (4.86 vs 1.29 t(7.0), p<0.01, 95% CI2.3-4.19) and a longer mean ttPeak (min) (14.26 vs 16.22, t(-3.2) p=0.02 95% CI-3.1- -0.76). In contrast, the presence of CTI did not affect ETP (1143 vs 1042, p=0.19 95% CI -54-256), mean Peak thrombin (252 vs 251, p=0.6 95%CI 27-46) or Velindex (118.54 vs 119.15 p= 0.95, 95%CI -23-12.9) in FIXa triggered TGA. There was a good correlation between FVIII:Chr2 and ETP (r=0.56, p=<0.001) Peak (r=0.6, p=<0.001) and Velindex (r=0.7, p=<0.001) in TF(CTI-) triggered TGA, however no relationship was seen between FVIII:C and TG parameters (ETP r=-0.01 p=0.9, Peak r=-0.003, p=0.97 and Velindex r=0.018, p=0.9) in TF(CTI+) triggered TGA. In both FIXa(CTI-) and FIXa (CTI+) triggered TGA, there was a good correlation seen between Lagtime (r=-0.6 p=<0.01), Peak (r=0.4-0.6, p=<0.01) ttpeak (r= -0.6, p=<0.01) and Velindex (r=0.69 <0.01) with FVIII:Chr2 but not with ETP. In patients with standard FVIII discrepancy (n=5), their ETP and Peak levels in TF and FIXa triggered TGA were in keeping with the ETP and Peak levels of non-discrepant patients with similar FVIII:C2 and significantly lower than that of non-discrepant patients with similar FVIII:C1. Conclusions Our study confirms that at low TF triggered TG, contact factor activation in vitro is an important preanalytical variable. Curiously any TG correlation with FVIII level is lost once the contact pathway is inhibited suggesting that TG remains largely determined by the extrinsic pathway in this system. In contrast, factor FIXa triggered TG is unaffected by inhibition of contact activation and demonstrates a good correlation to FVIII:C with or without CTI. This can be explained by suggesting that the supply of FIXa negates any effect of XIa from contact activation and that TG by this route is more completely dependent on FVIII. Therefore a FIXa triggered TGA may offer a better alternative in the assessment of haemophilia and further studies are underway to determine whether this is a better predictor of bleeding phenotypes. Disclosures Luo: Pfizer: Research Funding. Austin:Pfizer: Research Funding. Laffan:Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.

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Coagulation on Endothelial Cells: The Underexposed Part of Virchow’s Triad

Thrombosis and Haemostasis ◽

10.1160/th12-04-0269 ◽

2012 ◽

Vol 108 (11) ◽

pp. 863-871 ◽

Cited By ~ 8

Author(s):

Irma Geenen ◽

Mark Post ◽

Daniel Molin ◽

Geert Schurink ◽

Jos Maessen ◽

...

Keyword(s):

Endothelial Cells ◽

Thrombin Generation ◽

Factor Viia ◽

Bypass Graft ◽

Peak Height ◽

Factor Xii ◽

Contact Activation ◽

Extrinsic Pathway ◽

Serum Free ◽

Cabg Patients

SummaryThe process of thrombin generation involves numerous plasma proteases and cofactors. Interaction with the vessel wall, in particular endothelial cells (ECs), influences this process but data on this interaction is limited. We evaluated thrombin generation on EA.hy926, human coronary arterial ECs (HCAECs) and patient-derived human venous ECs (HVECs) by means of a modified calibrated automated thrombogram (CAT) method and especially looked into contribution of the intrinsic and extrinsic pathways. Thrombin generation was measured in presence of confluent ECs with normal pooled and factor XII-deficient (FXII-deficient) platelet-poor plasma, with/without active site inhibited factor VIIa (ASIS) to block the extrinsic pathway and corn trypsin inhibitor for blocking contact activation (intrinsic pathway). Fetal bovine serum (FBS) was removed from culture conditions as FXIIa from the serum retained on ECs apparently, thereby inducing strong contact activation. In serum-free conditions, EA.hy926 and patient-derived HVECs induced thrombin generation mainly via the contact activation pathway with minor influence of ASIS on peak height and very low thrombin generation curves in FXII-deficient plasma. HVECs derived from coronary arterial bypass graft (CABG) patients showed increased thrombin generation compared to control patients, which could be ascribed to increased contact activation. Contribution of the extrinsic pathway on patient-derived ECs was limited. We conclude that the CAT method in combination with serum-free cultured ECs offers a valuable high-throughput method to evaluate endothelial influences on thrombin generation, which appears to involve predominantly contact activation on ECs. Contact activation-mediated thrombin generation was increased on ECs from CABG patients compared to controls.

