Calibrated Automated Thrombography Research Articles

Antiretroviral Therapy Does Not Correct the Increased Thrombin Generation and Platelet Hyperactivity Associated with HIV

Background: Human Immunodeficiency Virus (HIV) infection is associated with an increased risk of thrombosis, and treatment with antiretroviral therapy (ART) does not decrease this risk. While the mechanism leading to thrombosis is unclear, HIV infection is associated with decreased plasma anticoagulant proteins, most commonly protein S (PS), expression of tissue factor (TF) on monocytes, platelet hyperactivity, and thrombocytopenia. Despite the prevalence of PS deficiency, its pathologic consequences are unclear because PS concentration does not correlate with thrombin generation in the standard calibrated automated thrombography (CAT) assay. As increased plasma thrombin generation is a strong predictor of mortality in these patients, we sought to determine which of the described procoagulant effects of HIV is associated with thrombin generation in this population. Method: Blood was collected from 27 consenting HIV+ patients (16 naive samples collected from patients on first diagnosis and 11 samples from patients on ART) and 10 healthy controls. Results: 63% (17/27) of HIV+ plasma samples were deficient in PS, compared to the control samples, as determined by a total PS ELISA (p=0.034). PS functions as a cofactor for the anticoagulant enzyme activated protein C (APC), whose activation requires endothelial cell thrombomodulin (TM). Thus, to correlate plasma PS with thrombin generation, we established a modified CAT assay that is sensitive to PS concentration, by supplementing plasma samples with 20nM TM. Plasma PS did not correlate with thrombin generation in the absence of TM, consistent with previous reports. In the presence of TM, there was a negative correlation, with decreased PS being associated with increased thrombin peak height (p=0.004) and endogenous thrombin potential (p=0.003). Since 2.5% of total circulating PS is stored in platelets and released upon platelet stimulation, we quantified platelet PS by immunoblotting, and found no correlation with plasma PS concentration, consistent with the physiologically distinct origins of these two pools. HIV is reported to promote TF expression in monocytes, and monocytes can release TF-bearing microvesicles into plasma, so we also measured circulating TF activity, but found no difference between the patients and controls. As anticipated, TF correlated with a shorter lag time for thrombin generation, but only when no exogenous TF was added (p=0.0097). Treatment with ART resulted in undetectable viral load and also decreased plasma markers of inflammation, including TNF-α, IL-6, and IL-1β, to the levels observed in controls. However, there was no effect of ART on PS concentration (p=0.521), and platelet-leukocyte aggregates (PLA) remained elevated in patients on ART (19.66%) compared to controls (4.62%, p=0.0001). Platelet hyperactivity, as measured by PLA, was independent of platelet count, and did not correlate with platelet or plasma PS concentration. Interestingly, the use of ART had no impact on plasma PS or TF, but did correlate with greater thrombin generation in assays performed in the absence of TM, and thus the absence of PS activity (10% higher total thrombin, p=0.039), suggesting a PS-independent procoagulant effect of ART. Conclusion: PS deficiency in HIV patients correlates with higher thrombin generation and likely contributes to the thrombotic risk associated with this condition, while microvesicle TF, which does not differ from that seen in healthy controls, is unlikely to be a contributing factor. ART treatment decreases viral load and inflammation, but does not decrease platelet hyperactivity or restore plasma PS concentration and is not associated with any decrease in thrombin generation. Rather, one or more of the ART drugs appear to be further increasing thrombin generation through a PS-independent mechanism. In summary, it appears that multiple, independent factors contribute to the thrombotic risk associated with HIV, including platelet hyperactivity, plasma PS, and the ART treatment itself. Disclosures No relevant conflicts of interest to declare.

Role of Molecular Drivers in Coagulation By Integral Assessment in Primary Myelofibrosis Patients

Background: Thrombosis in Primary Myelofibrosis (PMF) is one of the main but not fully covered problem. Thrombotic complications in PMF occur more frequently than in general population and could result to morbidity and lethality of patients. Several previous studies have demonstrated different thrombotic rates related to type molecular driver mutation (JAK2V617F, CALR, MPL) in myeloproliferative neoplasms, but this fact now is not fully understood. The standard clotting tests cover only initial stages of coagulation and could not evaluate anticoagulant system. Its results are not varied in PMF patients with different mutations. We have interested and initiated study of the coagulation system in PMF patients with integral hemostasis test - Calibrated Automated Thrombography (CAT). Here we present the results of pilot study group assessment.Aim: to investigate of the coagulation system in PMF patients with different molecular driver mutations (JAK2V617F, CALR, MPL) using thrombin generation test.Methods: The study included 13 MF patients (26 - 71 years, median = 40). There were 11 females and 2 males. Nine patients were JAK2V617F positive, 3 had CALR mutations and one patient was MPL-mutated. Thrombin generation was assessed by calibrated automated thrombinography (CAT) according to Hemker et al. Assessments were conducted in platelet poor plasma (PPP) with or without presence of thrombomodulin (PPP reagent +/- TM) as an activator of protein C system. The following parameters were evaluated: endogenous thrombin potential (ETP, nM*min), peak thrombin (Peak, nM), lag-time (Lag, min), and time to peak (TTP, min). Sensitivity ETP and Peak for TM were calculated as percent of decreasing of these parameters after adding to assay of TM (S ETP, % and S Peak, % respectively). STATISTICA 6.0 package was used in data analysis. Results were presented as median (Me) with 95% confidence intervals (CI).Results: ETP was highest in JAK2V617F patients (1213.57; min 781.60-max 1781.26 nM*min), intermediate in CALR (1058.47; min 933.14-max 1190.79 nM*min) and lowest in MPL group (972.49 nM*min). However, the Peak was higher in CALR (207.57; min 165.63-max 235.36 nM) than in JAK2V617F (160.9; (min 94.38-max 317.11 nM) and MPL (136.19 nM). We have observed disruption of protein C functioning in JAK2V617F (S-ETP 34%, min 8.65%-max 68.73%; S-Peak 20%, min 0.01%-max 44.64%) and MPL (S-ETP 46%; S-Peak 24%) groups compared to CALR patients (S-ETP 6.4%, min 2.5%-max 9.2%; S-Peak 1.8%, min 0.87%-max 1.8%).Conclusion: The results of thrombin production were differed in relation to driver mutation type. JAK2V617F-patients produced more thrombin and have protein C system dysfunction. It could be serve as an explanation of higher risk of thrombosis in MF patients with JAK2V617F mutation. The yielded results force us to continue research to confirm our findings on representative sample of PMF patients. DisclosuresNo relevant conflicts of interest to declare.

