Abstract P683: Thrombin Generation in Plasma of Stroke Patients With Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Sarina Falcione ◽  
Gina Sykes ◽  
Joseph Kamtchum Tatuene ◽  
Danielle Munsterman ◽  
Twinkle Joy ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Ischemic Stroke ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Lag Time ◽  
Thrombin Time ◽  
Stroke Patients ◽  
Time To Peak ◽  
Generation Capacity

Background and Purpose: Thrombus formation is central to pathophysiology of stroke in patients with atrial fibrillation. Whether factors in plasma contribute to thrombus generation in patients with atrial fibrillation remains unclear. In this study we sought to determine whether plasma contributes to thrombin generation in patients with atrial fibrillation. Methods: There were 78 acute ischemic strokes with atrial fibrillation and 37 non-stroke controls. Plasma thrombin generation was measured by thrombin generation assay, resulting lag time, peak thrombin, time to peak and area under the curve was assessed. Thrombin generation capacity was compared in stroke patients with atrial fibrillation to non-stroke controls. The relationship to anticoagulation was assessed. In vitro, the effect of anticoagulation on plasma thrombin generation was determined. Results: Thrombin generation capacity was increased (shorter lag time and time to peak) in ischemic stroke patients with atrial fibrillation compared to non-stroke atrial-fibrillation controls (p<0.05 and p<0.01, respectively). Anticoagulation decreased plasma induced thrombin generation. Ischemic stroke patients with atrial fibrillation treated with anticoagulation (DOAC or warfarin) had lower plasma induced thrombin generation compared to atrial-fibrillation patients not on anticoagulation (p<0.05). Thrombin generation by plasma could be further reduced by DOAC in an in-vitro assay. Conclusions: Stroke patients with atrial fibrillation have a higher plasma induced thrombin generation compared to atrial fibrillation controls. Factors in plasma such as leukocyte derived tissue factor likely contribute to thrombus formation in patients with atrial fibrillation. As such, components in plasma may represent new targets to reduce thrombus formation and stroke risk in patients with atrial fibrillation.

Factor VIII Supplementation Improves Recombinant VIIa Initiated Thrombin Generation in Hemophilia A Inhibitor Patient Plasmas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 28-28
Author(s):  
Bhavya Doshi ◽  
Courtney Cox ◽  
Bagirath Gangadharan ◽  
Christopher B Doering ◽  
Shannon L. Meeks
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Treatment Options ◽  
Lag Time ◽  
Time To Peak ◽  
Fviii Inhibitor

Abstract Abstract 28 Hemophilia A is an X-linked recessive disorder that is caused by a deficiency or defect of factor VIII (fVIII) coagulant protein. Approximately 20–30% of patients with severe hemophilia A develop antibodies (Abs) against fVIII (inhibitors) following fVIII replacement therapy, which makes bleeding episodes more difficult to control. Patients with inhibitors are treated with fVIII-bypassing agents such as recombinant factor VIIa (rfVIIa) or activated prothrombin-complex concentrate. However for unknown reasons, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Thrombin generation assays provide an in vitro methodology for monitoring bypass therapy in hemophilia (Turecek PL et al. Pathophysiol Haemost Thromb 2003; Varadi K et al. Haemophilia 2004). Recently, it was demonstrated by us and others that combination of fVIII and by-passing agents potentiates in vitro thrombin production in hemophilia A inhibitor plasma (Klintman J et al. Br J Haematol 2010). In our study we investigated the potentiation fVIII confers to fVIIa initiated in vitro thrombin generation using a panel of anti-fVIII Abs with known epitopes. We showed that kinetics of inhibition and Ab epitope were the dominant factors influencing ability of fVIII to potentiate in vitro thrombin production. Specifically, monoclonal Abs targeting only 2 of 11 epitopes, 1 of 3 non-overlapping A2 epitopes and 1 of 2 non-overlapping C2 epitopes, inhibited thrombin generation in a manner that could not be recovered by fVIII supplementation. Here, we analyzed in vitro thrombin generation in epitope-mapped plasmas from 10 patients with hemophilia A and long-standing inhibitors after addition of fVIIa alone or in conjunction with fVIII. Methods: FVIII inhibitor plasmas from 10 patients with hemophilia A were obtained as part of an IRB approved study at the Emory Comprehensive Hemophilia Center. FVIII inhibitor titers and inhibitor kinetics were determined using a modified Bethesda assay. Samples were classified as having type II inhibitors if undiluted plasma resulted in incomplete inhibition of residual fVIII activity (Meeks SL et al. Blood 2007). Thrombin generation assays were carried out in the presence of 2.25 μg/ml recombinant fVIIa in the presence or absence of 1 U/ml recombinant full-length fVIII using reagents purchased from DiaPharma (West Chester, OH). The parameters analyzed include endogenous thrombin potential (area under thrombin generation curve), peak thrombin concentration, time to peak thrombin, lag time (time to 1/6th of peak thrombin) and index velocity (Vi-peak thrombin divided by time to peak minus lag time). Domain specific epitope mapping was carried out using direct ELISA and human/porcine domain hybrid fVIII proteins. Results: Domain mapping of the Abs in the plasmas identified 2 plasmas with predominantly anti-A2 Abs, 4 with predominantly anti-C2 Abs, 2 with both anti-A2 and anti-C2 Abs, and 2 with antibodies that were porcine fVIII cross-reactive (see Table). Plasmas with inhibitor titers less than 25 BU/ml were more responsive to fVIII supplementation with 6 of 7 having increased thrombin generation. Plasmas harboring even trace anti-A2 Abs were more resistant to increased thrombin generation with fVIII supplementation than plasmas with anti-C2 Abs alone. Conclusion: This study suggests a more favorable response to fVIII supplementation of rfVIIa may be predicted by the presence of anti-C2 Abs or inhibitory titers less than 25 BU/ml. In conjunction with our previous monoclonal Ab data, further mapping of epitopes within the fVIII A2 and C2 domains may help improve the ability to predict positive responses to fVIII supplementation of by-passing agents.PatientInhibitor Titer (BU/ml)DomainFVIII InhibitorThrombin Generation (fVIII + fVIIa vs. fVIIa)122A2Type IIIncreased242A2Type IIEqual384C2, small A2Type IEqual47C2Type IIncreased58C2Type IIIncreased620C2Type IEqual78C2Type IIncreased842C2, small A2Type IEqual922Porcine cross-reactiveType IIIncreased105.2Porcine cross-reactiveType IIncreased Disclosures: No relevant conflicts of interest to declare.

