Thrombin Generation In Platelet-poor Plasma Research Articles

Measuring Thrombin Generation in Platelet Rich Plasma with the ST Genesia

Background: Thrombin generation (TG) is nowadays used more and more for diagnosing and monitoring of patients. The ST Genesia is a fully automated device for measuring TG in platelet poor plasma (PPP). As the samples are not being inverted prior to the measurement once loaded in the device, it was not known whether it was possible to measure TG in platelet rich plasma (PRP). Platelets can sediment in time possibly affecting TG results. The goal of this study was to evaluate the use of the ST Genesia for measuring TG in PRP in time and to investigate whether the assay is sensitive to platelet antagonists. Methods: Blood was taken from 6 healthy donors and PRP was immediately prepared by centrifuging the blood once for 15 minutes at 220g. The PRP was divided into 14 tubes: 11 tubes for measuring TG in time; i.e. after 0, 15, 30, 45, 60, 75, 90, 105, 120, 180, 240 minutes after the samples were loaded on board; 1 tube for platelet count measurement, 1 tube for TG with the Calibrated Automated Thrombogram (CAT) assay and 1 tube for freezing the PRP. The PRP reagent of the CAT assay was used for triggering TG that contains a low tissue factor concentration. Tukey's multiple comparisons test was used for statistical analysis. Results: The platelet count remained stable in time even without inverting the vial prior to the measurement up to 3 hours. After 4 hours, platelet count was significantly lower (p<0.01). TG in PRP remained stable in time up to 3 hours, after that lagtime and time-to-peak were extended by an average of 48% and 16%, respectively (p-values <0.0001 and 0.0121, respectively), while ETP and peak height remained stable (with low CV's of <6.0% and <7.3%, respectively). The ST Genesia proved to be sensitive to platelet antagonists aspirin (100µM) and Cangrelor (500nM) as peak height decreased with 22% and 24%, respectively. TG measured with the CAT and the ST Genesia was comparable. Measuring TG in PRP after being frozen at -80°C resulted TG kinetics similar to TG in PPP, with a shorter lagtime (-67%) and time-to-peak (-64%), with an increased peak height (+82%), and velocity index (+383%), and an unaffected ETP. Conclusion: The ST Genesia can be used for the reproducible measurement of TG in PRP up to two hours after loading the samples in the device.

Study of in vitro thrombin generation after neutralization of heparin.

Thrombin generation (TG) documentshypercoagulability. TG in platelet-poor plasma is exquisitely sensitive toheparins, which thus mustbe neutralized before testing. Heparinase and hexadimethrine bromide (polybrene) have been used for that purpose, but their effectsper seon TG have been poorly studied so far. (i) TG was studied in commercial normal pooled plasma (NPP; CryoCheck® , Cryopep) in absence or presence of neutralizing agents. (ii) NPP was spiked with increasing concentrations of unfractionated heparin (UFH; up to 1.0IU/mL) or low-molecular-weight heparin (LMWH; enoxaparin up to 1.2IU/mL) and TG studied after incubation of heparinase (Hepzyme® ; 15minutes) or polybrene (0.025mg/mL; 10minutes). (i) With ThromboScreen reagent to initiate TG, addition of heparinase was associated with increased peak, whereas polybrene caused lengthening of lag time and time to peak, compared with nonsupplemented NPP. (ii) With polybrene, TG was completely restored over the whole range of UFH and LMWH studied. By contrast, heparinase failed to fully restore TG in presence of UFH concentrations ≥0.8IU/mL or LMWH concentrations ≥1.0IU/mL. Those effects were matched with detectable tiny residual amounts of non-neutralized heparin (as assessed with an anti-Xa assay) and were less pronounced with a higher picomolar concentration of tissue factor (DrugScreen reagent). Polybrene fully restored TG of heparinized plasma at the expense of an alteration of TG, pointing to the need to use adapted reference ranges. Heparinase failed to do so in presence of high concentrations of both heparins.

Thrombin Generation as a Method to Identify the Risk of Bleeding in High Clinical-Risk Patients Using Dual Antiplatelet Therapy

Background: Patients using dual antiplatelet therapy after percutaneous coronary intervention are at risk for bleeding. It is currently unknown whether thrombin generation can be used to identify patients receiving dual antiplatelet therapy with increased bleeding risk.Objectives: To investigate whether thrombin generation measurement in plasma provides additional insight into the assessment of bleeding risk for high clinical-risk patients using dual antiplatelet therapy.Methods: Coagulation factors and thrombin generation in platelet-poor plasma were measured in 93 high clinical-risk frail patients using dual antiplatelet therapy after percutaneous coronary intervention. During 12-month follow-up, clinically relevant bleedings were reported. Thrombin generation at 1 and 6 months after percutaneous coronary intervention was compared between patients with and without bleeding events.Results: One month after percutaneous coronary intervention, the parameters of thrombin generation, endogenous thrombin potential, peak height, and velocity index were significantly lower in patients with bleeding in the following months compared to patients without bleeding. At 6 months follow-up, endogenous thrombin potential, peak height, and velocity index were still (significantly) decreased in the bleeding group as compared to non-bleeders. Thrombin generation in the patients' plasma was strongly dependent on factor II, V, and VIII activity and fibrinogen.Conclusion: High clinical-risk patients using dual antiplatelet therapy with clinically relevant bleeding during follow-up show reduced and delayed thrombin generation in platelet-poor plasma, possibly due to variation in coagulation factors. Thus, impaired thrombin-generating potential may be a “second hit” on top of dual antiplatelet therapy, increasing the bleeding risk in high clinical-risk patients. Thrombin generation has the potential to improve the identification of patients using dual antiplatelet therapy at increased risk of bleeding.