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Monitoring Recombinant FVIIa in Hemophilic and Normal Plasma Using a Thrombin Generation Assay

Blood ◽

10.1182/blood.v118.21.1207.1207 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1207-1207

Author(s):

Sarah T.B.G. Loubele ◽

Henri M.H. Spronk ◽

Rene van Oerle ◽

Hugo ten Cate ◽

Peter L.A. Giesen

Keyword(s):

Thrombin Generation ◽

Factor Viia ◽

Optimal Dose ◽

Peak Height ◽

Contact Activation ◽

Mean Values ◽

Thrombin Generation Assay ◽

Normal Plasma ◽

The Mean ◽

20 Nm

Abstract Abstract 1207 Background: Although the recombinant factor VIIa (rFVIIa) has been registered for use in hemophilia patients with inhibitors, there is still no method to monitor the effects of rFVIIa in restoring the coagulation balance in plasma. Hence, information is lacking about the individual optimal dose needed to normalize thrombin generation. Methods: The calibrated automated thrombogram (CAT) method was modified to increase sensitivity for rFVIIa addition to plasma at concentrations of 0, 2.5, 5, 10, 20, 40 and 80 nM, which covers the expected plasma concentration of 26 nM reached after standard administration. Thrombin generation was triggered using combinations of TF concentrations between 0 and 4 pM, and phospholipids concentrations between 0 and 4 μM. Endogenous thrombin potential (ETP), peak height, and velocity index were calculated in platelet poor plasmas (PPP) of different donors. All blood was collected in citrated tubes containing corn trypsin inhibitor (CTI) to minimize any contact activation. Results: The optimal conditions for discriminating rFVIIa (0–80 nM) in the CAT assay were determined in PPP: 0 or 0.25 pM TF with 4 μM of phospholipids. Also at higher TF concentrations, the CAT method was able to detect varying rFVIIa concentrations. The optimal concentration of phospholipids was 4 μM for all TF concentrations. In plasma of 6 healthy volunteers, thrombin generation triggered with 0.25 pM TF dose dependently increased using varying rFVIIa concentrations between 0 and 80 nM (Figure 1, left panel). The mean values for ETP, peak height and velocity index are depicted in Table 1. On average, addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in a 146, 156, 161, 174, 206, and 285 % of the peak height compared to 0 nM rFVIIa, which was set at 100 %. At 4 pM TF the maximum ETP, peak height, and velocity index were reached at concentrations less than 20 nM rFVIIa for all donors. The mean values are depicted in Table 2. Surprisingly, at 80 nM rFVIIa, thrombin generation was decreased compared to lower rFVIIa concentrations (Figure 1, right panel). Addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in 107, 109, 107, 103, 100, or 94 % of the peak height without addition of rFVIIa (0 nM set at 100 %). In FVIII deficient patient plasma (PPP), this effect was also present and even more pronounced. Here, a dose dependent effect of rFVIIa addition was visible at low (0 or 0.25 pM) TF trigger, whereas at 4 pM TF trigger ETP, peak height and velocity index were maximal in the presence of 10 nM rFVIIa. Overall, the peak height was 136, 142, 126, 102, 94, and 81 % upon addition of 5, 10, 20, 30, 40, or 80 nM rFVIIa respectively compared to 0 nM rFVIIa (set to 100 %). Discussion: In hemophilic as well as normal plasma, the addition of rFVIIa dose dependently altered thrombin generation triggered with a low TF trigger (0 or 0.25 pM). At a higher trigger of 4 pM TF, maximal thrombin generation was obtained at rFVIIa concentrations of less then 20 nM. Remarkably, thrombin generation was attenuated in the presence of 80 nM rFVIIa. This paradox may be explained by assuming that the endogenously activated VIIa is more active than the rVIIa that was added. At higher rVIIa dosages the fraction of TF occupied by endogenous VIIa will decrease resulting in less active TF:VIIa complexes. This effect will be more pronounced when FXa formation is dependent on TF:FVIIa alone without the involvement of the tenase complex, which shows from our analysis in hemophilic plasma. Overall, these data suggest that the assay is most sensitive to added rVIIa when the contribution to Xa formation of TF:VIIa complex is small compared to that of rVIIa alone, i.e., in conditions where there is no or very little TF present. Disclosures: Giesen: Thrombinoscope bv: Employment.