Use of DOAC Stop for elimination of anticoagulants in the thrombin generation assay

Calibrated automated thrombography (CAT) is useful in monitoring the anticoagulant status of patients treated with direct oral anticoagulants (DOACs). This as well as other applications of the CAT are hampered by the wide inter-individual variation, making the diagnosis of the anticoagulant status of a patient on DOAC difficult when using normal pooled plasma as a reference. With dabigatran, the CAT is further hampered, as this direct thrombin inhibitor also inhibits the calibrator that is used in CAT. In this study we examined the added value of the universal DOAC adsorbent DOAC Stop in CAT. For this, we used normal pooled plasma spiked with apixaban, dabigatran, edoxaban or rivaroxaban, and performed CAT with 5 pM tissue factor. DOAC Stop effectively removed DOACs from plasma, leaving the DOAC Stop-treated plasma slightly more procoagulant compared to sham treated, non-anticoagulated plasma. Examining levels of natural coagulation inhibitors revealed a slight reduction in tissue factor pathway inhibitor upon DOAC Stop treatment. When DOAC Stop-treated plasma was used in the calibrator wells, normal unaffected calibration curves were observed, even when dabigatran was present. In conclusion, DOAC Stop can be used to abolish dabigatran influences of anticoagulated plasma when used in the calibrator wells. Also, the anticoagulant status of a DOAC treated patient can be diagnosed simply by comparing untreated plasma with the same plasma sample treated with DOAC Stop. Using this approach, a minor DOAC-independent increase in CAT response in the DOAC Stop-treated sample should be taken into account.

Abstract 524: Fibrinogen Depletion Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm

Background: Fibrinogen and fibrin provide physical and biochemical support to a developing clot and is defined as one of the most crucial independent risk factors for cardiovascular diseases (CVDs). In addition to clot formation, fibrinogen promotes wound healing and powerful inflammatory and immune responses by engagement of leukocytes. Increased circulating fibrinogen and fibrin degradation products are correlated with increased diameter and progression of abdominal aortic aneurysm (AAA). However, a causal link between fibrinogen and AAA has not yet been established. The objective of this study was to determine the role of fibrinogen depletion in a mouse model of AAA. Methods and results: To determine whether aneurysm resulted in a procoagulant environment, we examined plasma levels of thrombin generation by calibrated automated thrombography (CAT), thrombin anti-thrombin (TAT), and fibrinogen in control and AAA plasma from mice and humans. Patients and mice with AAA had significant elevations in thrombin generation, TAT, and fibrinogen versus saline controls (mice) and control patients (human). To determine the effect of fibrinogen, in vivo, low density lipoprotein receptor deficient ( Ldlr -/- ) mice were injected with scrambled anti-sense oligonucleotide (ASO) or β-fibrinogen ASO (30 mg/kg) 3 weeks prior to experimentation and throughout the study. Fibrinogen ASO treatment achieved > 90% depletion of fibrinogen. After 3 weeks, mice were fed a fat and cholesterol enriched diet (42% milk fat; 0.2% cholesterol) 1 week prior to and throughout infusion with angiotensin II (AngII; 1,000 ng/kg/day) for 28 days. Fibrinogen ASO attenuated abdominal diameter (33% decrease; P = 0.001), and inflammatory cytokines (>75% decreased IL-1 and IL-6; P = 0.001) versus scrambled ASO control. Further, fibrinogen depletion significantly attenuated aneurysm incidence and rupture-induced death (P < 0.05). Conclusions: Our results demonstrate that AAAs augment procoagulant markers in both humans and mice. Importantly, fibrinogen depletion attenuates AAA incidence, diameter, rupture-induced death, and inflammation. Therefore, reduction of plasma fibrinogen may be a novel treatment strategy in patients with AAA.

Analytical and between-subject variation of thrombin generation measured by calibrated automated thrombography on plasma samples*

Background: The Calibrated Automated Thrombography (CAT) is an in vitro thrombin generation (TG) assay that holds promise as a valuable tool within clinical diagnostics. However, the technique has a considerable analytical variation, and we therefore, investigated the analytical and between-subject variation of CAT systematically. Moreover, we assess the application of an internal standard for normalization to diminish variation.Methods: 20 healthy volunteers donated one blood sample which was subsequently centrifuged, aliquoted and stored at −80 °C prior to analysis. The analytical variation was determined on eight runs, where plasma from the same seven volunteers was processed in triplicates, and for the between-subject variation, TG analysis was performed on plasma from all 20 volunteers. The trigger reagents used for the TG assays included both PPP reagent containing 5 pM tissue factor (TF) and PPPlow with 1 pM TF. Plasma, drawn from a single donor, was applied to all plates as an internal standard for each TG analysis, which subsequently was used for normalization.Results: The total analytical variation for TG analysis performed with PPPlow reagent is 3–14% and 9–13% for PPP reagent. This variation can be minimally reduced by using an internal standard but mainly for ETP (endogenous thrombin potential). The between-subject variation is higher when using PPPlow than PPP and this variation is considerable higher than the analytical variation.Conclusion: TG has a rather high inherent analytical variation but considerable lower than the between-subject variation when using PPPlow as reagent.

