scholarly journals Can We Measure the Individual Prothrombotic or Prohemorrhagic Tendency by Global Coagulation Tests?

2020 ◽  
Vol 40 (03) ◽  
pp. 364-378
Author(s):  
Sara Reda ◽  
Laure Morimont ◽  
Jonathan Douxfils ◽  
Heiko Rühl
Keyword(s):  
Anticoagulant Therapy ◽  
Thrombin Generation ◽  
Waveform Analysis ◽  
Laboratory Diagnostics ◽  
Coagulation Assays ◽  
Coagulation Process ◽  
Complex Process ◽  
Coagulation Tests ◽  
The Individual

AbstractHemostasis is a complex process in which abnormalities can cause shifts toward prothrombotic or prohemorrhagic states resulting in thrombosis or bleeding, respectively. Several coagulation tests may be required to characterize these defects but may yet not always reflect a patient's true hemostatic capacity. Thus, global coagulation tests aiming to simulate the coagulation process in vitro instead of measuring single components thereof are certainly of interest to assess prothrombotic or prohemorrhagic tendencies. This review describes the development and application of global coagulation tests, concentrating on the more widely used methods of viscoelastometry and thrombin generation. A focus is placed on conditions characterized by simultaneous changes of various components of hemostasis, such as anticoagulant therapy or hormone-induced coagulopathy, in which global coagulation tests are especially promising. If the key challenges of standardization and automation of these tests are solved, as is the case with automated thrombogram or clot waveform analysis, global coagulation assays will play an important role in the future of laboratory diagnostics of hemostasis and thrombosis.

scholarly journals Diagnostic Challenges in Newborns and Infants with Coagulation Disorders

2020 ◽  
Vol 40 (01) ◽  
pp. 084-087
Author(s):  
Wolfgang Eberl
Keyword(s):  
Children And Adolescents ◽  
Small Sample ◽  
Laboratory Diagnostics ◽  
Coagulation Disorders ◽  
Small Children ◽  
Preanalytical Phase ◽  
Coagulation Tests ◽  
Strategy Performance ◽  
Performance And Evaluation ◽  
The Individual

AbstractLaboratory diagnostics in children and adolescents, especially in newborns and very small children, differ considerably compared with laboratory diagnostics in adults. This applies to all individual steps of the examination (i.e., the indication, the preanalytical phase, the analysis itself, and interpretation of the findings). This is particularly true in the diagnostics of hemostasis, in which small sample volumes and relatively error prone coagulation tests are posing particular challenges to the strategy, performance, and evaluation of the tests. Differences in the individual steps are illustrated below.

Global Coagulation Assays in Transgender Women on Oral and Transdermal Estradiol Therapy

2020 ◽  
Vol 105 (7) ◽  
pp. e2369-e2377
Author(s):  
Hui Yin Lim ◽  
Shalem Y Leemaqz ◽  
Niloufar Torkamani ◽  
Mathis Grossmann ◽  
Jeffrey D Zajac ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Maximum Amplitude ◽  
Transgender Women ◽  
Cross Sectional ◽  
Platelet Poor Plasma ◽  
Coagulation Assays ◽  
Coagulation Tests ◽  
Transdermal Estradiol ◽  
Fibrinolytic Potential ◽  
General Community

Abstract Context The thrombotic effects of estradiol therapy in transgender women are unclear. Global coagulation assays (GCA) may be better measures of hemostatic function compared with standard coagulation tests. Objective To assess the GCA profiles of transgender women in comparison to cisgender controls and to compare how GCA differ between routes of estradiol therapy in transgender women. Design Cross-sectional case-control study. Setting General community. Participants Transgender women, cisgender male and cisgender female controls. Main outcome measures Citrated blood samples were analyzed for (i) whole blood thromboelastography (TEG®5000), (ii) platelet-poor plasma thrombin generation (calibrated automated thrombogram); and (iii) platelet-poor plasma fibrin generation (overall hemostatic potential assay). Mean difference (95% confidence intervals) between groups are presented. Results Twenty-six transgender women (16 oral estradiol, 10 transdermal estradiol) were compared with 98 cisgender women and 55 cisgender men. There were no differences in serum estradiol concentration (P = 0.929) and duration of therapy (P = 0.496) between formulations. Transgender women demonstrated hypercoagulable parameters on both thromboelastography (maximum amplitude + 6.94 mm (3.55, 10.33); P < 0.001) and thrombin generation (endogenous thrombin potential + 192.62 nM.min (38.33, 326.91); P = 0.009; peak thrombin + 38.10 nM (2.27, 73.94); P = 0.034) but had increased overall fibrinolytic potential (+4.89% (0.52, 9.25); P = 0.024) compared with cisgender men. No significant changes were observed relative to cisgender women. Route of estradiol delivery or duration of use did not influence the GCA parameters. Conclusion Transgender women on estradiol therapy demonstrated hypercoagulable GCA parameters compared with cisgender men with a shift towards cisgender female parameters. Route of estradiol delivery did not influence the GCA parameters.