Controlled Multifactorial Coagulopathy: Effects of Dilution, Hypothermia, and Acidosis on Thrombin Generation In Vitro.

Coagulopathy and hemostatic abnormalities remain a challenge in patients following trauma and major surgery. Coagulopathy in this setting has a multifactorial nature due to tissue injury, hemodilution, hypothermia, and acidosis, the severity of which may vary. In this study, we combined computational kinetic modeling and in vitro experimentation to investigate the effects of multifactorial coagulopathy on thrombin, the central enzyme in the coagulation system. We measured thrombin generation in platelet-poor plasma from 10 healthy volunteers using the calibrated automated thrombogram assay (CAT). We considered 3 temperature levels (31°C, 34°C, and 37°C), 3 pH levels (6.9, 7.1, and 7.4), and 3 degrees of dilution with normal saline (no dilution, 3-fold dilution, and 5-fold dilution). We measured thrombin-generation time courses for all possible combinations of these conditions. For each combination, we analyzed 2 scenarios: without and with (15 nM) supplementation of thrombomodulin, a key natural regulator of thrombin generation. For each measured thrombin time course, we recorded 5 quantitative parameters and analyzed them using multivariable regression. Moreover, for multiple combinations of coagulopathic conditions, we performed routine coagulation tests: prothrombin time (PT) and activated partial thromboplastin time (aPTT). We compared the experimental results with simulations using a newly developed version of our computational kinetic model of blood coagulation. Regression analysis allowed us to identify trends in our data (P < 10). In both model simulations and experiments, dilution progressively reduced the peak of thrombin generation. However, we did not experimentally detect the model-predicted delay in the onset of thrombin generation. In accord with the model predictions, hypothermia delayed the onset of thrombin generation; it also increased the thrombin peak time (up to 1.30-fold). Moreover, as predicted by the kinetic model, the experiments showed that hypothermia increased the area under the thrombin curve (up to 1.97-fold); it also increased the height of the thrombin peak (up to 1.48-fold). Progressive acidosis reduced the velocity index by up to 24%; acidosis-induced changes in other thrombin generation parameters were much smaller or none. Acidosis increased PT by 14% but did not influence aPTT. In contrast, dilution markedly prolonged both PT and aPTT. In our experiments, thrombomodulin affected thrombin-generation parameters mainly in undiluted plasma. Dilution with normal saline reduced the amount of generated thrombin, whereas hypothermia increased it and delayed the time of thrombin accumulation. In contrast, acidosis in vitro had little effect on thrombin generation.

Von Willebrand factor binding to myosin assists in coagulation

Abstractvon Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

Evaluation of Thrombin Generation in Patients with Stable Coronary Artery Disease with a Prior Myocardial Infarction

Background. Despite invasive technologies and the advancement of modern pharmacotherapy in the treatment of coronary artery disease (CAD), patients still have risks of complications. There is a special group of patients with CAD with a prior myocardial infarction (MI). As demonstrated, a prior MI increases risk of recurring cardiovascular events. Thus, there was a decrease in the frequency of such events to a greater extent than that with aspirin monotherapy in the clinical study on combined antiplatelet and anticoagulant therapy in this group. By contrast, there was an increase in the frequency of large bleeding, which is a significant limitation of the combined therapy. Therefore, it is relevant to identify subjects with CAD and high thrombotic risk for individualization of therapy. In this study, the activity of thrombin formation was assessed with help of an integral hemostasis assessment method — a thrombin generation test (TGT) in patients with CAD regarding the presence or absence of MI in anamnesis. Objective . Investigation of the thrombogenic activity with the help of TGT in patients with CAD with a prior MI. Materials and methods . A total of 59 patients with a diagnosis of stable CAD hospitalized for myocardial revascularization were examined. Of these, 28 patients who had a history of MI (group 1), and 31 patients without previous MI (group 2). The control group consisted of 31 people without clinical manifestations of atherosclerosis. In vitro thrombin generation in platelet-poor plasma was measured by means of the calibrated automated thrombogram method (Thrombinoscope BV, Maastricht, the Netherlands). Four parameters were derived from the thrombin generation curves: LT (lag time — initiation phase of coagulation, min), Peakthr (peak thrombin concentration, nmol), ttPeak (time to peak concentration, min), ETP (endogenous thrombin potential — area under the thrombin generation curve), and VI (velocity index of thrombin generation, nmol/min). Results. A significant increase in TGT parameters was revealed in Group 1 compared to Group 2 and control group: the peak height of thrombin (304.09 [279.97-353.91] nmol vs 283.38 [209.07-313, 54] and 258.86 [211.28-299.50] nmol, respectively (p &lt; 0.01)) and the VI thrombin (131.78 [111.98-158.38] nmol/min vs 100.64 [56.33-122.19] and 77.96 [62.45-122,54,] nmol/min, respectively, (p &lt; 0.001)). There was a significant positive correlation between ETP and total cholesterol level (r = 0.35, p = 0.01), LDL (r = 0.32, p = 0.02) in the general group of patients with CAD. Conclusion. In patients with CAD and prior MI, a higher quantity and rate of thrombin formation were found by TGT, determining the test perspectivity for assessment of thrombotic risk in order to isolate subjects with an indication for more strong antithrombotic therapy.

