Activated Protein C Generation Research Articles

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

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.

Role of the protein C system in aggravation of acute pancreatitis

The goal of the study was to identify risk factors for coagulation and fibrinolysis in aggravation of acute pancreatitis (AP) based on 11 markers and APACHE-II score. The subjects were 42 patients with AP (12 severe AP and 30 mild AP) and 20 healthy controls. Measurements of antithrombin III (AT-III), plasminogen (Plg), platelet counts, lipopolysaccaride (LPS), thrombomodulin (TM), protein C (PC), activated protein C (APC), APC/PC ratio, free-protein S (f-PS), thrombin-antithrombin complex (TAT), and tissue-type plasminogen activator·PAI-1 (t-pA·PAI-1) complex and clinical scores were performed using ELISA kits and clinical examinations of patients. Fluctuations of most markers in the course of AP indicated abnormal activation from onset to 3 days. After 7 days, non-survivor severe AP (SAP) cases showed abnormal marker reactivation, while survivor SAP and mild AP (MAP) cases had gradual normalization of markers. At 7 days after onset and during all stages of progression of AP, there were significant differences between non-survivor SAP and survivor SAP cases for 5 and 8 markers, respectively, but APC and t-pA·PAI-1 complex were common to both. The TAT/APC and t-pA·PAI-1/APC ratios, which reflect APC generation, were higher in survivor SAP than in non-survivor SAP cases. A decrease in APC generation leading to excessive thrombin synthesis may be a risk factor for coagulation and fibrinolysis in aggravation of AP.

An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice

AbstractAcute traumatic coagulopathy (ATC) occurs in approximately 30% of patients with trauma and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond can reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by activated partial thromboplastin time (APTT) prolongation and reductions of factor V (FV), factor VIII (FVIII), and fibrinogen but not factor II and factor X. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as a consequence of severe bleeding. superFVa prophylaxis before liver laceration reduced bleeding and prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. superFVa intervention started after the development of ATC stabilized bleeding, reversed prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

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.

Characterisation of a novel thrombomodulin c.1487delC,p.(Pro496Argfs*10) variant and evaluation of therapeutic strategies to manage the rare bleeding phenotype

IntroductionA novel variant in the thrombomodulin (TM) gene, c.1487delC,p.(Pro496Argfs*10), referred to as Pro496Argfs*10, was identified in a family with an unexplained bleeding disorder. The Pro496Argfs*10 variant results in loss of the transmembrane and intracellular segments of TM and is associated with an increase in soluble TM (sTM) in the plasma. The aim of this study was to characterise the effect of elevated sTM on thrombin generation (TG) and fibrinolysis, and to evaluate therapeutic strategies to manage the patients. MethodsPlasma samples were obtained from two patients carrying the variant. TG was triggered using 5 pM tissue factor and measured using the Calibrated Automated Thrombogram. A turbidity clot lysis assay was used to monitor fibrinolysis. TM antigen was quantified by ELISA. ResultsPatients with the Pro496Argfs*10 variant had significantly elevated plasma sTM compared to controls (372.6 vs. 6.0 ng/ml). TG potential was significantly lower in patients but was restored by inhibition of activated protein C (APC) or addition of activated Factor VII (FVIIa) or platelet concentrates. In vitro experiments suggested that activated prothrombin complex concentrates (APCC) posed a risk of thrombosis. The time to 50% lysis was significantly prolonged in patients compared to controls, 69.7 vs. 42.3 min. Clot lysis time was shortened by inhibition of activated thrombin activatable fibrinolysis inhibitor (TAFIa). ConclusionsOur data demonstrate that increased sTM enhances APC generation and reduces TG. Simultaneously, the rate of fibrinolysis is delayed due to increased TAFI activation by sTM. Treatment with platelet or FVIIa concentrates may be beneficial to manage this rare bleeding disorder.

Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation’s effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation–induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.

