Presence Of Activated Protein C Research Articles

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation

IntroductionThrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. Materials and methodsWe hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. ResultsIn plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. ConclusionsWe propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.

Selective modulation of monocyte and neutrophil responses with activated protein C in preterm infants

Background Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease, and necrotizing enterocolitis. Activated protein c (APC) has shown positive immunomodulatory effects. Objectives We aimed to study neutrophil and monocyte function in response to lipopolysaccharide (LPS) and APC stimulation ex vivo in preterm infants <32 weeks gestation over the first week of life compared to neonatal and adult controls. Methods Peripheral blood was taken on day 1, 3, and 7 and stimulated with LPS in the absence or presence of APC. Expression of toll-like receptor 4 (TLR4) and CD11b and reactive oxygen intermediate (ROI) release from neutrophils and monocytes was examined by flow cytometry. Results LPS induced neutrophil ROI in adults and preterm infants and was significantly reduced by APC. Baseline and LPS-induced monocyte ROI production in preterm neonates was increased compared to adult and term controls. Neutrophil TLR4 baseline expression was higher in term controls compared to preterm infants. Conclusion Increased systemic ROI release in preterm infants may mediate tissue damage, ROI was reduced by APC. However, due to the high risk of hemorrhage further examination of APC mutant forms with anti-inflammatory but decreased anticoagulant properties is merited.

Activated Protein C-Catalyzed Inactivation Pathway Down-Regulates NXT007-Mediated Hemostatic Function

Activated protein C (APC) together with protein S (PS) inactivates factor (F)Va and FVIIIa and efficient FVIIIa inactivation requires not only PS but also cofactor function of FV. Any impairment in these inactivation pathways is one of the most common risks of venous thrombosis. NXT007, emicizumab (Emi)-based engineered therapeutic bispecific antibody (Ab), is currently undergoing the phase 1 clinical trial. NXT007 increased coagulation potential of FVIII-deficient plasma up to the similar level to healthy controls in tissue factor (TF)-triggered thrombin generation (TG; Yamaguchi, ASH 2020). Additionally, concomitant use of NXT007 and bypassing agents could be useful for improving coagulation potentials in FVIII-deficient samples (Ogiwara, ASH 2021). However, the role of APC-dependent inactivation in NXT007-mediated hemostasis reactions remains to be investigated. Here, we examined the regulatory effects by APC in NXT007-driven hemostasis using a purified FXa generation assay and TF/ellagic acid-triggered thrombin generation assay (TGA). We firstly investigated coagulation potentials in FVIII-deficient plasma (ΔFVIII) spiked with NXT007 (3-100 μg/mL) or Emi (50 μg/mL). Peak thrombin (PeakTh) in ΔFVIII spiked with NXT007 was increased in a NXT007 dose-dependent manner. ΔFVIII spiked with NXT007 (3 μg/mL) showed higher PeakTh (355±74 nM) than emicizumab (50 μg/mL; 274±48 nM). PeakTh in ΔFVIII after the addition of NXT007 (10, 30, and 100 μg/mL) were 419±12, 454±13, and 484±10 nM, respectively. The coagulant effects of NXT007 (30 and 100 μg/mL) were comparable to pooled normal plasma (PNP). To evaluate the impact of APC-mediated down regulation on the hemostatic enhancement by NXT007 in ΔFVIII, TGA in ΔFVIII reacted with NXT007 (10 μg/mL) in the presence of APC (0-16 nM) was examined. PeakTh was decreased in an APC-dose dependent manner. Comparing with PeakTh without and with APC (16 nM), PeakTh was reduced from 457±11 nM to 331±1.4 nM and the %inhibition was 28%. We next evaluated the effects of APC on TG in PNP with or without an anti-FVIII polyclonal Ab (FVIIIAb) (10 BU/mL). The %inhibition of PeakTh in PNP with APC (16 nM) was 30%, indicating that the effect of APC-mediated inactivation was not disturbed by the presence of NXT007. To clarify the association of NXT007 alone with APC-catalyzed inactivation, we examined a purified FXa generation with NXT007 (10 μg/mL) or with FVIIIa (2 nM) after the addition of APC (0-2 nM) and PS (5 nM). FXa generation with FVIIIa was reduced by ~60% at maximum level in an APC dose-dependent manner, whereas that with NXT007 was not impaired, indicating that NXT007 itself was not affected by APC-catalyzed inactivation. To elucidate the contribution of APC-catalyzed inactivation of FV(a) in NXT007-catalyzed hemostatic regulation, we evaluated the effect of APC on the TG with NXT007 (10 μg/mL) and FV (7.5-30 nM; corresponded to 25-100% of physiological concentration) in the FV-deficient plasma with FVIIIAb (10 BU/mL). The PeakTh in the presence of NXT007 and FV (7.5-30 nM) without APC was 310-340 nM and the %inhibition of PeakTh by the addition of APC (16 nM) was ~35%, showing that NXT007 showed sufficient coagulation potential even at low concentration of FV and that the inhibition of TG by APC-catalyzed FV(a) inactivation was not disturbed by the presence of NXT007 even at FV (7.5 nM). In addition, the influence on TG with APC in the FV-Leiden (FV-L) plasma without or with FVIIIAb and NXT007 (10 μg/mL) were investigated. The %inhibition of PeakTh by APC (16 nM) in FV-L plasma was ~20%. While, the %inhibition of PeakTh by APC (16 nM) in FV-L plasma with FVIIIAb and NXT007 was similar to FV-L plasma alone (~20%), indicating that APC could regulate NXT007-catalyzed hemostatic reaction by inactivation of FV(a) under the FV-L condition. We conclude that APC-catalyzed inactivation mechanisms could play a significant role on the down-regulation of coagulation in NXT007-mediated hemostasis.