Diminished coagulation capacity assessed by calibrated automated thrombography during acute Puumala hantavirus infection.

: Coagulation abnormalities are associated with Puumala-virus-induced hemorrhagic fever with renal syndrome (PUUV-HFRS). We evaluated the coagulation capacity of plasma during acute PUUV-HFRS by measuring thrombin generation using calibrated automated thrombography (CAT). The study cohort comprised 27 prospectively collected, consecutive, hospital-treated patients with acute PUUV infection. Blood samples were drawn in the acute phase and at the control visit approximately 5 weeks later. To evaluate thrombin generation, the lag time of initiation, endogenous thrombin potential (ETP), and peak and time to peak thrombin concentration were assessed by CAT in platelet poor plasma without corn trypsin inhibitor. Plasma levels of D-dimer, fibrinogen and prothrombin fragments (F1 + 2) were also evaluated. When the acute phase was compared with the control phase, ETP was decreased (median 1154 nmol/l/min, range 67-1785 vs. median 1385 nmol/l/min, range 670-1970; P < 0.001), while the lag time was prolonged (median 3.8 min, range 2.1-7.7 vs. median 2.9 min, range 2.0-4.1; P < 0.001). Low ETP correlated with low peak thrombin concentration (r = 0.833, P < 0.001). Prolonged time to peak associated with the lag time (r = 0.78, P < 0.001). ETP was associated with thrombocytopenia (r = 0.472, P = 0.015) and weakly with fibrinogen level (r = 0.386, P = 0.047). The measured CAT parameters did not associate with D-dimer and F1 + 2 levels. Decreased ETP together with low peak and prolonged lag time indicate decreased plasma potential for thrombin generation in vitro. Together with low platelet count and enhanced fibrinolysis, this further refers to altered blood coagulation and increased propensity toward bleeding in acute PUUV-HFRS.

Differing mechanisms of thrombin generation in live haematological and solid cancer cells determined by calibrated automated thrombography.

: Calibrated automated thrombography (CAT) is emerging as a reliable tool for real-time estimation of thrombin generation potential. There is a clinical need for knowledge about the pathways underlying the thrombotic phenotype of different malignancies. Cells from solid (e.g. pancreatic cancer; n = 7) and malignant haematological cell lines (e.g. multiple myeloma; n = 5) were evaluated for thrombin generation, using CAT, with the addition of control plasma (NormTrol; Helena Biosciences, Gateshead, UK)) or plasma deficient in coagulation factors VII and XII. In addition, tissue factor (TF) cell surface expression was determined by flow cytometry. In platelet-free plasma, thrombin generation in all cancer cell lines was cell concentration dependent, with the pancreatic cancer line CFPAC-1 producing the highest thrombin of 220 nmol/l at 5 × 10-cells/ml concentration. Lag times and times to peak reflected most significant differences out of all thrombin generation parameters measured and were inversely correlated with cell surface TF surface expression. Solid tumour cell lines had higher thrombin peaks, faster lag times, and a thrombin generation profile of overall greater magnitude than haematological cell lines. In the absence of factor VII in platelet-free plasma, thrombin generation in solid pancreatic cancer cell lines was significantly reduced unlike in haematological cell lines. However, in the absence of factor XII, thrombin generation was reduced more in haematological cells but had little or no effect on solid cell lines. The CAT assay identified characteristic differences in thrombin generation kinetics between solid tumour and haematological cancer cell lines, of which lag time and time to peak correlated with TF cell surface expression.

Implication of Free Fatty Acids in Thrombin Generation and Fibrinolysis in Vascular Inflammation in Zucker Rats and Evolution with Aging.

Background: The metabolic syndrome (MetS) and aging are associated with modifications in blood coagulation factors, vascular inflammation, and increased risk of thrombosis.Objectives: Our aim was to determine concomitant changes in thrombin generation in the blood compartment and at the surface of vascular smooth muscle cells (VSMCs) and its interplay with adipokines, free fatty acids (FFA), and metalloproteinases (MMPs) in obese Zucker rats that share features of the human MetS.Methods: Obese and age-matched lean Zucker rats were compared at 25 and 80 weeks of age. Thrombin generation was assessed by calibrated automated thrombography (CAT).Results: Endogenous thrombin potential (ETP) was increased in obese rats independent of platelets and age. Clot half-lysis time was delayed with obesity and age. Interleukin (IL)-1β and IL-13 were increased with obesity and age respectively. Addition of exogenous fibrinogen, leptin, linoleic, or palmitic acid increased thrombin generation in plasma whereas adiponectin had an opposite effect. ETP was increased at the surface of VSMCs from obese rats and addition of exogenous palmitic acid further enhanced ETP values. Gelatinase activity was increased in aorta at both ages in obese rats and MMP-2 activity was increased in VSMCs from obese rats.Conclusions: Our study demonstrated in MetS an early prothrombotic phenotype of the blood compartment reinforced by procoagulant properties of dedifferentiated and inflammatory VSMCs. Mechanisms involved (1) increased fibrinogen and impaired fibrinolysis and (2) increased saturated fatty acids responsible for additive procoagulant effects. Whether specifically targeting this hypercoagulability using direct thrombin inhibitors would improve outcome in MetS is worth investigating.

