Evaluation of thrombin generation assay in factor XI deficiency

AbstractThe prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to β-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by β-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, β-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.

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Blood Coagulation in Hemophilia A and Hemophilia C

Blood ◽

10.1182/blood.v91.12.4581 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4581-4592 ◽

Cited By ~ 144

Author(s):

Kevin M. Cawthern ◽

Cornelis van ‘t Veer ◽

Jennifer B. Lock ◽

Maria E. DiLorenzo ◽

Richard F. Branda ◽

...

Keyword(s):

Factor Viii ◽

Platelet Activation ◽

Thrombin Generation ◽

Hemophilia A ◽

Recombinant Factor Viii ◽

Normal Blood ◽

Factor V ◽

Fibrinopeptide A ◽

Factor Xi ◽

Factor Xi Deficiency

Abstract Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed (∼37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantly delayed clot formation only at sufficiently low TF concentrations. However, even at these low TF concentrations, significant thrombin is generated in the propagation phase after formation of the initial clot in hemophilia C blood.

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Monitoring low dose recombinant factor VIIa therapy in patients with severe factor XI deficiency undergoing surgery

Thrombosis and Haemostasis ◽

10.1160/th10-12-0816 ◽

2011 ◽

Vol 106 (09) ◽

pp. 521-527 ◽

Cited By ~ 29

Author(s):

Anne Riddell ◽

Rezan Abdul-Kadir ◽

Debra Pollard ◽

Edward Tuddenham ◽

Keith Gomez

Keyword(s):

Thrombin Generation ◽

Low Dose ◽

Recombinant Factor Viia ◽

Factor Viia ◽

Venous Blood ◽

Optimal Dose ◽

Factor Xi ◽

Factor Xi Deficiency

SummaryAlthough factor XI (FXI) concentrate is an effective replacement therapy in severe FXI deficiency without inhibitors, some patients are unwilling to receive it because it is plasma-derived. We report on the use and monitoring of low dose, recombinant factor VIIa (rFVIIa, NovoSeven®), to cover surgery (caesarean section, cholecystectomy and abdominoplasty) in four female patients (FXI:C 2–4 IU/dl, aged 32–51 years) who wished to avoid exposure to plasma. None of our patients had inhibitors to FXI. Our aim was to find the optimal dose of rFVIIa by in vitro spiking of patient samples and to correlate this with the response to rFVIIa in vivo. Prior to surgery, venous blood was collected into sodium citrate with corn trypsin inhibitor and spiked with 0.25–1.0 μg/ml rFVIIa in vitro, equivalent to a 15–70 μg/kg dose of rFVIIa in vivo. Analysis using thromboelastometry and thrombin generation assays, triggered with tissue factor, showed that the thrombin generation assay was insufficiently sensitive to the haemostatic defect in these patients. A concentration of 0.5 μg/ml was as effective as 1.0 μg/ml FVIIa in normalising thromboelastometry in vitro in all four patients. Therefore, patients received 15–30 μg/kg rFVIIa at 2–4 hourly intervals with tranexamic acid 1g every six hours. Post treatment samples were taken at 10–240 minutes and showed initial normalisation of thromboelastometry with gradual return to baseline after 2–4 hours. In conclusion, low-dose rFVIIa therapy was successfully used in four patients with severe FXI deficiency undergoing surgery to prevent bleeding and can be monitored using thromboelastometry.

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Blood Coagulation in Hemophilia A and Hemophilia C

Blood ◽

10.1182/blood.v91.12.4581.412k12_4581_4592 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4581-4592 ◽

Cited By ~ 2

Author(s):

Kevin M. Cawthern ◽

Cornelis van ‘t Veer ◽

Jennifer B. Lock ◽

Maria E. DiLorenzo ◽

Richard F. Branda ◽

...

Keyword(s):

Factor Viii ◽

Platelet Activation ◽

Thrombin Generation ◽

Hemophilia A ◽

Recombinant Factor Viii ◽

Normal Blood ◽

Factor V ◽

Fibrinopeptide A ◽

Factor Xi ◽

Factor Xi Deficiency

Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed (∼37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantly delayed clot formation only at sufficiently low TF concentrations. However, even at these low TF concentrations, significant thrombin is generated in the propagation phase after formation of the initial clot in hemophilia C blood.

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Elevated factor VIII enhances thrombin generation in the presence of factor VIII-deficiency, factor XI-deficiency or fondaparinux

Thrombosis Research ◽

10.1016/j.thromres.2010.10.017 ◽

2011 ◽

Vol 127 (2) ◽

pp. 135-140 ◽

Cited By ~ 8

Author(s):

Fania Szlam ◽

Gautam Sreeram ◽

Cristina Solomon ◽

Jerrold H. Levy ◽

Ross J. Molinaro ◽

...

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Factor Xi ◽

Factor Xi Deficiency ◽

Factor Viii Deficiency

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Thrombin generation in patients with factor XI deficiency and clinical bleeding risk

Haemophilia ◽

10.1111/j.1365-2516.2010.02246.x ◽

2010 ◽

Vol 16 (5) ◽

pp. 771-777 ◽

Cited By ~ 48

Author(s):

L. RUGERI ◽

F. QUÉLIN ◽

B. CHATARD ◽

P. DE MAZANCOURT ◽

C. NEGRIER ◽

...

Keyword(s):

Thrombin Generation ◽

Bleeding Risk ◽

Factor Xi ◽

Factor Xi Deficiency

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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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Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657717 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1215-1220 ◽

Cited By ~ 25

Author(s):

D Prasa ◽

L Svendsen ◽

J Stürzebecher

Keyword(s):

Thrombin Generation ◽

Factor Xa ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Thrombin Generation Assay ◽

Ic50 Values ◽

Fxa Inhibitor ◽

20 Nm ◽

Tick Anticoagulant Peptide ◽

Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

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Acquired Factor XI Inhibitors in Two Patients with Hereditary Factor XI Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661104 ◽

1984 ◽

Vol 51 (03) ◽

pp. 371-375 ◽

Cited By ~ 10

Author(s):

Kangathevy Morgan ◽

Sandra Schiffman ◽

Donald Feinstein

Keyword(s):

Protein A ◽

Lambda Light Chain ◽

Factor Xi ◽

Spontaneous Bleeding ◽

Factor Xi Deficiency ◽

Hereditary Factor ◽

Coagulation Mechanism ◽

Factor Xia ◽

Polyclonal Igg ◽

Glass Surfaces

SummaryTwo patients with hereditary factor XI deficiency developed inhibitors following plasma transfusions. Neither had severe spontaneous bleeding. The patients’ plasmas neutralized both factor XI in plasma, purified factor XI, and purified factor XIa. The inhibitor in both patients’ plasmas adsorbed to Protein A- Sepharose. The inhibitors eluted from Protein A-Sepharose were partially neutralized by kappa and lambda light chain antisera indicating that they were polyclonal IgG antibodies. Both inhibitors markedly decreased adsorption of factor XI to glass surfaces. The cleavage of factor XI by trypsin was unaffected by the inhibitors. The lack of severe spontaneous bleeding in both of these patients strongly suggests that an alternate coagulation mechanism bypassing factor XI must compensate for this severe defect.

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