Von Willebrand Factor Binding to Myosin Assists in Coagulation

Introduction: Von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factors V and X (Deguchi et al, Blood 2016;128:1807-1878). We hypothesized that VWF could also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation.Methods: Recombinant wild-type (WT) was transfected into HEK293T cells and supernatants collected for VWF to use in experiments. VWF variants containing point mutations at specific sites were constructed via site directed mutagenesis and expressed as above. Myosin from rabbit skeletal muscle was plated on maleic anhydride plates, recombinant VWF or plasma samples added and presence of VWF detected using anti-VWF monoclonal antibodies. Competition assays were performed with antibodies blocking various sites on VWF. FVIII activity was measured using a chromogenic substrate (Chromogenix Coatest). Thrombin generation was performed using a fluorogenic substrate from the Technothrombin TGA kit with either tissue factor +/- myosin or the RCL reagent +/- myosin added to platelet poor plasma from healthy controls.Results: WT VWF and human plasma VWF from healthy controls bound myosin, while plasma lacking VWF failed to demonstrate binding. Binding was multimer dependent, with a dose dependent decrease in binding seen with increasing loss of high molecular weight multimers. When myosin binding to VWF:Ag ratios were compared, a solution of ultra high molecular weight multimers demonstrated a mean ratio of 1.6, a solution of high molecular weight multimers a ratio of 1.4, a solution of medium molecular weight multimers a ratio of 0.4 and binding was undetectable with only low molecular weight multimers. A polyclonal anti-VWF antibody (DAKO) completely blocked VWF binding to myosin as did an antibody directed against the VWF A1 domain (AVW-3). Antibodies directed against other VWF sites including the N and C terminal ends failed to affect VWF binding to myosin. Since collagen IV also binds in this region, collagen IV was added to the assay as competition and also completely blocked VWF binding to myosin. VWF variants p.R1395A and p.R1399A showed undetectable binding, while a variant in the A3 domain (that abrogates type III collagen binding) showed normal binding to myosin (95% of WT). However, additional residues that affect VWF-collagen IV interactions were tested and had minimal effect on myosin binding, including p.1392A and p.1402A. Taken together, these results suggest a binding site for myosin in the VWF A1 domain similar but not identical to that of collagen IV.FVIII activity was detected when a VWF concentrate containing both VWF and FVIII was bound to myosin, with a dose-dependent increase in activity. However, no increase in FVIII activity was seen using recombinant FVIII alone in the absence of additional VWF. Because previous results from J. Griffin and coworkers showed a role for myosin in enhancing thrombin generation, we also looked at thrombin generation in platelet-poor plasma with and without myosin, but no difference was seen in the absence of additional phospholipids. Peak thrombin with myosin was 80 nM vs 62 nM without myosin (p=NS), and the area under the curve was 1022 vs 775 (p=NS). With additional phospholipids, there was a trend towards increased thrombin generation with myosin but the difference was again not statistically significant. Peak thrombin with myosin was 70 nM vs 64 nM without myosin (p=NS), and the area under the curve was 974 vs 867 (p=NS).Discussion: Myosin can also serve as a surface for VWF binding. This may help facilitate delivery of FVIII to sites of injury and improve thrombin generation, although in our hands myosin did not substantially increase thrombin generation in the presence of additional phospholipid. Unlike factor V, FVIII does not intrinsically interact with myosin to accelerate thrombin generation. However, myosin, similar to collagen, can increase local accumulation of VWF-FVIII and indirectly accelerate thrombin generation and clot formation. DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

Interindividual Variability and Normal Ranges of Whole Blood and Plasma Thrombin Generation.

Assays measuring thrombin generation (TG) in plasma increasingly gained attention in the field of thrombosis and hemostasis. Adaptation of the method enabled the measurement of TG in whole blood (WB). Despite their potential, TG assays did not reach the stage of universal clinical application, partly because of the absence of normal ranges. Our study aimed to accurately determine normal ranges and interindividual variability of TG and correlate results with coagulation factor levels, sex, and oral contraceptive usage. The study protocol was evaluated by the local medical ethical board. In total, 129 healthy volunteers gave full informed consent. Normal ranges of TG in platelet-poor plasma (PPP), platelet-rich plasma (PRP), and WB were determined according to CLSI guidelines. Our study is the first to measure normal ranges of TG in PPP, PRP, and WB in a large healthy cohort. Significant correlations were found between TG in plasma and WB. Interindividual variability of TG in WB was comparable to that of plasma. Oral contraceptive use increased TG in PPP, PRP, and WB. The inhibitory effect of thrombomodulin on TG was significantly lower in females than in males. This effect was more pronounced upon oral contraceptive use. Primary clotting factor determinants for TG parameters depended on the tissue factor concentration, but were similar in WB and plasma. Establishing normal ranges for TG brings us 1 step closer to clinical use. Good correlations between plasma and WB (including clotting factor determinants for TG) suggest that WB TG can be reliably used in clinic.