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Monitoring thrombin generation: Is addition of corn trypsin inhibitor needed?

Thrombosis and Haemostasis ◽

10.1160/th08-10-0670 ◽

2009 ◽

Vol 101 (06) ◽

pp. 1156-1162 ◽

Cited By ~ 59

Author(s):

Arne Dielis ◽

Marina Panova-Noeva ◽

René van Oerle ◽

José Govers-Riemslag ◽

Karly Hamulyák ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Thrombin Generation ◽

Lag Time ◽

Peak Height ◽

Blood Collection ◽

Contact Activation ◽

In Vitro Measurements ◽

Analytical Variable ◽

Corn Trypsin Inhibitor

SummaryThrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the near future. However, robust pre-analytical conditions may be required, and one factor that has been reported is in-vitro contact activation that might influence in-vitro measurements of thrombin generation and thereby act as an unpredictable pre-analytical variable. The aim of the current study was to investigate the influence of contact activation and the necessity of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurements at low tissue factor (TF) concentrations. Thrombin generation was performed using the calibrated automated thrombinoscopy (CAT), thereby determining the endogenous thrombin potential (ETP), peak height, and the lag time, in plasma obtained from healthy volunteers. Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.5 pM TF or higher, as demonstrated by unaltered ETP and lag time values between analyses with and without CTI. Addition of CTI before blood collection reduced thrombin generation triggered with 0.5 pM TF: both the ETP and peak height were significantly reduced compared to no CTI addition. In contrast, thrombin generation remained unaltered at a 1 pM TF trigger or above. This study demonstrates that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.5 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring thrombin generation at TF concentrations of ≥1 pM.

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

Blood ◽

10.1182/blood.v126.23.1085.1085 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1085-1085

Author(s):

Florian Langer ◽

Minna Voigtländer ◽

Katharina Holstein ◽

Brigitte Spath ◽

Walter Fiedler ◽

...

Keyword(s):

Plasma Levels ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Antigen Expression ◽

Compensatory Mechanism ◽

Bleeding Tendency ◽

Fibrin Deposition ◽

Significant Difference ◽

Support Research

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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Thrombin Generation Test Can Predict Bleeding Tendency In Patients With Severe Factor XI Deficiency

Blood ◽

10.1182/blood.v122.21.3600.3600 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3600-3600 ◽

Cited By ~ 2

Author(s):

Tami Livnat ◽

Uriel Martinowitz ◽

Rachel Mansharov ◽

Zivelin Ariella ◽

Ophira Salomon

Keyword(s):