Protection of Human Factor V from Activated Protein C Using a Monoclonal Antibody-Biochemical Characterization and Evaluation Using a Mouse Injury Model

Hemophilia is an X-linked bleeding disorder resulting in deficiency of FVIII (hemophilia A; HA) or FIX (HB). Despite treatment with infused FVIII or FIX, people with hemophilia still experience joint bleeding and impaired quality of life. Furthermore, the development of neutralizing antibodies to FVIII or FIX remains a major complication of therapy. To this end, several innovative therapeutic advances which rebalance the coagulation system by targeting anticoagulant pathways (antithrombin, tissue factor pathway inhibitor, and activated protein C (APC)) are particularly interesting as they should be effective for both HA and HB, with and without inhibitors, and may also be useful to treat certain rare bleeding disorders. Many of these approaches are in clinical trials and show promise. However, each of these anticoagulant pathways target multiple coagulation factors, and APC plays an important cellular signaling role. In the current study, we developed a monoclonal antibody (GB5) that targets human FV/FVa which could serve as an alternative therapeutic approach to enhance thrombin generation and prevent bleeding in different clinical situations.Monoclonal antibody GB5 was identified following screening of hybridoma clones derived from mice immunized with human FV. The initial screen identified GB5 as an antibody that could promote thrombin generation (TG) in the presence of APC. To characterize the antibody, purified GB5 was attached to a biosensor tip and evaluated using biolayer interferometry. Analysis of biosensor tracings revealed GB5 bound specifically and with high affinity to both FV (K d = 0.6 nM; 2 experiments) and FVa (K d = 0.7 nM; 2 experiments). A TG assay with pooled normal human plasma (NHP) or HA plasma in the absence or presence of APC was used to assess the functional effects of GB5. In the absence of APC, GB5 had a minimal effect on the TG profile initiated with low tissue factor (0.2 pM). As expected, the addition of APC (2 nM, final) to NHP or HA plasma substantially reduced peak thrombin. However, GB5 dose-dependently increased peak thrombin in the presence of APC in NHP or HA plasma and restored TG to normal levels at ~20 nM. GB5 also protected FV from APC when assessed using a purified prothrombin activation assay. Western blotting experiments using FV proteolyzed with thrombin, APC, or plasmin revealed that GB5 binds the light chain A3-C1-C2.Preliminary experiments revealed that GB5 does not cross react with mouse FV. To evaluate the effect of GB5 in vivo, wild-type (WT) mice were treated with a liver targeted antisense oligonucleotide (ASO) to knockdown mouse plasma FV (ASO-FV; 40 mg/kg; FV-ASO mice). Mouse platelet FV was not impacted. To supplement the plasma compartment with FV, the FV-ASO mice were administered human FV (hFV-mice). This experimental set-up allowed for clot formation to largely be dependent on hFV using the mouse laser injury model. In these studies, we compared the kinetics of thrombus formation in WT mice, FV-ASO mice, or hFV-mice (n=2-3/group; 7-10 thrombi per group) for 10-15 minutes following laser injury. We found that platelet and fibrin accumulation was robust in WT mice and almost undetectable in FV-ASO mice (consistent with the lack of plasma FV), while clot formation was low but detectable in hFV-mice. There was no obvious difference in thrombus size when GB5 was given to WT or FV-ASO mice. However, in hFV-mice (200 μg/kg hFV), GB5 increased platelet and fibrin accumulation to levels seen with WT mice. Quantitative analysis revealed that compared to hFV mice alone, GB5 increased platelet accumulation 3-fold and fibrin (3-6-fold accumulation. Significantly greater accumulation of platelets and fibrin (~30-fold) was observed when higher amounts of hFV (400 μg/kg) were co-infused with GB5 compared to hFV-mice without antibody.Together, these results demonstrate that monoclonal antibody GB5 binds to hFV/FVa with high affinity and confers APC resistance. Using a unique mouse model to assess human FV, we found that GB5 enhanced clot formation in vivo using the laser injury model. From a mechanistic standpoint, these data show that protecting FV from APC translates to greater thrombin generation in vivo and suggest this approach may be useful to treat bleeding disorders such as HA and HB. Additional injury models using the hFV-mice and hemophilic mice to assess the effectiveness of GB5 are ongoing. DisclosuresCrosby: Ionis Pharmaceuticals: Other: Jeffrey is a current employee of Ionis Pharmaceuticals. Revenko: Ionis Pharmaceuticals: Other: Alexey is a current employee of the company.. MacLeod: Ionis Pharmaceuticals: Other: Robert is a current employee of the company. Monia: Ionis Pharmaceuticals: Other: Brett is a current employee of the company. .

