Enzyme Capture Assay Research Articles

Decreased Protein C Pathway Activity in COVID-19 Compared to Non-COVID Sepsis: An Observational and Comparative Cohort Study.

Sepsis-associated coagulopathy increases risk of mortality. Impairment of the anticoagulant protein C (PC) pathway may contribute to the thrombotic phenotype in coronavirus disease 2019 (COVID-19) sepsis. This study assessed the functionality of this pathway in COVID-19 and non-COVID sepsis by measuring its key enzymes, thrombin and activated PC (APC). The study population included 30 patients with COVID-19, 47 patients with non-COVID sepsis, and 40 healthy controls. In healthy controls, coagulation activation and subsequent APC formation was induced by 15 µg/kg recombinant activated factor VII one hour before blood sampling. APC and thrombin in plasma were measured using oligonucleotide-based enzyme capture assays. The indirect thrombin markers prothrombin-fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) were also measured. Compared with stimulated healthy controls, median thrombin, F1+2, and TAT levels were higher in patients with COVID-19 (up to 6-fold, p < 2 × 10-6) and non-COVID sepsis (up to 4.7-fold, p < 0.010). APC levels were 2.4-fold higher in patients with COVID-19 (7.44 pmol/L, p = 0.011) and 3.4-fold higher in non-COVID sepsis patients (10.45 pmol/L, p = 2 × 10-4) than in controls (3.08 pmol/L). Thrombin markers and APC showed correlation in both COVID-19 (r = 0.364-0.661) and non-COVID sepsis patients (r = 0.535-0.711). After adjustment for PC levels, median APC/thrombin, APC/F1+2, and APC/TAT ratios were 2-fold (p = 0.036), 6-fold (p = 3 × 10-7) and 3-fold (p = 8 × 10-4) lower in the COVID-19 group than in the non-COVID sepsis group, and the latter two were also lower in the COVID-19 group than in stimulated healthy controls. In conclusion, it was found that a comparatively lower anticoagulant APC response in COVID-19 patients as compared to non-COVID sepsis patients, potentially linked to endothelial dysfunction, contributes to the prothrombotic phenotype of COVID-19 sepsis.

Increased Activated Protein C Response Rates to Thrombin Formation in Asymptomatic Factor V Leiden Carriers Are Driven By the Endothelium

Background The thrombophilic factor V Leiden (FVL) mutation shows a highly variable clinical expressivity. We have shown in vivo that asymptomatic FVL carriers respond to extrinsic coagulation activation with higher activated protein C (APC) formation rates than those with a history of venous thromboembolism (VTE). Recently we have established an ex vivo model for personalized assessment of the protein C (PC) pathway combining endothelial colony forming cells (ECFCs) and autologous plasma. Aim of this study was to investigate potential modulating factors of the APC response in the endothelium unraveling the enigma of the variable expressivity of the FVL phenotype. Methods ECFCs were isolated from FVL carriers with (FVL VTE+) or without (FVL VTE-) a history of unprovoked VTE and healthy controls (non-FVL) (n=7 each). Quality control of cultured cells included cobblestone morphology as examined by light microscopy, positive staining for CD31, CD309, CD201, and CD141, CD34, and negative staining for CD45 analyzed by flow cytometry. We either added autologous or pooled normal defibrinated plasma to the confluent cell culture and inititated thrombin formation by addition of tissue factor (1 pmol/L) and CaCl 2 (16.6 mmol/L) ( Fig. 1A). Thrombin and APC formation were measured over time using oligonucleotide-based enzyme capture assays (OECAs) and the ratio between the area under the curve (AUC) of APC formation and the AUC of thrombin formation was calculated as measure for APC response to thrombin. Moreover, we quantified thrombomodulin (TM) and endothelial protein C receptor (EPCR) expression in cell-based enzyme-linked immunosorbent assays (cell-ELISAs) and measured activation of purified PC on ECFCs in the APC-OECA. Results ECFC morphology in light microscopy and characteristic surface marker expression in flow cytometry did not differ between non-FVL, FVL VTE-, and FVL VTE+ subgroups. In the ECFC-based ex vivo model, the APC response in plasma on autologous cells was significantly higher in asymptomatic FVL VTE- carriers compared to non-FVL carriers (P = .0072) and compared to the FVL VTE+ subgroup (P = .0292) whereas it did not differ significantly between non-FVL and FVL VTE+ carriers. EPCR expression on ECFCs measured by cell-ELISA was higher in non-FVL carriers compared to FVL VTE+ subjects (P = .0374) but did not differ significantly when comparing non-FVL versus FVL VTE-, and FVL VTE+ versus FVL VTE-. Similarly, we showed that TM expression as well as activation of purified PC on ECFCs did not differ between cohorts. However, when the APC response was assessed on FVL VTE- and FVL VTE+ ECFCs using non-FVL plasma, the difference in the APC response between asymptomatic FVL carriers and FVL carriers with a history of VTE persisted (P = .0111) ( Fig. 1B). Conclusion Consistent with previous in vivo experiments, APC response rates to thrombin formation were increased on ECFCs obtained from asymptomatic FVL carriers compared to those from FVL carriers with a history of VTE in autologous plasma. Although TM and EPCR expression did not differ between both groups, we showed that the endothelial APC response in asymptomatic FVL carriers remained increased when using plasma from healthy controls. Our observations suggest that the increased APC response to thrombin in asymptomatic FVL carriers is driven by the endothelium. Further studies are warranted to elucidate the underlying mechanism for a better understanding of endogenous protective factors in FVL carriers. These findings may pave the way towards personalized, precision medicine approaches for managing the thrombotic risk in FVL and other disorders related to the PC pathway.

