Procoagulant Status In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3528-3528 ◽  
Author(s):  
Ihosvany Fernández Bello ◽  
Mayte Álvarez Román ◽  
Elena G. Arias Salgado ◽  
Monica Martin Salces ◽  
Miguel Canales ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Red Cells ◽  
Procoagulant Activity ◽  
Platelet Counts ◽  
Significant Difference ◽  
The Mean ◽  
Spearman Test

Abstract Introduction Immune thrombocytopaenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopaenia caused by autoantibodies against platelet proteins. Bleeding risk in patients with ITP is increased with platelet counts less than 20 or 30 x 109/L. However, patients with ITP often have few bleeding symptoms despite very low platelet counts suggesting the existence of compensatory mechanisms. Moreover, an increased risk for thrombosis in patients with ITP has been described (Nørgaard M, 2012). It has been recently reported that increased production of platelet- and red cells-derived microparticles (MP) might be one of the causes of increased thrombotic risk in ITP patients (Sewify, 2013). Objective The aim of this study was to evaluate the microparticle-associated and plasma procoagulant activities in ITP patients with thrombocytopaenia. Methods Sixty-eight patients with chronic ITP and platelet count less than 50 x 109/L and twenty-two healthy controls were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 1,500 g for 15 min at 23°C. Platelet-poor plasma obtained was additionally centrifuged twice at 23°C (15 min at 1,500 g, and 2 min at 13,000 g) and aliquots were stored at -70ºC until analysis. Phosphatidylserine-MP (Ph-MP) and tissue factor-MP (TF-MP) dependent procoagulant activities were determined with the ZYMUPHEN kits (Hyphen BioMed, Neuville sur Oise, France) following the manufacturer’s instructions. Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) test as described by Hemker et al (2000) at a final concentration of 1 pM tissue factor and 4 μM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak. To test resistance to protein C, CAT experiments were performed without and with the addition of thrombomodulin (TM) (PPP and PPP with thrombomodulin reagents, Thrombinoscope BV, Maastricht, The Netherlands). Results were expressed as the ratio [(ETP with TM)/(ETP without ETP)]x100. Results were expressed as mean±SD. Comparisons of quantitative variables were made with Mann-Whitney test and correlations with Spearman test. Values of p≤0.05 were considered statistically significant. Results Ph-MP associated procoagulant capacity in ITP patients was higher than in controls (p<0.05) whereas MP-TF associated procoagulant activity was practically negligible in both groups. Plasma procoagulant activity was higher in ITP patients than in controls (ETP: 1604±616 nM x min in ITP patients and 1302±416, p=0.012 in controls; Peak: 328±123 nM in ITP patients and 203±74 nM in controls, p<0.001). We tested whether the higher procoagulant activity of plasma from ITP patients was due to a resistance to protein C. We observed that the mean Ratio value in ITP patients was slightly higher than the mean Ratio of controls (60±18 and 50±13 respectively, p=0.034). Despite this significant difference in the Ratio, no correlation was found between this value and the CAT parameters. Conclusion ITP patients with thrombocytopaenia had a higher Ph-MP associated and plasma procoagulant activity than controls. The fact that the increased MP-procoagulant activity was not accompanied by a higher TF-MP associated procoagulant activity brings further support to the previous observation that MPs in ITP patients are from platelets and red cells, as both cells express very low levels of TF (Sewify, 2013). Regarding the increased plasma procoagulant capacity observed in ITP patients, our results suggest that resistance to protein C does not seem to be the main mechanism involved. References • Nørgaard M. Thromb Res. 2012;130 Suppl 1:S74-75. • Sewify EM, et al. Thromb Res. 2013;131:e59-63. Hemker HC, et al. Thromb Haemost 2000;83:589-9. Disclosures: No relevant conflicts of interest to declare.

Thrombin Generation Is Normal in Most Patients with Cirrhosis despite a Prolonged INR.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1826-1826
Author(s):  
Alexander Gatt ◽  
Anne Riddell ◽  
Vincenza Calvaruso ◽  
Michael Makris ◽  
Edward Tuddenham ◽  
...  
Keyword(s):  
Liver Disease ◽  
Tissue Factor ◽  
Patient Group ◽  
Protein C ◽  
Thrombin Generation ◽  
Control Sample ◽  
Normal Subjects ◽  
Sensitivity Index ◽  
Clotting Factors ◽  
The Mean