Inhibitory effects of amniotic fluid on the activated protein C anticoagulation system in maternal plasma.

Pulmonary thromboembolism (PTE) is one of the leading causes of maternal mortality. We previously reported that possible contamination of amniotic fluid (AF) into maternal circulation accelerated thrombin production and activated platelet function in maternal blood through the extrinsic pathway, which may be associated with the high incidence of PTE in early puerperium. However, it remains unclear whether the maternal anticoagulation system, e.g., the activated protein C (APC) pathway, contributes to the hypercoagulable condition induced by AF. Our previous study using an endogenous thrombin potential (ETP)-based assay revealed that sensitivity to APC was reduced during the postpartum first day, i.e., immediately after delivery, when parturients were supposed to be exposed to AF. Our aim is to investigate the susceptibility of maternal plasma to APC when mixed with AF. We collected plasma from 51 pregnant females and mixed with AF as well as APC. APC-sensitivity ratio (APC-sr) was calculated using the ETP-based assay. Addition of AF to maternal plasma showed a significant increase of ETP in the presence of APC. APC-sr was significantly increased, indicating decreased sensitivity to APC, after AF mixture to maternal plasma. The present APC-sr difference with AF contamination was smaller than that we reported previously in venous thromboembolism cases. The inhibitory effects of AF on the APC anticoagulation pathway may contribute, at least partly, to further promotion of thrombin production induced by AF. Combined with other classical thrombophilic risk factors, the present findings support possible involvements of AF exposure in the high incidence of PTE in early puerperium.