In Vitro Effect of Danaparoid Sodium (Orgaran®) on Thrombin Generation after Minimal Tissue Factor Pathway Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4151-4151
Author(s):  
Ismail Elalamy ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Grigoris T. Gerotziafas
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Dependent Manner ◽  
Pathway Activation ◽  
Coagulation Tests ◽  
Vitro Effect ◽  
Routine Coagulation

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

scholarly journals Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Blood ◽  
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.

scholarly journals Sufficient Thrombin Generation Despite 95% Hemodilution: An In Vitro Experimental Study

2020 ◽  
Vol 9 (12) ◽  
pp. 3805
Author(s):  
Johannes Gratz ◽  
Christoph J. Schlimp ◽  
Markus Honickel ◽  
Nadine Hochhausen ◽  
Herbert Schöchl ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Prothrombin Complex Concentrate ◽  
Oral Anticoagulants ◽  
Coagulation Factor ◽  
Severe Bleeding ◽  
Crystalloid Solution ◽  
Coagulation Tests ◽  
Coagulation Factor Concentrates ◽  
Dilution Level

Guidelines for the treatment of severe bleeding comprise viscoelastic-test-guided use of coagulation factor concentrates as part of their recommendations. The aim of this study is to investigate the effects of substituting fibrinogen, prothrombin complex concentrate, and a combination of both on conventional coagulation tests, viscoelastic test results, and thrombin generation. Blood was drawn from seven healthy volunteers to obtain platelet-free plasma, which later was diluted by replacing 40%, 60%, 80%, 90%, 95%, and 99% with a crystalloid solution. The diluted samples were spiked with fibrinogen concentrate, prothrombin complex concentrate, a combination of both, or a corresponding amount of crystalloid solution. Up to a dilution level of 95%, viscoelastically determined clotting time was significantly shorter in the group substituted with fibrinogen only in comparison with the additional use of prothrombin complex concentrate. Clot firmness and endogenous thrombin potential remained at relatively stable values up to a dilution level of 95% with the substitution of fibrinogen but not prothrombin complex concentrate. Substitution of prothrombin complex concentrate led to an excessive overshoot of thrombin generation. The results of our study question currently propagated treatment algorithms for bleeding patients that include the use of prothrombin complex concentrate for patients without former intake of oral anticoagulants. Even in severely bleeding patients, thrombin generation might be sufficient to achieve adequate hemostasis.

Interference of the new oral anticoagulant dabigatran with frequently used coagulation tests

2012 ◽  
Vol 50 (9) ◽  
Author(s):  
Walter-Michael Halbmayer ◽  
Guenter Weigel ◽  
Peter Quehenberger ◽  
Josef Tomasits ◽  
Alexander C. Haushofer ◽  
...  
Keyword(s):  
Oral Anticoagulant ◽  
Dabigatran Etexilate ◽  
The Elderly ◽  
Multicenter Trial ◽  
Plasma Samples ◽  
Activity Assay ◽  
Coagulation Assays ◽  
New Oral Anticoagulant ◽  
Coagulation Tests

AbstractDabigatran etexilate is a new oral anticoagulant for the therapy and prophylaxis of venous thromboembolism and stroke prevention in patients with atrial fibrillation. To investigate the extent of interactions of this new anticoagulant with frequently used coagulation assays, we completed a multicenter in vitro trial with Conformité Européenne(CE)-labeled dabigatran-spiked plasma samples.Lyophilized plasma samples with dabigatran concentrations ranging from 0.00 to 0.48 μg/mL were sent to the coagulation laboratories of six major Austrian hospitals for evaluation. Coagulation assays were performed under routine conditions using standard reagents and analyzer.Dabigatran led to a dose-dependent prolongation of the clotting times in coagulometric tests and influenced the majority of the parameters measured. Statistically significant interference could be observed with the prothrombin time (PT), activated partial thromboplastin time (aPTT) and PT/aPTT-based assays (extrinsic/intrinsic factors, APC-resistance test) as well as lupus anticoagulant testing. Even non-clotting tests, such as the colorimetric factor XIII activity assay and to a minor extent the amidolytic antithrombin activity assay (via factor IIa) were affected.This multicenter trial confirms and also adds to existing data, demonstrating that laboratories should expect to observe strong interferences of coagulation tests with increasing concentrations of dabigatran. This finding might become particularly important in the elderly and in patients with renal impairment as well as patients whose blood is drawn at peak levels of dabigatran.

