Peak Thrombin Generation Research Articles

Factor XIII Contributes to Clot Formation and Thrombin Generation

BackgroundFactor XIII (FXIII) stabilizes fibrin clots, minimizing the amount of thrombin generation required to stop bleeding. It also has a long half-life (>7 days) and supratherapeutic levels are not associated with thrombosis making it an attractive therapeutic agent to augment factor replacement in hemophilia. FXIII is a transglutaminase that cross-links fibrin and localizes alpha 2-antiplasmin to fibrin inhibiting fibrinolysis. It also interacts with numerous other proteins, most of which have not been well studied and their physiologic significance is unknown. FXIII is classically considered to be activated by thrombin, which may limit its utility in low thrombin states such as hemophilia. However, activation also occurs physiologically through other mechanisms including calpain, neutrophil elastase and calcium influx. In addition, low levels of thrombin are generated by Factor Xa in the absence of the Factor VIII (FVIII)/Factor IX (FIX) complex that may contribute to both FXIII activation and clot formation. Thus, it is not clear if thrombin generated through the participation of FVIII and FIX is necessary for FXIII activation.HypothesisWe hypothesize that FXIII (recombinant FXIII-A2) may contribute to both clot formation and thrombin generation in hemophilia.MethodsAnimal models include Exon 16-deleted FVIII-deficient mice (FVIII-KO), wild-type mice (WT), and exon 16-deleted Factor VIII deficient/GP1bα-FVIII knock-in mice (PF). The PF mice are FVIII-deficient mice expressing human FVIII driven from the glycoprotein 1bα promoter resulting in approximately 3% circulating FVIII.Various combinations of factors were given via tail-vein injection. Citrated blood was collected by cardiac puncture 1.5 - 4 hours post-injection, depending on the factor type. Clotting was characterized using thromboelastography (TEG). Thrombin generation was measured on a fluorescence reader using a reagent comprised of a low concentration of phospholipid micelles containing tissue factor in HEPES buffer.ResultsTEG characterization shows differences in clotting times (R), speed of clot formation (K), and the kinetics of the formation of the clot (α angle) between the various groups without changes in overall clot stability (MA) or degree of fibrinolysis (LY30). R, K, and α angle are all measurements of clot formation, with prolongation of R and K and reduced α angle characteristic of hemophilia. PF mice have similar R, K, and α angle compared to FVIII-KO (p = 0.5, 0.68, and 0.89, respectively), and elongated R and K, and reduced α angle compared to WT (p = 4.0E-3, 2.0E-3, and 1.37E-6, respectively). Giving supratherapeutic FXIII to PF mice results in normalization of these values compared to WT, with a trend towards elongated K and α angle (p = 0.21, 0.08, and 0.13, respectively), and differences compared to FVIII-KO (p = 8.1E-3, 7.4E-3, and 2.1E-3, respectively). Administering FXIII to FVIII-KO mice did not alter R, K, and α angle compared to untreated FVIII-KO mice (p = 0.25, 0.37, and 0.67, respectively).PF mice have similar peak thrombin generation compared to FVIII-KO (p = 0.56) and reduced peak thrombin generation compared to WT (p = 6.69E-5). Giving supratherapeutic FXIII to the PF mice results in peak thrombin generation similar to that of WT (p = 0.97). In contrast, giving supratherapeutic FXIII to FVIII-KO mice did not alter peak thrombin generation levels compared to untreated FVIII-KO mice (p = 0.72). The administration of a cocktail containing both FVIII (2.5 U/kg) and FXIII resulted in a trend for improved peak thrombin generation when compared to an injection of FVIII alone (p = 0.12).ConclusionsThe function of FXIII has classically been considered to be secondary to its transglutaminase activity. With a direct impact on early clot formation and thrombin generation, these data suggest that FXIII has other roles beyond its known activities. The implication of these findings is that FXIII may be an effective hemostatic agent in mild and moderate hemophilia. DisclosuresTaylor:Novo Nordisk: Research Funding; Kedrion: Research Funding; Baxalta/Shire: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding.

Tissue Factor Pathway Inhibitor Mediates Attenuation of Tissue Factor-Stimulated Thrombin Generation in Early Onset Preeclampsia