Regulation of Factor VIII By Activated Protein C: Assessment In Vitro and In Vivo

Factor VIII (FVIII) plays an essential role in the activation of factor X (FX), acting as a factor IXa (FIXa) cofactor in the intrinsic Xase complex. Activated protein C (APC) is a serine protease that inactivates FV/Va. Resistance of FV/FVa to APC due to the Leiden mutation is the most common inherited thrombophilia. By cleaving FVIII/FVIIIa at Arg336 and Arg 562, APC can also inactivate FVIII; however, the role of this reaction, especially in vivo, is not well defined. Further, thrombophilic mutations at either of these sites have yet to be identified, calling into question the importance of this reaction. Understanding the role of APC in FVIII regulation is important as there are several novel therapeutics in development for hemophilia A (HA) that either bypass FVIII regulatory pathways or target inhibition of APC. Additionally, while data using a high-specific activity factor IX variant has been successful in a hemophilia B gene transfer trial, there is no such described hyperactive FVIII protein. In principle, manipulation of APC cleavage sites may result in a more procoagulant FVIII protein with enhanced hemostastic function that may serve as a novel therapeutic for HA in the setting of a recombinant protein treatment or gene therapy.We sought to further understand the role of APC regulation of FVIII and to evaluate the in vivo hemostatic effect of an APC-resistant FVIII as a potential treatment strategy for HA. We conducted site-directed mutagenesis to introduce missense arginine to glutamine mutations at FVIII APC cleavage sites (R336Q and R562Q) on a B-domain deleted FVIII (FVIII-WT) backbone. The resulting recombinant, purified FVIII variant protein (FVIII-QQ) was then compared to FVIII-WT. Following incubation with APC, SDS-PAGE analysis of FVIII-QQ demonstrated no apparent cleavage by APC. In contrast, FVIII-WT demonstrated expected cleavage at R336 and R562, thereby confirming FVIII-QQ is resistant to APC cleavage. Surprisingly, one-stage clotting assay determination of FVIII-QQ specific activity was approximately 30% higher than FVIII-WT; this was unexpected given the absence of thrombomodulin or APC. We speculate this may be related to the ability of FXa to cleave FVIIIa at Arg336 and Arg562 inactivating FVIIIa that is likely disrupted in the FVIII-QQ variant (Eaton D et al. Biochemistry 1986 and Plantier JL et al. J Thromb Haemost 2010). To test the role of APC regulation of FVIII, we performed plasma-based in vitro thrombin generation assays in the presence of APC. Human FVIII deficient plasma was reconstituted to 1 nM (normal plasma FVIII concentration) with FVIII-QQ or FVIII-WT wherein increasing concentrations of APC were then added and thrombin generation was measured. Consistent with the known role of APC being an anticoagulant, the amount of thrombin generation decreased with increasing APC concentrations. However, the amount of APC necessary to inhibit 50% peak thrombin generation with FVIII-WT was 5.2 nM ± 0.3 nM versus FVIII-QQ was 11.3 nM ± 0.8 nM, which again demonstrated FVIII-QQ is resistant to APC inhibition. We suspect the observed decrease in thrombin generation with increasing APC concentrations is due to the role of APC in FV/FVa inhibition. Overall, our data support a role for APC inactivation of FVIIIa in this in vitro system. Interestingly, FVIII-QQ reliably demonstrated an approximate 2-fold increase in peak thrombin in the thrombin generation assay. This is being explored, but we again speculate this may be related to the effects of FXa on FVIII-WT at R336 and R562.We next evaluated the in vivo hemostatic efficacy of FVIII-WT vs. FVIII-QQ in HA mice. HA mice were infused with FVIII-WT or FVIII-QQ protein prior to tail clip assay. Comparison of both proteins at a dose of 3 µg/kg demonstrated greater blood loss in HA mice treated with FVIII-WT vs. FVIII-QQ (p=0.006). Further, blood loss in HA mice treated with FVIII-WT was greater than wild type mice (p=0.007); in contrast, there was no difference between wild type and HA mice treated with 3 µg/kg of FVIII-QQ (p=0.26).Our data demonstrate FVIII-QQ has a higher specific activity, requires 2-fold greater APC concentrations to inhibit thrombin generation in vitro and an approximate 2-fold greater in vivo hemostastic efficacy. Our results support that APC has physiologic significance in FVIII/FVIIIa inactivation and provide rationale for the study of APC in FVIII/FVIIIa regulation to develop potential novel HA therapeutics. DisclosuresDavidson:Spark Therapeutics: Consultancy. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Bayer: Consultancy, Research Funding. George:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B.