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach.

The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1β (interleukin 1β). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). The median peak APC concentration was 1.12 nmol/L in experiments with IL-1β stimulated ECFCs and 3.66 nmol/L without IL-1β. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy.

Protein C (PC) activity tests are routinely performed in a thrombophilia workup to screen for PC deficiency. Currently used tests combine conversion of PC to activated PC (APC) by the snake venom Protac with subsequent APC detection through hydrolysis of a chromogenic peptide substrate or prolongation of a clotting time. In this prospective cohort study, we analyzed how different modes of PC activation and subsequent APC determination influence the diagnostic accuracy of PC activity testing in a cohort of 31 patients with genetically confirmed PC deficiency. In addition to chromogenic and clot-based measurement, an oligonucleotide-based enzyme capture assay utilizing a basic exosite-targeting aptamer was used for APC detection. To study the influence of the PC activation step on diagnostic sensitivity, PC activation through Protac and through the thrombin-thrombomodulin (TM) complex were compared. Twenty-six (84%) and 24 (77%) PC deficient patients were identified as true-positive using the chromogenic and the clot-based PC activity assay, respectively. True-positive results increased to 27 (87%) when the basic exosite-targeting aptamer approach was used for APC measurement. Additional replacement of the PC activator Protac by thrombin-TM gave true-positive results in all patients. These data indicate that the mode of PC activation is crucial in determining the accuracy of PC activity testing and that diagnostic sensitivity can be significantly improved by replacing the PC activator Protac with thrombin-TM. APC detection using a basic exosite-targeting aptamer achieves high sensitivity toward mutations outside the active center while being less subject to interfering factors than clot-based PC activity assays.

Evaluation of the Thrombophilic Response to rFVIIa-Induced Hemostasis Activation in Factor V Leiden Carriers By Monitoring of Activated Protein C (APC) In Vivo

Background: The majority of patients with unprovoked venous thromboembolism (VTE) does not exhibit laboratory risk factors. Monitoring a limited and standardized activation of the hemostatic system in these patients might help to identify prothrombotic phenotypes in VTE. Promising biomarkers to monitor subclinical changes of the hemostatic activation status are plasma levels of active enzymes including thrombin and APC. Aim of this study was to characterize the prothrombotic phenotype in factor V (FV) Leiden carriers as a prerequisite for the identification of novel prothrombotic mechanisms.Methods: Subclinical activation of extrinsic hemostasis was induced by i.v. injection of recombinant activated factor VII (rFVIIa, 15 µg/kg) into 14 asymptomatic FV Leiden carriers (thereof 12 heterozygous, five males) and 12 healthy FV wild type (WT) carriers (six males). rFVIIa-induced hemostatic activation was monitored by measuring plasma levels of free thrombin and APC at baseline and repeatedly during an eight-hour follow-up period using oligonucleotide-based enzyme capture assays (OECA). In addition, prothrombin fragment F1+2 (F1+2), thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, soluble fibrin monomer, and D-dimer were determined.Results: Administration of rFVIIa was well tolerated in all subjects. Its pharmacokinetics showed an expected course with no differences between FV Leiden carriers and WT carriers (Figure 1A). At baseline, median (interquartile range, IQR) APC plasma levels were 0.081 (0.068-0.117) ng/ml in heterozygous FV Leiden carriers and 0.035 (0.028-0.067) ng/ml in WT carriers. Following rFVIIa injection, a significant (p<0.05) elevation of APC levels from baseline was observed between t=10 min and t=5 h (3 h for WT carriers). Peaks of 0.509 (0.431-0.721) ng/ml (p=10-4) in heterozygous FV Leiden carriers and of 0.164 (0.139-0.194) ng/ml (p=3·10-4) in WT carriers were observed at t=1 h (Figure 1B). In addition to higher peaks (p=2·10-4) in heterozygous FV Leiden carriers than in WT carriers, the area under the APC generation curve (AUC) was greater (p=4·10-6) with 2.40 (1.84-2.84) vs. 0.77 (0.63-0.97) ng/ml·h. Interestingly, the AUC of both homozygous FV Leiden carriers (2.82 and 2.67 ng/ml·h) and their APC peak levels (0.717 and 1.123 ng/ml) lay within the ranges of the heterozygous cohort. Among the other biomarkers, only F1+2 showed an increase from baseline (0.178, 0.122-0.197 nmol/l in heterozygous FV Leiden carriers; 0.125, 0.087-0.188 nmol/l in WT carriers) that became statistically significant at t=2 h (0.229, 0.187-0.264 nmol/l, p=0.001), t=3 h (0.230, 0.190-0.281 nmol/l, p=0.006), and t=5 h (0.230, 0.172-0.274 nmol/l, p=0.02) in heterozygous FV Leiden carriers, and at t=2 h (0.179, 0.111-0.200 ng/ml, p=0.03) in WT carriers. All other biomarkers remained unchanged, and no statistically significant differences were observed between FV carriers and WT carriers.Conclusion: Increased thrombin generation rates are counterbalanced by increased APC generation rates in clinically asymptomatic FV Leiden carriers after subclinical activation of extrinsic coagulation. Furthermore, the data presented demonstrate that subclinical activation of the hemostatic system by rFVIIa combined with serial APC testing is a new diagnostic tool to assess the functionality of the anticoagulant APC pathway in vivo. [Display omitted] DisclosuresRühl:Grifols: Research Funding; Sobi: Consultancy. Winterhagen:Sobi: Consultancy.