IntroductionPregnancy increases the risk of venous thromboembolism (VTE) in women. Interestingly, preeclampsia, an extremely pro-inflammatory disorder specific to pregnancy is associated with a lower than expected increase in thrombotic risk compared to other proinflammatory disorders. The mechanism underling this lower thrombotic risk is unknown. The aim of this study was to investigate the coagulation balance, a major determinant of VTE risk, in early onset preeclampsia (EOP) patients. EOP is the most inflammatory form of preeclampsia, characterised by the development of hypertension and proteinuria prior to 34 weeks gestation.MethodsPlatelet-poor plasma was collected from patients with early onset preeclampsia (EOP n=26), matched pregnant (n=20) and non pregnant controls (n=16). Calibrated automated thrombography, an assay of thrombin generation and TFPI assays were performed. Data are expressed as mean ± standard deviation.ResultsDuring the study period, 15,299 women delivered and 40 patients developed EOP, of whom 26 were successfully recruited with consent. For comparison, 16 non pregnant controls and 20 pregnant controls were also recruited. When artefactual contact activation was inhibited by using corn trypsin inhibitor as anticoagulant, EOP patients were characterised by a decrease in the rate and extent of Tissue Factor (TF) thrombin generation compared to pregnant controls. There was a prolongation of the time to peak thrombin generation (16 ± 4 minutes vs. 13 ± 2 minutes, p < 0.05), a decrease in the velocity index (25 ± 17nM/minute vs. 41 ± 27nM/minute, p < 0.05), and a decrease in peak thrombin generation (150 ± 80 nM IIa vs. 210 ± 70 nM IIa, p < 0.05 ) in EOP compared to pregnant controls. This reduction in the rate and extent of thrombin generation was most amplified in patients with severe EOP (multi-organ involvement) compared with moderate EOP. This lower overall procoagulant state seen was further emphasised by the increase in sensitivity to the anticoagulant activity of exogenously added activated protein C and thrombomodulin observed in EOP. Again, the increased sensitivity to APC and thrombomodulin was most apparent in severe EOP cases. Previous studies have shown that preeclampsia is characterised by a increase in plasma TFPI activity. As such, we investigated whether increases in plasma TFPI activity explained the reduction in TF-dependent thrombin generation. Consistent with previous studies, plasma tissue factor pathway inhibitor (TFPI) levels and plasma TFPI activity significantly increased in EOP, most notably in severe EOP cases. There was a significant inverse correlation between total TFPI levels and peak thrombin generation (r2 = -0.64, p < 0.05) and TFPI activity and peak thrombin generation (r2= -0.52, p < 0.05). The inhibition of TFPI with a polyclonal anti-TFPI antibody abolished the attenuation in thrombin generation seen in severe EOP.ConclusionIn conclusion, TF-dependent thrombin generation is reduced in patients with early onset preeclampsia due to increases in plasma TFPI activity. These findings may partially explain the lower thrombosis risk observed in patients with early onset preeclampsia relative to comparable systemic proinflammatory conditions. These data also have future potential in helping clinicians to manage competing bleeding and thrombotic risks in these very high-risk patients. DisclosuresMaguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

Thrombin Generation and Microparticles As Promising Biomarkers for Venous Thromboembolism in Children with Central Venous Lines

Background: Pediatric hospital-acquired venous thromboembolism (HA-VTE) has increased over the past ten years (Raffini et al. Pediatrics 2009; 124; 1001-1008), with an associated increase in both VTE-associated complications and treatment-related adverse events. Children with VTE have a four-fold increase in length of admission compared to children without VTE. There are many known clinical risk factors but the presence of central venous lines (CVLs) has been shown to be among the strongest risk factors in children (Mahajerin et al. Haematologica 2015 Aug; 100(8): 1045-50). Current tools, including clinical risk-prediction scoring systems as well as genetic risk factors, are not sufficient to predict VTE risk at this time. Identification of dependable, rapid and cost-effective biomarkers for the prediction of VTE in children with CVLs is essential in guiding anti-coagulation prophylaxis, thus reducing VTE occurrence among hospitalized children with CVLs.Objectives: Determine the ability of thrombin generation (TG) and microparticle-based phospholipid-dependent procoagulant potential (MP-PPL) to predict VTE occurrence in hospitalized children with CVLs.Methods: Plasma sample were collected from 34 hospitalized acutely ill children between the age of 1 month and 21 years, who are at risk for VTE due to multiple known clinical risk factors, including a newly placed non-tunneled CVL. Children that had undergone a recent cardiac catheterization or on dialysis, plasmapheresis or ECMO were excluded. Samples from 16 age matched healthy controls were also collected. Venous samples were collected prior to CVL placement. Platelet poor plasma (PPP) was obtained according to a strict protocol to minimize pre-analytical variable. All pertinent clinical and laboratory information was extracted, including CVL-related variables and details of VTE in affected subjects. TG was measured by means of the calibrated automated thrombogram (CAT). MP-PPL was measured using two methods: 1) Functional assay that detects the ability of circulating MPs to indirectly facilitate thrombin generation (Zymuphen MP-Activity kit, Hyphen BioMed, France), 2) Clot-based assay that measures phospholipid-dependent, factor Xa initiated clotting time in relation to normal controls (STA-Procoag-PPL, Diagnostica Stago).Results: The median age of our cohort was 10 yrs (IQR 12, range 0 - 19), with a slight male predominance (53%) and increased representation of adolescents (41%). CVLs were placed in an intensive care setting in 38% of subjects and 39% of subjects had an underlying condition associated with chronic inflammation. The median duration of CVL placement from admission was 2 days (IQR 5, range 1 - 19) and the median total duration of CVLs were 14 days (IQR 20, range 5 - 84). Line-associated VTE occurred in 9% (3/34) of children with CVL and one subject had a VTE unrelated to CVL. There was no observed significant difference between children with or without line-related VTE in terms of age, gender, lag of CVL placement from admission, duration of CVLs, caliber of initial vessel of insertion, number of lumens, size of CVL and internal length of the CVL. However, all subjects with line-associated VTE were admitted for acute infection to an intensive care setting, with the majority of VTE occurring within 48 hours from line placement (2/3). Correlation analysis of the two MP-PPL assays was significant (r = -0.659). Even though the MP-PPL functional assay did not show a significant difference between subjects with and without line-associated VTE (p-value 0.189), the MP-PPL clot-based assay showed measurable difference between the two groups, both as an absolute value and as a ratio (p-value: 0.04 and 0.038 respectively, Table 1). Peak thrombin generation (p-value 0.001), estimated thrombin potential (ETP) - (p-value 0.02) and velocity index (p-value 0.001) were significantly higher in subjects with line-associated VTE (see Figure 1). Subject recruitment and sample analyses, involving these assays and other potential biomarker candidates, are ongoing.Conclusion: Given the rising incidence of pediatric VTE, the ability to use biomarkers to stratify patients according to their VTE risk will provide a vital tool to guide preventative efforts, and minimizing unnecessary expense and toxicities. TGA and MP-PPL have shown great potential for differentiating children with CVLs that may develop a line-related VTE as a HA-VTE. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Antibody to Tissue Factor Pathway Inhibitor (PF-06741086) in Combination with Recombinant Factor VIIa Increases Hemostasis in Hemophilia Plasma without Excessive Thrombin Generation

