Background: 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. Methods: 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). Results: 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. Conclusions: 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.

BACKGROUND: Clinical expressivity of the thrombophilic factor V Leiden (FVL) mutation is highly variable. Recently, we demonstrated an increased APC (activated protein C) response in asymptomatic FVL carriers compared with FVL carriers with a history of venous thromboembolism (VTE) after in vivo coagulation activation. Here, we further explored this association using a recently developed ex vivo model based on patient-specific endothelial colony-forming cells (ECFCs). METHODS: ECFCs and citrated plasma were obtained from FVL carriers with previous VTE (VTE+, n=9), FVL carriers without previous VTE (VTE−, n=8), and 7 healthy controls. Coagulation was activated by TF (tissue factor) in defibrinated recalcified plasma added to confluent cell cultures. Thrombin and APC concentration were measured over time, and the respective areas under the curve (AUCs) were calculated. Additionally, inhibition kinetics of exogenously added APC, APC inhibitor levels, and APC sensitivity ratios were measured in plasma. Expression of TM (thrombomodulin) and EPCR (endothelial protein C receptor) on ECFCs was assessed using cell-based ELISAs. RESULTS: In autologous plasma on ECFCs, the APC response (AUC APC/AUC thrombin) was higher in FVL VTE− than in FVL VTE+ patients (0.138 versus 0.028; P =0.026). APC inhibitor levels, APC inactivation kinetics, and APC sensitivity ratios did not differ between cohorts. Crossover experiments, which combined pooled plasma from FVL VTE− patients with FVL VTE+ ECFCs in the ex vivo model, and vice versa, showed increased APC response rates when FVL VTE− ECFCs were used, regardless of the plasma component. In cell-based ELISAs, TM and EPCR expression showed no significant difference. CONCLUSIONS: Although the FVL gene product induces an almost identical APC resistance phenotype in plasma, the endothelial cell–dependent APC response rates differ significantly, with a higher APC response in asymptomatic FVL carriers. Further studies are warranted to elucidate the disease-modulating role of the endothelium in FVL carriers at the molecular level.

Splanchnic vein thrombosis (SVT), which is particularly prevalent in myeloproliferative neoplasms (MPNs), has a multifactorial pathomechanism involving the anticoagulant protein C (PC) pathway. To better characterize the hypercoagulable state in SVT we assessed its key enzymes thrombin and activated PC (APC). The study population included 73 patients with SVT, thereof 36 MPN+, confirmed by bone marrow biopsy, 37 MPN−, and 30 healthy controls. Direct measurement of the active enzyme forms of thrombin and APC in the circulation was achieved by using oligonucleotide-based enzyme capture assays (OECA). Additionally, activation markers of coagulation and fibrinolysis were measured. Plasma levels of free thrombin and APC were higher in the MPN+ than in the MPN− cohort, with 0.49 vs. <0.46 pmol/L (p = 0.0057), respectively, 1.23 vs. 0.58 pmol/L (p = 0.0122), and in healthy controls (vs. <0.46 pmol/L, p = 0.0012; vs. 0.54 pmol/L, p = 0.0035). The indirect activation markers prothrombin fragment 1+2, thrombin-antithrombin complex, and D-dimer did not differ between groups. Receiver operating characteristic analysis suggested that SVT patients with MPN can be better distinguished by APC than by conventional indirect thrombin markers. A potential application of these biomarkers to guide anticoagulant therapy and to investigate the role of the PC pathway in MPN-associated hypercoagulability should be further studied.

Introduction: The standard therapy for hemophilia A patients is the replacement of deficient factor (F) VIII with plasma-derived or recombinant FVIII concentrates. This replacement therapy can be complicated due to the potential occurrence of (neutralizing) antibodies and subsequent short half-life or even completely diminished activity of FVIII. Several strategies have been followed to optimize the pharmacokinetics of therapeutic FVIII, including coupling to polyethylene glycol (PEG). Presumably, the use of PEG should increase the half-life of FVIII and may reduce the frequency of administration to the patients. Method: For detection and characterization of antidrug antibodies (ADAs), an in-house multiplex microsphere-based immunoassay based on Luminex (LumiTope), an in-house ELISA, and a flow cytometry assay were established. The specificity of the identified ADAs was confirmed by competitive immunoassays. Based on a modified Nijmegen-Bethesda assay (NBA), the neutralizing nature of the ADAs was determined. Results: A mild hemophilia A patient developed ADAs after treatment with an extended half-life FVIII product PEGylated with a 40-kDa PEG. Our in-house immunoassays detected ADAs raised against both, the FVIII and PEG fraction of the therapeutic FVIII. The anti-FVIII antibodies were of immunoglobulin G (IgG) isotype only, whereas IgG, IgM, and IgA anti-PEG antibodies were detected. Competitive assays confirmed the respective specificity of the ADAs against FVIII or PEG. NBA revealed that anti-PEG antibodies were neutralizing while anti-FVIII antibodies were nonneutralizing. The PEG antibody cross-reacted with other PEGylated FVIII modified with different PEG reagents. The ADAs against PEG were persistent and could be detected several months after discontinuation of treatment with the PEGylated FVIII. Conclusion: Inhibitors against the PEG moiety of FVIII therapeutics may abolish the efficacy of the product. Accordingly, screening for antibodies against PEGylated FVIII proteins may be useful before and during the treatment. Article published online: 26 October 2022 © 2022. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

