The Influence of Lepirudin, Bivalirudin and Dabigatran on the Calibrated Automated Thrombogram (CAT)

Abstract In a two centre study (laboratories in Diagnostica Stago and Biomnis) we compared the in vitro effect on thrombin generation (TG) of Dabigatran and Bivalirudin (reversible direct anti-IIa inhibitors) with that of Lepirudin (an irreversible direct anti-IIa inhibitor) spiked into normal pool plasma. The effect of Lepirudin, Bivalirudin and Dabigatran were evaluated in both centres using the CAT (Diagnostica Stago, France) TG method in a concentration ranges up to 5, 20 and 1 μg/mL respectively. Testing was done in triplicate and repeated over 2 days. To reduce assay variability both centres used the same reagents lots and the same normal pool plasma (George King, USA). The range of each drug tested extended well above the therapeutic range concentrations normally found in patient plasma (0.5 to 1.0 μg/mL, 5 to 10 μg/mL and 0.1 to 0.3 μg/mL respectively for Lepirudin, Bivalirudin and Dabigatran). To see the effect of increasing activation forces, TG was performed at 3 different final concentrations of Tissue Factor (TF) - 1, 5 and 20 pM. All reagents were used as recommended by the manufacturer (Thrombinoscope, The Netherlands). A prolongation in the lag time (LT) is observed with all 3 drugs with all 3 concentrations of TF, but this is more marked for Lepirudin and Bivalirudin than it is for Dabigatran. In the therapeutic range Dabigatran (at 5pM TF) shows both an increase in LT and a decrease in peak thrombin and the ETP. At low concentration of Bivalirudin or Lepirudin, there is a paradoxical increase in peak height, which is even more pronounced at low TF concentration. At 1pM TF, this paradoxical peak increase is also observed with Dabigatran. Results obtained in both laboratories are similar and complement our previous results and those reported elsewhere (1–4). The effect of Lepirudin and Bivalirudin on TG is different from that of Dabigatran. We also note that at lower TF concentration the anticoagulant effect on TG initiation is more intense but the test becomes less reproducible.

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

Blood ◽

10.1182/blood.v106.11.4151.4151 ◽

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%

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

Thrombosis and Haemostasis ◽

10.1160/th09-03-0180 ◽

2009 ◽

Vol 102 (11) ◽

pp. 936-944 ◽

Cited By ~ 64

Author(s):

Kellie Machlus ◽

Emily Colby ◽

Jogin Wu ◽

Gary Koch ◽

Nigel Key ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Relative Sensitivity ◽

Epidemiological Studies ◽

Peak Height ◽

Clotting Factor ◽

Time To Peak ◽

High Throughput Method ◽

Assay Conditions

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

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The Rate of Prothrombin Conversion Is Increased in Antiphospholipid Syndrome and Is Insensitive to Thrombomodulin

Blood ◽

10.1182/blood.v128.22.718.718 ◽

2016 ◽

Vol 128 (22) ◽

pp. 718-718

Author(s):

Romy Kremers ◽

Stéphane Zuily ◽

Hilde Kelchtermans ◽

Tessa Peters ◽

Saartje Bloemen ◽

...

Keyword(s):

