Faculty Opinions recommendation of Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test - Demonstrated in vitro and ex vivo.

2017 ◽  
Author(s):  
Coen Hemker
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Generation Test

The Influence of Infusions of 1-Desamino-8-D-Arginine Vasopressin (DDAVP) In Vivo on Thrombin Generation In Vitro

1992 ◽  
Vol 68 (01) ◽  
pp. 037-039 ◽  
Author(s):  
S H Ibbotson ◽  
J A Davies ◽  
P J Grant
Keyword(s):  
Arginine Vasopressin ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Occlusive Disease ◽  
Lag Phase ◽  
Computer Assisted ◽  
Chromogenic Substrate ◽  
Generation Test

SummaryTo investigate the effect of increasing FVIII:C in-vivo on coagulation ex-vivo, DDAVP was infused over 15 min in 10 volunteers and in-vitro thrombin generation measured. FVIII:C rose from 0.42 and 0.43 IU/ml before DDAVP to 1.38, 1.73 and 1.78 IU/ml at 15, 30 and 60 min respectively (p <0.001).A computer-assisted thrombin generation test was performed in defibrinated plasma using chromogenic substrate, S2238. Time to reach 50% maximal thrombin activity (T50/s) and lag phase of thrombin generation (lag/s) were measured. Lag shortened from 75 and 60 s before to 45 s during and after infusion (p <0.001). T50 shortened from 78.5 and 76.0 to 62.5, 60.0 and 58.5 s at times 15 (p <0.01), 30 (p <0.001) and 60 (p <0.001) min. FVIII:C correlated inversely with lag and T50 (r = –0.847, p <0.001, r = –0.826, p <0.001, n = 10) respectively.These findings show that acute elevations of FVIII:C in-vivo accelerate in-vitro thrombin production. This work suggests that elevated FVIII:C levels in-vivo may be important in thrombo-occlusive disease.

scholarly journals Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test – Demonstrated in vitro and ex vivo

PLoS ONE ◽  
2017 ◽  
Vol 12 (4) ◽  
pp. e0175030 ◽  
Author(s):  
Daniel Elenius Madsen ◽  
Timothy C. Nichols ◽  
Elizabeth P. Merricks ◽  
Emily K. Waters ◽  
Bo Wiinberg
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Generation Test

Experimental Studies on a Recombinant Hirudin, CGP 39393

1991 ◽  
Vol 65 (04) ◽  
pp. 355-359 ◽  
Author(s):  
E Gray ◽  
J Watton ◽  
S Cesmeli ◽  
T W Barrowcliffe ◽  
D P Thomas
Keyword(s):  
Thrombin Generation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Experimental Studies ◽  
Venous Stasis ◽  
Antithrombotic Agent ◽  
Recombinant Hirudin ◽  
Excessive Bleeding ◽  
Generation Test

SummaryThe in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test : systems. In the APTT at concentrations below 5 μg/ml, r-hirudin showed a dose-response curye. At concentrations above 5 μg/ml, the plasma became unclottable, but in the thrombin generation test , at least 10 μg/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 μg/ml r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.

Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies

1997 ◽  
Vol 78 (04) ◽  
pp. 1173-1177 ◽  
Author(s):  
Jacek Musiał ◽  
Jakub Swadźba ◽  
Miłosz Jankowski ◽  
Marek Grzywacz ◽  
Stanisława Bazan-Socha ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Skin Incision ◽  
Anticardiolipin Antibodies ◽  
Small Blood Vessel ◽  
Anionic Phospholipids ◽  
Increased Risk ◽  
High Concentrations

SummaryAntiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and (β2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were signiF1cantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of F1brinopeptide A were detected already in the F1rst samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated signiF1cantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalciF1ed plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the F1rst time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4197-4205 ◽  
Author(s):  
J.M. Herbert ◽  
J.P. Hérault ◽  
A. Bernat ◽  
R.G.M. van Amsterdam ◽  
J.C. Lormeau ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Therapeutic Index ◽  
Inhibitory Effect ◽  
Experimental Models ◽  
Treatment And Prevention ◽  
Long Acting

Abstract SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.

scholarly journals Sulfated agaran with 4,6-pyruvated form from red seaweed Acanthophora muscoides attenuates thrombin formation: in vitro and ex vivo studies

2021 ◽  
Vol 43 ◽  
pp. e55043
Author(s):  
José Ariévilo Gurgel Rodrigues ◽  
Johnny Peter Macedo Feitosa ◽  
Sandra de Aguiar Soares ◽  
Norma Maria Barros Benevides
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Continuous Model ◽  
Sulfated Polysaccharides ◽  
Extrinsic Pathway ◽  
Plasma Fraction ◽  
Thrombin Formation