Pattern of Coagulation Activation in Patients with Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF), are associated with a high incidence of venous thromboembolism (VTE), especially splanchnic vein thrombosis (SVT), but the etiology of a hypercoagulable state in MPN remains elusive. JAK2V617F-associated development of acquired protein C (APC) resistance has been reported, suggesting an alteration of the thrombin-protein C (PC) axis.Methods: To further study the impact of the thrombin-PC axis on hypercoagulability in MPN, plasma levels of thrombin and APC were measured using oligonucleotide-based enzyme capture assays (OECA) in prospectively collected samples from 21 MPN patients (10 females) with a history of VTE (thereof 17 with SVT) and 24 MPN patients (13 females) without VTE. In addition to the OECA, markers of coagulation and fibrinolysis were measured including prothrombin activation fragment F1+2, D-dimer, plasmin-α2-antiplasmin complex (PAP), and thrombin-antithrombin-complex (TAT). All but one patient with prior VTE and 21 patients (88%) without prior thrombosis were positive for the JAK2V617F or the CALR mutation. Due to repeated study visits over a period of 21 months, a total of 35 samples from patients with a history of thrombosis and 46 from patients without VTE were available for analysis. Another comparison was performed between patients under anticoagulant treatment (17 patients, 27 samples) vs no anticoagulant treatment (28 patients, 54 samples). Furthermore, the results obtained in MPN patients were compared to those in 43 healthy subjects (22 females) who acted as a control group.Results: All biomarkers were significantly increased in MPN patients compared to healthy controls (Table 1). ANOVA neither revealed a difference between the disease subgroups of MF, PV, and ET nor between MPN patients with and without a history of VTE. In both patients with and without prior VTE APC, D-Dimer, F1+2, and PAP were significantly increased: Plasma levels of APC (median, IQR) of 0.078 (0.033-0.146) ng/mL, D-Dimer of 0.45 (0.29-0.67) mg/L, F1+2 of 0.219 (0.138-0.499) nmol/L, and PAP of 195 (118-453) ng/mL were measured in MPN patients with prior VTE. In patients without a history of thrombosis APC was 0.063 (0.026-0.108) ng/mL, D-dimer 0.33 (0.27-0.54) mg/L, F1+2 0.247 (0.146-0.381) nmol/L, and PAP 321 (175-526) ng/mL. The same pattern was observed in samples from patients without anticoagulant treatment, whereas in MPN patients under anticoagulants only APC (0.103, 0.027-0.137 ng/mL, p=0.0014), D-dimer (0.35, 0.23-0.60 mg/L, p=0.0022) and PAP (202, 144-423 ng/mL, p=0.0108), but not F1+2 (0.152, 0.098-0.219 nmol/L) were increased in comparison to the healthy control group. Consistent with these findings, F1+2 was the only biomarker, that differed significantly (p=0.0021) between MPN patients with anticoagulant therapy and patients not receiving anticoagulant medication (0.296, 0.185-0.502 nmol/l).Conclusion: The obtained data indicate, that MPN-induced thrombin formation, as demonstrated by increased levels of F1+2, causes activation of the anticoagulant PC system. In addition, the increase of PAP indicates activation of fibrinolysis which might contribute to elevated plasma levels of D-dimer. Thus, F1+2 might be better suited than D-dimer to monitor the effect of anticoagulant therapy in MPN. [Display omitted] DisclosuresRühl:Grifols: Research Funding; Sobi: Consultancy.

Functional Characterization of Antithrombin Mutations by Monitoring of Thrombin Inhibition Kinetics.

Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.

Activated Factor XI is Increased in Plasma in Response to Surgical Trauma but not to Recombinant Activated FVII-Induced Thrombin Formation.