Anti-thrombotic properties of a CD31-derived peptide coated on a surface

Event Abstract Back to Event Anti-thrombotic properties of a CD31-derived peptide coated on a surface Charlotte Rasser1, Frédéric Chaubet1, Caroline Jobin1, Giuseppina Caligiuri1 and Véronique Ollivier1 1 Laboratory for Vascular Translational Science - Inserm U1148, France Introduction: Thrombosis is a major concern in the design of most biomaterials, especially in those for vascular applications. Coating the biomaterial surface with a bioactive agent is one of the possible strategies to avoid thrombosis. CD31 (also known as PECAM-1) is a transmembrane glycoprotein present on endothelial cells, lymphocytes, neutrophils and platelets. CD31 has been demonstrated to inhibit platelet activation and thrombus formation[1],[2] and to modulate thrombin-induced tissue factor expression on endothelial cells[3].We therefore assessed the potential of a CD31-agonist peptide as an anti-thrombotic coating for biomaterials. Materials and Methods: The CD31-agonist peptide was left to adsorb on a 96-well plate with high binding affinity (Immulon® 2 HB, Thermo Scientific). Excess peptide was rinsed with phosphate buffered saline (PBS). Unmodified wells of the same plate were used as control. In a first set of experiments, thrombin generation in platelet-rich plasma (PRP) was performed using a Calibrated Automated Thrombogram (CAT) in presence of 0.5 pM tissue factor (TF), in a Fluoroskan Ascent (Thermo Scientific). In a second set, human coronary artery endothelial cells (HCAEC) were cultured in a 96-well plate with or without peptide coating until confluence was reached. Then, thrombin generation was measured at the surface of the cells in platelet-poor plasma (PPP) and in presence of 1 pM TF. To estimate cell viability, a tetrazolium (MTT, Sigma Aldrich) colorimetric assay was performed in parallel. Figure 1. Typical curve of thrombin generation in PRP in presence of 0.5 pM TF, in control wells or peptide-coated wells. n=6. Figure2. a) Typical curve of thrombin generation in PPP, in presence of 1 pM TF, at the surface of endothelial cells cultivated either in control wells or peptide-coated wells. b) MTT assay. n=3. Results and Discussion: Figure 1 presents the anti-platelet action of the peptide (thrombograms acquired in PRP, without endothelial cells). Thrombin generation is significantly reduced (-40%) in the peptide-coated wells, suggesting that the CD31-derived peptide is able to control the activation of the platelets coming into contact with the surface. In addition, thrombin generation at the surface of endothelial cells was reduced by 50% when the cells were cultured onto the peptide-coated wells (Figure 2a) as compared to unmodified wells. Since the number of endothelial cells in the two types of wells was not significantly different, as demonstrated by the MTT assay (Figure 2b), the peptide has most likely acted on the phenotype of the endothelial cells, promoting their anti-thrombotic phenotype. Conclusion: Our data show that coating surfaces with a CD31-agonist peptide results in a dual anti-thrombotic action: on the platelets coming into direct contact with the coated surface as well as on endothelial cells that shall grow on it. The CD31-derived peptide thus appears as a promising bioactive agent for biomaterial coating in applications such as vascular stents.

Hypobaric Hypoxia Induces Hypercoagulability Due to an Increase in Microparticles

Studies investigating the role of hypoxia on coagulation, either by going to high altitude or after a long-haul flight, reveal contrasting results. Previous work from our group suggests that the cellular part of the blood is involved in causing a prothrombotic phenotype. Therefore this study investigated the effect of hypobaric hypoxia on blood coagulation, focusing on the role of cellular components.After approval from the local medical ethics committee, 16 healthy participants (aged 20 to 50 years old) were included in this study. Exclusion criteria were a history of cardiovascular disease or pulmonary disease, impaired mobility, and medication known to interfere with coagulation. Participants ascended by cable car to 3,883 meters above sea level, after acclimatizing to altitude for 6 days. At 50 meters and 3,883 meters above sea level, blood was drawn and vital signs (SpO2, heart rate) and the Lake Louise acute mountain sickness questionnaire (LLQ) were recorded. The following tests were performed on whole blood: hemoglobin (Hb), hematocrit (Ht), leucocyte count (L), platelet count (PC) and mean platelet volume (MPV); thrombin generation (TG) in whole blood (WB-TG) with 0.5 pM tissue factor (TF); platelet activation test (PAc-T) triggered by adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP) and collagen related peptide (CRP) in end concentrations of respectively 10 µM, 30 µM and 1 µg ml-1. The remainder of the blood was centrifuged to obtain platelet rich plasma (PRP) (once for 15 minutes at 230 g) and platelet poor plasma (PPP) (twice for 10 minutes at 2,821 g). PRP was used to test PRP-TG (1 pM TF) and plasma was used for PPP-TG (1 pM TF) and microparticles (MP)-TG (0 pM TF). The paired t-test with p <0.05 was used to determine statistical significance within participants.At 3,883 meters above sea level, oxygen saturation decreased and heart rate increased significantly. LLQ scores revealed mild acute mountain sickness (AMS) symptoms. One participant was withdrawn from the study at 3,030 meters due to moderate AMS. After applying TG in PPP, PRP and whole blood, we found that peak height and endogenous thrombin potential (ETP) were increased. Interestingly, we found a decrease in platelet activation and a decreased MPV. To find an explanation for the increased TG in the different media, we performed a TG assay specially designed to detect microparticles. As with the PPP-TG, PRP-TG and WB-TG, we found an increase in ETP and peak height, proving increased content of MPs.In conclusion, we found that exposure to hypobaric hypoxia increased TG in PPP, PRP and in whole blood. In contrast we found that platelet activation was decreased, indicating that platelets do not play a role in hypoxia-induced hypercoagulability. The increase in peak and ETP in the MP-sensitive TG assay indicates that MPs play a major role in hypoxia-induced hypercoagulability. DisclosuresDe Laat:Synapse bv: Employment.