Tissue Factor ◽

Antiphospholipid Syndrome ◽

Conversion Rate ◽

Healthy Subjects ◽

Thrombin Generation ◽

Lag Time ◽

Peak Height ◽

Thrombin Inhibitors ◽

Time To Peak ◽

Glycoprotein I

Abstract Background: The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies directed mainly against prothrombin and β2-glycoprotein I. The syndrome is associated with an increased risk of thrombosis. The global hemostatic state in a patient can be tested by measuring thrombin generation (TG). Recently, we developed a method to study the main pro- and anticoagulant processes at the basis of TG, called the thrombin dynamics method. Aim: In this study we investigated the dynamics of thrombin generation in healthy subjects and APS patients. Materials and methods: Healthy subjects (n=129) and antiphospholipid syndrome (APS) patients (n=31) were included in the study. Sixty-eight percent of the APS patients were lupus anticoagulant positive, anti-cardiolipin antibodies were detected in 84% of the patients, and 52% presented with anti-β2-glycoprotein I antibodies. Patients on anticoagulant therapy were excluded from the study. Thrombin generation was measured at 1 pM tissue factor (TF) and activated protein C (APC) system sensitivity was tested by measuring TG in the presence and absence of 20 nM thrombomodulin (TM). Results: Thrombin generation was measured in platelet poor plasma at 1 pM tissue factor. The lag time and time-to-peak were significantly prolonged in APS patients compared to healthy subjects (lag time: 3.30 ± 0.59 vs. 6.69 ± 4.26 min, p<0.001; time-to-peak: 8.33 ± 1.29 vs. 10.76 ± 4.51 min, p<0.001). The peak height was significantly higher in APS patients (240 ± 84 vs. 214 ± 58 nM, p<0.05) and the velocity index was elevated in APS patients (134 ± 66 vs. 70 ± 32 nM/min, p<0.001) compared to healthy subjects. The ETP values were comparable between healthy subjects and APS patients (1260 ± 235 vs. 1176 ± 362 nM*min). The pro- and anticoagulant processes underlying thrombin generation were studied separately. The total amount of prothrombin converted during thrombin generation (PCtot) did not differ between healthy subjects and patients (1234 vs. 1165 nM). However, the maximum rate of prothrombin conversion (PCmax) was significantly elevated in APS patients (291 vs 425 nM/min; p<0.001). The amount of thrombin-antithrombin (T-AT) complexes formed was comparable between patients and controls (1169 vs. 1098 nM), and the thrombin decay capacity (TDC) was comparable as well (0.675 vs. 0.674 min-1). These results are in line with the finding that the plasma levels of the main thrombin inhibitors are unchanged in APS patients. Antithrombin levels are on average 2.31 ± 0.44 μM in healthy subjects and 2.36 ± 0.56 μM in APS patients, and the mean α2-macroglobulin levels were 3.22 ± 0.77 μM in healthy subjects and 3.23 ± 1.11 μM in patients. Thrombomodulin reduced the ETP by 45% in healthy subjects, but had significantly less effect in APS patients (10%). The addition of TM decreased total prothrombin conversion by 40% and the maximum prothrombin conversion rate by 50% in healthy subjects. In patients, TM only slightly reduced total prothrombin conversion (8%) and the maximum prothrombin conversion rate (7%). Discussion: The thrombin generation results indicate a predisposition to thrombosis in APS patients, as the TG parameters peak height and the velocity index are increased. Examination of the underlying pro- and anticoagulant processes of prothrombin conversion and thrombin inactivation revealed that although the same amount of prothrombin is converted in patients, the maximum activity of the prothrombinase complex is higher, indicating that patients can generate thrombin faster. In addition, APS patients have a dysfunctional APC system, as prothrombin conversion and thrombin generation could be only slightly inhibited by the addition of thrombomodulin. Disclosures No relevant conflicts of interest to declare.

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Reverting Rivaroxaban Effect By Activated Factor X: Assessment Using Thrombin Generation Assay

Blood ◽

10.1182/blood.v124.21.2868.2868 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2868-2868 ◽

Cited By ~ 1

Author(s):

Dominique Grenier ◽

Meyer Michel Samama ◽

Sami Chtourou ◽

Jean-Luc Plantier

Keyword(s):