Aim: Feedback activation of factor XI (FXI) by thrombin is believed to play a critical role in the amplification phase of thrombin generation and to contribute to thrombosis development and hemostasis. However, the activation of FXI by thrombin has been shown in vitro to require a cofactor. In this study, the role of thrombin in activated FXI (FXIa) formation in vivo is investigated. Methods: The study population comprised probands in whom coagulation activation was triggered by low-dose (15 µg/kg) recombinant activated factor VII (rFVIIa, n =89), of whom 34 with (VTE+) and 45 without a history of venous thromboembolism (VTE−), and patients undergoing major orthopedic surgeries ( n =45). FXIa was quantified via an enzyme capture assay using a monoclonal FXI-specific antibody. Thrombin formation was monitored using an oligonucleotide-based enzyme capture assay and the thrombin activation markers prothrombin fragment 1＋2 (F1＋2) and thrombin–antithrombin complex (TAT). Results: In the rFVIIa cohort, FXIa and thrombin remained below their lower limit of quantification of 3.48 and 1.06 pmol/L, respectively. By contrast, during the surgeries, median FXIa levels increased from 3.69 pmol/L pre-operatively to 9.41 pmol/L mid-operatively ( P =4·10 −4 ) and remained significantly elevated 24 h thereafter, with 9.38 pmol/L ( P =0.001). Peak levels of F1+2 were comparable in the VTE+, VTE−, and surgery cohort (235, 268, and 253 pmol/L), whereas peak TAT levels were higher in the surgery cohort (53.1, 33.9, and 147.6 pmol/L). Conclusions: Under in vivo conditions, the activation of FXI requires specific local features that are present at the wounded site including potential cofactors of thrombin.

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Circulating Plasminogen Activator Inhibitor-1 (PAI-1) Is Reduced By In Vivo Thrombin Generation and Subsequent Formation of Activated Protein C (APC)

Background: APC has been suggested to contribute to a hyperfibrinolytic state in various acquired coagulation disorders, including coagulopathies induced by trauma and sepsis. However, in vivo evidence for the proposed underlying mechanism of proteolytic degradation of PAI-1 by APC remains inconclusive. Recently, we have shown increased APC generation in response to in vivo thrombin formation in carriers of the factor V Leiden mutation (FVL). In this approach of stimulated hemostasis activity pattern evaluation (SHAPE), in vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to investigate the resulting effects on the fibrinolytic system. Methods: The study population consisted of 30 FVL carriers (thereof 13 with a history of venous thromboembolism) and 15 healthy controls. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of APC were quantified using an oligonucleotide-based enzyme capture assay (OECA). Other monitored parameters included plasma levels of prothrombin activation fragment 1+2 (F1+2), tissue-type plasminogen (t-PA), PAI-1, plasminogen, α2-antiplasmin, plasmin-α2-antiplasmin complexes (PAP), soluble fibrin monomers, d-dimer, and thrombin-activatable fibrinolysis inhibitor (TAFI). Results: At baseline, FVL carriers showed higher median levels of APC in comparison to the controls (1.39 vs. 0.86 pmol/L, P=.001), higher PAI-1 levels (30.1 vs. 15.3 ng/mL, P=.002) and lower plasminogen levels (94.8 vs. 110.4%, P=6·10-4). The other baseline parameters did not differ significantly. In both cohorts a comparable increase of F1+2 was observed after administration of rFVIIa, whereas APC increased more (P=.004) in FVL carriers (by 6.40 pmol/L) than in healthy controls (by 2.14 pmol/L), Concurrently, median PAI-1 levels decreased more (P=.007) in FVL carriers (by 19.8 pmol/L) than in healthy controls (by 8.0 pmol/L) (Figure 1). TAFI levels decreased temporarily, from 104.8 to 94.4% (P=.007) in FVL carriers and from 105.0 to 92.1% (P=2·10-4) in healthy controls. PAP levels increased from 164 to 209 ng/mL (P&lt;10-4) in FVL carriers and from 154 to 189 ng/mL (P=.023) in the control group. The extent of these changes did not differ between the two cohorts. D-dimer level increased only in FVL carriers, from 0.34 to 0.41 mg/L (P=.008). t-PA and the other parameters did not show significant changes after rFVIIa administration. Conclusion: Increased APC formation rates in FVL carriers were associated with a greater decline of PAI-1 levels in the absence of interfering changes in t-PA levels. These data provide further in vivo evidence that APC down-regulates PAI-1. Overall, the SHAPE approach utilized here does not induce a significant profibrinolytic response, even in patients with thrombophilia. Disclosures Reda: Grifols: Honoraria; Shire: Honoraria. Winterhagen:SOBI: Honoraria. Berens:Pfizer: Honoraria; Shire: Honoraria; Biotest: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Baxter: Honoraria. Müller:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Oldenburg:CSL Behring: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Research Funding, Speakers Bureau; Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; Grifols: Consultancy, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau. Rühl:CSL Behring: Honoraria; Bayer: Honoraria; Shire: Honoraria; SOBI: Honoraria; Sanofi Genzyme: Honoraria; Grifols: Honoraria.