EPCR Deficiency Prevents Development of Hemophilic Arthropathy

Recent studies from our laboratory showed that clotting factor VIIa (FVIIa), whose primary function is to initiate the coagulation cascade following its binding to procoagulant cofactor tissue factor (TF), also binds anticoagulant cofactor, endothelial cell protein C receptor (EPCR). EPCR plays a key role in the activated protein C (APC)-mediated anticoagulant pathway by promoting the activation of protein C bound to it. Pharmacological concentrations of rFVIIa could compete with protein C for the EPCR and downregulate APC generation by preventing protein C binding to the EPCR. Our recent studies showed that FVIIa binding to EPCR also induces barrier-protective and anti-inflammatory effects both in vitro and in vivo model systems. Bio-distribution of rFVIIa in mice revealed that a small fraction of rFVIIa administed to mice i.v. accumulates in knee joints and retained there for three to seven days. The present study was conducted to investigate the relevance of FVIIa binding to EPCR in pharmacological FVIIa treatment of joint disease in hemophilia. For this, we first generated hemophilia A mice lacking EPCR or overexpressing EPCR. To generate EPCR deficient FVIII-/- mice, Procrflox/flox and Procr+/floxMeox2+/cre mice were first backcrossed with FVIII-/- mice to generate Procrflox/flox/FVIII-/- and Meox2+/cre/FVIII-/- mice. EPCR deficient FVIII-/- mice (Procr-/-/FVIII-/-) were generated by breeding female Procrflox/flox/FVIII-/- with male Procr+/floxMeox2+/cre /FVIII-/- mice. EPCR- overexpressing FVIII-/- were generated by crossing Tie2-EPCR mice with FVIII-/- mice. We induced joint bleeding in FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice by needle puncture injury. Right knee joint intra articular space was pierced with 30G needle to induce joint bleeding; left knee joint served as an uninjured control. Injured mice were treated with a single dose of rFVIIa (1 mg/kg) at 20 min following the injury or three doses of rFVIIa (1 mg/Kg) at 20 min, 24 h and 72 h following the injury. In controls, saline was administered in place of rFVIIa. The knee injury was evaluated by measuring knee joint diameter and visual bleeding score. At the end of 14 days post injury, mice were euthanized, and the knee joints were excised and fixed for immunohistochemical analysis. Hemophilic arthropathy was evaluated by scoring a set of pathological parameters (e.g., synovial hyperplasia, neoangiogenesis, presence of blood, iron score, villus formation, and cartilage degeneration). A subset of mice was used to collect synovial fluid at 7 days post injury. Assessment of joint bleeding at 5 h following the needle injury, by extracting and measuring hemoglobin levels in joint tissues and monitoring hematocrit, showed an equal amount of bleeding in all three genotypes of mice (FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice). Administration of three doses of FVIIa was effective in preventing hemophilic arthropathy in FVIII-/- and EPCR-overexpressing FVIII-/- mice, whereas a single dose of rFVIIa had a minimal effect in reducing hemophilic arthropathy. Interestingly, a single dose of FVIIa fully corrected the needle injury-induced hemophilic arthropathy in EPCR-deficient FVIII-/- mice. More importantly, EPCR-deficient FVIII-/- mice failed to develop full-blown hemophiic arthropathy even in the absence of any treatment. Measurement of inflammatory cytokines in the synovial fluid showed a robust increase in IL-6 levels in FVIII-/- mice and a single dose of FVIIa substantially reduced IL-6 levels. Interestingly, IL-6 levels in the synovial fluid of injured EPCR-overexpressing FVIII-/- mice were markedly higher compared to injured FVIII-/- mice, three doses of rFVIIa was required to attain a significant reduction in IL-6 levels in these mice. In contrast to these data, we found very little IL-6 levels in the synovial fluids of injured EPCR-deficient FVIII-/- mice. Overall, these data indicate that EPCR levels profoundly influence hemophilic arthorpathy. EPCR-deficiency protects from development hemophilic arthropathy because down-regulation of APC generation in EPCR deficiency could allow sufficient thrombin generation in hemophilia to prevent joint bleeding and bleeding-associated inflammation. Disclosures Rao: Takeda: Research Funding.

Superfactor Va, an Engineered Factor V Molecule, Effectively Reduces Bleeding and Acute Coagulopathy in Mouse Models of Trauma and Shock