Abstract T MP20: A Rapid Clot Lysis Assay to Predict Response to IV rt-PA in Stroke

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Irina Y Sazonova ◽  
Francesco Janes ◽  
Jennifer L Waller ◽  
Julia E Brittain ◽  
Gian Luigi Gigli ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Clinical Outcome ◽  
Clinical Response ◽  
Clot Lysis ◽  
Thrombin Time ◽  
Stroke Patients ◽  
Favorable Clinical Response ◽  
Pai 1 ◽  
D Dimer

Background: The cornerstone of acute stroke therapy is intravenous rt-PA, with the goal of rapid thrombolysis and restoration of blood flow. Unfortunately, less than half of patients treated have a favorable outcome. Since recanalization is closely correlated with the clinical response, endogenous resistance to rt-PA may inhibit thrombolysis and neurologic improvement. The primary objective of this research study was to evaluate whether an in vitro clot lysis assay and thrombolytic biomarkers could predict response to IV rt-PA in acute ischemic stroke patients Methods: Ischemic stroke patients who received IV rt-PA were recruited from two sites (Augusta, GA and Udine, Italy). Blood samples were collected prior to rt-PA administration and a favorable clinical response to rt-PA was specified as an NIHSS score of two or less at 24 hours. The time required for 50% platelet-poor clot lysis (C50%) was measured by an in vitro turbidimetric assay. The endogenous major fibrinolytic inhibitors (tissue plasminogen inhibitor, PAI-1; and α2-antiplasmin, α2-AP), D-dimer, fibrinogen and pro-thrombin time (PT), were assessed and correlated with clinical outcome. Results: We enrolled 54 rt-PA recipients (63±15 years; 72% male, and 85% white). The mean NIHSS was 11.2±5.1 at admission and 8.2±8.1 at 24 hours; 36% of patients had a favorable clinical response. There was a moderate reduction in C50% among patients with a favorable clinical outcome compared to those with an unfavorable outcome (669.5±46.3 vs. 704.7±91.6; p=0.075). There was no significant difference in PT (13.6±0.8 sec vs. 13.8±1.2 sec; p=0.444), PAI-1(28.2±34.1 pg/mL vs. 19.8±25.9 pg/mL; p=0.321), α2-AP (85.1±7.2 % vs. 88.8±10.1 %; p=0.153), D-dimer (269.5±232.2 ng/mL vs. 368.2±444.6 ng/mL; p=0.303) or fibrinogen (318.1±71.4 mg/dL vs. 343.0±173.1 mg/dL; p=0.465) levels between subjects with favorable and unfavorable response to rt-PA. Summary: Lack of response to IV rt-PA in stroke patients may reflect endogenous resistance to fibrinolysis reflected by prolonged clot lysis in vitro. The utility of clot lysis to predict rt-PA unresponsiveness should be tested in a larger clinical study.