Patients with Familial Thrombophilia of Unknown Origin Show Low APC Response to In Vivo Thrombin Formation in Stimulated Hemostasis Activity Pattern Evaluation (SHAPE)

Background: In a large proportion of patients with unprovoked venous thromboembolism (VTE), no risk factors can be identified. Recently, we have shown that an increased activated protein C (APC) response to in vivo thrombin formation reduces the thrombotic risk of factor V Leiden (FVL) carriers but not of prothrombin G20210A mutation (PTM) carriers. In vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa) followed by hemostasis biomarker monitoring. Aim of the present study was to extend this SHAPE approach to patients with thrombophilia of unknown origin. Methods: The study population consisted of 21 subjects with a positive self-history of unprovoked VTE (VTE+) as well as a positive family history, in whom no established thrombophilic risk factor was detectable (thrombophilia of unknown origin, TUO). A second cohort included 27 VTE+ patients tested positive for FVL (n=14) or PTM (n=13). None of the subjects was under anticoagulant treatment at the time of analysis. The control group consisted of 18 healthy volunteers. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of free thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally. Results: Injections of rFVIIa were well-tolerated by all subjects and median D-dimer levels remained within the reference range in all three cohorts. Plasma levels of F1+2 increased after rFVIIa injection, showing no significant differences between the groups. A comparable increase of TAT was observed in both TUO patients and VTE+ mutation carriers, from a median of 21.3 to 51.1 pmol/L (P=.022), and from 21.3 to 43.9 pmol/L, respectively (Figure 1A). Median levels of TAT in the controls and median levels of free thrombin in all cohorts remained below their respective quantifiable limits after rFVIIa injection. APC increased from 0.80 to 3.84 pmol/L (P=.001) in TUO patients, from 1.14 to 5.82 pmol/L (P&amp;lt;10-4) in VTE+ mutation carriers, and from 0.87 to 3.39 pmol/L (P=.001) in the control group. The APC increase was significantly smaller in the TUO cohort than in the VTE+ mutation carriers (P=.019) (Figure 1B). Conclusion: A low APC response to an increased in vivo thrombin formation is a frequent finding among patients with thrombophilia of unknown origin. This finding is in line with previous data suggesting that higher APC levels protect FVL carriers from thrombosis development. The results of the present study further suggest that a low APC response might be an independent risk factor of VTE. Further studies are warranted to identify the factors that modulate the APC response. Disclosures Rühl: Sanofi Genzyme: Honoraria; SOBI: Honoraria; Shire: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria; Grifols: Honoraria. Reda:Shire: Honoraria; Grifols: Honoraria. Winterhagen:SOBI: Honoraria. Berens:Pfizer: Honoraria; Shire: Honoraria; Biotest: Honoraria; CSL Behring: Honoraria; NovoNordisk: Honoraria; Baxter: Honoraria. Müller:Roche: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Membership on an entity's Board of Directors or advisory committees. Oldenburg:Grifols: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau.

Characterization of circulating thrombin in patients with septic shock: a prospective observational study.

Septic shock is characterized by a dysregulated response to infection, hypotension and activation of the coagulation system. Markers of coagulation activation are commonly used to diagnose and monitor ensuing coagulopathies. In this study, we sought to determine levels of circulating thrombin in patients with septic shock. To characterize levels of circulating, active thrombin in patients with septic shock. 48 patients with septic shock were included in this prospective, observational study. Blood samples were obtained on admission, day 1, day 3 and day 6. Levels of active thrombin were measured using a standardized, clinically applicable oligonucleotide (aptamer)-based enzyme-capture assay (OECA). Thrombin levels were correlated with established indirect thrombin parameters, conventional coagulation tests, laboratory parameters, patient characteristics and outcome. Elevated levels of thrombin were detected in 27 patients (56.3%) during the course of the study. Thrombin levels were positively correlated with thrombin-antithrombin complexes (r = 0.30, p < 0.05) and negatively associated with FVII levels (r = - 0.28, p < 0.05). Thrombin levels on admission did not predict 30-day mortality (OR 0.82, 95% CI 0.23-2.92, p = 0.77). Circulating levels of active thrombin can be measured in a subset of patients with septic shock. Although thrombin levels are correlated with established markers of coagulation, they do not provide additional prognostic information.

The Variable Clinical Expressivity of Factor V Leiden, but Not of Prothrombin G20210A, Correlates with the Extent of the APC Response to In Vivo Thrombin Formation