Activated protein C inhibits lipopolysaccharide‐mediated acetylation and secretion of high‐mobility group box 1 in endothelial cells

Essentials APC elicits cytoprotective responses in endothelial cells via EPCR-dependent cleavage of PAR1. APC inhibits LPS-mediated translocation and extracellular secretion of HMGB1 in endothelial cells. Signaling activity of APC inhibits LPS-mediated acetylation of HMGB1 by epigenetic mechanisms. APC inhibits LPS-mediated HMGB1 expression in CD31-positive endothelial cells in cremaster muscle. SUMMARY: Background Activated proteinC (APC) inhibits high-mobility group box1 (HMGB1) signaling and its lipopolysaccharide (LPS)-mediated release by endothelial proteinC receptor (EPCR)-dependent activation of protease-activated receptor1 (PAR1) in endothelial cells. Post-translational acetylation is known to modulate the subcellular localization of HMGB1, and its hyperacetylated form is translocated to the cytoplasm of innate immune cells before being secreted into the extracellular space. Objective To determine whether APC inhibits LPS-mediated HMGB1 secretion from endothelial cells by modulating its acetylation status. Methods The subcellular localization of HMGB1 in LPS-treated endothelial cells was monitored in the absence and presence of APC by western blot analysis of fractionated cell lysates and confocal immunofluorescence microscopy. Results Both western blot and immunofluorescence data indicated that APC effectively inhibits LPS-mediated translocation of HMGB1 from the nucleus to the cytoplasm by EPCR-dependent and PAR1-dependent mechanisms. When EPCR was ligated by the Gla-domain of proteinC/APC, thrombin also inhibited LPS-mediated HMGB1 translocation. Further studies revealed that APC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm by inhibiting LPS-mediated hyperacetylation of HMGB1 by (de)acetylating enzymes. Furthermore, the translocated HMGB1 was found to be associated with lysosome-associated membrane protein1 in LPS-treated endothelial cells. The invivo relevance of these findings was investigated in the mouse cremaster muscle, and this demonstrated that both wild-type APC and a signaling-selective mutant of APC inhibit LPS-mediated HMGB1 expression and translocation in CD31-positive endothelial cells. Conclusion These results suggest that APC inhibits LPS-mediated cytoplasmic translocation and secretion of HMGB1 in endothelial cells by epigenetic mechanisms.

Emicizumab-mediated haemostatic function in patients with haemophilia A is down-regulated by activated protein C through inactivation of activated factor V.

Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti-FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once-weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab-mediated haemostasis remain to be explored. We investigated the role of APC-mediated reactions in these circumstances. APC dose-dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII-deficient plasmas, and in normal plasmas preincubated with an anti-FVIII antibody (FVIII-depleted). FVIIIa-independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC-induced down-regulation of emicizumab-dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII-depleted FV-deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab-related TG in FVIII-depleted FVLeiden plasma was decreased by APC more than that observed with native FVLeiden plasma. The findings indicated that emicizumab-driven haemostasis was down regulated by APC-mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.

Abstract 520: Skeletal Muscle Myosin Supports the Anticoagulant Functions of Activated Protein C and Protein S but not Protein C Activation by Thrombin:Thrombomodulin

Skeletal muscle myosin was reported by Deguchi et al to promote thrombus formation ex vivo for flowing human whole blood, to support prothrombinase activity in plasma and to promote prothrombin activation by factors (F) Xa and Va in purified systems. Skeletal muscle myosin binds FXa and FVa and effectively replaces the procoagulant activity of phospholipid vesicles. We therefore assessed whether myosin might replace phospholipids in support of the anticoagulant function of activated protein C (APC) and its cofactor protein S (ProS) for inactivation of purified FVa. When purified component prothrombinase assays were used to monitor FVa inactivation by APC, FVa activity was significantly reduced by APC in the presence of myosin compared to controls, and FVa inactivation in the presence of 40 nM myosin over 25 min was much greater than that in the presence of 40 nM phospholipids and similar to the rate of FVa inactivation in the presence of 25 μM phospholipids (see Figure). Inhibition of the APC-enhancing effects of myosin were confirmed to be specific for myosin because the anti-myosin inhibitor, myoVin-1, inhibited myosin’s effects. ProS at ≥ 25 nM enhanced the myosin-stimulated inactivation of FVa by APC. The ProS anticoagulant enhancement effect was most obvious at lower APC concentrations, as expected. Immunoblots showed that myosin enhanced Arg506 cleavage in FVa by APC alone; but notably in the presence of ProS, myosin enhanced both Arg506 and Arg306 cleavages in FVa. Myosin did not enhance activation of protein C by thrombin in the presence or absence of thrombomodulin. Thus, in purified reaction mixtures, skeletal muscle myosin remarkably can support both procoagulant thrombin generation, as previously reported, and compensatory anticoagulant mechanisms in the presence of APC and ProS, as shown here. Further studies are needed to determine the molecular basis for these reactions and to assess the physiologic and pathologic relevance of these reaction mechanisms.