Hemophilia is a hereditary bleeding disorder caused by intrinsic coagulation pathway deficiencies of Factor VIII (hemophilia A) or Factor IX (hemophilia B). Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. In hemophilia patients the extrinsic pathway remains intact and thus augmentation of this pathway may circumvent the clotting deficiency in hemophilia. PF-06741086, a monoclonal antibody that binds to and neutralizes the inhibitory activity of TFPI is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors.Currently, treatment of inhibitor patients is managed by bypass treatments, such as recombinant Factor VIIa (rFVIIa). The effect of PF-06741086 on thrombin generation in the presence of increasing concentrations of rFVIIa (0.0002 to 20 µg/mL) was studied in severe hemophilia A plasma. A dose-dependent increase in thrombin generation was observed over vehicle control with the addition of rFVIIa to the hemophilia plasma. Addition of a fixed concentration of PF-06741086 (16 µg/mL) in combination with rFVIIa resulted in an increase in thrombin generation including higher peak thrombin and shortening of lag time compared to rFVIIa alone. The TGA profiles with the combination of PF-06741086 and rFVIIa at 0.2, 2, and 20 µg/mL were similar suggesting a saturation of mechanism at these concentrations. The combination of PF-06741086 and rFVIIa restored thrombin generation to normal plasma levels at all rFVIIa concentrations examined.The TFPI inhibitory activity of PF-06741086 on thrombin generation in the presence and absence of rFVIIa was further studied in additional hemophilia A plasmas, including hemophilia A plasmas with inhibitors and hemophilia B plasma. All donors had less than 1% coagulation factor activity. A rFVIIa concentration of 2 µg/mL was selected because it corresponded to plasma levels that could be observed following dosing of FVIIa and because the thrombin generation response in hemophilia plasma was similar with FVIIa added to hemophilia A plasma at 0.2, 2 and 20 µg/mL. The concentration of PF-06741086 was 16 µg/mL in these studies. The effect of PF-06741086 on thrombin generation was also measured in non-hemophilic plasma which would have the full complement of coagulation factors. The addition of PF-06741086 alone or in combination with rFVIIa to hemophilia A and B plasma resulted in an increase in thrombin generation including higher peak thrombin concentration and shortening of lag time compared to addition of rFVIIa alone. In hemophilic plasma samples with inhibitors (3 - 1261 Bethesda Units), PF-06741086 alone also restored thrombin generation. A minimal additive effect in peak thrombin generation was observed with the combination of PF-06741086 (16 µg/mL) and 2 µg/mL rFVIII. The midpoint peak thrombin levels achieved with PF-06741086 alone or in combination with rFVIIa were similar to those observed in non-hemophilic plasma and did not exceed the level observed in non-hemophilic plasma dosed with PF-06741086. To summarize, use of rFVIIa in combination with PF-06741086 results in increased thrombin generation in hemophilia A, hemophilia B and inhibitor plasmas without inducing excessive coagulation. DisclosuresRakhe:Pfizer: Employment. Hett:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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.