Thrombin Generation ◽

Lag Time ◽

Peak Height ◽

Factor X ◽

Thrombin Generation Assay ◽

Lag Times ◽

Dose Dependent ◽

Control Plasma

Abstract Specific anti-activated factor X molecules are currently used for the prevention and the treatment of various thromboembolic disorders. However, despite a growing use of these molecules, they are still devoid of a reliable antidote. Rivaroxaban is a specific anticoagulant targeting activated factor X (FXa). Its potential in inhibiting FXa in vitro and in vivo was demonstrated during the characterization of the molecule. However, the use of FXa to revert the effect of Rivaroxaban in plasma was never studied. To do so the measurement of thrombin generation (TG) using the calibrated automatic thrombinoscope was performed. The ability of purified human FXa (Haematologic Technologies at 10, 50, 100, 500 and 1000 ng/ml) to induce TG in a platelet-poor plasma (PPP) without the induction of the coagulation was first evaluated. There was a FXa dose-dependent TG. The TG profile at concentrations up to 50 ng/ml of FXa was similar than the control profile obtained by a PPP activated by tissue-factor (0.5 pM) and phospholipids. Above 50 ng/ml FXa, the lag time decreased and the endogeneous thrombin potential (ETP) increased with the dose. This pattern revealed the thrombogenic potential of FXa and demonstrated that a dose of 50 ng/ml (or ≈1 nM) FXa was the maximum safer dose identified by this assay. A similar experiment was performed following the activation of plasma with 0.5 pM Tissue-Factor (TF) and 4 µM phospholipids (PL) and adding FXa at 31, 62, 125, 250 and 500 ng/ml. The kinetics of TG in the presence of the different amounts of FXa differed less than when coagulation was not induced. The lag times varies from 3 to 1.83 min with the increasing concentrations of FXa and the peak heights from 120 to 212 nM, being the two most affected parameters. Following the addition of 62 ng/ml (or ≈1.25 nM) FXa, the TG was more effective than a control plasma identically stimulated. Rivaroxaban was then spiked in the PPP at the therapeutic dose of 0.35 µg/ml (or 0.8 µM). Following 0.5 pM TF/4 µM PL stimulation, this dosage completely inhibits the TG. Increasing doses of FXa (31, 62, 125, 250 and 500 ng/ml) were then added and dose-dependently restores the TG. All the parameters of the TG profile were affected by the presence of FXa. The normalization was attained at the dose of 250 ng/ml (or 5 nM) FXa. A similar set of experiment was repeated by activating the plasma with cephalin, used as a model to mimic the initiation of the contact phase coagulation. The pattern of TG was different than following FT/PL activation. With cephalin and for all FXa concentrations identical peak aspects (velocity, ETP and peak height) were obtained differing only by their lag times and times-to-peak. Lag times and times to peak were shortened by the addition of FXa from 10.7 to 3.7 min and 13.2 to 6 min respectively. Plasma were then spiked by Rivaroxaban (0.35 µg/ml) and activated by cephalin in the presence of various concentrations of FXa (31, 62, 125, 250 and 500 ng/ml). A dose-dependent TG was demonstrated with the ETP, the peak height and the velocity increasing with the amount of FXa spiked whereas the lag time and time to peak were shortened. Following the induction by cephalin, the presence of FXa systematically shortened the TG when Rivaroxaban was present or not, when compared to the TG from control plasma. This work aimed to establish the antidote potential of the natural substrate of the anti-Xa molecules and limiting the risk in promoting a thrombotic response. The calibrated thrombin generation assay was used to determine the in vitro efficiency of FXa to induce a normal thrombin generation without primary induction or following an induction by TF/PL or cephalin. The doses of FXa required to normalize coagulation in the presence of Rivaroxaban and following induction were identified. These conditions will now be assessed in vivo in Rivaroxaban treated-mice. In addition of establishing the antidote properties of FXa, this data paved the way to compare its capacities, which are optimal to inhibit such inhibitor, to further antidote in development. Disclosures Grenier: LFB BIotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.

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A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Blood ◽

10.1182/blood-2018-99-119831 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3779-3779

Author(s):

Swapnil Rakhe ◽

Sheryl Bowley ◽

John E. Murphy ◽

Debra D Pittman

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Prothrombin Complex Concentrate ◽

Hemophilia A ◽

Lag Time ◽

Hemophilia B ◽

Activated Prothrombin Complex Concentrate ◽

Tissue Factor Pathway

Abstract Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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Evaluation of the Antithrombin Potential in a Thrombin Generation Assay Performed at the Surface of Endothelial Cells

Blood ◽

10.1182/blood.v124.21.4168.4168 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4168-4168

Author(s):

Béatrice Catieau ◽

Sami Chtourou ◽

Jean-Luc Plantier

Keyword(s):