Effect of Hemodilution In Vitro with Hydroxyethyl Starch on Hemostasis.

BackgroundHydroxyethyl starch (HES) solutions are used for volume expansion during surgery. We aimed to investigate how 6%HES 130/0.4 affects hemostasis.Material/MethodsBlood samples were collected from 12 healthy adult volunteers, diluting with 6%HES 130/0.4 (HES group) or Ringer lactate solution (RL control group). The hemodilution ratio (HR) of citrated blood volume to plasma substitute volume was 10: 0 (undiluted), 10: 2, 10: 4, and 10: 6. Clotting factors activity was measured. Thrombin generation was monitored. Platelet function was analyzed.Results1) Activity of coagulation factor was decreased with increasing HR compared to undiluted baseline, and the activity of FVIII was significantly decreased in HES vs. RL. 2) Calibrated automated thrombography (CAT) results showed HES extended lag time, time to peak (ttpeak), start tail, and decreased peak of thrombin generation. Although lag time and ttpeak were significantly prolonged in HES vs. RL, endogenous thrombin potential (ETP) did not change. 3) Flow cytometric (FCM) analysis showed that HES reduced platelet phospholipids serine (PS) vs. baseline and RL. 4) HES significantly decreased antithrombin activity (AT: A) of the anticoagulant system with increasing HR vs. baseline and RL. 5) For fibrinolytic system, HES did not affect fibrinogen degradation products (FDP) and D-dimers (D-D) vs. baseline, or α2-antiplasmin (α2-AP) vs. RL.ConclusionsBy reducing FVIII activity and platelet PS expression, HES interfered with PS combining to FXIa, FVIIIa, and FVa, which affected the acceleration and explosion stage of thrombin. The decreased velocity and peak of thrombin generation delays and reduces clot formation. Combined 6%HES 130/0.4 decreased anticoagulant activity and may have clinical utility.

Near-Patient Thrombin Generation in Patients Undergoing Elective Cardiac Surgery.

Measuring thrombin generation (TG) in plasma increasingly gained attention as a diagnostic tool in the field of thrombosis and hemostasis. To include the contribution of all blood cells, recently, the whole blood TG method was developed. We changed the calculation method of the standard calibrated automated thrombography (CAT) to a method only taking into account the data until the peak of TG, thereby considerably reducing the time from blood draw to result. By redesigning the method, the blood volume per test was reduced to 15 μL. For all TG parameters, the interassay variation proved to be below 15%. The interindividual variation of all parameters was comparable to the CAT method. Thirty-three patients undergoing cardiothoracic surgery were included to investigate whether our assay correlates with postoperative blood loss. On dividing patients into severe and mild bleeders, significant differences between both groups were found for the peak endogenous thrombin potential (peakETP) and peak values determined by our near-patient device. Importantly, patients with a peakETP below the median experienced significantly more blood loss compared to those with a peakETP above the median. A similar division based on the peak as well as the body mass index of the patient yielded similar significant differences. A combination of the peakETP, the body mass index, and the lag time even resulted in a better predictor of blood loss compared to each parameter separately. Our adapted whole blood TG assay can be used near patients and is indicative for the amount of blood loss post cardiothoracic surgery.

Neutrophil Extracelluar Traps Contributes to the Hypercoagulable State and Liver Damage in Liver Cirrhosis

Background: We recently reported that phosphatidylserine on blood and endothelial cells played an important role in the hypercoagulable state of liver cirrhosis (Wu et al, Liver Int 2016). Recent studies showed that enoxaparin could reduce the incidence of portal vein thrombosis and alleviate liver decompensation. Thus, we speculate that there may be other mechanisms involved in the hypercoagulability of cirrhosis. Neutrophil extracelluar traps (NETs) play an important role in thrombosis and organ dysfunction. Whether the benefit of enoxaparin is due to inhibiting NETs in cirrhotic patients remains unknown. Our objectives were to study the formation of NETs and their role in the hypercoagulable state and liver damage in cirrhotic patients.Methods:Cirrhotic patients (n = 36), healthy controls (n = 10), and mice treated with CCl4 (n = 50) were studied. Immunofluorescence confocal microscope was used to analyze and quantify NETs formation in neutrophil cells from human and mice. TAT and fibrin formation assays in normal and cirrhotic plasma were performed to analyze the procoagulant activity of NETs. Calibrated automated thrombography (CAT) was performed with and without 6 nM thrombomodulin I to analyze TM resistance changes induced by NETs. Liver injury was assessed by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, determined spectrophotometrically, and by morphometric evaluation of left lateral liver lobes. DNase I and activated protein C (APC) were used to abolish NETs.Results: We observed that cirrhotic platelets induced 1.9 fold higher NETs formation in neutrophil cells (p = 0.021). Increased NETs promoted higher TAT (Fig 1A, B) and fibrin formation in normal and cirrhotic plasma (p < 0.05). DNase I abolished the procoagulant activity of NETs in corn trypsin inhibitor (CTI ) or anti-TF pre-treated plasma (p < 0.001) (Fig 1C). TM pre-induced with NETs for 24 h showed a higher endogenous thrombin potential (ETP) than no treatment in normal plasma using CAT (p = 0.032). APC and de-N-sulfated heparin (de-N-Hep), but not DNase I, can decrease the ETP ratio (with/without TM) in cirrhotic plasma (p < 0.05) (Fig 1D). Mice treated with CCl4 showed a higher ALT, AST, necrotic area (Fig 2), and NETs formation than control (p < 0.05). DNase I and enoxaparin decreased ALT, AST, necrotic area (Fig 2) and NETs formation (p < 0.05).Conclusions:Cirrhotic patients have enhanced NETs formation compared to healthy controls. NETs initiated intrinsic and extrinsic coagulation pathway and impaired anticoagulant function of TM. Additionally, NETs partly contributed to tissue injury in the liver of cirrhotic mice. DNase I and enoxaparin can disconnect NETs and decreased the toxin to liver. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Beneficial Role for Macitentan in Patients at High Thrombosis Risk: Attenuation of Platelet-Mediated Derangements of Blood Coagulation in Eisenmenger Syndrome