scholarly journals The in vitro addition of idarucizumab to plasma samples from patients increases thrombin generation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mojca Božič Mijovski ◽  
Rickard E. Malmström ◽  
Nina Vene ◽  
Jovan P. Antovic ◽  
Alenka Mavri
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Lag Time ◽  
Plasma Samples ◽  
Blood Samples ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Coagulation Tests ◽  
Before And After

AbstractDabigatran interferes with many coagulation tests. To overcome this obstacle the use of idarucizumab as an in vitro antidote to dabigatran has been proposed. The aim of this study was to test the effect of idarucizumab as an in vitro antidote to dabigatran in ex vivo plasma samples from routine clinical patients examined by a thrombin generation assay (TGA). From 44 patients with atrial fibrillation five blood samples were collected. Thrombin generation was measured in all samples before and after the addition of idarucizumab. When idarucizumab was added to baseline plasma (no dabigatran), it caused a significantly shorter Lag Time and Time to Peak Thrombin, and a higher Peak Thrombin and Endogenous Thrombin Potential (ETP) of TGA. Similar results were obtained when idarucizumab was added to dabigatran-containing plasma, with TGA parameters comparable to baseline + idarucizumab plasma, but not to baseline plasma. In summary, our study showed that in vitro addition of idarucizumab to plasma samples from patients increases thrombin generation. The use of idarucizumab to neutralize dabigatran in patient plasma samples as well as the clinical relevance of in vitro increased thrombin generation induced by idarucizumab needs further investigation.

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.

scholarly journals Large extracellular vesicles in the left atrial appendage in patients with atrial fibrillation—the missing link?

2021 ◽  
Author(s):  
Andreas Zietzer ◽  
Baravan Al-Kassou ◽  
Paul Jamme ◽  
Verena Rolfes ◽  
Eva Steffen ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Extracellular Vesicles ◽  
Left Atrial ◽  
Left Atrial Appendage ◽  
Thrombus Formation ◽  
Flow Cytometric Analysis ◽  
Heart Rhythm ◽  
Atrial Appendage ◽  
Peripheral Embolization

AbstractAtrial fibrillation (AF) is the most frequent arrhythmic disease in humans, which leads to thrombus formation in the left atrial appendage and stroke through peripheral embolization. Depending on their origin, large extracellular vesicles (lEVs) can exert pro-coagulant functions. In the present study, we investigated how different types of AF influence the levels of large EV subtypes in three distinct atrial localizations. Blood samples were collected from the right and left atrium and the left atrial appendage of 58 patients. 49% of the patients had permanent AF, 34% had non-permanent AF, and 17% had no history of AF. Flow cytometric analysis of the origin of the lEVs showed that the proportion of platelet-derived lEVs in the left atrial appendage was significantly higher in permanent AF patients compared to non-permanent AF. When we grouped patients according to their current heart rhythm, we also detected significantly higher levels of platelet-derived lEVs in the left atrial appendage (LAA) in patients with atrial fibrillation. In vitro studies revealed, that platelet activation with lipopolysaccharide (LPS) leads to higher levels of miR-222-3p and miR-223-3p in platelet-derived lEVs. Treatment with lEVs from LPS- or thrombin-activated platelets reduces the migration of endothelial cells in vitro. These results suggest that permanent atrial fibrillation is associated with increased levels of platelet-derived lEVs in the LAA, which are potentially involved in LAA thrombus formation.

scholarly journals Incorporation of α2-Plasmin Inhibitor into Fibrin Clots and Its Association with the Clinical Outcome of Acute Ischemic Stroke Patients

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 347
Author(s):  
Zsuzsa Bagoly ◽  
Barbara Baráth ◽  
Rita Orbán-Kálmándi ◽  
István Szegedi ◽  
Réka Bogáti ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Intracranial Hemorrhage ◽  
Intravenous Thrombolysis ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Stroke Patients ◽  
Plasmin Inhibitor ◽  
Fibrin Clots

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.

scholarly journals Heparin is more effective than apixaban in inhibiting in vitro contact activated coagulation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M.M Engelen ◽  
C Van Laer ◽  
M Jacquemin ◽  
C Vandenbriele ◽  
K Peerlinck ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Heart Valves ◽  
Thrombus Formation ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Mechanical Heart Valves ◽  
Contact Activation

Abstract Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

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.

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