Natural IgM Against Oxidation-Specific Epitopes Inhibit Microvesicle-Driven Coagulation

Increasing evidence demonstrates an important role for immune functions in coagulation and thrombosis, and both microbial and sterile triggers have been implicated in these responses. Oxidation-specific epitopes (OSE) are products of lipid peroxidation that are present on oxidized low-density lipoproteins and dying cells, and represent important sterile triggers of inflammation in cardiovascular diseases. We have previously shown that a large part of naturally occurring IgM antibodies have specificity for OSEs and that 50% of circulating procoagulant microvesicles (MV) carry OSEs, such as malondialdehyde-adducts (MDA), on their surface. Because OSE-specific natural IgM bind MVs and increased levels of circulating MVs have been implicated in an increased thrombotic risk, we studied the capacity of these antibodies in directly modulating the procoagulant potential of MVs in vitro and in vivo. We first showed that increasing concentrations of three different monoclonal IgM antibodies with specificity for MDA (LR04, NA17, E014) significantly delayed MV-induced fibrin formation in plasma, while an MDA-specific IgG antibody or F(ab')2 fragments of LR04 had no effect. Moreover, peak thrombin generation of normal, factor VII- or factor XII- deficient microvesicle poor plasma (MVPP) substituted with tissue factor positive MVs was significantly reduced in the presence of LR04 but not a control IgM antibody, indicating that it exerts its effect on the common pathway of the coagulation cascade. A similar anticoagulatory effect of LR04 was observed in freshly drawn whole blood analyzed by rotational thrombelastometry. Addition of LR04, compared to a control IgM antibody, increased clotting time, clot formation time, and the time to reach maximum clot firmness, and decreased maximum clot firmness. Finally, in a murine pulmonary embolism model, we could show that 30-minute survival of mice that received intravenous injections of epinephrine together with pro-coagulant MVs derived from the pancreatic cancer cell line HPAFII was significantly increased in mice co-injected with LR04 compared to controls. Our study identifies an inhibitory role of MDA-specific IgM antibodies in MV-induced coagulation. These effects are exerted on the common pathway of the coagulation cascade and require the intact structure of IgM antibodies. The findings may offer novel diagnostic and therapeutic approaches in the prevention and treatment of thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 13514: Serial Quantitative Assessment of Thrombogenicity in LVAD Recipients: A Case for Personalized Antithrombotic Therapy

Introduction: Bleeding and thrombotic complication affect between 25-40% of left ventricular assist device (LVAD) recipients. Measures of thrombogenicity such as maximum platelet-fibrin clot strength (MAKH), endogenous thrombin potential (ETP) and platelet reactivity may indicate thrombotic and bleeding risk in patients on LVAD support and may vary after LVAD implantation. Methods: We prospectively assessed thrombogenicity using the novel point-of-care (TEG-6S) with PlateletMapping and the calibrated automated thrombogram (CAT) in 31 patients before and serially after LVAD implantation (HeartWare HVAD, n = 17; Heartmate II, n = 14). All LVAD patients were on aspirin 325mg/daily and warfarin (INR 2.0 to 2.5) therapy. Standard hematologic and hemolysis laboratories were compared. Results: Markers of thrombogenicity were highly variable post-LVAD. Overall, in the acute phase, MAKH, and maximum ADP-, and arachidonic acid (AA)-induced clot strength increased (p &lt; 0.05, Table) and returned to near baseline by ≥3 months. Despite target INR values at ≥ 3 months (2.0±0.3) and antiplatelet therapy, the R-time (7.1±1.8min) remained within the normal range. Hypercoagulable patients (MAKH≥69mm) had higher endogenous thrombin potential, peak thrombin generation, WBC count, and LDH (p≤0.05). Conclusions: A state of heightened thrombogenicity indicated by high MAKH and high platelet reactivity are present in the acute phase after LVAD implantation and in selected patient during the chronic phase despite therapy with warfarin/aspirin. These findings suggest that intensification of antithrombotic therapy in a personalized fashion based on sensitive thrombogenicity testing, rather than INR should be investigated to mitigate adverse events.

Ex vivo reversal of effects of rivaroxaban evaluated using thromboelastometry and thrombin generation assay

BackgroundIn major bleeding events, the new direct oral anticoagulants pose a great challenge for physicians. The aim of the study was to test for ex vivo reversal of the direct oral anticoagulant rivaroxaban with various non-specific reversal agents: prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and fibrinogen concentrate (FI). MethodsBlood was obtained from healthy volunteers and from patients treated with rivaroxaban. Blood samples from healthy volunteers were spiked with rivaroxaban to test the correlation between rivaroxaban concentration and coagulation tests. Patient blood samples were spiked with various concentrations of the above-mentioned agents and analysed using thromboelastometry and thrombin generation. ResultsWhen added in vitro, rivaroxaban was significantly (P<0.05) correlated with ROTEM® thromboelastometry EXTEM (extrinsic coagulation pathway) clotting time (CT), time to maximal velocity (MaxV−t), and with all measured thrombin generation parameters. In vivo, CT, MaxV−t, lag time, and peak thrombin generation (Cmax) were significantly correlated with rivaroxaban concentrations. Regarding reversal of rivaroxaban, all tested agents significantly (P<0.05) reduced EXTEM CT, but to different extents: rFVIIa by 68%, aPCC by 47%, PCC by 17%, and FI by 9%. Only rFVIIa reversed EXTEM CT to baseline values. Both PCC (+102%) and aPCC (+232%) altered overall thrombin generation (area under the curve) and increased Cmax (+461% for PCC, +87.5% for aPCC). ConclusionsThromboelastometry and thrombin generation assays do not favour the same reversal agents for rivaroxaban anticoagulation. Controlled clinical trials are urgently needed to establish doses and clinical efficacy of potential reversal agents. Clinical trial registrationEudracCT trial no. 213-00474-30.