IntroductionEisenmenger syndrome is a rare manifestation of congenital cardiac disease with pulmonary hypertension. Potentially dangerous thrombotic and haemorrhagic complications frequently occur in patients with ES and raise significant clinical dilemmas. Underlying mechanisms are unknown but are thought to be linked to the underlying pulmonary vascular disease. Endothelin-1 (ET-1) signalling in the pulmonary vasculature is implicated in the pathogenesis of ES and Macitentan (a novel dual ET-1 receptor antagonist) has been shown to improve clinical outcomes. However the effect of Macitentan on reversing abnormal blood coagulation is unknown.We aimed to characterise derangements in physiological procoagulant and anticoagulant pathways in ES and to assess the effect of Macitentan therapy in modulating haemostasis in this disorder. We hypothesized that an enhanced understanding of pro-and anticoagulant mechanisms in these patients has the potential to improve risk stratification and targeting of antithrombotic therapy to patients at high risk of thrombosis.MethodsBlood was collected from subjects with ES and age/gender-matched healthy controls into citrated tubes, some containing corn trypsin inhibitor (CTI, a contact pathway inhibitor; final concentration 50µg/mL).Parameters of plasma thrombin generation in platelet-rich and platelet-poor plasma were assessed by calibrated automated thrombography (CAT) in the presence or absence of exogenous tissue factor (TF), anionic phospholipid (PL) and activated protein C (APC).ResultsPlatelet-dependent blood coagulation measured by CAT was enhanced in ES platelet-rich plasma (PRP) relative to healthy volunteers despite lower platelet counts: peak thrombin generation (TG) was significantly higher in ES subjects v controls in PRP (230.6±10.9 vs 188.3±13.1 nM; p=0.025) and lagtime to TG (6.1±0.3 vs 7.8±0.5 mins, p=0.005), time to peak TG (11.5±0.5 vs 15.2±0.6 mins, p=0.0003) and TG velocity index (43.9±3.7 vs 26.3±2.4 nM/min, p=0.001) were significantly accelerated. No significant differences in TG parameters in PPP were observed between groups suggesting a critical mechanistic role for platelet activity in supporting hypercoagulability in ES. TG in PRP was also found to be significantly enhanced in ES in the presence of CTI, suggesting that in vitro or in vivocontact pathway activation does not contribute to the enhanced coagulation activation observed in this disorder.The anticoagulant activity of APC is PL dependent and in healthy PPP incubated with exogenous PL (as a substitute for platelet PL), TG was potently inhibited. Conversely, in healthy PRP (in the absence of exogenous PL), APC activity was attenuated. Remarkably, this effect was not observed among the ES subjects, where APC anticoagulant activity was significantly increased in PRP, with significantly lower residual peak plasma thrombin generation and endogenous thrombin potential relative to that observed in healthy volunteer PRP. APC anticoagulant activity in PPP (both in the presence and absence of exogenous PL) was similar between groups. These findings suggest that platelets also modulate the activity of the APC pathway in ES, but in a manner which could promote bleeding.Among the 7 subjects who commenced macitentan therapy, at 6 months peak TG in PRP was significantly lower (234.5±9.8 vs 204.6±14.1 nM, p=0.002) and the enhanced sensitivity to APC in PRP was also attenuated. No significant difference in any parameter of TG was observed in PPP following macitentan therapy.ConclusionPlatelet activity appears to contribute significantly to hypercoagulability in ES but also appears to modulate the activity of endogenous anticoagulant pathways reflecting the clinical phenotype where bleeding and thrombotic risks frequently co-exist. Our findings suggest that targeted pharmacological agents such as the dual endothelin-1 antagonists (which crucially do not confer a bleeding risk) may attenuate platelet-mediated derangements in blood coagulation. Given the significant clinical dilemmas posed by the presence of competing thrombotic and haemorrhagic tendencies in ES, these finding are likely to be of significant translational relevance. DisclosuresWalsh:Actelion UK: Research Funding. Maguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

Endothelial Protective Properties of Low Molecular Weight Heparin (LMWH) Tinzaparin in Combination with Statin: A Novel Potential Therapeutic Tool in Preventing Cancer Metastasis?

