Tissue factor up-regulation in proinflammatory conditions confers thrombin generation capacity to endothelial colony-forming cells without influencing non-coagulant properties in vitro

Abstract Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

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The Impact of Factor IX Level on Thrombin Generation and Bleeding During Warfarin Anticoagulation

Blood ◽

10.1182/blood.v118.21.541.541 ◽

2011 ◽

Vol 118 (21) ◽

pp. 541-541

Author(s):

Yesim Dargaud ◽

Maureane Hoffman ◽

Claude Negrier ◽

Leana Lefrapper ◽

Dougald M. Monroe

Keyword(s):

Tissue Factor ◽

Vitamin K ◽

Intracranial Haemorrhage ◽

Thrombin Generation ◽

Factor Ix ◽

Target Range ◽

Significant Difference ◽

Generation Capacity ◽

The Impact

Abstract Abstract 541 Bleeding occurs in from 10 – 16% of warfarin-treated patients. Having a PT-INR in the target range is associated with better outcomes. However, even patients with an INR in the target range of 2–3 can suffer bleeding, suggesting that INR does not perfectly reflect the therapeutic effect of warfarin. The goal of our studies was to determine whether the level of specific coagulation factors could predict the risk of bleeding while the INR was in the target range. We modeled warfarin anticoagulation in our previously published in vitro cell based-model by adjusting the levels of vitamin K-dependent factors to those of patients with an INR of 2–3. We then examined the effect of variations in the level of FIX. The cogulation reactions were initiated by monocyte-expressed tissue factor (assayed at 1pM). Variation in FIX had a marked effect on thrombin generation. However, in plasma with the same levels of factors, as expected, variations in FIX had no effect on the PT-INR. Thus, we hypothesized that a subject with a lower FIX level than average may have a lower level of thrombin generation than is indicated by the INR. The INR might, therefore, underestimate the level of anticoagulation in such a subject. If s/he is maintained in the “therapeutic range” as measured by the INR, s/he will actually be over-anticoagulated and prone to hemorrhage. A prospective, single centre clinical study has been carried out to test this hypothesis in warfarinized patients. Between October 2010 and June 2011, 312 consecutive patients admitted to the emergency department of Edouard Herriot Hospital in Lyon, with an INR between 1.8 and 3.2, were included in the study after obtaining informed consent. Twenty six patients were admitted for a bleeding episode, 18 for recurrent thrombosis and 268 for other medical reasons. Patients presenting with bleeding, 17 males and 9 females, were aged 74±14 years old compared to the rest of the patients aged 76±14. Among the 26 bleeders, 7 had a spontaneous intracranial haemorrhage, 2 had a trauma-induced intracranial haemorrhage, 12 presented a gastrointestinal bleeding and 5 exhibited muscle hematomas, severe epistaxis or urinary tract bleeding. PT-INR and vitamin K-dependent factor levels were determined in all patients. Thrombin generation capacity in platelet poor plasma was measured using Calibrated Automated Thrombin generation assay (Thrombinoscope bv, Maastricht, The Netherlands), with tissue factor 1pM and phospholipids PC:PS:PE 4μM. No statistically significant difference was observed in the PT-INR of bleeding patients (INR=2.4±0.4) and those having a thrombosis (INR=2.5±0.5) or patients admitted for other reasons (INR=2.6±0.2). Plasma prothrombin and factor × levels were also similar in all three groups. However, a statistically lower plasma factor IX activity was observed in bleeders (p=0.01, Mann Whitney test) compared to other groups, 47.6±20 IU/dL vs. 63±33 IU/dL. In all the warfarinized subjects with an INR between 1.8 and 3.2, no correlation was found between thrombin generation capacity and PT-INR results (p=0.85, Spearman correlation test). However, a statistically significant correlation was observed between thrombin generation capacity and factor IX levels (p=0.0002). In patients, presenting with warfarin-related haemorrhage, the endogenous thrombin potential (ETP) was significantly lower at 340±335 nM.min (p=0.05) then that of warfarinized subjects who did not suffer bleeding (ETP 406±215 nM.min). These data support our hypothesis based on our in vitro results and show that patients who bleed when their PT-INR is in the target range 2 – 3 might have defective thrombin generation related to a lower level of factor IX than expected. Thus, our results suggest that the appropriate target INR level might not be the same for all patients. Those with factor IX levels that differ significantly from the mean of the population might be managed best by selecting a target INR that is based on the level of thrombin generation. Of course, a “target range” for parameters of thrombin generation during warfarin therapy would need to be developed if the assay were to be used for this purpose. Disclosures: No relevant conflicts of interest to declare.

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Effect of Argatroban versus Recombinant Hirudin on Tissue-Factor-Mediated Thrombin Generation: An in Vitro Study

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0028-1086087 ◽

2008 ◽

Vol 34 (S 01) ◽

pp. 087-090

Author(s):

Meyer Samama ◽

Léna Le Flem ◽

Céline Guinet ◽

François Depasse

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

In Vitro Study ◽

Vitro Study ◽

Recombinant Hirudin

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

Stroke ◽

10.1161/str.52.suppl_1.p683 ◽

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.