Only minor changes in thrombin generation of children and adolescents with type 1 diabetes mellitus – A case-control study

BackgroundMicro- and macrovascular diseases are frequent complications in patients with diabetes. Hypercoagulability may contribute to microvascular alterations. ObjectiveIn this study, we investigated whether type 1 diabetes in children is associated with a hypercoagulable state by performing a global function test of coagulation – the thrombin generation assay. Subjects75 patients with type 1 diabetes aged between 2 and 19years were compared to an age-matched healthy control group. Diabetes patients were divided into high-dose and low-dose insulin cohorts with a cut-off at 0.8Ukg−1d−1. MethodsMeasurements were performed with platelet poor plasma using Calibrated Automated Thrombography and 1 pM or 5 pM tissue factor. Additionally, we quantified prothrombin fragments F1+2, thrombin-antithrombin complex, prothrombin, tissue factor pathway inhibitor, and antithrombin. ResultsPatients with type 1 diabetes exhibited a significantly shorter of lag time as well as decreased thrombin peak and endogenous thrombin potential compared to control subjects with 5 pM but not with 1 pM tissue factor. In high-dose insulin patients peak thrombin generation was higher and time to peak shorter than in low-dose patients. Thrombin-antithrombin complex was decreased in patients with type 1 diabetes, whereas prothrombin fragments F1+2 was comparable in both groups. Thrombin generation parameters did not correlate with parameters of metabolic control and the duration of diabetes. ConclusionsTaken together, we found only minor changes of thrombin generation in children and adolescents with type 1 diabetes which - in contrast to type 2 diabetes - do not argue for a hypercoagulable state.

Early thrombin formation capacity in trauma patients and association with venous thromboembolism

Incidence of venous thromboembolism (VTE) in adult trauma patients is high despite mechanical and pharmacologic prophylaxis. We hypothesized that thrombin formation capacity as measured by calibrated automated thrombogram (CAT) is increased early in hospitalization and is associated with the development of VTE. MethodsWe conducted a prospective study in adult, critically ill trauma patients. Plasma was generated from whole blood samples collected within the first 3days of hospital admission. CAT was used to determine lag time, thrombin peak, time to thrombin peak, endogenous thrombin potential (ETP), and velocity index in plasma samples from patients, and in control samples of platelet-poor, pooled normal plasma. ResultsThere were 35 trauma patients and 35 controls included in this pilot analysis. Patients were a mean (SD) age of 45 (19) years, and 23 (66%) were male. The most common mechanism of injury was motor vehicle crash followed by falls, and the median (IQR) injury severity score was 17 (12–27). Three patients (8.6%) had deep vein thrombosis (DVT) confirmed by Doppler ultrasound on median hospital day 7. Compared to control samples, patients had significantly longer lag times (3.1min vs. 2.7min, p=0.02) and significantly higher ETP (1136nM∗min vs. 1019nM∗min, p=0.007), peak thrombin generation (239nM vs. 176nM, p<0.001), and velocity index (108nM/min vs. 57nM/min, p<0.001) (Fig. 1). There was no difference in the time to peak thrombin generation between the two groups (5.5min vs. 5.7min, p=0.22). In the 3 patients with VTE compared to controls, lag times were shorter and velocity index was higher while ETP and peak thrombin generation were similar. There were no statistically significant differences in thrombin generation parameters in patients with or without VTE, but lag time was numerically shorter, and thrombin peak, time to peak and area-under-the-curve (ETP) were numerically lower in patients with DVT. ConclusionsWe observed a thrombin generation profile in critically ill trauma patients consistent with an early hypercoagulable state; however, thrombin generation parameters did not discriminate patients with VTE.

Evaluation of thrombin generation in classical Philadelphianegative myeloproliferative neoplasms / Evaluarea generării trombinei în neoplasmele mieloproliferative Philadelphia- negative

Abstract Introduction: Patients with Philadelphia-negative chronic myeloproliferative neoplasms (Ph-MPN), polycytemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), are prone to develop thrombotic events. We aimed to investigate the coagulation status in their plasma using thrombin generation assay (TGA), a functional global assay, on Ceveron® Alpha. Materials and methods: The samples were collected from 89 consecutive Ph-negative MPN patients and from 78 controls into K2EDTA and CTAD tubes for blood cell counts, TGA and coagulation screening tests. Thrombin generation was analysed in platelet-poor plasma using Technothrombin® TGA assay kit. Results: We found a significantly increased peak thrombin generation (p=0.049) and velocity index (VI) (p=0.012) in patients in comparison with controls, especially in ET patients, and a significantly higher values for peak thrombin (p=0.043) and VI (p=0.042) in patients receiving anagrelide in comparison with those treated with hydroxyurea. We also noticed an inverse correlation between the length of cytoreductive therapy and TGA parameters, (peak thrombin R=-0.25, p=0.018, AUC R=-0.257, p=0.015, and VI R=-0.21, p=0.048). Conclusion: Our results suggest that Ph-MPN patients, and especially those with ET, are predisposed to thrombotic events due to their higher peak thrombin and VI values and their risk may decreases as treatment is longer. Patients treated with hydroxyurea generate less thrombin and could be less prone to develop thrombotic events in comparison with those treated with anagrelide.