The Effects Of An Anticoagulant Phospholipase A2 On The Procoagulant Activity Of Phospholipids And Platelets

1981 ◽  
Author(s):  
A C Cox
Keyword(s):  
Phospholipase A2 ◽  
Thrombin Generation ◽  
Coagulation Factors ◽  
Procoagulant Activity ◽  
Activation Process ◽  
Blood Coagulation Tests ◽  
Normal Platelet ◽  
Coagulation Tests ◽  
Lag Period

Although phospholipids readily substitute for platelets in many in vitro blood coagulation tests, their participation in normal platelet procoagulant activity is uncertain. Phospholipase A2 (PLA2) from Naja nigricollis nigricollis venom, a known anticoagulant, blocked the enhancement of the rate of prothrombin conversion to thrombin by both phospholipids and platelets. The rate of inhibition of the phospholipid procoagulant activity by PLA2 was reduced by indomethacin, an inhibitor of PLA2. At concentrations of PLA2 which inhibited phospholipid procoagulant activity, conversion of purified prothrombin by factors Xa and Va without phospholipid was unaffected.Unactivated, washed platelets combined with coagulation factors II, Va and Xa initially produce thrombin at a slow rate before the platelets become activated but after this lag period the rate of thrombin generation increases. PLA2 added at the same time as the coagulation factors increased the lag period and decreased the rate of thrombin generation during and after the lag. Preincubation of platelets with PLA2 further decreased only the lag rate and this inhibition was partially blocked by 300 uM indomethacin. At a concentration of about one ng/ml, PLA2 reduced the post-lag thrombin generation rate of 3 × 10-9 platelets/ml in half but had no effect on platelet aggregation induced by thrombin, ADP or collagen.These results combined with the known specificity of PLA2 support the theory that phospholipids are involved in platelet procoagulant activity. Furthermore, the ineffectiveness of preincubating PLA2 with platelets on the post-lag procoagulant activity suggests that the phospholipids involved in this post-activation process become accessible during the lag period.

scholarly journals Coagulation Proteins Influencing Global Coagulation Assays in Cirrhosis: Hypercoagulability in Cirrhosis Assessed by Thrombomodulin-Induced Thrombin Generation Assay

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Nam Youngwon ◽  
Ji-Eun Kim ◽  
Hae Sook Lim ◽  
Kyou-Sup Han ◽  
Hyun Kyung Kim
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Lag Time ◽  
Activated Partial Thromboplastin Time ◽  
Coagulation Assays ◽  
Thrombin Generation Assay ◽  
Coagulation Tests ◽  
Coagulation Proteins ◽  
Cirrhotic Patients ◽  
Coagulation Status

Background. Liver disease is accompanied by profound hemostatic disturbances. We investigated the influences of pro- and anticoagulation factors on global coagulation tests including prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin generation assay (TGA) in cirrhosis. We also investigated whether cirrhotic patients exhibit hypo- or hypercoagulability using the TGA.Methods. The TGA was performed on a calibrated automated thrombogram, given lag time, endogenous thrombin potential (ETP), and peak thrombin in 156 cirrhotic patients and 73 controls.Results. PT was determined according to the factor (F) II, FV, FVII, FIX, and protein C levels. We observed that aPTT was dependent on FII, FIX, and FX levels. The ETP was dependent on FII, antithrombin, and protein C with 5 pM tissue factor (TF) stimulation, and FIX and protein C at 1 pM TF. The ETP ratio with 1 pM TF increased significantly in cirrhosis, indicating hypercoagulability, whereas that with 5 pM TF did not increase in cirrhosis.Conclusion. PT and the TGA are sensitive to protein C levels. Even with prolonged PT, the TGA can detect hypercoagulability in cirrhosis. Further studies should evaluate global coagulation status in cirrhosis patients using the newly devised TGA system.