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The in Vitro Effect on Thrombin Generation of Adding rFVIII or rFIX to Hemophilia a or b Plasma in the Absence or Presence of Concizumab

Blood ◽

10.1182/blood-2020-140272 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Marianne Kjalke ◽

Søren Andersen

Keyword(s):

Drug Interaction ◽

Thrombin Generation ◽

Hemophilia A ◽

Plasma Samples ◽

Publicly Traded ◽

Plasma Pool ◽

Generation Capacity ◽

Drug Drug Interaction ◽

Vitro Effect

Introduction: Lack of factor VIII/IX (FVIII/FIX) in hemophilia A/B (HA/HB), respectively, results in reduced thrombin generation, leading to recurrent/spontaneous bleeds. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody, currently under clinical investigation for subcutaneous prophylaxis of HA/HB patients with/without inhibitors. Breakthrough bleeds occurring in HA/HB patients while on concizumab prophylaxis may be treated with FVIII/FIX. We aimed to compare the in vitro effect of recombinant FVIII (rFVIII) and FIX (rFIX) in HA and HB plasma, respectively, in the absence or presence of concizumab. Methods: rFVIII/rFIX was added to HA/HB pooled plasma at 0.25, 0.5 or 1 IU/mL (corresponding to post-administration plasma concentrations of 12.5, 25 and 50 IU/kg rFVIII and 12.5−25, 25−50 and 50−100 IU/kg rFIX) in the absence or presence of concizumab (1,500, 4,500 or 15,000 ng/mL). In a separate experiment, 33 plasma samples from eight HA patients, who were on concizumab prophylaxis as part of the phase 2 explorer5 trial (NCT03196297), were spiked with 0.5, 1 and 1.5 IU/mL rFVIII. Pre-dose samples (before concizumab prophylaxis) from seven of these patients were also included. Thrombin generation was measured after initiation with 1 pM tissue factor (PPP-Low, Thrombinoscope). Statistical analysis of the effects conferred by each (combination of) drug(s) was performed by ANOVA analyses. Results: A significant (p<0.001) and concentration-dependent increase in thrombin peak was observed when HA plasma pool samples were spiked with rFVIII, both in the absence and presence of concizumab. Likewise, concizumab increased the thrombin peak both in the absence and in presence of rFVIII. Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL only slightly increased the thrombin peak further, demonstrating that a close-to-maximal effect on thrombin peak was achieved at 1,500 ng/mL concizumab. The effects of concizumab and rFVIII were mainly additive with an up to 20% additional effect caused by drug-drug interaction. The addition of rFVIII to explorer5 patient plasma samples resulted in a significant and concentration-dependent increase in thrombin peak. The effects observed for rFVIII and concizumab were exclusively additive. The thrombin peak obtained with 1.0 IU/mL rFVIII before concizumab administration was lower than with 0.5 IU/mL rFVIII in the presence of concizumab. This suggests that a 2-fold reduced rFVIII dose may be sufficient to achieve the same plasma thrombin generation capacity as with the standard rFVIII dose in the absence of concizumab. The addition of rFIX to a HB plasma pool increased the thrombin peak significantly (p<0.001) and in a concentration-dependent manner both in the absence and presence of concizumab (1,500 ng/mL). Likewise, concizumab increased the thrombin peak at all rFIX concentrations (p<0.001). Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL had no or limited further effect. The effects of concizumab and rFIX were mainly additive with an up to 10% effect conferred by negative drug-drug interaction for 1 IU/mL rFIX combined with concizumab >1,500 ng/mL and 0.5 IU/mL rFIX combined with 15,000 ng/mL concizumab, i.e., a 10% smaller effect of rFIX was observed in the presence of concizumab than in its absence. The thrombin peak obtained upon adding 1.0 IU/mL rFIX to plasma without concizumab was similar to the thrombin peak in the presence of concizumab and 0.5 IU/mL rFIX. This suggests that in the presence of concizumab, a 2-fold reduced dose of rFIX would be sufficient to obtain the same plasma thrombin generation capacity as with 1.0 IU/mL rFIX in the absence of concizumab. Conclusion: rFVIII/rFIX increased the thrombin peak in HA and HB plasma, respectively, both in the absence and presence of concizumab. The combined effects of rFVIII/rFIX with concizumab were mainly additive with an up to 20% additional effect caused by drug-drug interaction with rFVIII and a 10% reduction with rFIX. No signs of exaggerated thrombin generation were observed by combining concizumab with rFVIII/rFIX. Therefore, the data support rFVIII/rFIX use for bleed treatment in patients on concizumab prophylaxis. As rFVIII/rFIX and concizumab have additive effects in terms of thrombin generation capacity, data suggest that clinical effectiveness could be achieved with rFVIII/rFIX doses in the lower range recommended for such products. Disclosures Kjalke: Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company. Andersen:Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company.

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In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

Thrombosis and Haemostasis ◽

10.1160/th09-10-0690 ◽

2010 ◽

Vol 104 (09) ◽

pp. 514-522 ◽

Cited By ~ 3

Author(s):

Thomas Lecompte ◽

Agnès Tournier ◽

Lise Morlon ◽

Monique Marchand-Arvier ◽

Claude Vigneron ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Time Course ◽

Anticoagulant Activity ◽

Cathepsin G ◽

Coagulation Cascade ◽

Clotting Factor ◽

Factor V ◽

Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

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Thrombin generation and fibrin clot formation under hypothermic conditions: An in vitro evaluation of tissue factor initiated whole blood coagulation

Journal of Critical Care ◽

10.1016/j.jcrc.2013.10.010 ◽

2014 ◽

Vol 29 (1) ◽

pp. 24-30 ◽

Cited By ~ 9

Author(s):

Matthew F. Whelihan ◽

Armin Kiankhooy ◽

Kathleen E. Brummel-Ziedins

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Whole Blood ◽

Thrombin Generation ◽

Fibrin Clot ◽

Clot Formation ◽

In Vitro Evaluation

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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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