Thrombin Generation ◽

Prophylactic Treatment ◽

Conflicts Of Interest ◽

Peak Height ◽

Activity Level ◽

Bleeding Tendency ◽

Bleeding Disorders ◽

Normal Range ◽

Factor Xi ◽

Factor Xi Deficiency

Abstract Introduction Factor XI (FXI) is a rare bleeding disorder defined as severe deficiency when FXI activity level is less than 20IU/dL. Unlike hemophilia A or B, patients with severe FXI deficiency do not bleed spontaneously and their bleeding tendency is unpredictable and poorly correlated with FXI level. Therefore, almost all patients with severe FXI deficiency are being treated similarly unrelated to their inert bleeding tendency. Lately there is a growing interest in introducing global coagulation tests to assess the risk of bleeding in trauma patients as well as in patients with congenital bleeding disorders. Thrombin generation (TG) test is a global assay that can provide information regarding hemostasis in healthy individuals or in patients with congenital and acquired bleeding disorders. Our group had previously shown that recalcification induced TG is a useful tool to determine the optimal dose of recombinant factor VIIa for patients with severe FXI deficiency and inhibitors going through major surgery (Livnat et al. Thromb Haemost 2009). Aim In the present study we aimed to characterize the capability of TG to serve as an ideal tool to define upfront bleeders and non-bleeders among FXI deficient patients and find the optimal conditions of TG that could distinguish between bleeders and non-bleeders thus eventually leading to efficient personalized treatment. Methods Case control study composed of 16 unrelated patients with FXI levels range >1-8dL-1and 14 healthy controls. For TG assay blood was taken from all participants simultaneously in both buffered citrate and corn trypsin inhibitor (CTI) tubes after obtaining informed consent. TG was performed in platelet poor plasma (PPP) in the presence of 4 µM phospholipids and initiated by recalcification in the presence and absence of 1pM tissue factor (TF). Three TG parameters were analyzed: lag time, thrombin peak and endogenous thrombin potential (ETP). Results Table 1 summarizes FXI activity, FXI genotype, thrombin peak height and bleeding status (i.e, bleeding following challenges when prophylactic treatment was not given) of patients in the study group. As expected, FXI levels poorly correlated with bleeding tendency. Good correlation between FXI levels, bleeding tendency and TG peak height was found when blood was taken in citrated tubes and not in CTI containing tubes. While the normal range of peak height in recalcification-induced TG (without TF) was 421±161 nM, no TG was initiated with recalcification in PPP of FXI patients with less than 1%. FXI levels 2-4% were sufficient to induce TG with recalcification but thrombin peak height was remarkable lower in comparison to controls. In FXI levels above 5%, the thrombin peak height induced by recalcification varied between low to normal range. Interestingly, when TG was initiated by 1pM TF the TG peak of non-bleeders reached normal values (normal peak height in the presence of 1pM TF=411±121), while in the bleeders the peak was reduced unrelated to FXI levels (range 74-205). Conclusions In summary TG induced by recalcification in the presence of low TF but not when performed in CTI tubes may efficiently distinguish between bleeders and non-bleeders in FXI deficient patients going through major trauma unrelated to patients' FXI level. This observation permits to consider less aggressive prophylactic treatment to patients with reduced risk for bleeding thus lowering the risk of thrombosis due to over treatment. Disclosures: No relevant conflicts of interest to declare.

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Tissue Factor Pathway Inhibitor Mediates Attenuation of Tissue Factor-Stimulated Thrombin Generation in Early Onset Preeclampsia

Blood ◽

10.1182/blood.v128.22.1431.1431 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1431-1431

Author(s):

Karl Egan ◽

Hugh O'Connor ◽

Barry Kevane ◽

Fergal Malone ◽

Amani Al Zadjali ◽

...