Background: The factor V Leiden mutation (FVL) and the prothrombin G20210A mutation (PRO) are the most frequent thrombophilic mutations among Caucasians. Both mutations show a highly variable expressivity, ranging from a lifelong asymptomatic course to severe venous thromboembolism (VTE). Recently, we have shown increased generation rates of thrombin and activated protein C (APC) in response to in vivo thrombin formation in asymptomatic FVL carriers (VTE-). In vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to extend this stress test approach to carriers of PRO and to FVL carriers with a history of VTE (VTE+).Methods: The study population consisted of 29 FVL carriers (thereof 25 heterozygotes: 13 VTE- and 12 VTE+) and 21 PRO carriers (thereof 19 heterozygotes: 12 VTE- and 7 VTE+). None of the subjects was under anticoagulant treatment at time of analysis. The control group consisted of 13 healthy volunteers tested negative for FVL and PRO. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of free thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally.Results: Injections of rFVIIa were well-tolerated by all subjects and median D-dimer levels remained within the reference range in all groups. Compared with the controls, peak plasma levels of F1+2 and TAT were significantly higher after rFVIIa injection in FVL carriers (p=.009 and p=1.0∙10-4, respectively), and in PRO carriers (p=.045 and p=6.0·10-4, respectively), but they did not differ between the FVL and PRO cohorts, and between the VTE+ and VTE- subgroups. Median plasma concentrations of thrombin were below the limit of detection at baseline, and rFVIIa did not induce statistically significant changes of thrombin levels in any group. APC increased to peak levels 30 minutes after rFVIIa injection, from 0.63 (0.54-1.16) to 3.14 (2.55-3.55) pmol/L (p=.017) in the controls, and from 1.39 (1.14-1.86) to 7.86 (5.55-11.55) pmol/L (p<1.0∙10-4) in the FVL group. In PRO carriers, plasma levels of APC increased from 1.07 (0.86-1.23) pmol/L at baseline to a peak of 5.86 (4.44-6.41) pmol/L 1 hour after rFVIIa injection p=4.0·10-4). Peak plasma levels of APC were higher in both FVL carriers and PRO carriers in comparison to the controls (p<1.0∙10-4 and p=4.0∙10-4, respectively), and lower in PRO carriers compared with those in FVL carriers (p=.003). With peak levels of 8.11 (7.59-11.77) vs. 5.62 (4.94-6.49) pmol/L, APC was significantly higher in the VTE- subgroup than in the VTE+ subgroup (p=.004) in heterozygous FVL carriers, while in heterozygous PRO carriers, APC peak levels did not differ between the VTE- subgroup and the VTE+ subgroup (Figure 1).Conclusion: The APC response to in vivo thrombin formation is significantly lower in symptomatic than in asymptomatic FVL carriers, implying that higher APC levels protect FVL carriers from thrombosis development. This hypothesized effect appears to be specific for FVL as prothrombin G20210A carriers do not show differences in the APC response related to VTE history. Further studies are warranted to identify the factors that modulate the APC response in FVL carriers. [Display omitted] DisclosuresRühl:Swedish Orphan Biovitrum: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; Grifols: Research Funding; CSL-Behring: Research Funding. Berens:Pfizer: Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; CSL-Behring: Research Funding; Biotest: Research Funding. Winterhagen:Swedish Orphan Biovitrum: Consultancy, Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; Grifols: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria; Biotest: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Swedish Orphan Biovitrum: Consultancy, Honoraria.

Platelet-Dependent Thrombin Generation Induced By ADP or Activated Factor X Does Not Contribute to Increased In Vivo Thrombin Formation in Myeloproliferative Neoplasms

Introduction: Myeloproliferative Neoplasms (MPN), including the clinical entities Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by an increased thrombotic risk, the pathomechanisms of which are not well-understood. It has been suggested that an increased sensitivity of platelets to adenosin diphosphate (ADP) contributes to the hypercoagulable state in PV and ET through increased thrombin generation. In the present study we analyzed plasma levels of thrombin and platelet-dependent thrombin generation in MPN patients with an additional focus on prothrombin activation by the prothrombinase complex.Methods: A total of 33 blood samples were obtained from patients with MPN (PV, n=18; ET, n=5; PMF, n=10) and from 33 healthy blood donors that served as controls. In vitro thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was assessed using the Calibrated Automated Thrombogram (CAT) assay. To induce thrombin generation either ADP (1 µmol/L final concentration) or activated factor X (FXa, 10 ng/mL final concentration) were applied. To further characterize the MPN-associated hypercoagulable state in vivo, plasma levels of free thrombin were measured using an oligonucleotide-based enzyme capture assay (OECA). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally.Results: In PRP of MPN patients a slightly higher ADP-induced peak thrombin concentration (Cpeak) was observed than in the healthy controls, with 106 (79-130) vs. 84 (65-110) nmol/L (median and interquartile range, p=.026). There was no statistically significant difference in the ADP-induced endogenous thrombin potential (ETP) in MPN patients (1445, 1194-1643 nmol/L·min) compared with the controls (1417, 1258-1814 nmol/L·min). There was no statistically significant difference in the FXa-induced Cpeak and ETP between MPN patients and controls, with 106 (79-127) vs. 97 (82-128) nmol/L, and 1424 (1165-1560) vs. 1641 (1193-1841) nmol/L·min, respectively. With 0.68 (<0.46-1.20) pmol/L, plasma levels of free thrombin were significantly higher (p=.025) in MPN patients than in the control group, in which median thrombin levels were below the limit of detection. Plasma levels of F1+2 and TAT were also higher in the MPN group than in healthy controls, with 0.31 (0.17-0.50) vs. 0.18 (0.13-0.25) nmol/L (p=.002) and 4.36 (2.53-6.76) vs. 2.36 (<2.00-2.68) ng/mL (p=.003), respectively.Conclusion: Increased plasma levels of thrombin, F1+2, and TAT indicate enhanced in vivo thrombin formation in MPN patients. Despite a slightly increased ADP sensitivity of MPN-platelets, the total amount of thrombin generated in PRP from MPN patients is not increased. This makes it unlikely that a ‘hyperreactivity’ of MPN platelets, resulting in increased activities of the prothrombinase complex on the platelet surface, contributes to the increased thrombin formation in MPN patients. DisclosuresBerens:Shire: Research Funding; Biotest: Research Funding; Pfizer: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Rühl:Shire: Research Funding; Swedish Orphan Biovitrum: Consultancy, Research Funding; Grifols: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding.