In vitro studies have described the sulfated agaran from Acanthophora muscoides as an intrinsic inhibitor of thrombin generation (TG), but not in ex vivo assay. This investigation partially characterized a pyruvate fraction with in vitro and ex vivo effects on an intrinsic/extrinsic pathway-induced thrombin generation (TG) continuous model using 36 or 60-fold diluted mice or defibrinated, normal human plasma. Fraction separated by DEAE-cellulose chromatography exhibited charge homogeneity and non-sulfated polysaccharides (<100 kDa) by agarose and polyacrylamide gel electrophoresis, respectively, using Stains-all alone. Fourier Transform Infrared and Nuclear Magnetic Resonance studies indicated a 4,6-pyruvated agaran-structure. The fraction and heparin had no effect on prothrombin time, but there was a preponderant intrinsic rather than extrinsic pathway inhibition in TG assay; themselves, acting on both free and fibrin bound thrombin activity without chromogenic substrate interaction. Both fractions, desulfated and native, anticipated and induced thrombin formation in activators-devoid or normal plasma. In addition, mice pretreated with fraction (20 mg kg-1, intraperitoneally) reduced intrinsically plasma TG ex vivo after 2h. Heparin suppressed TG in vitro, but induced it ex vivo. Therefore, agaran from A. muscoides blocks TG on in vitro and ex vivo studies, suggesting to evaluate the blood coagulability status.

Trace FVIII Augments Rfviia Induced Thrombin Generation and Improves Hemostasis in Hemophilia A Patients with Inhibitors: A clinical Cohort Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 40-40
Author(s):  
Tami Livnat ◽  
Uri Martinowitz ◽  
Shirley Azar-Avivi ◽  
Ariela Zivelin ◽  
Gili Kenet
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Therapy Response ◽  
Response To Therapy ◽  
Individual Therapy ◽  
Severe Bleeding ◽  
Inhibitor Level

Abstract Abstract 40 Treatment of Hemophilia A patients with inhibitors is challenging, as correlation between inhibitor level and hemostatic response to therapy may be limited. Thrombin generation (TG) assays may be used to monitor hemostasis and/or predict patients' response to various bypass agents. Since combination of excess FVIII and bypassing agents may potentiate improved TG in inhibitor plasma tested in-vitro, we aimed to define the therapeutic feasibility of co-administration of rFVIIa and FVIII in hemophilia A patients with inhibitors. Patients and Methods: Following consent, blood was sampled from 15 hemophilia patients (age: 0.5–46y) with inhibitor (0.5–668 BU). Platelet poor plasma (PPP) was prepared, spiked and incubated with excess FVIII. Ex-vivo kinetics of FVIII neutralization over time was evaluated by sequential measurements of residual FVIII activity. We then used recalcification induced-TG (performed in PPP supplemented with 4μM phospholipids), to measure the ex-vivo response to increasing concentrations of FVIII (0–200%) and rFVIIa (0–6.8μg/ml), alone or in combination. Based upon these ex-vivo studies, an individually tailored therapeutic regimen of concomitant bolus doses of rFVIIa and FVIII was applied to nine hemophilia patients with inhibitors. Results: FVIII ex- vivo measurements post incubation detected either rapid or slow neutralization- not correlating with inhibitor level. Flat baseline TG curves were recorded for all inhibitor patients, with variable responses to FVIII and/or rFVIIa. Combined spiking with FVIII and rFVIIa dramatically increased rFVIIa induced ETP (762.7 ±305.7 as compared to 339.3±179.9 nM/min with rFVIIa only) and peak height (48.7±23.6 vs 23.7±16.6) in all patients' plasma samples. Based upon individual ex vivo assays, concomitant bolus doses of rFVIIa (120–200 mcg/kg) and FVIII (50–100 U/Kg), were applied to 9 patients, for a total of 333 episodes during study period (February 2010-Septemeber 2012). Patients during immune tolerance received rFVIIa prophylaxis with combined rFVIIa/FVIII dosing applied 3 times weekly. For most mild- moderate joint bleeds hemostasis was defined as satisfactory following a single combined dose. Severe bleeding episodes or target joint bleeds responded to 2–8 (median:3) combined doses, applied every 12 hours. During study period the median number of spontaneous joint bleeds decreased from 4 to 1 per month. Neither thrombosis nor any other complications evolved. Conclusions: Prediction of individual therapy response may be achieved by pre-analytical studies, assessing FVIII neutralization kinetics as well as ex-vivo TG responses to combined bypass/FVIII therapy. Such studies enabled treatment of inhibitor patients according to individually tailored regimens. We confirmed for the first time that the in- vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved hemostasis and may safely be applied to inhibitor patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ex Vivo Evaluation of the Effect of Plasma-Derived Factor VIII/Von Willebrand Factor in Patients with Severe Hemophilia_A on Prophylaxis with Emicizumab By Thrombin Generation Assay