Introduction8.2 million people die from cancer worldwide every year. LMWH prolongs survival in newly-diagnosed, limited stage lung cancer. Interestingly, trials recruiting patients with other cancers have also demonstrated improved survival in patients with long life-expectancy but not in advanced malignancy, suggesting that the benefit may be mediated by metastasis prevention. LMWHs inhibit metastasis in vivo but may cause bleeding complications when used at effective doses. Consequently, LMWH is unlikely to be used for metastasis prevention unless bleeding side-effects can be urgently addressed. Enhanced endothelial barrier (EB) permeability is a hallmark of metastasis and permits the extravasation of circulating tumour cells into the tissues of the target organ.AimsTo characterise LMWH Tinzaparin-mediated EB protection and underlying mechanisms and to optimise its cytoprotective properties in a manner that may reduce the associated bleeding risk.MethodsEA.hy926 endothelial monolayers were exposed to LMWH (including a novel LMWH formulation comprised of a fraction of LMWH tinzaparin consisting of short polysaccharide chains, mean MW 2.8KDa), simvastatin (a lipid-lowering agent shown to exhibit EB protective properties) and either metastasis-associated agonists (thrombin & VEGF) or calcein blue-AM labelled metastatic tumour cells (DU145, prostate carcinoma). EB function was assessed through the measurement of the migration of an Evans blue-conjugated albumin solution/labelled tumour cells through the monolayers. The role of PAR-1 signalling in modulating permeability was assessed by flow cytometry and permeability assays using an anti-PAR-1 cleavage site antibody and PAR-1 activating/inhibitory peptides. Endothelial myosin light chain-2 diphosphorylation status (MLC-2; the key determinant of endothelial cell actin cytoskeleton contraction) was determined by SDS-PAGE and western blotting. The anticoagulant activity of the LMWHs was determined by calibrated automated thrombography (CAT) and plasma anti-factor Xa activity assays.ResultsTinzaparin treatment prior to thrombin (IIa) and VEGF exposure significantly attenuated agonist-induced EB permeability in a concentration-dependent manner. This cytoprotective effect was observed within a clinically relevant concentration range (0-2IU/mL). Incubation of endothelial monolayers with LMWH also significantly reduced the migration of metastatic carcinoma cells (fluorescence of culture media containing labelled migrated tumour cells reduced to 73.9±5.7% of baseline; p<0.01).IIa-induced permeability was found to be entirely mediated through PAR-1 cleavage. IIa-induced permeability was lost in the presence of an anti-PAR-1 receptor antibody and a PAR-1 signalling inhibitory peptide. Tinzaparin did not alter EA.hy926 PAR-1 expression and did not inhibit IIa-mediated PAR-1 cleavage but reduced IIa-induced MLC-2 phosphorylation and attenuated PAR-1 activating peptide-induced permeability, suggesting that the protective effect of LMWH is not mediated through an inhibition of IIa proteolytic activity (and is therefore independent of its anticoagulant activity).The 2.8KDa LMWH fraction exhibited diminished anticoagulant activity relative to Tinzaparin but retained EB protective properties and attenuated tumour cell trans-migration (at a concentration equivalent to 0.5IU/mL, tumour cell migration was reduced to 76.4±4.7% of baseline and IIa-induced albumin permeability was attenuated to 66.9±8.9%).Remarkably, the cytoprotective properties of Tinzaparin were significantly enhanced when combined with a statin. The combination of a sub-anticoagulant concentration of Tinzaparin (0.1IU/mL) and a clinically relevant concentration of simvastatin (20nM), attenuated EB permeability to a significantly greater extent that that induced by each agent alone (IIa-induced EB permeability was reduced to 7.9±0.2% of baseline (p<0.05)).ConclusionLMWH enhances the barrier function of endothelium in vitro. This effect is not linked to its anticoagulant activity. Strategies which permit using either non-anticoagulant LMWH or sub-anticoagulant LMWH concentrations in combination with other barrier-protective agents may represent a means of delivering this potentially anti-metastatic activity of LMWH to patients without conferring a bleeding risk. DisclosuresMaguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

New Functional Assays to Selectively Quantify APC- and TFPI-Cofactor Activities of Protein S in Plasma

Background: Protein S is an important anticoagulant in the down-regulation of blood coagulation by acting as cofactor for two main regulators of coagulation. Protein S acts as a cofactor for activated protein C (APC) in the inactivation of the procoagulants FVa and FVIIIa and for TFPI in the formation of the TFPI-FXa encounter complex that subsequently inactivates TF-FVIIa. Several studies have indicated that protein S deficiency is associated with increased risks of venous thrombosis.Aims: Current protein S functional assays are influenced by plasma determinants such as FVLeiden. The aim of the study was to develop thrombin generation based assays that enable quantification of both the APC- and TFPI-cofactor activities of protein S in plasma whereas the outcome of the assays should not be affected by the presence of FVLeiden or other plasma variables.Methods: APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography (CAT) in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISA's.Results: Average APC-cofactor activities of protein S were 113%, 122%, and 87% in plasma from normal individuals (n=10), FV Leiden heterozygotes (n=4), and FV Leiden homozygotes (n=2), respectively, while the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n=21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 108%, 112%, and 97% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, while TFPI-cofactor activities correlated less with protein S antigen levels.Summary/Conclusion: We have developed functional protein S assays that measure both the TFPI- and APC-cofactor activities of protein S in plasma and that are not affected by the FV Leiden mutation or other plasma influences. DisclosuresNo relevant conflicts of interest to declare.