Endothelial Cells ◽

Thrombin Generation ◽

Peak Height ◽

Factor Ix ◽

Flat Bottom ◽

Thrombin Generation Assay ◽

Heparan Sulfates ◽

Vitro Effect ◽

Control Plasma

Abstract Thrombin generation assay (TGA) was recently evaluated on a living endothelial-derived cell line (Coll et al. J. Thromb. Haemost. 2013; 11, 1916). This innovative assay brought into an hemostasis assay the cellular components of the anticoagulation pathway (APC and TFPI pathways) as well as a activated cell surface. It might help elucidate the relationship between hemostasis and inflammation in a more complex system. In the aim of evaluating the potential of antithrombin (AT) connecting both processes we set-up a similar assay on human vein endothelial cells (HUVEC). We first demonstrated that thrombin generation can be measured in flat-bottom 96-wells in factor IX-or factor VIII-deficient plasma substituted by either 0.1 or 1 U/ml of FIX or FVIII, respectively. Next, HUVEC were grown and expanded in a complete commercial medium (EndoGRO-LS, Millipore) for no more than 6 passages. Wells were then coated with gelatin 1% and cells seeded at 10,000 cells/well. The binding of plasma-derived AT (Aclotine ®, LFB; France dialyzed in cell culture medium) to HUVEC was demonstrated as being dose- (0.5; 1; 2.5 and 5 U/ml) and time- (0-6 hours) dependent. Saturating conditions were found using 2.5 U/ml AT for a 2h incubation. We also showed that the binding was moderately affected in the presence of heparin at concentrations up to 50 U/ml (loss of 19% of the signal) and not at all following an heparanase I+II+III treatment suggesting that another receptor(s) than cellular heparan sulfates being responsible for this interaction. The effect of AT on coagulation was then compared in the presence of cells or not. To do this cells were grown to confluence, washed with non-supplemented medium and incubated in the presence of the TGA mix (plasma containing AT or not, 0.5 pM Tissue Factor, 4 µM Phospholipids). The reaction was initiated by injection of the FluCa kit thrombin substrate (Stago). In the presence of HUVEC, the efficiency of thrombin generation from a control plasma (Unicalibrator, Stago) was decreased with a lag time increased (from 5.67 min to 6.83 min), the peak height diminished from 204.4 nM thrombin to 150.4 nM and the velocity from 55.8 nM/min to 33.4 nM/min. However, the overall amount of thrombin generated was less affected, diminishing from 1515.5 nM to 1482 nM. These data confirms that the presence of the HUVEC anticoagulants pathways can effectively diminish the thrombin generation. Without cells, the presence of 0.5, 1 or 2 U/ml AT dose-dependently decreased the generation of thrombin from the control plasma. The velocity was decreased by 23.2%, 57.6% and 75.5% and the peak height by 33.5%, 61.5% and 78.8%, respectively. When the same experiment was performed in the presence of HUVEC cells, the concentrations of AT similarly decreased the velocity by 34.2%, 54% and 70 % and the peak height by 39%, 59.1% 74.3%, respectively. There was no difference in the TGA parameters if AT was pre-incubated at the surface of the cells for up to 2h prior the TGA or if it was added extemporaneously. These results indicate that the presence of HUVEC did not modulate the in vitro effect of AT during coagulation. The effect of AT on the cell response during this process are in the process of being investigated with a particular focus on the anti-inflammatory properties of AT. Disclosures Catieau: LFB Biotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.

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Evidence of Different Subspecies of Microparticles (MP) Derived from Red Blood Cells (RMP) and Comparison of Their Phenotypes and Procoagulant Activity.

Blood ◽

10.1182/blood.v108.11.1763.1763 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1763-1763

Author(s):

Wenche Jy ◽

Jaehoon Bang ◽

Loreta Bidot ◽

Andrew Lin ◽

Joaquin J. Jimenez ◽

...

Keyword(s):

High Speed ◽

Thrombin Generation ◽

Calcium Ionophore ◽

Lag Time ◽

Peak Height ◽

Calcium Ionophore A23187 ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Differential Centrifugation