Abstract Introduction: Advanced liver disease is associated with prolongation of the prothrombin time (PT). In order to decrease the inter-laboratory variability of PT measurement, the international normalised ratio (INR) calculated as the ratio of patient’s PT to a normal (control) sample, raised to the power of the international sensitivity index (ISI) of the particular thromboplastin used was developed. However, the ISI is derived from PT results of patients on warfarin and results cannot be extrapolated to liver patients. Despite this, the INR is still commonly performed to assess bleeding risk in patients with liver disease worldwide. Furthermore the INR is only affected by factors I, II, V, VII, and X and is not influenced by other factors such as factor VIII which is usually raised in hepatic cirrhosis. Recently it has been reportd that thrombomodulin addition (to take into account the protein C pathway) normalises thrombin generation (TG)1 despite these patients having a low TG if thrombomodulin is not used. Aim: We speculated that TG, which is a global assay of coagulation and sensitive to all coagulation factors, when triggered by a low tissue factor (TF) concentration might not correlate with the INR in patients with liver disease and that contact inhibition with corn trypsin inhibitor (CTI) might better reflect the coagulation potential in this patient group. Results: 73 unselected patients with liver cirrhosis due to various diseases and 25 normal subjects were studied. INR and TG using the calibrated automated thrombogram (CAT) at 1pM tissue factor (TF) with CTI, 5pM without CTI and with and without Protac (a Protein C activator) were performed using platelet poor plasma (PPP). The INR range was 0.8–4.0 (mean 1.6). At 5pM TF without Protac, the patient group had a significantly lower endogenous thrombin potential (ETP) than the controls (mean ETP difference 752nM/min; P &lt;0.0001). With Protac, no significant differences could be detected between the 2 groups. However, if the ETP without Protac was divided by the ETP with Protac x 100, the liver group showed more resistance to PC activation (mean % difference 25.4; P 0.0002). At 1pM TF, the mean ETP in the cirrhosis cohort was slightly lower than the normal group (difference between means 216nM/min; P 0.03). However, only 7 (9.6%) patients had ETP values less than the normal range (mean±2SD). No correlation was found between the ETP at 1pM and the INR. The mean FVIII:C was raised at 185.6 (78–420U/dl). Conclusion: TG measured at low TF with CTI is normal in the majority of patients with cirrhosis. These patients are also more resistant to PC activation and have supranormal FVIII:C. Thus most patients have a normal or high thrombin potential despite an abnormal INR. These findings have important implications as in the absence of bleeding, “prophylactic” plasma and clotting factors are unnecessary.

Detection of Circulating Tissue Factor and Factor VII in a Normal Population

1996 ◽  
Vol 75 (05) ◽  
pp. 772-777 ◽  
Author(s):  
Sybille Albrecht ◽  
Matthias Kotzsch ◽  
Gabriele Siegert ◽  
Thomas Luther ◽  
Heinz Großmann ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Normal Population ◽  
Mean Value ◽  
Factor Vii ◽  
Significant Difference ◽  
Age Dependent ◽  
The Mean ◽  
Highly Correlated ◽  
And Gender

SummaryThe plasma tissue factor (TF) concentration was correlated to factor VII concentration (FVIIag) and factor VII activity (FVIIc) in 498 healthy volunteers ranging in age from 17 to 64 years. Immunoassays using monoclonal antibodies (mAbs) were developed for the determination of TF and FVIIag in plasma. The mAbs and the test systems were characterized. The mean value of the TF concentration was 172 ± 135 pg/ml. TF showed no age- and gender-related differences. For the total population, FVIIc, determined by a clotting test, was 110 ± 15% and the factor VIlag was 0.77 ± 0.19 μg/ml. FVII activity was significantly increased with age, whereas the concentration demonstrated no correlation to age in this population. FVII concentration is highly correlated with the activity as measured by clotting assay using rabbit thromboplastin. The ratio between FVIIc and FVIIag was not age-dependent, but demonstrated a significant difference between men and women. Between TF and FVII we could not detect a correlation.

Phospholipid-bound Tissue Factor Modulates both Thrombin Generation and APC-mediated Factor Va Inactivation

1999 ◽  
Vol 82 (12) ◽  
pp. 1673-1679 ◽  
Author(s):  
Katalin Váradi ◽  
Jürgen Siekmann ◽  
Peter Turecek ◽  
H. Peter Schwarz ◽  
Victor Marder
Keyword(s):  
Tissue Factor ◽  
Surface Density ◽  
Protein C ◽  
Thrombin Generation ◽  
Feedback Inhibition ◽  
Activated Protein C ◽  
Reaction System ◽  
Inverse Correlation ◽  
High Concentration ◽  
Factor Va

SummaryHemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

Whole Blood Tissue Factor Procoagulant Activity Is Elevated in Patients With Sickle Cell Disease

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4216-4223 ◽  
Author(s):  
Nigel S. Key ◽  
Arne Slungaard ◽  
Luke Dandelet ◽  
Stephen C. Nelson ◽  
Christopher Moertel ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Mononuclear Cell ◽  
Sickle Cell ◽  
Whole Blood ◽  
Procoagulant Activity ◽  
Cell Disease ◽  
Blood Samples ◽  
Significant Difference ◽  
Whole Blood Samples

Abstract We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P &lt; .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.