Traumatic bleeding is a leading cause of death and disability, without targeted interventions available outside blood component resuscitation and tranexamic acid. Acute traumatic coagulopathy (ATC) in association with bleeding occurs in ~30-50% of patients and carries a high mortality risk. Recent evidence suggests a role of activated protein C (APC) in the development of ATC, whereby excessive generation of APC leads to rapid depletion of Factor(F) V, FVIII and hyperfibrinolysis causing depletion of fibrinogen. Here, we investigated to what extent targeted intervention with an engineered activated FV variant (superFVa), resistant to inactivation by APC, can reduce traumatic bleeding and prevent or normalize hemostasis parameters indicative of ATC, a process thought to be different from disseminated intravascular coagulation (DIC). SuperFVa was engineered to withstand cleavage by APC through mutations of 3 APC cleavage sites (Arg506/306/679Gln), with additional stability provided by introduction of a disulfide bond (Cys609-Cys1691) between the A2 and A3 domains. Materials and Methods First, an established trauma/shock model for ATC (Chesebro et al. 2009) was used. Here, trauma is induced by laparotomy and shock is induced by blood withdrawal (~500ul) to reduce and maintain the mean arterial pressure [MAP] from 75±5 to 35±5 mmHg for 1 hour. SuperFVa (0.4-0.8 mg/kg) was administered by continuous infusion 30 minutes after trauma/shock induction. Second, a liver laceration model was used where ATC develops as the consequence of severe bleeding. SuperFVa was administered either as bolus (0.4-0.8mg/kg) before injury or as continuous infusion (0.8mg/kg) started 30 minutes after injury. Bleeding, APTT clotting times, clotting factor activity levels (FII, FV, FVIII, FX, fibrinogen) and Thrombin-Antithrombin (TAT) complexes were assessed to determine the extent of ATC at various time points. Results In the trauma/shock model, only mice subjected to both trauma and shock developed ATC, evident from a significant APTT prolongation (25.4±0.2 to 36.1±0.2 seconds), associated with a severe reduction of FV (14%) and FVIII( 6%), a partial reduction of fibrinogen (44%), and no effect on FX (89%) and FII (89%). Administration of superFVa normalized the APTT (27.1±0.2 seconds) despite the persistence of shock, returned FV and FVIII levels to their normal range, but had no effect on fibrinogen. TAT levels increased significantly upon trauma/shock (1522 ±125 ng/mL) and were reduced by superFVa administration (511±125 ng/mL), suggesting improved hemostasis. After liver laceration severe bleeding occurred within 60 minutes, causing shock (MAP decreased to 35±5), selective reductions of FV, FVIII, and fibrinogen levels, and prolongation of the APTT clotting time, indicating the development of ATC. SuperFVa prophylaxis prior to liver laceration reduced blood loss significantly (non-treated mice 0.54±0.01 vs. pre-treated mice 0.34±0.01 mL, p&lt;0.0001), prevented APTT prolongation and depletion of FV and FVIII. Thus, prophylactic administration of superFVa prevented the development of ATC. Most bleeding after liver laceration occurred in the first 15-30 minutes (0.44±0.01mL at 30 minutes) and ATC became evident at 30 min (APTT prolongation to 32.63±0.2 seconds; partial depletion (~50%) of FV and FVIII). To determine effects of superFVa on the reversal of ongoing ATC, superFVa intervention was started therefore at the 30-minute time point. superFVa blunted additional blood loss after 30 minutes, normalized prolonged APTT clotting times and reversed FV and FVIII levels to their normal range. Reversal of ATC by superFVa was associated with a reduction of TAT levels (646.5±125 ng/mL) that were increased by ATC (1577.0 ±125 ng/mL). Conclusion SuperFVa restored hemostasis in two mouse models of ATC where coagulopathy was induced by either trauma/shock or by severe bleeding. Improved hemostasis in ATC by superFVa was achieved by correction of APTT clotting times and normalization of depleted FV and FVIII levels and was associated with a reduction of elevated TAT levels. Because superFVa rescued traumatic bleeding to prevent the development of ATC and was also efficacious for the reversal of ongoing ATC, targeted interception of ATC by superFVa therefore may be an attractive strategy to reduce complications associated with ATC. Disclosures Mosnier: The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. von Drygalski:University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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.

Resistin impairs activation of protein C by suppressing EPCR and increasing SP1 expression

Endothelial cells are vital to blood coagulation and maintain whole body hemostasis. Binding of endothelial cells to endothelial protein C receptor (EPCR) and thrombomodulin (TM) is essential to the formation of activated protein C (APC), one of the key factors regulating blood coagulation. In our study, we showed that resistin, an adipocyte hormone, suppresses thrombin-induced protein C activation in endothelial cells. Resistin treatment results in a reduction in EPCR expression, but not TM. Mechanistically, we demonstrate that resistin induces expression of the nuclear transcription factor SP-1, which could lead to downregulation of EPCR. Both inhibition and silencing of SP1 protein abolishes abnormal APC generation induced by resistin. Collectively, our data support a new role of resistin in disturbing APC formation.