Abstract BACKGROUND: The potential roles of cell derived microparticles (MP) such as those derived from platelets (PMP), endothelium (EMP), leukocytes (LMP), and red cells (RMP) have been receiving increasing attention in disorders of hemostasis/thrombosis and inflammation and they are emerging as valuable biomarkers. However among these MP, little is known about RMP. Our recent clinical studies indicate that RMP play a role in hemostasis and thrombosis in patients with thrombocytopenia and in thrombocytosis. However, the phenotypes and procoagulant activity of their subspecies remain unknown. We report evidence for heterogeneity of RMP following differential centrifugation. METHODS: RMP were prepared by exposure of washed RBC to the calcium ionophore, A23187, and the RBC were removed by low-speed centrifugation. The RMP were washed twice at 20,000xg for 15 min. Procoagulant activity of RMP was measured by the calibrated automated thrombogram (CAT) system (Hemker et al Pathophysiol Haemost Thromb.2002;32:249) using thrombin substrate Z-Gly-Gly-Arg-AMC on a fluorescence plate reader. The lag time and peak height (nM) of thrombin generation were recorded. Markers used for labeling RMP were PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). RESULTS: In thrombin generation assay, RMP induced a long lag time (24±3 min) but high thrombin peak (330±37 nM). These data were consistent with the flow cytometric finding that RMP carried very little TF (<0.1%) but very high AnV binding (88±6%). By high speed centrifugation (15,000xg for 10 min), two populations of RMP were studied: the larger RMP in the pellet expressed GlyP, AnV and Ulex while the smaller or lighter RMP remaining in the supernatant, did not express GlyP and AnV but do express Ulex. The smaller RMP accounted for 30–40% of total Ulex+ RMP. These two subspecies (large and small) of RMP showed distinct thrombin generation profiles. The lag time and peak height of thrombin generation for large RMP (GlyP+/AnV+/Ulex+) was 23–28 min and 300–335 nM, respectively, which is close to values of whole RMP. On the other hand, the smaller RMP (Ulex+/GlyP−/AnV−) produced much longer lag time (31–38 min) and lower peak (60–75 nM), indicating that the majority of the procoagulant activity of RMP is associated with larger RMP. SUMMARY: The present study demonstrates that RMP are rich in anionic phospholipids and effective in generating thrombin in vitro. We have identified 2 distinct subpopulations of RMP by differential centrifugation: One larger RMP express binding of anti-GlyP, AnV and Ulex, and carry the majority of procoagulant activity. The smaller RMP expressing only Ulex binding exhibit much weaker procoagulant activity. The roles of these two species of RMP remain to be elucidated. We speculate that smaller RMP may represent the nanovesicles described by Allen et al [Biochem J 188:881, 1980] and that Ulex may be a novel and convenient means for the study of these small vesicles.

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Laboratory detection of the antiphospholipid syndrome via calibrated automated thrombography

Thrombosis and Haemostasis ◽

10.1160/th08-06-0393 ◽

2009 ◽

Vol 101 (01) ◽

pp. 185-196 ◽

Cited By ~ 29

Author(s):

Katrien Devreese ◽

Kathelijne Peerlinck ◽

Jef Arnout ◽

Marc Hoylaerts

Keyword(s):

Protein C ◽

Thrombin Generation ◽

Oral Anticoagulants ◽

Activated Protein C ◽

Lag Time ◽

Peak Height ◽

Time Ratio ◽

Plasma Pool ◽

Normal Plasma

SummaryLupus anticoagulants (LAC) consist of antiphospholipid antibodies, detected via their anticoagulant properties in vitro. Strong LAC relate to thromboembolic events, a hallmark of the anti-phospholipid syndrome. We have analyzed whether detection of this syndrome would benefit from thrombin generation measurements. Therefore, calibrated automated thrombography was done in normal plasma (n=30) and LAC patient plasma (n=48 non-anticoagulated, n=12 on oral anticoagulants), diluted 1:1 with a normal plasma pool. The anti-β2-glycoprotein I monoclonal antibody 23H9, with known LAC properties, delayed the lag time and reduced the peak height during thrombin generation induction in normal plasma dose-dependently (0–150 μg/ml). At variance, LAC patient 1:1 plasma mixtures manifested variable lag time prolongations and/or peak height reductions. Coupling these two most informative thrombin generation parameters in a peak height/lag time ratio, and upon normalization versus the normal plasma pool, this ratio distributed normally and was reduced in the plasma mixtures, for 59/60 known LAC plasmas. The normalized peak height/lag time ratio correlated well with the normalized dilute prothrombin time, diluted Russell’s viper venom time and silica clotting time, measured in 1:1 plasma mixtures (correlation coefficients 0.59–0.72). The anticoagulant effects of activated protein C (0–7.5 nM) or 23H9 (0–150 μg/ml), spiked in the 1:1 LAC plasma mixtures were reduced for the majority of patients, compatible with functional competition between patient LAC and activated protein C and LAC and 23H9, respectively. Hence, the normalized thrombin generation-derived peak height/lag time ratio identifies LAC in plasma with high sensitivity in a single assay, irrespective of the patient’s treatment with oral anticoagulants.

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

Thrombosis and Haemostasis ◽

10.1160/th08-10-0670 ◽

2009 ◽

Vol 101 (06) ◽

pp. 1156-1162 ◽

Cited By ~ 59

Author(s):

Arne Dielis ◽

Marina Panova-Noeva ◽

René van Oerle ◽

José Govers-Riemslag ◽

Karly Hamulyák ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Thrombin Generation ◽

Lag Time ◽

Peak Height ◽

Blood Collection ◽

Contact Activation ◽

In Vitro Measurements ◽

Analytical Variable ◽

Corn Trypsin Inhibitor

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

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