Procoagulant Profile in Patients with Immune Thrombocytopaenia

Background: Patients with platelet counts less than 20 or 30 x 109/L have an increased risk of bleeding. Nevertheless, some patients with immune thrombocytopenia (ITP) have fewer bleeding symptoms than expected. In a previous communication (ASH 2014) we reported that these patients presented high microparticles (MP)-associated procoagulant activity to compensate bleeding risk and that cellular origin of these MPs were platelets and red cells. However, other mechanisms might be involved.Objective: The aim of this work was to analyse the involvement of other factors to compensate bleeding risk in thrombocytopenic ITP patients. Moreover, the feasibility of using the coagulation global assays thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT) to test haemostasis in these patients was evaluated.Methods: Fifty patients with chronic ITP with platelet count less than 50 x 109/L and twenty-five 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 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP) and aliquots were stored at -70ºC until analysis.To assess the kinetics of clot formation, non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness,which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded.Plasma thrombin generation was measured in PPP using the Calibrated Automated Thrombogram (CAT) test at a final concentration of 1 pM tissue factor and 4 mM 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.Fibrinolytic proteins and E-selectin was tested in PPP using commercialized kits.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: PRP from ITP patients showed a prolonged CT (control: 550+ 95 sec, ITP: 890+165 sec, p<0.01), diminished alpha angle (control: 62.8+4.3, ITP: 53.5+7.5, p<0.05), and increased MCF (control: 46.7+3.1mm, ITP: 52.4+6.1 mm, p<0.05) and LI60 (control: 90.6+3.0% , ITP: 95.5+3.4, p<0.05) when compared with controls.In order to evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed between patients and healthy controls except for PAI-1 which level was increased in ITP patients (control: 14.7 ng/ml+11.7 ng/ml, ITP: 30.4+17.5, p<0.05). Since plasma PAI-1 might be increased as consequence of endothelial damage, plasma concentration of E-selectin, marker of endothelial injury, was determined. E-selectin was increased in samples from ITP patients (control: 10.5 ng/ml+3.9 ng/ml, ITP: 31.6+14.0, p<0.05). Moreover, MCF and LI60 ROTEM parameters correlated to E-selectin plasma concentration (Spearman r values 0.6643, p<0.001 for MCF; 0.6053, p<0.001 for LI60).Thrombin generation in PPP was also measured and a shorter time to peak (control: 9.3+1.2 sec, ITP: 8.3+1.7 sec,p<0.05) and increased ETP (control: 1223.8+257.7 nMxmin, ITP: 1696.4+524 nMxmin,p<0.05) and peak (control: 225.7+82.8.1 nM, ITP: 330.4+106.1 nM,p<0.05) were observed in ITP patients.Conclusions: We demonstrated that ITP patients presented a hypercoagulable profile that might be related, at least in part, to a reduced fibrinolysis mainly caused by an increase in PAI-1 level that seemed to be related to endothelial damage. Moreover ROTEM and CAT appeared to be useful tools for evaluating coagulant profile in ITP patients. DisclosuresNo relevant conflicts of interest to declare.

Synergistic Anticoagulant Effect of Activated Protein C and Tissue Factor Pathway Inhibitor As a Mechanism for Acute Traumatic Coagulopathy