Circulating activated protein C levels are not increased in septic patients treated with recombinant human soluble thrombomodulin

BackgroundRecombinant human soluble thrombomodulin (rTM) has been used for the treatment of disseminated intravascular coagulation in Japan, and an international phase III clinical trial for rTM is currently in progress. rTM mainly exerts its anticoagulant effects through an activated protein C (APC)-dependent mechanism, but the circulating APC levels after rTM treatment have not been clarified. This prospective observational study investigated plasma APC levels after rTM treatment.MethodsPlasma levels of soluble thrombomodulin, thrombin-antithrombin complex (TAT), protein C, and APC were measured in eight septic patients treated with rTM. APC generation in vitro was assessed in the presence or absence of rTM.ResultsrTM significantly increased thrombin-mediated APC generation in vitro. In septic patients, soluble thrombomodulin levels were significantly increased during a 30–60-min period of rTM treatment and TAT levels were decreased. However, APC activity was not increased during the treatment period.ConclusionsPlasma APC activity is not increased in septic patients treated with rTM. It is possible that APC acts locally and does not circulate systemically.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

AbstractCarriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs.

Thrombomodulin alfa attenuates the procoagulant effect and cytotoxicity of extracellular histones through the promotion of protein C activation

IntroductionExtracellular histones are reported to increase thrombin generation in the plasma and induce endothelial cell death in vitro. These effects of histones were suggested to involve histone-induced inhibition of TM-dependent activated protein C (APC) generation. Therefore, we hypothesized that TM alfa, a recombinant human soluble TM, attenuates these effects of histones by promoting the generation of APC. In the present study, we investigated the effects of TM alfa on the histone-induced decrease in APC generation, an increase in thrombin generation, and endothelial cell death in vitro. MethodsAPC generation was investigated using a chromogenic substrate based assay. Thrombin generation in plasma was studied by using a calibrated automated thrombogram method. Histone cleavage was detected by western blot analysis. Histone-induced endothelial cell death was evaluated by the trypan blue exclusion test. ResultsHistones decreased APC generation and increased thrombin generation in the presence of endothelial cells. TM alfa increased APC generation and decreased thrombin generation in the presence of histones and endothelial cells. TM alfa with thrombin and protein C cleaved histone H3, and attenuated histone-induced endothelial cell death. Antithrombin, an endogenous thrombin inhibitor, and gabexate mesilate, a synthetic protease inhibitor, inhibited thrombin generation, decreased APC generation, and did not have any effect on histone H3 cleavage or histone-induced endothelial cell death. ConclusionsTM alfa attenuated the histone-induced increase in thrombin generation and endothelial cell death by promoting APC generation in vitro.

The impact of the endothelial protein C receptor on thrombin generation and clot lysis

IntroductionWhen thrombin is bound to thrombomodulin (TM), it becomes a potent activator of protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI). Activation of PC is enhanced when PC is bound to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin generation while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of diminished EPCR function on thrombin generation and fibrinolysis we generated cells that expressed TM and a variant of EPCR (R96C) that does not bind PC. MethodsTo determine the impact of EPCR on the generation of APC and TAFIa and how this affects thrombin generation and fibrinolysis we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type TM and the R96C variant of EPCR (R96C cells). ResultsIn the presence of R96C cells, thrombin generation in normal plasma is increased, as a result of impaired PC activation when compared to WT cells. In addition, clot lysis is delayed in normal plasma in the presence of R96C cells, despite no increase in TAFI activation. In PC deficient plasma, clot lysis is delayed in the presence of WT and R96C cells as a result of increased TAFI activation. ConclusionsWe demonstrate that impaired EPCR function can be detected by thrombin generation and clot lysis assays on cells expressing TM and EPCR. We also demonstrated that deficiency in EPCR has procoagulant effects that lead to a delay in clot lysis.

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.

Bioactive Anti-Thrombotic Modification of Decellularized Matrix for Vascular Applications.