Rivaroxaban versus warfarin to treat patients with thrombotic antiphospholipid syndrome, with or without systemic lupus erythematosus (RAPS): a randomised, controlled, open-label, phase 2/3, non-inferiority trial

SummaryBackgroundRivaroxaban is established for the treatment and secondary prevention of venous thromboembolism, but whether it is useful in patients with antiphospholipid syndrome is uncertain.MethodsThis randomised, controlled, open-label, phase 2/3, non-inferiority trial, done in two UK hospitals, included patients with antiphospholipid syndrome who were taking warfarin for previous venous thromboembolism, with a target international normalised ratio of 2·5. Patients were randomly assigned 1:1 to continue with warfarin or receive 20 mg oral rivaroxaban daily. Randomisation was done centrally, stratified by centre and patient type (with vs without systemic lupus erythematosus). The primary outcome was percentage change in endogenous thrombin potential (ETP) from randomisation to day 42, with non-inferiority set at less than 20% difference from warfarin in mean percentage change. Analysis was by modified intention to treat. Other thrombin generation parameters, thrombosis, and bleeding were also assessed. Treatment effect was measured as the ratio of rivaroxaban to warfarin for thrombin generation. This trial is registered with the ISRCTN registry, number ISRCTN68222801.FindingsOf 116 patients randomised between June 5, 2013, and Nov 11, 2014, 54 who received rivaroxaban and 56 who received warfarin were assessed. At day 42, ETP was higher in the rivaroxaban than in the warfarin group (geometric mean 1086 nmol/L per min, 95% CI 957–1233 vs 548, 484–621, treatment effect 2·0, 95% CI 1·7–2·4, p<0·0001). Peak thrombin generation was lower in the rivaroxaban group (56 nmol/L, 95% CI 47–66 vs 86 nmol/L, 72–102, treatment effect 0·6, 95% CI 0·5–0·8, p=0·0006). No thrombosis or major bleeding were seen. Serious adverse events occurred in four patients in each group.InterpretationETP for rivaroxaban did not reach the non-inferiority threshold, but as there was no increase in thrombotic risk compared with standard-intensity warfarin, this drug could be an effective and safe alternative in patients with antiphospholipid syndrome and previous venous thromboembolism.FundingArthritis Research UK, Comprehensive Clinical Trials Unit at UCL, LUPUS UK, Bayer, National Institute for Health Research Biomedical Research Centre.

Plasma Thrombin Generation and Sensitivity to Activated Protein C Among Patients With Myeloma and Monoclonal Gammopathy of Undetermined Significance

The etiology of the prothrombotic state in myeloma has yet to be definitively characterized. Similarly, while recent evidence suggests that patients with monoclonal gammopathy of undetermined significance (MGUS) may also be at increased risk of thrombosis, the magnitude and the etiology of this risk have also yet to be defined. The present study aims to characterize patterns of plasma thrombin generation and sensitivity to the anticoagulant activity of activated protein C (APC) at the time of initial diagnosis of myeloma and in response to therapy in comparison to that observed among patients with MGUS and matched, healthy volunteers. Patients presenting with newly diagnosed/newly relapsed myeloma (n = 8), MGUS (n = 8), and matched healthy volunteers (n = 8) were recruited. Plasma thrombin generation was determined by calibrated automated thrombography. Peak thrombin generation was significantly higher in patients with myeloma (383.4 ± 33.4 nmol/L) and MGUS (353.4 ± 16.5 nmol/L) compared to healthy volunteers (276.7 ± 20.8 nmol/L; P < .05). In the presence of APC, endogenous thrombin potential was significantly lower in control plasma (228.6 ± 44.5 nmol/L × min) than in either myeloma (866.2 ± 241.3 nmol/L × min, P = .01) or MGUS plasma (627 ± 91.5 nmol/L × min, P = .003). Within the myeloma cohort, peak thrombin generation was significantly higher at diagnosis (353.2 ± 15.9 nmol/L) than following completion of the third cycle of therapy (282.1 ± 15.2 nmol/L; P < .005). Moreover, sensitivity to APC increased progressively with each cycle of chemotherapy. Further study of the etiology and evolving patterns of hypercoagulability among patients with these conditions is warranted and may have future implications for thromboprophylaxis strategies.

A Snapshot of Coagulopathy After Cardiopulmonary Bypass.