PP.16.24] EFFECTS OF ANTIPHOSPHOLIPID ANTIBODIES ON VASCULAR SMOOTH MUSCLE CELLS

Objective: Activation of platelets, monocytes and endothelial cells antiphospholipid antibodies (aPL) has been proposed as one of the pathogenic mechanisms contributing to the antiphospholipid syndrome (APS). We determined effects of aPL on characteristic functions of human vascular smooth muscle cells (hVSMCs). Design and method: Total IgG were purified from sera of 10 patients with APS by chromatography on protein G. Thrombin generation was monitored using calibrated automated thrombography (CAT) at the surface of adherent cultured normal hVSMCs incubated with IgG (250 μg/ml) from APS patients or healthy controls, and with or without activated protein C (APC). Proliferation of hVSMCs in response to stimulation by IgG from patients or controls was studied in medium without fetal bovine serum (FBS). Negatively-charged phospholipids were quantified in the cell supernatant. hVSMC microparticles (MPs) release was measured by flow cytometry in medium without FBS. Results: Thrombin generation was markedly increased after incubation with IgG from all patients irrespective of the aPL concentrations in sera compared to control IgG or to medium without IgG. The increase ranged from 2-fold to 4-fold for triple positive patients (anti-beta2GPI antibodies, anticardiolipin and lupus anticoagulant). In addition, IgG from the majority of APS patients induced a resistance to APC. For all patients, procoagulant activity increase without FBS in parallel with increased apoptosis (75%) compared with unstimulated cells or cells stimulated with IgG control. IgG of all patients induced significant increase of MPs released from hVSMCs (from 200% to 300%). Conclusions: The results show the activating effects of aPL on hVSMCs: thrombin generation increased and this hypercoagulability depends on aPL antibody profiles. The aPL-induced release of MPs and apoptotic cells induction may represent crucial mechanisms driving increased thrombin generation.

Abstract 531: In Patients with Uncontrolled Arterial Hypertension Platelets and Coagulation Factor XI are Responsible of Modifications of Thrombin Generation

Background: Hypertensive crisis is an extreme phenotype of increased blood pressure that can lead to organ failure and thrombotic complications. We recently found in experimental hypertension an angiotensin II driven factor XI (FXI)-thrombin amplification loop leading to vascular injury. Objective: We wanted to evaluate, in patients with uncontrolled hypertension (HT), the role of platelet and factor XI in thrombin generation (TG). Patients and methods: Prospectively, 52 patients with uncontrolled HT (16 grade I and 36 grade II/III) were examined at the outpatient clinic and emergency room of the University Medical Center Mainz, and 16 controls. Calibrated automated thrombography (CAT) was used to measure TG in platelet rich plasma (PRP, adjusted to 150.109/L of platelets), platelet free plasma (PFP), with platelets from controls and uncontrolled HT patients resupsended in a pool of healthy controls and in the presence or absence of an antibody blocking FXI activation pathway. Results: We found an increased systolic blood pressure in HT patients, compared to control patients (179±1.9 mmHg for grade II/III patients, 139±1.2 mmHg for grade I patients and 123±2.5 mmHg for controls); age, BMI as well as weight were not different. TG in PRP showed a positive and significant correlation between blood pressure and the other TG parameters as Endogenous thrombin potential (ETP), peak TG and velocity of TG. ETP was significantly increased in grade II/III patients compared to the control group. TG parameters in HT patients and controls were completely blunted by the inhibition of FXI pathway by blocking its apple 3 domain. This result indicates an involvement of the FXI thrombin loop in thrombin generation in PRP of uncontrolled HT patients. Without platelets TG showed no difference between HT patients and controls. Platelets resuspended from HT or controls restored the differences in ETP, peak and velocity between the 2 groups indicating that increased TG in HT patient mostly depends on platelet overreactivity. Conclusion: These results point out the important role of platelet overreactivity as well as FXI-dependent thrombin activation pathway in hypertension. Monitoring the prothrombotic state of platelets might add to risk stratification of patients with HT.

Procoagulant microparticles promote coagulation in a factor XI‐dependent manner in human endotoxemia

Essentials The procoagulant effects of microparticles (MPs) on coagulation in endotoxemia are not known. MPs from endotoxemia volunteers were evaluated for procoagulant activity in a plasma milieu. MPs from endotoxemia volunteers shortened clotting times and enhanced thrombin generation. MP procoagulant effects were mediated in a factor XI-dependent manner. Background Human endotoxemia is characterized by acute inflammation and activation of coagulation, as well as increased numbers of circulating microparticles (MPs). Whether these MPs directly promote coagulation and through which pathway their actions are mediated, however, has not been fully explored. Objectives In this study, we aimed to further characterize endotoxin-induced MPs and their procoagulant properties using several approaches. Methods Enumeration and characterization of MPs were performed using a new-generation flow cytometer. Relative contributions of the extrinsic and intrinsic pathways in MP-mediated procoagulant activity were assessed using plasmas deficient in factor (F) VII or FXI or with blocking antibodies to tissue factor (TF) or FXIa. Results Total MPs and platelet MPs were significantly elevated in plasma at 6 h after infusion of endotoxin in healthy human subjects. MPs isolated from plasma following endotoxin infusion also demonstrated increased TF activity in a reconstituted buffer system. When added to recalcified platelet-poor plasma, these MPs also promoted coagulation, as judged by a decreased clotting time with shortening of the lag time and time to peak thrombin using calibrated automated thrombography (CAT). However, the use of FVII-deficient plasma or blocking antibody to TF did not inhibit these procoagulant effects. In contrast, plasma clotting time was prolonged in FXI-deficient plasma and a blocking antibody to FXIa inhibited all MP-mediated parameters in the CAT assay. Conclusions The initiation of coagulation by cellular TF in endotoxemia is in contrast to (and presumably complemented by) the intrinsic pathway-mediated procoagulant effects of circulating MPs.