Keyword(s):

Tissue Factor ◽

Early Onset ◽

Research Funding ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Inflammatory Disorder ◽

Contact Activation ◽

Thrombotic Risk ◽

Tissue Factor Pathway ◽

Early Onset Preeclampsia

Abstract Introduction Pregnancy increases the risk of venous thromboembolism (VTE) in women. Interestingly, preeclampsia, an extremely pro-inflammatory disorder specific to pregnancy is associated with a lower than expected increase in thrombotic risk compared to other proinflammatory disorders. The mechanism underling this lower thrombotic risk is unknown. The aim of this study was to investigate the coagulation balance, a major determinant of VTE risk, in early onset preeclampsia (EOP) patients. EOP is the most inflammatory form of preeclampsia, characterised by the development of hypertension and proteinuria prior to 34 weeks gestation. Methods Platelet-poor plasma was collected from patients with early onset preeclampsia (EOP n=26), matched pregnant (n=20) and non pregnant controls (n=16). Calibrated automated thrombography, an assay of thrombin generation and TFPI assays were performed. Data are expressed as mean ± standard deviation. Results During the study period, 15,299 women delivered and 40 patients developed EOP, of whom 26 were successfully recruited with consent. For comparison, 16 non pregnant controls and 20 pregnant controls were also recruited. When artefactual contact activation was inhibited by using corn trypsin inhibitor as anticoagulant, EOP patients were characterised by a decrease in the rate and extent of Tissue Factor (TF) thrombin generation compared to pregnant controls. There was a prolongation of the time to peak thrombin generation (16 ± 4 minutes vs. 13 ± 2 minutes, p < 0.05), a decrease in the velocity index (25 ± 17nM/minute vs. 41 ± 27nM/minute, p < 0.05), and a decrease in peak thrombin generation (150 ± 80 nM IIa vs. 210 ± 70 nM IIa, p < 0.05 ) in EOP compared to pregnant controls. This reduction in the rate and extent of thrombin generation was most amplified in patients with severe EOP (multi-organ involvement) compared with moderate EOP. This lower overall procoagulant state seen was further emphasised by the increase in sensitivity to the anticoagulant activity of exogenously added activated protein C and thrombomodulin observed in EOP. Again, the increased sensitivity to APC and thrombomodulin was most apparent in severe EOP cases. Previous studies have shown that preeclampsia is characterised by a increase in plasma TFPI activity. As such, we investigated whether increases in plasma TFPI activity explained the reduction in TF-dependent thrombin generation. Consistent with previous studies, plasma tissue factor pathway inhibitor (TFPI) levels and plasma TFPI activity significantly increased in EOP, most notably in severe EOP cases. There was a significant inverse correlation between total TFPI levels and peak thrombin generation (r2 = -0.64, p < 0.05) and TFPI activity and peak thrombin generation (r2= -0.52, p < 0.05). The inhibition of TFPI with a polyclonal anti-TFPI antibody abolished the attenuation in thrombin generation seen in severe EOP. Conclusion In conclusion, TF-dependent thrombin generation is reduced in patients with early onset preeclampsia due to increases in plasma TFPI activity. These findings may partially explain the lower thrombosis risk observed in patients with early onset preeclampsia relative to comparable systemic proinflammatory conditions. These data also have future potential in helping clinicians to manage competing bleeding and thrombotic risks in these very high-risk patients. Disclosures Maguire: Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

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Two Factor XI Concentrates Correct Impaired Thrombin Generation in Major FXI Deficiency but Are Not Equivalent in Their Effect

Blood ◽

10.1182/blood.v124.21.1519.1519 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1519-1519

Author(s):

Gillian N Pike ◽

Anthony M Cumming ◽

Charles R.M Hay ◽

Brenda Sempasa ◽

Megan Sutherland ◽

...

Keyword(s):