Cardiac surgery involving cardiopulmonary bypass (CPB) is often associated with important blood loss, allogeneic blood product usage, morbidity, and mortality. Coagulopathy during CPB is complex, and the current lack of uniformity for triggers and hemostatic agents has led to a wide variability in bleeding treatment. The aim of this review is to provide a simplified picture of the data available on patients' coagulation status at the end of CPB in order to provide relevant information for the development of tailored transfusion algorithms. A nonsystematic literature review was carried out to identify changes in coagulation parameters during CPB. Both prothrombin time and activated partial thromboplastin time increased during CPB, by a median of 33.3% and 17.9%, respectively. However, there was marked variability across the published studies, indicating these tests may be unreliable for guiding hemostatic therapy. Some thrombin generation (TG) parameters were affected, as indicated by a median increase in TG lag time of 55.0%, a decrease in TG peak of 17.5%, and only a slight decrease in endogenous thrombin potential of 7%. The most affected parameters were fibrinogen levels and platelet count/function. Both plasma fibrinogen concentration and FIBTEM maximum clot firmness decreased during CPB (median change of 36.4% and 33.3%, respectively) as did platelet count (44.5%) and platelet component (34.2%). This review provides initial information regarding changes in coagulation parameters during CPB but highlights the variability in the reported results. Further studies are warranted to guide physicians on the parameters most appropriate to guide hemostatic therapy.

PS110 GSTM1 Null Genotype Isn’t Associated With Increased Risk of Chronic Heart Failure Among Patients With Diabetes Mellitus

Concizumab is a humanized mAb targeting tissue factor pathway inhibitor (TFPI), leading to enhanced thrombin generation (TG) potential. explorer™3 (NCT02490787) was a phase 1b, double‐blind, multiple‐dose escalation trial of subcutaneous concizumab in people with severe hemophilia A without inhibitors.The primary objective was to evaluate safety. Assessments of pharmacokinetics, pharmacodynamics and subcutaneous concizumab immunogenicity were secondary objectives.Adverse events (AEs), clinical assessments and bleeding episodes were recorded. Plasma concizumab levels and unbound TFPI levels were measured with ELISAs; residual TFPI activity was measured with a chromogenic assay. Standardized assays were used to assess TG, D‐dimer and prothrombin fragment 1 + 2 (F1 + 2) levels. explorer™3 was completed after investigation of three dose cohorts (0.25, 0.5 and 0.8 mg kg−1, once every 4 days) had been completed. Twenty‐four patients received 12 doses of concizumab or placebo in a 3 : 1 randomization over a 42‐day period.No serious AEs and no anti‐drug antibodies were observed. Fifty‐four mild and two moderate AEs were observed in 19 patients. Concizumab exposure increased with dose in a non‐linear manner, confirming target‐mediated drug disposition. D‐dimer and F1 + 2 levels were increased mostly in the highest dose cohort, in line with previous observations. The level of unbound TFPI decreased in a dose‐dependent manner, and was accompanied by a residual TFPI activity decrease and an increase in peak TG. Although the trial was not powered to evaluate efficacy, a trend towards lower bleeding rates was observed in patients in the highest dose cohort.explorer™3 data support further clinical development of concizumab for use in people with hemophilia, with or without inhibitors.

Differential Contributions of Intrinsic and Extrinsic Pathways to Thrombin Generation in Adult, Maternal and Cord Plasma Samples.

BackgroundThrombin generation (TG) is a pivotal process in achieving hemostasis. Coagulation profiles during pregnancy and early neonatal period are different from that of normal (non-pregnant) adults. In this ex vivo study, the differences in TG in maternal and cord plasma relative to normal adult plasma were studied.MethodsTwenty consented pregnant women and ten consented healthy adults were included in the study. Maternal and cord blood samples were collected at the time of delivery. Platelet-poor plasma was isolated for the measurement of TG. In some samples, anti-FIXa aptamer, RB006, or a TFPI inhibitor, BAX499 were added to elucidate the contribution of intrinsic and extrinsic pathway to TG. Additionally, procoagulant and inhibitor levels were measured in maternal and cord plasma, and these values were used to mathematically simulate TG.ResultsPeak TG was increased in maternal plasma (393.6±57.9 nM) compared to adult and cord samples (323.2±38.9 nM and 209.9±29.5 nM, respectively). Inhibitory effects of RB006 on TG were less robust in maternal or cord plasma (52% vs. 12% respectively) than in adult plasma (81%). Likewise the effectiveness of BAX499 as represented by the increase in peak TG was much greater in adult (21%) than in maternal (10%) or cord plasma (12%). Further, BAX499 was more effective in reversing RB006 in adult plasma than in maternal or cord plasma. Ex vivo data were reproducible with the results of the mathematical simulation of TG.ConclusionNormal parturient plasma shows a large intrinsic pathway reserve for TG compared to adult and cord plasma, while TG in cord plasma is sustained by extrinsic pathway, and low levels of TFPI and AT.

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.