PO-27 - Thrombin generation in pancreatic cancer and multiple myeloma with use of calibrated automated thrombography

The calibrated automated thrombography (CAT) assay is emerging as a reliable tool for real time estimation of thrombin generation (TG) potential. As a time-dependent colorimetric assessment of thrombin quantity generated per sample, it measures the amount of thrombin-cleaved fluorogenic substrate produced, and so is regarded as a better overall indicator of the clotting efficiency and function of the haemostatic process than one stage clotting-time based assays. We already recognise that the pathways underlying the thrombotic phenotype for different malignancies may be driven by different factors of the coagulation cascade with TG has a common denominator. Two such malignancies with high venous thromboembolism (VTE) incidence are Multiple myeloma (MM) and Pancreatic cancer (PC). Understanding the underlying variations in these two distinct cancer models using patient samples and cell lines might potentially allow individual approaches to identifying thrombotic risk and relevant prevention strategies. Citrated blood samples were taken from healthy controls, pre-surgical pancreatic cancer and pre-chemotherapy multiple myeloma patients enrolled into ongoing clinical trials. The clotting ability was tested using platelet free plasma (PPP) on a one-step clotting time-based (CT) assay and the TG profiles were evaluated on a Thrombinoscope™ software (Thrombinoscope BV, Maastricht, Netherlands). Solid tumour cells of pancreatic cancer and malignant haematological cell lines were used at various cell concentrations for the CAT assay, which was performed with the addition of platelet-free control plasma or control plasma deficient in coagulation factors VII and XII. At TF concentration conditions of 1 pmol/L, the peak height of thrombin generated on thrombogram curves strongly correlated with CT of patient samples. Compared to healthy controls, pancreatic cancer had higher thrombin peaks (P), shorter lag times (LG), and an overall stronger TG profile than MM. Pancreatic cancer cell lines exhibited higher concentration-dependent TG profiles in control plasma than haematological cell lines, with higher peaks, endogenous thrombin potential (ETP), shorter lag times (LG) and faster times-to-peak (ttPeak). This study demonstrates that the CAT assay is a useful predictor of the thrombotic phenotype in cancer patients as it gives a more comprehensive overall coagulation profile than one stage CT-based assays. It shows that for patient samples and cell lines, the similarities and differences that exists in the TG potential, significantly depends on specific coagulation factors present in the intrinsic or extrinsic arms of the clotting cascade.

Thrombin generation and international normalized ratio in inherited thrombophilia patients receiving thromboprophylactic therapy

Thrombin generation assay (TGA) is useful as a global functional test for assessing bleeding or thrombotic risk and its modification with therapy. We investigated TGA to assess anticoagulation status compared with the international normalized ratio (INR) system in patients with primary thrombophilia receiving and not undergoing thromboprophylaxis. We studied 50 patients with at least one thrombotic event and a confirmed diagnosis of inherited thrombophilia. Thrombin generation was measured in platelet-poor plasma by calibrated automated thrombography (CAT). Patients in optimal anticoagulation (INR: 2.0-3.0) showed an endogenous thrombin potential (ETP) of 14-56% of normal and a peak of 18-55% of normal. A significant inverse relationship between INR and thrombin generation parameters (ETP, peak and velocity index) and a linear correlation for lag time was found in patients treated with vitamin-K antagonists (VKA). Receiver-operating characteristics (ROC) analysis showed that the optimal cutoff for ETP was 1600.2 nM · min (111.6% of normal, with a sensitivity of 96.6% and a specificity of 92.9%) and for the peak was 298.3 nM (112.1% of normal, with a sensitivity of 96.4% and a specificity of 100%). According to this analysis, ETP was able to identify patients with increased thrombotic and hemorrhagic risk, correlating with severe clinical complications. TGA showed excellent sensitivity and specificity for assessing anticoagulation status in patients with primary thrombophilia receiving VKA, with significant advantages with regard to INR. Clinical data strongly support ETP as a valuable indicator of thrombotic or hemorrhagic risk in patients receiving or not receiving thromboprophylaxis.

Effects of Exercise and Nutrition on the Coagulation System During Bedrest Immobilization.

Immobilization in hospitalized medical patients or during simulation of spaceflight induced deconditioning has been shown to be associated with loss of muscle mass and bone. Resistance vibrating exercise (RVE) and/or high protein diet are countermeasures, which are capable of mitigating the adverse effects of immobilization. We investigated the effect of these countermeasures on the coagulation system. Two groups of volunteers, each of whom performed such countermeasures, were enrolled in the study. Volunteers, who did nothing while bed rested, served as controls.The berest and the intervention protocols were carried out at Clinique d’ Investigation, MEDES, Toulouse, France.Eleven healthy men volunteered for this randomized crossover study. The subjects underwent 21 day of 6° head down bed rest (HDBR) followed by a washout period of 4 months. The first group followed an exercise schedule using resistance-vibrating exercise (RVE group). The second group also used the RVE but complemented it with high-protein supplement diet (NeX group). The third group only did bed rest.The highly sensitive methods calibrated automated thrombography (CAT) and thrombelastometry (TEM) were applied to monitor hemostatic changes.In all 3 groups, the hemostatic system shifted toward hypocoagulability during bed rest. For example, peak and thrombin formation velocity (VELINDEX) reduced in this period. Interestingly, a tendency toward hypercoagulation was observed during re-ambulation. In all 3 groups, ttPeak and StartTail were reduced, and Peak and VELINDEX (except in the RVE group) were significantly higher in relation to baseline values.Influence of bed rest on the coagulation system in the 2 groups performing countermeasures (RVE and NeX group) was the same as in the control bed-rested group. Clotting does not seem to be worsened by prolonged immobilization, or by countermeasures such as RVE/exercise or high-protein supplementation during immobilization. Therefore, only hospitalized medical patients at an elevated risk for thrombosis should be treated with anticoagulants. However, clinicians have to be aware that the re-ambulation period following immobilization might be associated with an elevated risk of thrombotic events.