Activity Pattern Analysis Indicates Increased but Balanced Systemic Coagulation Activity in Response to Surgical Trauma.

In the nonbleeding patient, constant low-level activation of coagulation enables a quick procoagulant response upon an injury. Conversely, local activation of coagulation might influence the systemic activity level of coagulation. To characterize this interaction in more detail, activity pattern analysis was performed in patients undergoing elective surgeries. Blood samples were taken before, during, and 24 hours after surgery from 35 patients undergoing elective minor ( n = 18) and major ( n = 17) orthopaedic surgeries. Plasma levels of thrombin and activated protein C (APC) were measured using oligonucleotide-based enzyme capture assays, while those of prothrombin fragment 1.2, thrombin–antithrombin-complexes, and D-dimer were measured using commercially available enzyme-linked immunosorbent assays. In vitro thrombin generation kinetics were recorded using calibrated automated thrombography. Results showed that median plasma levels of up to 20 pM thrombin and of up to 12 pM APC were reached during surgery. D-dimer levels started to increase at the end of surgery and remained increased 24 hours after surgery, while all other parameters returned to baseline. Peak levels showed no significant differences between minor and major surgeries and were not influenced by the activity state at baseline. In vitro thrombin generation kinetics remained unchanged during surgery. In summary, simultaneous monitoring of the procoagulant and anticoagulant pathways of coagulation demonstrates that surgical trauma is associated with increased systemic activities of both pathways. Activity pattern analysis might be helpful to identify patients at an increased risk for thrombosis due to an imbalance between surgery-related thrombin formation and the subsequent anticoagulant response.

Aptamer Display on Diverse DNA Polyhedron Supports.

DNA aptamers are important tools for molecular recognition, particularly for a new generation of tools for biomedicine based on nucleic acid nanostructures. Here, we investigated the relative abilities of different shapes and sizes of DNA polyhedra to display an aptamer which binds to the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH). The aptamer was shown to perform an Aptamer-Tethered Enzyme Capture (APTEC) assay with the hypothesis that the display of the aptamer above the surface through the use of a polyhedron may lead to better sensitivity than use of the aptamer alone. We compared different numbers of points of contact, different shapes, including tetrahedron, square, and pentagon-based pyramids, as well as prisms. We also investigated the optimal height of display of the structure. Our results demonstrated that the display of an aptamer on an optimized nanostructure improved sensitivity up to 6-fold relative to the aptamer alone in the APTEC assay. Other important factors included multiple basal points of contact with the surface, a tetrahedron proved superior to the more complex shaped structures, and height above the surface only made minor differences to efficacy. The display of an aptamer on a nanostructure may be beneficial for higher sensitivity aptamer-mediated malaria diagnosis. Aptamer displays using DNA nanostructure polyhedron supports could be a useful approach in a variety of applications.

The evolution of activated protein C plasma levels in septic shock and its association with mortality: A prospective observational study

PurposeSeptic shock is commonly associated with hemostatic abnormalities. The endothelium-activated serine protease activated protein C (APC) plays a pivotal role in limiting coagulation and possesses anti-apoptotic and anti-inflammatory properties. We hypothesized that APC levels correlate with established coagulation parameters and provide prognostic information in patients with septic shock. MethodsWe conducted a prospective, observational cohort study in patients with septic shock. APC was measured on admission (day 0) and on days 1, 3, and 6 by a clinically applicable oligonucleotide (aptamer)-based enzyme-capture assay (OECA). The primary endpoint was defined as sepsis-associated 30-day mortality. Furthermore, we analyzed the correlation of APC levels with established coagulation markers. Results48 consecutive patients admitted with septic shock were included (mortality 39.6%). APC levels were elevated upon admission (0.59 ng/ml, IQR 0.26–0.97) and showed a strong correlation with established markers of coagulation and lactate. Multivariable logistic regression identified APC (OR 4.3, 95% CI 1.1–17.8, p = 0.04) and lactate levels (OR 7.0, 95% CI 4.1–18.2, p = 0.04) as independent predictors of 30-day mortality. ConclusionsAPC levels are increased in patients with septic shock and are correlated with established markers of coagulation. Elevated APC levels on admission are an independent predictor of mortality.