Venous thromboembolism (VTE) is multifactorial, and established hereditary risk factors explain only part of heritable risk. The protein C (PC) pathway is central to anticoagulant control, and common variants in thrombomodulin and endothelial PC receptor genes (THBD, PROCR) have been proposed as modulators. In 73 participants, we used standardized in vivo coagulation activation with recombinant activated factor VII (15 μg/kg) and measured thrombin markers and activated PC (APC) over 8 hours. Endothelial colony-forming cell–based ex vivo experiments were performed in 43 participants. In vivo, the ratio of APC area under the curve (AUC) to thrombin-antithrombin complex (TAT) AUC, which reflects the endogenous anticoagulant response relative to thrombin generation, provided the best model fit (adjusted R2 = 0.345). The APC response was lower in individuals with previous VTE (median, 0.13 vs 0.26) and PROCR 655A>G carriers (0.11 vs 0.26), but higher in factor V Leiden (FVL) carriers (0.32 vs 0.14) and THBD 1418C>T carriers (0.38 vs 0.13). The THBD 1418C>T effect was driven by reduced TAT AUC (30.6 vs 82.7 pmol × h/L), whereas the PROCR 655A>G effect reflected a lower APC AUC (8.6 vs 10.7 pmol × h/L). Ex vivo, the APC AUC/thrombin AUC ratio was lower with previous VTE and PROCR 655A>G, higher with FVL, and not associated with THBD 1418C>T. No in vivo or ex vivo association was observed for PROCR 4678C>G. This study provides in vivo evidence that common THBD and PROCR variants modulate PC pathway function, establishing the APC response as a sensitive end point for subtle genetic effects beyond FVL.

Background: Clinical expressivity of the thrombophilic factor V Leiden (FVL) mutation is highly variable. Increased activated protein C (APC) formation in response to thrombin formation has been observed in asymptomatic FVL carriers in vivo. Here we further explored this association using a recently developed endothelial colony forming cell (ECFC)-based ex-vivo model. Methods: ECFCs and citrated plasma were obtained from FVL carriers with/without previous venous thromboembolism (VTE+/-, n=7 each) and seven healthy controls. Coagulation was activated by tissue factor in defibrinated recalcified plasma added to confluent cell cultures. Thrombin and APC concentration were measured over time and the respective areas under the curve (AUC) calculated. Additionally, inhibition kinetics of exogenously added APC and APC sensitivity of the prothrombinase complex were measured in plasma. Expression of thrombomodulin and endothelial protein C receptor (EPCR) on ECFCs was assessed using cell-based enzyme-linked immunosorbent assays. Results: In autologous plasma on ECFCs, the APC response to thrombin formation (AUC APC/AUC thrombin), was higher in FVL VTE-than FVL VTE+ patients (0.138 versus 0.028, P =0.026). APC inactivation kinetics, APC sensitivity, and thrombomodulin/EPCR expression on ECFCs did not differ between these cohorts and compared to healthy controls. Cross-over experiments with plasma from FVL VTE- and FVL VTE+ patients on non-FVL ECFCs yielded indistinguishable results. In contrast, in normal plasma on FVL VTE-ECFCs the APC response remained significantly higher than on FVL VTE+ ECFCs (0.052 versus 0.022, P =0.011). Conclusions: Consistent with results from previous in vivo experiments, APC response rates to thrombin formation were higher in asymptomatic FVL carriers compared to those with previous VTE. Our observations suggest that this increased APC response is driven by the endothelium. Further studies are warranted to elucidate yet unknown endothelial mechanisms that might modulate the clinical expressivity of FVL.