PO-24 - Determinants of thrombin generation in gynaecological malignancies

Increased thrombin production is associated with malignancy and is a marker for venous thromboembolism (VTE). Our group has shown that thrombin generation is increased in gynaecological malignancies. Although tumour derived Tissue Factor (TF) has been implicated, the precise mechanism by which thrombin production is increased is not fully understood. Our group has shown that gynaecological cancers can alter tumour expression of cogulation proteases. These changes may be implicated in the increased thrombin generation observed. Previous studies in normal control patients by other groups have shown that multiple coagulation factors are implicated in thrombin generation and that the effects depend on assay conditions however free Tissue Factor Pathway Inhibitor (TFPI), Factors V, VIIIc and protein S, were significant determinants of thrombin generation. The aim of this study was to determine the effect of factor V factor, VIIIc, TFPI and free protein S on the thrombin generation in patients with endometrial and ovarian cancer compared with benign controls. Patients with a gynaecological malignancy (n=43) (ovarian or endometrial) were matched with patients with a benign tumour (n=43) gave full and informed consent. Venous blood samples were obtained prior to surgery and chemotherapy. Thrombin generation was was measured using a fluorogenic assay. Lag time, peak thrombin and area under the thrombin generation curve (ETP) was determined and reported for each sample. Free protein S, free TFPI and factor V were determined using ELISA. Factor VIIIc was determined using a hromogenic assay. Factor V and factor VIIIc were significantly increased in the malignant group compared with the benign group (P<0.03; P<0.006). Increased free TFPI levels were also observed in the malignant group but this did not reach significance (P<0.06). There was no difference in free Protein S levels between the groups. Highest levels of peak thrombin generation were observed in the high grade serous and clear cell ovarian cancer patients. Changes were less marked in the endometrial patients. Free TFPI, factor V and factor VIIIc were important determinants of thrombin generation in the malignant group. Coagulation factors V and VIIIc and free TFPI are altered in patients with gynaecological malignancies and contribute to the increased thrombin generation found in these cancer patients. We have previously found increased expression of factor V in tumours from patients with ovarian cancer in addition to increased TF expression. These changes may explain the high rate of venous thromboembolism found in these patients.

Patients Referred for Bleeding Symptoms of Unknown Cause: Does Evaluation of Thrombin Generation Contribute to Diagnosis?

IntroductionPatients with mild to moderate bleeding symptoms referred for coagulation investigation sometimes never receive a definitive diagnosis. Bleed assessment tools have been developed and validated to assess the severity of symptoms. Global coagulation assays, e.g., the thrombin generation test (thrombogram) have a potential to identify hemostatic defects that are not detected in specific assays.Material and MethodsOne hundred and eighty-five patients referred to our centre because of bleeding symptoms were evaluated using the bleeding assessment tool (BAT) described by Tosetto and colleagues in 2006. Blood samples were investigated for thrombin generation (TG) capacity (Technoclone), in platelet poor (PPP) plasma, and specific clotting factors, i.e., von Willebrand factor, factor VIII and IX, as well as INR, APTT, platelet count, and platelet adhesion.ResultsOf the 185 patients, five women were diagnosed with mild von Willebrand disease and one male with mild hemophilia A. The remaining 179 subjects (76% females and 24% males with average ages of 33 and 28 years, respectively) were evaluated further. In the total cohort and among women, peak TG, and lag time correlated with bleeding score (p=0.01 and p=0.04, respectively with correlation coefficients). No such correlations were found among males.Discussion and ConclusionAlthough our study showed some correlation between TG and bleeding score, results are generally consistent with a previous report which failed to demonstrate the value of TG measurement in a similar setting. In conclusion, the complexity of the mechanisms underlying clinical bleeding complicates the ability to use TG tests as reliable predictors of bleeding. Mild congenital bleeding disorders, especially VWD, should be specifically screened for in patients with mild/moderate symptoms.

Computational simulation and comparison of prothrombin complex concentrate dosing schemes for warfarin reversal in cardiac surgery

Prothrombin complex concentrate (PCC) is increasingly used for acute warfarin reversal. We hypothesized that computational modeling of thrombin generation (TG) could be used to optimize the timing and dose of PCC during hemodilution induced by cardiopulmonary bypass (CPB). Thrombin generation patterns were modeled in anticoagulated patients (n=59) using a published computational model. Four dosing schemes were evaluated including single full dose (median, 41.2IU/kg) of PCC before or after CPB, ½-dose before and after CPB, or 1/3-dose before CPB plus 2/3-dose after CPB. Hemodilution was modeled as 40 or 60% dilution of factors from baseline. The lag time (s) of TG, and peak thrombin level (nM) were evaluated. Prolonged lag time, and reduced peak TG were due to low vitamin K-dependent (VKD) factors, and pre-CPB PCC dose-dependently restored TG to near-normal or normal range. After 40% dilution, TG parameters were similar among 4 regimens at the end of therapy. The recovery of VKD factors was less when PCC was given before CPBafter 60 % dilution, but TG parameters were considered hemostatically effective (>200nM) with any regimen. Withholding the full dose of PCC until post-CPB resulted in severely depressed TG peak (median, 47nM) after 60 % dilution, and some supra-normal TG peaks after treatment. Pre-CPB administration of full or divided doses of PCC prevents extremely low TG peak during surgery, and maintains hemostatic TG peaks in both 40 and 60% hemodilution models. Although PCC's hemostatic activity appears to be highest using the full dose after CPB, hypercoagulability may develop in some cases.