Aptamer-mediated Plasmodium-specific diagnosis of malaria

There is a critical need for better malaria rapid diagnostic tests to discriminate Plasmodium falciparum and Plasmodium vivax infection given the recent observation of HRP2 deletions in P. falciparum parasites. We previously identified a DNA aptamer, 2008s, that targets P. falciparum lactate dehydrogenase (PfLDH) and developed a sensitive aptamer-tethered enzyme capture (APTEC) assay. Here, we characterise two different LDH-binding DNA aptamers in their species-specific activities, then integrate within biochemical diagnostic assays and test in clinical samples. An enzyme-linked oligonucleotide assay demonstrated that aptamer pL1 bound with high affinity to both PfLDH and P. vivax lactate dehydrogenase (PvLDH), whereas aptamer 2008s was specific to PfLDH. An aptamer-tethered enzyme capture (APTEC) assay confirmed the specificity of 2008s in binding and capturing the enzyme activity of PfLDH which could be observed colorimetrically. In malaria patient samples, the 2008s APTEC assay was specific for P. falciparum blood samples and could discriminate against P. vivax blood samples. An aptamer for specific detection of falciparum malaria holds promise as a new strategy for species-specific malaria diagnosis rather than the conventional HRP2 immuno-assay.

A portable microfluidic Aptamer-Tethered Enzyme Capture (APTEC) biosensor for malaria diagnosis

There is a critical need for better biosensors for the detection and diagnosis of malaria. We previously developed a DNA aptamer that recognises the Plasmodium falciparum lactate dehydrogenase (PfLDH) enzyme with high sensitivity and specificity. The aptamer was integrated into an Aptamer-Tethered Enzyme Capture (APTEC) assay as a laboratory-based diagnostic approach. However, a portable equipment-free point-of-care aptamer-mediated biosensor could have a significant impact on malaria diagnosis in endemic regions. Here, we present a new concept for a malaria biosensor whereby aptamers are coated onto magnetic microbeads for magnet-guided capture, wash and detection of the biomarker. A biosensor incorporating three separate microfluidic chambers was designed to enable such magnet-guided equipment-free colorimetric detection of PfLDH. A series of microfluidic biosensor prototypes were optimised to lower rates of inter-chamber diffusion, increase sensitivity, and provide a method for point-of-care sample testing. The biosensor showed high sensitivity and specificity when detecting PfLDH using both in vitro cultured parasite samples and using clinical samples from malaria patients. The high performance of the biosensor provides a proof-of-principle for a portable biosensor that could be adaptable for a variety of aptamer-mediated diagnostic scenarios.

Evidence of Thrombin-Independent Generation of Activated Protein C

Introduction: In a recent study, we performed autologous serum infusions to evaluate the elimination kinetics of hemostasis-related biomarkers in healthy human subjects. In order to monitor a serum-induced activation of coagulation, we measured free thrombin in the infused serum and in plasma samples taken during and after infusion, but did not detect any de novo thrombin formation [PLoS One. 2015; 10(12): e0145012]. To study if the low levels of free thrombin in the infused serum induce generation of activated protein C (APC) we additionally measured APC in samples drawn after autologous serum infusion and in vitro in a purified system.Methods: Autologous serum was infused (50 mL/30 min) into 19 healthy volunteers. Four of them were simultaneously receiving infusions of the thrombin inhibitor argatroban (1 µg/kg/min), initiated 1 h before and ceased 1 h after starting the infusion of serum. Thrombin and APC were measured in serum and in plasma samples drawn before and in 15-min intervals during the infusion of serum, using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) platform. In in vitro experiments, APC formation was induced by addition of purified thrombin or serum to buffer containing protein C and thrombomodulin in excess, and CaCl2 at physiological concentrations. The formation of APC was subsequently measured by OECA.Results: In the autologous serum median (interquartile range) concentrations of thrombin and APC were 6.68 (4.63 - 8.73) ng/mL and 9.17 (7.63 - 13.91) ng/mL, thus doses of 0.12 (0.07 - 0.15) ng/mL of thrombin and 0.16 (0.14 - 0.22) ng/mL of APC were infused per mL of the subjects' plasma volume. In the plasma of probands, that did not receive argatroban, peak thrombin levels of 0.04 (0.00 - 0.08) ng/mL were measured, indicating a rapid inactivation of thrombin by endogenous inhibitors present in the plasma. However, with 1.41 (0.76 - 2.97) ng/mL peak APC levels exceeded the infused APC doses by a multiple. This was also true for the plasma samples from the probands that received argatroban, in which peak levels of APC of 0.94 (0.79 - 1.22) ng/mL were measured despite thrombin inhibition indicated by prolongation of the aPTT of 42.9 (40.1 - 44.4) s and thrombin time of 78.3 (69.3 - 87.2) s. In the in vitro experiments addition of argatroban at the concentrations achieved in the probands completely abolished APC generation up to a thrombin concentration of 5 ng/ml. Addition of human serum as a source for thrombin in the same purified system consistently induced generation of greater amounts of APC than expected on the basis of the amount of thrombin present in the serum samples.Conclusions: The data obtained provide evidence for a thrombin-independent mechanism of APC formation. Further in vitro studies with endothelial cells are required to identify the components that are involved in this alternative way of APC generation. DisclosuresRühl:CSL Behring: Research Funding; Bayer: Consultancy, Honoraria. Müller:Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.