IntroductionSevere trauma with tissue damage and shock can rapidly (<30 min) result in abnormal coagulation function which is independent of consumption or dilution effects; this acute traumatic coagulopathy (ATC) has been linked to increased transfusion requirements, morbidity and mortality. It has been suggested that ATC is caused by an increase in activated protein C (aPC; from 40 pM in normal to 175 pM) which has been identified in trauma patients [Cohen MJ, Ann Surg 255:379 (2012)].aPC inactivates factor Va (FVa) as part of normal hemostasis, but our previous results indicate that aPC at trauma levels is not sufficient to prevent coagulation, particularly when platelets are present, as platelet fVa is resistant to aPC [Campbell JE, PLoS ONE 9:e99181 (2014); Camire RM, Blood 91:2818 (1998)].Tissue Factor Pathway Inhibitor (TFPI) is an endothelial-bound protein which inhibits coagulation through effects on factor Xa and the factor VIIa-tissue factor complex. It is released in the plasma (normal level 2.5nM, pathophysiologic states including trauma up to 10nM) and is typically cleared by the liver or kidneys. TFPIα accounts for 20% of circulating TFPI and binds FV and FVa [Ndonwi M, J Thromb Haemost 10:1944 (2012)]. Recently, a truncated FV (termed FV-short) produced by an autosomal dominant mutation was found to form complexes with TFPIα, resulting in retention of TFPIα in a 10-fold increase over normal levels in affected individuals [Vincent LM, J Clin Invest 123:3777 (2013)]. FV-short has significantly reduced thrombogenic potential and, by concomitantly raising TFPIα, causes a bleeding disorder.We hypothesize that the activation of PC in acute trauma may result in the production of FV-degradation products which could stabilize TFPIα similarly to the effect of FV-short. The combination of reduced FVa and increased TFPIα may contribute to ATC.MethodsWhole blood from healthy volunteers was drawn by phlebotomy into ACD-containing tubes. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were collected by centrifugation (200g for 10 min and 1000g for 15 min, respectively). Calibrated automated thrombogram (CAT) assays and thromboelastography (TEG) were conducted using PRP and PPP with exogenously delivered aPC (0, 100pM, or 1nM) and TFPI (0, 2.5nM, or 10nM). An immunodepleted FV-deficient (<1% normal) plasma (FVdp) was used as a reference.ResultsCAT assays verified that aPC and TFPI each delay and suppress thrombin generation in PPP in a dose-dependent manner, but the combination of aPC and TFPI had a synergistic effect at the highest doses tested. The endogenous thrombin potential (ETP) was eliminated in PPP (control: 1663 nM-min; 1nM aPC + 10nM TFPI: 0 nM-min; P<0.001); lag time was similarly affected (control: 2.5 min; 1nM aPC + 10nM TFPI: >60 min; P<0.001). FVdp also featured a delayed lag time (11.17 min, P<0.001 versus control), but the delay induced by the addition of 10nM TFPI was not nearly as severe in FVdp (19.67 min) as in the PPP + 10nM aPC sample (>60 min, P<0.001). For PRP, there was no statistical difference between control and the highest doses of aPC and TFPI in ETP (1636 nM-min versus 1388 nM-min) or lag time (8.72 min versus 9.13 min).ConclusionsIn vitro studies suggest that PC activation is not the sole cause of ATC; the concentrations of aPC measured in trauma patient blood have little effect on the coagulation potential of plasma with or without platelets when aPC is delivered exogenously. The results here demonstrate that there is a synergistic effect between aPC and TFPI. When TFPI is added to FV deficient plasma, the thrombin generation is delayed and suppressed; however, in normal plasma digested with aPC (containing fragmented FVa), the addition of TFPI is sufficient to suppress the generation of thrombin beyond the window of measurement (>60 min). The addition of platelets to the milieu eliminated the effects of aPC, TFPI, and the combination on thrombin generation, highlighting the central role of platelets in maintaining hemostatic function. DisclosuresNo relevant conflicts of interest to declare.

Detecting Bleeding Risk in Patients Taking Vitamin K Antagonists Using Thrombin Generation Tests

Introduction:During the last 50 years vitamin K antagonists (VKAs) have been widely used for the primary prevention of thromboembolism in patients with atrial fibrillation, or for the secondary prevention in patients with a history of venous thromboembolism. The most prevalent adverse effect of anticoagulant therapy is an increased risk of bleeding. Annually approximately 1-4% of patients treated with VKAs suffer from major bleeding episodes.In the past several attempts have been made to predict the bleeding risk for these patients. Among the methods used at this moment are clinical decision scores, of which the HAS-BLED score is the most common one. Up to this point there are no laboratory methods available to predict patients at risk for bleeding. Thrombin generation (TG) tests have been shown to provide an intermediate phenotype for thrombosis. Additionally, TG has the capacity to detect the anticoagulant effect of many, if not all, anticoagulants, including VKAs. This method was only applicable in plasma, but recently this TG test has been introduced in whole blood (WB). With this study we aimed to investigate whether TG, in plasma or WB, could detect bleeding in patients taking VKAs.Materials & methods:Blood samples were collected in citrate tubes from 129 patients taking VKAs for over 3 months, who were older than 18 years of age and followed over time for bleeding episodes. The patients signed informed consent forms and the study was approved by the local medical ethical committee. TG was determined in WB, platelet rich plasma (PRP) and platelet poor plasma (PPP) by means of calibrated automated thrombinography (CAT). Tissue factor was used as a trigger at a final concentration of 1 pM in WB and PRP and at both 1 pM and 5 pM with 4µM phospholipids in PPP. Hematocrit and the hemoglobin concentration were determined in WB. The PPP of the patients was used to define the International normalized ratio (INR).Results:In our study the average INR value of all patients was 2.95 (± 0.92 (SD)). Twenty six patients (20.2 %) suffered from 44 clinically relevant bleeding episodes during a mean follow-up of 15.5 months.Applying TG in PPP and PRP we found no differences in either endogenous thrombin potential (ETP), an indication for the total amount of thrombin that can be converted, nor peak height (the highest amount of active thrombin present during coagulation). Interestingly, when we applied TG in whole blood, so including the blood cells, we found a significantly lower ETP (p < 0.01) and peak (p < 0.05) in the patients that suffered from bleeding (median: 182.5 nM.min and 23.9 nM, respectively) compared to patients that did not bleed (median: 256.2 nM.min and 39.1 nM). When examining the INR, hematocrit and hemoglobin levels, no significant differences were detected. A receiver operating curve (ROC) was constructed for the ETP and peak (figure 1). By determining the area under the curve (AUC) of the ROC we found that the ETP and peak are significantly (p < 0.05) associated with the tendency to bleed (ETP, AUC: 0.700; peak, AUC: 0.642). This compares favorably with the AUC reported for the HAS-BLED score related to clinically relevant bleeding in patients on VKAs (0.60).Conclusions:The TG test in whole blood was only recently developed and this is the first study implementing it to test patients using VKAs. In our study TG measured in WB proved to be the first laboratory test that is able to detect patients at risk of bleeding when treated with VKAs. The INR and plasma TG (CAT-based method), on the other hand, did not discriminate between bleeding and non-bleeding patients. For WB TG, based on the AUC of the ROC, the ETP and peak in WB have a predictive value for bleeding in patients taking VKAs superior over the currently used HAS-BLED score. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms.

Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from patients with sepsis. NETs released from phorbyl myristate-activated neutrophils enhance thrombin generation in platelet-poor plasma. This effect was DNA dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with coagulation factor XII- and coagulation factor XI-depleted plasma). In platelet-rich plasma treated with corn trypsin inhibitor, addition of phorbyl myristate-activated neutrophils increased thrombin generation and shortened the lag time in a toll-like receptor-2- and toll-like receptor-4-dependent mechanism. Addition of DNase further augmented thrombin generation, suggesting that dismantling of the NET scaffold increases histone-mediated, platelet-dependent thrombin generation. In platelet-poor plasma samples from patients with sepsis, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation. These studies examine the procoagulant activities of CFDNA and histones in the context of NETs. Our studies also implicate a role for the intrinsic pathway of coagulation in sepsis pathogenesis.

Abstract 569: Neutrophil Extracellular Traps Promote Thrombin Generation Through Platelet-Dependent and -Independent Mechanisms

Objective: Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) which are comprised of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from septic patients. Approach and Results: NETs released from phorbyl myristate (PMA) activated neutrophils enhance thrombin generation in platelet-poor plasma (PPP). This effect was DNA-dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with FXII- and FXI-depleted plasma). In platelet-rich plasma treated with corn-trypsin inhibitor, addition of PMA-activated neutrophils increased thrombin generation and shortened the lag time in a TLR-2- and TLR-4-dependent mechanism. Addition of DNase further augmented thrombin generation, suggesting that dismantling of the NET scaffold increases histone-mediated, platelet-dependent thrombin generation. In PPP plasma samples from septic patients, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation. Conclusions: These studies are the first to examine the procoagulant activities of CFDNA and histones in the context of NETs. Our studies also implicate a role for the intrinsic pathway of coagulation in sepsis pathogenesis.

Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice

Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 μg/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation.

Inhibition of tissue factor pathway inhibitor by the aptamer BAX499 improves clotting of hemophilic blood and plasma

Tissue factor pathway inhibitor (TFPI) is the major inhibitor of tissue factor-initiated coagulation, making it an interesting and novel therapeutic target in hemophilia treatment. The aptamer BAX499 (formerly ARC19499) is designed to improve hemostasis by specifically inhibiting TFPI. The aim of the study was to examine the concentration-dependent augmentation of clotting by BAX499. Whole blood clot formation was quantified by rotational thromboelastometry and thromboelastography, and thrombin generation in platelet-poor plasma was assessed with the calibrated automated thrombogram, in samples from patients with congenital hemophilia A (N=55) and B (N=11), patients with acquired hemophilia A (N=1), and healthy controls (N=37). BAX499 significantly improved clotting of samples from hemophilic patients in a concentration-dependent manner, resulting in clotting profiles in samples from patients with severe hemophilia that were similar to those of healthy controls. BAX499 improved ex vivo clotting parameters in blood and plasma from patients with hemophilia A and B with different severity of disease, and also in a patient with acquired hemophilia. These results further support the contention that anti TFPI strategies may be an effective treatment for hemophilic patients.