The decellularized matrix derived from porcine small intestinal submucosa (SIS) is a widely used biomaterial being investigated for numerous applications. Currently, thrombus deposition and neointimal hyperplasia have limited the use of SIS in some vascular applications. To limit these detrimental processes, this work applies bioactive, endothelial-inspired properties to the material. SIS is modified with the endothelial cell membrane protein thrombomodulin and the glycosaminoglycan heparin to facilitate protein C activation and anticoagulant activity, respectively. Modifying SIS with thrombomodulin alone enables robust activated protein C (APC) generation, and thrombomodulin activity is maintained after prolonged exposure to fluid shear and blood plasma. Heparin-modified SIS has a potent anticoagulant activity. When both modifications are applied sequentially, SIS modified first with thrombomodulin then with heparin retains the full activity of each individual modification. Tubular SIS devices are connected to a baboon arteriovenous shunt to quantify thrombus deposition on these materials. After being exposed to flowing whole blood for 60 min, SIS devices modified first with thrombomodulin then with heparin have significantly less platelet accumulation compared to unmodified SIS devices. These studies demonstrate that modifying SIS with thrombomodulin and heparin confers APC generation and anticoagulant activity that results in reduced thrombogenesis.

Thalidomide and multiple myeloma serum synergistically induce a hemostatic imbalance in endothelial cells in vitro

BackgroundThalidomide (Thal) treatment of patients with multiple myeloma (MM) is associated with vascular thrombosis, but the underlying mechanism is unknown. ObjectivesTo evaluate the hypothesis that Thal, dexamethasone (Dex) and MM serum perturb the hemostatic balance on human umbilical vein endothelial cells (HUVECs). MethodsDrugs with or without the serum of MM patients or healthy controls were incubated with HUVECs. Analyses of phosphatidylserine (PS), tissue factor (TF), endothelial protein C receptor (EPCR) and thrombomodulin (TM) were performed using flow cytometry. The production of thrombin and activated protein C (APC) were measured by chromogenic assay. The roles of IL-6 and TNFα in regulating these indicators were also investigated. ResultsWe found that Thal or Dex alone could not increase TF and PS expression in HUVECs. However, when pretreated with MM serum, their expression was significantly increased by either Thal or Dex. Concurrent changes were also detected in thrombin generation. In contrast, Thal and Dex had a direct inhibitory effect on the expression of EPCR and TM, and this inhibitory effect was especially significant when MM serum was added. The generation of APC paralleled the expression of EPCR and TM. All of the above outcomes were reversed to a variable extent by anti-IL-6R and anti-TNFα antibodies. ConclusionsThese findings suggest Thal may act as a procoagulant by altering the balance between procoagulant and anticoagulant proteins on the surface of HUVECs, thereby contributing to thrombogenesis. MM serum plays a synergistic role in this process.

FGFR1 mediates recombinant thrombomodulin domain-induced angiogenesis.

The recombinant epidermal growth factor-like domain plus the serine/threonine-rich domain of thrombomodulin (rTMD23) promotes angiogenesis and accelerates the generation of activated protein C (APC), which facilitates angiogenesis. The aim of this study was to elucidate the molecular mechanisms underlying the angiogenic activity of rTMD23. We prepared rTMD23 and its mutants that did not possess the ability to promote APC generation and investigated their angiogenic activities in vitro and in vivo. rTMD23 mutants promoted proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro and induced neovascularization in vivo; these effects were similar to those exerted by wild-type rTMD23. To investigate its interaction with rTMD23, Type I fibroblast growth factor receptor (FGFR1) was precipitated along with syndecan-4 by rTMD23-conjugated Sepharose in human umbilical vein endothelial cells and FGFR1-expressing human embryonic kidney 293 cells. Additionally, the kinetics of the interaction between rTMD23 and FGFR1 were analysed using surface plasmon resonance. rTMD23-induced FGFR1 activation and tube formation were inhibited by an FGFR1-specific tyrosine kinase inhibitor, PD173074, or by knockdown of FGFR1 using siRNA technology. We observed an improvement in rat hindlimb recovery in an ischaemic model following rTMD23 treatment, and this was associated with increased neovascularization and FGFR1 phosphorylation. rTMD23 induced angiogenesis via FGFR1, a process that is independent of the APC pathway.

Secretory group V phospholipase A2: a new player in thrombosis?

The protein C anticoagulant system plays a critical role in the regulation of haemostasis and inflammation. Binding of protein C to endothelial cell protein C receptor (EPCR) and the subsequent activation of EPCR-bound protein C by thrombin/thrombomodulin are the key steps in the protein C anticoagulant system. Activated protein C (APC) -mediated cell signalling, supported by its binding to EPCR or independent of EPCR, activates anti-inflammatory and cytoprotective pathways [1]. Therefore, pathophysiologic conditions that impair the interaction of protein C with EPCR have the potential to decrease APC generation. Such reduced binding to EPCR may not only contribute to thrombotic disorders, but also result in the loss of endogenous APC-mediated cytoprotection..