Red Cells Release Microparticles Heterogeneous in Phenotypes, Procoagulant Activities, and Size under Different Conditions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1726-1726
Author(s):  
Wenche Jy ◽  
Jaehoon Bang ◽  
Joaquin J. Jimenez ◽  
Lawrence L. Horstman ◽  
Loreta Bidot ◽  
...  
Keyword(s):  
Shear Stress ◽  
Thrombin Generation ◽  
Calcium Ionophore ◽  
Red Cells ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Flip Flop ◽  
Increased Risk ◽  
Thrombotic Complications ◽  
Inside Out

Abstract BACKGROUND. Hemolytic anemias (HA) such as autoimmune hemolytic anemia (AIHA), thalassemia, and sickle cell disease are associated with increased risk for thrombosis. Although exposure of procoagulant phospholipids on the RBC membrane in HA has been implicated, the pathogenesis remains to be elucidated. We recently showed that microparticles released from red cells (RMP) are procoagulant and significantly elevated in HA, suggesting a link of RMP to thrombotic complications. In this study, we report the release of RMP, heterogeneous in size, phenotypes and procoagulant activity by different methods. METHODS. RMP were prepared from washed RBC (2×109/mL) by three methods, exposure to 10 μM calcium ionophore A23187 (CaIo) for 60 min, 20 μM lysophosphatidic acid (LPA) for 60 min, or shear stress induced by a rotating Teflon pestle for 5 min at 160 rpm in a tissue grinder. After removing residual whole cells, the supernatant was centrifuged at 15,000×g for 10 min (large RMP) and the supernatant was further centrifuged at 150,000×g for 30 min (small RMP). The resulting RMP were labeled for flow cytometry using PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). Thrombin generation of RMP was measured by the calibrated automated thrombogram (CAT) system using fluorescent thrombin substrate on a fluorescence plate reader. RESULTS. The two-step centrifugation revealed two distinct populations, the large RMP expressing both GlyP and Ulex binding while the smaller RMP expressed only Ulex binding. Electron micrography showed diameters for the large RMP of 200 - 800 nM while the small RMP were 40 - 80 nm. Shear stress produced the greatest number of large GlyP + RMP (5.2 ± 1.3 × 106/μL), followed by CaIo (3.8 ± 0.7 × 106/μL), and LPA (2.5 ± 0.6 × 106/μL). However, CaIo produced the greatest number of small GlyP −/Ulex + RMP (4.6 ± 0.9 × 106/μL), followed by LPA (1.4 ±0.3 × 106/μL), and very few by shear tress (0.3 ±0.1 × 106/μL). The methods also gave different AnV binding: CaIo yielded &gt;90% of RMP (large and small) that were positive, whereas RMP induced by LPA or shear stress gave only 45% and 18% positive, respectively. No TF + RMP were detected in any procedure. The RMP also differed in thrombin generation. Adjusting concentrations to equal numbers of Ulex + MP, CaIo RMP displayed the strongest activity (375 ± 62 nM) followed by LPA-induced (227 ± 58 nM) and shear-induced (136 ±33 nM). Thrombin generation correlated well with degree of AnV binding. CONCLUSIONS. We demonstrate that RBC release different species of RMP, heterogeneous in size, phenotypes and procoagulant activity. Both CaIo and LPA induced two distinct species of RMP, the larger expressing GlyP and Ulex binding while the smaller phenotype was negative for GlyP, possibly indicating that the small RMP are inside-out vesicles. In contrast, shear stress produced mainly large rightside-out vesicles with both GlyP expression and Ulex binding, and very low AnV binding. Thus, calcium influx appears necessary to release small inside-out RMP, and to induce the membrane flip-flop bringing negatively charged AnV binding sites to the plasma membrane. We postulated that highly procoagulant RMP that bind AnV may contribute to the thrombotic complications of HA and other RBC disorders associated with thrombosis.

Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3736-3736
Author(s):  
Anna Falanga ◽  
Alfonso Vignoli ◽  
Marina Marchetti ◽  
Laura Russo ◽  
Marina Panova-Noeva ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Coagulation Activation ◽  
Allele Burden ◽  
Generation Capacity

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.