Impaired glucose metabolism and type 2 diabetes are associated with hypercoagulability: potential role of central adiposity and low-grade inflammation – The Hoorn Study

IntroductionType 2 diabetes (DM2) is associated with greater risk for cardiovascular disease (CVD), which may, at least partially, be explained by prothrombotic alterations. We therefore investigated; first, the extent to which individuals with impaired glucose metabolism (IGM) and/or DM2 had greater levels of thrombin generation than those with normal glucose metabolism (NGM); and second, whether any differences were independent of other cardiovascular risk factors, such as smoking, hypertension, dyslipidaemia, (micro)albuminuria, glycemic control and (central) adiposity, and/or were potentially ‘mediated’ by low-grade inflammation (high-sensitivity C-reactive protein (hsCRP)). Materials and methodsWe studied 744 individuals from the Hoorn Study (275 NGM, 176 IGM and 293 DM2, mean age 68.6±7.1years). Thrombin generation in platelet-poor plasma was measured using the Calibrated Automated Thrombogram and three parameters were derived: lag time, peak height and endogenous thrombin potential (ETP). Data were analyzed with multiple linear regression analyses. ResultsAfter adjustment for age, sex, prior CVD and smoking status, individuals with IGM or DM2 had a longer lag time [ß=0.14min (95% CI: 0.02; 0.26)], higher peak height [ß=7.29 nM (−1.33; 15.91)] and ETP [ß=35.65nM*min (0.97; 70.34)] than those with NGM. These differences were attenuated to ß=0.06min (−0.07; 0.19), 3.82nM (−5.46; 13.10) and 16.34nM*min (−20.92; 53.59), respectively, when further adjusted for waist circumference and hsCRP. ConclusionIndividuals with IGM or DM2 had up to 4% higher thrombin generation compared with NGM, which may be explained, to a great extent, by the greater levels of central adiposity and related low-grade inflammation characterizing these individuals.

The Anticoagulant Effect of Protamine Sulfate Is Attenuated in the Presence of Platelets or Elevated Factor VIII Concentrations

Protamine sulfate is the antidote for heparin, but in excess it exerts weak anticoagulation. We evaluated the effects of increasing protamine concentrations (0 to 24 microg/mL) on prothrombin time and diluted Russell's viper venom time measurements on thrombin generation in platelet-poor and platelet-rich plasma after activation by tissue factor or actin, and on thromboelastometry in platelet-poor plasma and whole blood from 6 healthy volunteers. The reversibility of excess protamine (24 microg/mL) by recombinant factor VIIa or factor VIII/von Willebrand factor concentrate was also tested. Protamine prolonged prothrombin time and Russell's viper venom time, concentration dependently. Protamine also increased lag time and decreased peak of thrombin generation in platelet-poor plasma after tissue factor and actin activation. In platelet-rich plasma with platelets at 50 to 200 x 10(3)/microL, protamine (24 microg/mL) prolonged the lag time, but had no effect on peak thrombin generation. The addition of factor VIII/von Willebrand factor (1.5-3.0 U/mL) to platelet-poor plasma with protamine (24 microg/mL) decreased lag time and increased peak thrombin generation with actin activation. A therapeutic concentration of recombinant factor VIIa (60 nM) only affected the lag time of thrombin generation triggered with actin. In agreement, protamine increased coagulation time evaluated by thromboelastometry significantly more in platelet-poor plasma than in whole blood. We demonstrated that protamine affects the propagation of thrombin generation, which is partially reversed by platelets or increased factor VIII/von Willebrand factor concentrations. The present data suggest that excess protamine might potentially increase bleeding in the case of severe thrombocytopenia or low factor VIII.

Abstract 5482: Unexpected Role for Connexin37 in Circulating FVIII and Coagulation

The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. Cx37 is expressed in endothelial cells, platelets and megakaryocytes. We have recently shown that Cx37 limits thrombus propensity by permitting intercellular signaling between aggregating platelets. Here, we have performed high throughput phage display to identify potential binding partners for the regulatory intracellular C-terminus of Cx37 (Cx37CT). We retrieved 2 consensus binding motifs for Cx37CT: WHK…[K,R]XP… and FH-K…[K,R]XXP…. Sequence alignment against the NCBI protein database indicated 66% homology of one the selected peptides with FVIII B-domain. We performed cross-linking reactions using BS3 and confirmed that an 11-mer peptide of the FVIII B-domain sequence linked to recombinant Cx37CT. In vitro binding of this peptide to Cx37CT was also confirmed by surface plasmon resonance. The dissociation constant of FVIII B-domain peptides to Cx37CT was ~20 uM. Other peptide sequences, designed upstream or downstream of the FVIII B-domain sequence, showed very low or no affinity for Cx37CT. Finally, in vivo studies revealed that thrombin generation in platelet-poor plasma from Cx37−/− mice (endogenous thrombin potential: 634±11 nM min, mean±SEM) was increased compared to Cx37+/+ mice (427±12, P&lt;0.001). Moreover, partial activated thromboplastin time (aPTT) was shorter in Cx37−/− (39.7±1.5 s) than in Cx37+/+ mice (45.9±1.8, P=0.03), whereas prothrombin time was comparable. The shorter aPTT in Cx37−/− mice correlated with higher circulating FVIII activity (46.0±0.7 vs. 53.5±2.7 s for Cx37+/+, P=0.03). Overall, our data show for the first time a functional interaction between FVIII and Cx37. This interaction may be relevant for the control of FVIII secretion and, thereby, in the regulation of levels of FVIII circulating in blood. In addition, these results may open new perspectives to improve the efficiency of recombinant FVIII manufacturing.