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4233-4233
Author(s):  
Maria-Isabel Bravo ◽  
Aida Raventós ◽  
Alba Pérez ◽  
Elena G Arias-Salgado ◽  
María Teresa Alvarez Román ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Research Funding ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Concomitant Treatment ◽  
Healthy Controls ◽  
Normal Range ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Introduction: Hemophilia A (HA) patients under emicizumab prophylaxis treatment may require the concomitant use of procoagulant factors for breakthrough bleedings or immune tolerance induction. Thromboembolic events have been described with the concomitant use of emicizumab and activated prothrombin complex concentrate (aPCC), but not with recombinant activated factor VII (rFVIIa). Previous studies showed that the in vitro combination of emicizumab and plasma-derived Factor VIII/Von Willebrand Factor (pdFVIII/VWF) had a non-additive effect on thrombin generation (TG)(Bravo M-I, et al J Thromb Haemost. 2020;18:1934-39). The aim of this study was to evaluate the TG resulting from ex vivo combination of plasma samples from HA patients treated with emicizumab, with a pdFVIII/VWF concentrate (Fanhdi ®, Grifols). Methods: Twelve adult patients with severe HA without inhibitors on prophylaxis with emicizumab and nine healthy controls were included in the study. Blood samples were drawn in citrate plus corn trypsin inhibitor tubes. Then, platelet poor plasma (PPP) was collected for the TG assay, which measures the whole kinetics of TG. Thrombin peak (TP) and endogenous thrombin potential (ETP) were calculated using calibrated automated thrombogram (Thrombinoscope ™ software, Stago) after in vitro activation of coagulation by trigger solution, PPP Reagent LOW TM (4 μM phospholipids/1 pM tissue factor), fluorogenic substrate and CaCl 2 (FLUKAkit TM) reagents (Diagnostica Stago). Fluorescence was read in a Fluoroskan Ascent reader (Thermo) equipped with a 390/460 filter set. Samples were spiked with increasing concentrations of pdFVIII/VWF (10 to 400 IU/dL), rFVIIa (0.9 µg/mL) or aPCC (0.5 U/mL). Results: TG from healthy control samples was measured to establish TP and ETP normal ranges. TP and ETP results obtained from HA plasma with emicizumab were lower than in healthy controls. The addition of pdFVIII/VWF as of 25 IU/kg (prophylaxis dose in HA w/o inhibitors) to samples from HA patients concomitantly treated with emicizumab restored TP and ETP levels within healthy controls normal range (Table 1). Increasing ex vivo concentrations of pdFVIII/VWF maintained TP and ETP similar to healthy controls. The highest concentration of concomitant treatment with pdFVIII/VWF (200 IU/kg) and emicizumab did not result in excessive TP and, importantly, ETP levels were always within the normal range. The combination with the bypassing agent rFVIIa moderately increased TP and ETP values up to normal range. However, when HA plasma was spiked with aPCC in the presence of emicizumab, TP and ETP dramatically increased above normal range resulting in a synergistic procoagulant profile. Conclusions: The concomitant use of pdFVIII/VWF in patients with prophylaxis with emicizumab did not trigger a multiplying effect on TG. These results were aligned with previous in vitro data and suggested the low risk of overdose and thrombotic events of concomitant treatment emicizumab with the pdFVIII/VWF concentrate in HA patients. Figure 1 Figure 1. Disclosures Bravo: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Raventós: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Pérez: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Bayer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Costa: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Willis: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®.

Evaluation of the In Vitro Detection of the Hypercoagulable State Using the Thrombin Generation Test and Plasma Clot Impedance Test

1979 ◽  
Vol 42 (02) ◽  
pp. 764-777 ◽  
Author(s):  
Sanford D Peck
Keyword(s):  
Hip Replacement ◽  
Thrombin Generation ◽  
Clinical Evidence ◽  
Clinical Signs ◽  
Rate Of Change ◽  
Hypercoagulable State ◽  
Plasma Clot ◽  
Over 40 Years ◽  
Generation Test

SummaryThis study reports the correlation of the thrombin generation test and the plasma clot impedance test with clinical evidence of hypercoagulability. Thrombin generation is increased and the rate of change of plasma from a liquid to a gel (clot impedance) is increased in situations where the risk of thrombosis is increased.These situations include increasing clinical signs and/or symptoms of thromboembolism, positive lung scans, postoperative total hip replacement, patients over 40 years old, low serum antithrombin III, thrombocytosis, transient cerebral ischemia, and positive isotope venogram for thrombosis. The two tests failed to indicate a significant effect of antiplatelet drugs on the hypercoagulable state.This study shows that the thrombin generation and plasma clot impedance tests are practical, rapid and useful tests for the detection and monitoring of the hypercoagulable state.

scholarly journals Antiplatelet drugs and generation of thrombin in clotting blood

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2006-2011 ◽  
Author(s):  
A Szczeklik ◽  
M Krzanowski ◽  
P Gora ◽  
J Radwan
Keyword(s):  
Oral Administration ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Coagulation Factors ◽  
Antiplatelet Drugs ◽  
Specific Marker ◽  
Rate Of Increase ◽  
Synthase Inhibitor ◽  
Thrombin Formation

Abstract Platelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro--in recalcified plasma-- and ex vivo--in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.

Sign in / Sign up

Export Citation Format

Share Document