Procoagulant Activity of Laminaria Japonica Derived Fucoidan (BAX513) Depends on the Interaction with the C-Terminus of Tissue Factor Pathway Inhibitor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4417-4417 ◽  
Author(s):  
Michael Palige ◽  
Christoph Redl ◽  
Sabine Knappe ◽  
Hartmut J. Ehrlich ◽  
Michael Dockal ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mechanism Of Action ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Laminaria Japonica ◽  
Full Length ◽  
Procoagulant Activity ◽  
C Terminus ◽  
Tissue Factor Pathway ◽  
Dose Dependent

Abstract Abstract 4417 BAX513, a fucoidan derived from the brown seaweed Laminaria japonica, and other non-anticoagulant sulfated polysaccharides (NASPs) improve coagulation in hemophilic blood and plasma. Fucoidans are heterogeneous, polysulfated molecules with procoagulant activities in a wide concentration range. Tissue factor pathway inhibitor (TFPI) has been described as a potential target for the procoagulant activity of NASPs (Liu et al. Thromb Haemost 2006; 95:68). In the current study, we investigated the interaction of BAX513 with TFPI proteins to gain a detailed understanding of the mechanism of action of BAX513. We used calibrated automated thrombography to monitor the activity of BAX513 in normal, FX and TFPI-deficient plasma. TFPI plasma levels were varied by the addition of truncated TFPI (TFPI1-160) and TFPI-domain specific antibodies. Initiating thrombin generation by addition of FXa to plasma deficient in both, FX and FVIII-showed a BAX513-dose dependent increase of thrombin generation, which was completely abolished when TFPI-specific polyclonal antibodies were present. Furthermore, when full-length TFPI was inhibited in plasma and instead supplemented with increasing amounts of TFPI 1–160, BAX513 did not show any activity. The data are further supported by surface plasmon resonance experiments (BiaCore) exploring the BAX513-TFPI interaction. A high affinity interaction was only observed for BAX513 with full-length TFPI but not for BAX513 with TFPI1-160. Our findings support a mechanism of action in which BAX513 acts as a potent dose-dependent TFPI antagonist that requires the highly charged C-terminus of TFPI to unfold its full potential. Understanding the mechanism of action of BAX513 supports the development of BAX513 as a promising new therapeutic for hemophiliacs and FVIII-inhibitor patients. Disclosures: Palige: Baxter Innovations GmbH: Employment. Redl:Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Dockal:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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

Blood ◽  
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.

scholarly journals Increased thrombin generation and activity in patients with systemic lupus erythematosus and anticardiolipin antibodies: evidence for a prothrombotic state [see comments]

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2958-2963
Author(s):  
JS Ginsberg ◽  
C Demers ◽  
P Brill-Edwards ◽  
M Johnston ◽  
R Bona ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Anticardiolipin Antibodies ◽  
Laboratory Evaluation ◽  
Prothrombotic State ◽  
Systemic Lupus ◽  
Cross Sectional ◽  
Significant Difference ◽  
The Mean

The objective of this study is to determine whether patients with systemic lupus erythematosus (SLE) and anticardiolipin antibodies (ACA) have biochemical evidence of an ongoing prothrombotic state. Using a cross-sectional analysis of a cohort design in an outpatient SLE clinic setting, 43 consecutive patients with SLE participated. Patients underwent clinical and laboratory evaluations on two separate occasions at least 3 months apart. As part of the clinical evaluation, the following were ascertained: (1) the ongoing use of warfarin therapy; (2) the presence of prior venous and arterial thromboembolic disease by history, critical review of objective tests, and examination for reflux in the deep veins of the legs as an indicator of venous thrombosis; and (3) disease-related activity by performing a lupus activity criteria count (LACC). As part of the laboratory evaluation, blood was taken on both occasions and assayed for prothrombin fragments (F1 + 2) and fibrinopeptide A (FPA), as indices of thrombin generation and activity, respectively, and ACA. For the analyses, patients were classified as ACA+ if the assay was abnormal on both occasions and ACA- if the assay was negative on both occasions or negative on one occasion and positive on the other. ACA+ patients had: (1) a significantly higher mean level of F1 + 2 (1.07 nmol/L) than ACA- patients (0.79 nmol/L; P = .02) and patients receiving warfarin (0.47 nmol/L; P = .009) and (2) a significantly higher mean level of FPA (1.01 nmol/L) than ACA- patients (0.45 nmol/L; P = .02). When patients with prior thromboembolism were excluded from the analysis, significant differences in the mean levels of F1 + 2 and FPA between ACA+ and ACA- patients were still seen, whereas when patients with prior thromboembolism and/or active disease were excluded from the analysis, a significant difference in the mean level of FPA and a nonsignificant trend in the mean level of F1 + 2 were seen. The results of this study support the hypothesis that the presence of ACA in SLE patients is associated with an ongoing prothrombotic state.

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