Distinct features of bivalent direct thrombin inhibitors, hirudin and bivalirudin, revealed by clot waveform analysis and enzyme kinetics in coagulation assays

2019 ◽  
Vol 72 (12) ◽  
pp. 817-824 ◽  
Author(s):  
Masatoshi Wakui ◽  
Yuta Fujimori ◽  
Shoko Nakamura ◽  
Yoshino Kondo ◽  
Yuko Kuroda ◽  
...  
Keyword(s):  
Enzyme Kinetics ◽  
Waveform Analysis ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Asymptotic Line ◽  
Coagulation Assays ◽  
Drug Concentrations ◽  
The Hill ◽  
Dose Dependent

AimsBivalent direct thrombin inhibitors (DTIs), hirudin and bivalirudin, bind to the active site and exosite 1 of thrombin irreversibly and reversibly, respectively. The present study aims to assess in vitro effects of hirudin and bivalirudin through clot waveform analysis (CWA) and enzyme kinetics in coagulation assays.MethodsThe pooled normal plasma and its dilutions were spiked with hirudin or bivalirudin. The activated partial thromboplastin time (APTT) assay and the Clauss fibrinogen assay were performed using the CS-5100 (Sysmex). The APTT-CWA data were automatically gained by the CS-5100 programme.ResultsIn APTT-CWA, the maximum coagulation velocity, acceleration and deceleration were decreased dependently on the drug concentrations, demonstrating evidence for the blockade of thrombin-positive feedback by hirudin or bivalirudin. The Hill plot analysis was applied to the dose-dependent curves in bivalirudin. The Hill coefficients were greater than 1, showing positive anticoagulant cooperativity. Regarding the dose-dependent curves in hirudin, all the parameters dropped to almost zero without making an asymptotic line. In the Clauss fibrinogen assay, the Lineweaver-Burk plots demonstrated that both drugs exhibit mixed inhibition mimicking uncompetitive binding. The Dixon plots in bivalirudin were linear and supported the inhibition type described above. The Dixon plots in hirudin were non-linear and inappropriate to use for determination of the inhibition type. In addition, the inverse function of the clotting time appeared to drop to zero without making an asymptotic line, suggesting complete loss of thrombin activity by irreversible binding.ConclusionsThe results provide insights into anticoagulation with bivalent DTIs.

Monitoring of FVIII Replacement Therapy with Cell-Based Monitoring Tests.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3098-3098
Author(s):  
Steffen Bassus ◽  
Wolfgang Wegert ◽  
Manuela Krause ◽  
Inge Scharrer ◽  
Thomas Scholz ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Coagulation Factors ◽  
One Stage ◽  
Interindividual Variations ◽  
Fviii Activity ◽  
Before And After ◽  
The Individual

Abstract Although FVIII replacement therapy is clinically safe and effective, there is still a debate about the individually sufficient efficacy of treatment. With routinely performed one-stage and chromogenic assays individual haemostatic effects of FVIII therapy are poorly detectable due to artificial test conditions, e.g. strong dilution and adding contact activators, phospholipids or activated coagulation factors to patients plasma. Moreover, the crucial relevance of platelets for coagulation characteristics can not be investigated. For our study cell-based coagulation assays (thrombin generation in platelet rich plasma and thrombelastography in whole blood) as well as an analytic strategy were adapted to make them suitable for measuring individual haemostatic effects of FVIII therapy. 40 patients (age 7–79) with haemophilia A replacements therapies were investigated. We measured FVIII activity resp. efficacy before and after substitution with one-stage, chromogenic, thrombin generation and thrombelastography assay. 2/3 of patients received a recombinant, 1/3 of patients a plasma-derived concentrate. The results showed an average FVIII activity, measured with chromogenic and one-stage assays, of 8% before and 76% after substitution of an average of 27,3 U/kg body weight. Thrombin generation and clot firmness showed almost maximum values for relatively low FVIIIactivities (20 to 30%). Moreover, the results of the cell-based tests showed large interindividual variations in pre-substitution residual activities and in haemostatic effects after substitution for same FVIII plasma activities. This indicates the need for different interindividual FVIII activities for achieving sufficient thrombin activity and clot firmness. Therefore it seems to be advantageous to measure patients FVIII efficacy profiles before treatment and use them as their own standard to assess FVIII efficacy after treatment instead of using standard curves from pooled plasma. According to this we measured thrombin generation and clot firmness before treatment. Than we added in vitro one unit FVIII to the same sample to determine patients individual maximum of thrombin generation and clot firmness (100%). After FVIII replacement therapy we determined the same parameters and calculated the relative haemostatic effect of FVIII therapy compared to in vitro results in relative per cent of the maximum FVIII efficacy. With thrombin generation and thrombelastography assays and our revised strategy of analysis we are able to determine the individual haemostatic effect of FVIII replacement therapy and, moreover, to predict the required FVIII-amount for successful treatment with pre-substitutional in vitro spiking of patients’ own blood.

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