Research Funding ◽

Thrombin Generation ◽

Reference Range ◽

Ex Vivo ◽

Peak Height ◽

Factor Xi ◽

Time To Peak ◽

Post Infusion

Abstract Introduction: Factor XI (FXI) deficiency is an autosomally inherited bleeding disorder characterised by an increased risk of excessive bleeding following trauma or surgery. However, considerable phenotypic heterogeneity of the bleeding tendency is observed between individuals with this disorder. Treatment options for patients requiring FXI replacement are fresh frozen plasma (preferably pathogen-inactivated) or FXI concentrate. Two FXI concentrates are available: Hemoleven® ( LFB Biomedicaments, Les Ulis, France) and Factor XI Concentrate (Bio-Products Laboratory (BPL), Elstree, UK). Guidelines previously recommended a maximum replacement dose of 30 U/Kg for both concentrates but more recently a lower dose of 10-15 U/kg has been advised. The two FXI concentrates may reduce bleeding risk in FIX deficiency following surgery but indications for their use are unclear and treatment in some cases has been associated with thrombosis. Aims: To quantify thrombin generation in major FXI deficiency (FXI:C <15 IU/dL) and to compare the in vitro effects of both FXI concentrates on thrombin generation parameters in this population with each other and with reference range values. To assess the clinical relevance of in vitro results through comparison of thrombin generation following in vitro and in vivo FXI replacement in individuals requiring surgery. Methods: Thrombin generation (TG) was measured in controls (n=50), in individuals with FXI deficiency ( FXI < 15 IU/dL) pre and post in vitro spiking with both FXI concentrates (n=10), and in ex vivo samples following treatment with BPL FXI concentrate (n=3). Blood was drawn into S-Monovette® tubes (Sarstedt, Leicester, U.K.) containing 0.106 M trisodium citrate (1:9, V:V) and corn trypsin inhibitor (CTI) (Haematologic Technologies Inc., Essex Junction, VT, U.S.A.), at a concentration of 20 µg/mL whole blood. Thrombin generation was measured in platelet rich plasma using the Calibrated Automated Thrombography method with a tissue factor trigger of 0.5pM. Statistical analysis was performed using GraphPad prism, version 6 software package (GraphPad, san Diego, CA, USA) using student’s t-test, Mann-Whitney U test or a Wilcoxon signed rank test according to data distribution. P value <0.05 was considered significant. Results: Major FXI deficiency (FXI:C <15 IU/dL) was associated with significantly impaired TG compared to controls, demonstrating reduced endogenous thrombin potential (ETP), peak height and velocity (all p<0.0001) and prolonged time to peak (p = 0.021). All TG parameters significantly improved from baseline with FXI replacement with both concentrates in vitro (equivalent in vivo dose 10 U/Kg). Comparison of the two FXI concentrates demonstrated that LFB Hemoleven® had greater effect on TG than BPL FXI in vitro at all doses (equivalent in vivo doses 10, 20 and 30 U/Kg): higher ETP (p < 0.0001), peak height (p < 0.01) velocity (p < 0.0002) and shorter lag time and time to peak (both p < 0.003). However, some measurements with LFB Hemoleven® exceeded the reference range. At lower doses both FXI concentrates normalised TG parameters in vitro (equivalent in vivo dose 2.5 IU/Kg LFB Hemoleven® or 5 U/Kg BPL FXI). Three patients received in vivo treatment with BPL FXI concentrate prior to surgery. TG was compared between baseline, in vitro spiked and post infusion ex vivo samples. Comparable ETP and peak height results were obtained from in vitro spiked and post infusion ex vivo samples. Conclusions: Individuals with FXI:C levels <15 IU/dL show impaired thrombin generation. Both FXI concentrates improve thrombin generation in vitro in patients with FXI deficiency. However, when tested in vitro with the TG assay, the concentrates differ in potency and dose response and for both concentrates, doses lower than present recommendations normalised thrombin generation. Comparison of in vitro spiked and ex vivo samples suggest that in vitro results could be used to estimate an expected in vivo response to FXI replacement for the BPL product. Acknowledgments: This work is supported by a Fellowship Project Award from the Bayer Hemophilia Awards Program, an unrestricted grant from LFB Biotechnologies and a Wycherley Fellowship grant. Hemoleven® concentrate was kindly provided by LFB Biotechnologies. Disclosures Pike: Bayer: Honoraria, Research Funding; LFB Biotechnologies: Honoraria, Research Funding. Bolton-Maggs:Bio-Products Laboratory (U.K.): Consultancy; LFB Biotechnologies: Consultancy.

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