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

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

PO-47 - Microparticles derived from ovarian cancer cell line contained genomic and biologically active proteins, including tissue factor involved in coagulation

The microparticles (MPs) are sized vesicles of less than 1 μm, from different cell types upon activation or subsequent to apoptosis. They are involved in the thrombotic process, particularly in cancer. The role of MPs in ovarian cancer and their involvement in thrombosis being poorly understood. The aim of this study was to identify in vitro the generation of MPs by an human ovarian adenocarcinoma cell line (OVCAR-3). OVCAR-3 cells were cultured in three conditions [without stimulation, with protein C (PC), and with activated protein C (APC)]. Then, the MPs present in the supernatant, were isolated by ultracentrifugation and were analyzed for their shape and properties by flow cytometry, electron microscopy, cryofracture analysis, DNA and RNA, and proteomic analysis. The level of tissue factor (TF) on MP was evaluated by TF-induced shortening of Ca(2+) plasma coagulation time. Our results demonstrated that 1) 92% of MPs derived from OVCAR-3 were less than 100 nm. 2) As tested by flow cytometry, the MPs contained b2 microglobulin, annexin, DNA fragments and TF that induces a shortening of Ca(2+) -induced plasma coagulation time. When OVCAR-3 were cultured for 18H with PCA, MPs were generated in greater amount than those generated by OVCAR-3 in its absence and their level of TF was increased of 20%. Curiously, in contrast with intact OVCAR-3 cells, the endothelial protein C receptor (EPCR) was not detected in MPs 3) Proteomic analysis show that the MPs contain proteins involved in cancer progression such as mucins (5A and 5B), keratin type-1, actin, annexin (A1, A2, A4), CD44, glypican, heat shock (70kDa and HS90a) proteins, Agrin associated with heparan sulfate proteoglycan abundant in the tumor-specific basement membrane, Ephrin type A receptor, coronin-1C, catenin α, integrin β-1 and also p-selectin responsible of platelet activation. They also express several DNA associated proteins includingtranscription factors, various polymerases, nucleases, and histones involved in chromosome packaging and transcription in the cell nucleus. MPs derived from OVCAR-3 have an apoptotic character. They expressed several biologically active proteins, DNA and their associated proteins. Despite the absence of EPCR expression on MPs that was expressed on intact OVCAR-3 cells, they expressed procoagulant TF activity already found on intact ovarian cancer cells. This activity is greater extent in the presence of APC.

Influence of single nucleotide polymorphisms on thrombin generation in factor V Leiden heterozygotes

Carriership of the factor V (FV) Leiden mutation increases the risk of venous thromboembolism (VTE) ~4-fold, but the individual risk of each FV Leiden carrier depends on several co-inherited risk and protective factors. Under the hypothesis that thrombin generation might serve as an intermediate phenotype to identify genetic modulators of VTE risk, we enrolled 188 FV Leiden heterozygotes (11 with VTE) and determined the following parameters: thrombin generation in the absence and presence of activated protein C (APC); plasma levels of prothrombin, factor X, antithrombin, protein S and tissue factor pathway inhibitor; and the genotypes of 24 SNPs located in the genes encoding these coagulation factors and inhibitors. Multiple regression analysis was subsequently applied to identify the (genetic) determinants of thrombin generation. The endogenous thrombin potential (ETP) showed a striking inter-individual variability among different FV Leiden carriers and, especially when measured in the presence of APC, correlated with VTE risk. Several SNPs in the F2 (rs1799963, rs3136516), F10 (rs693335), SERPINC1 (rs2227589), PROS1 (Heerlen polymorphism) and TFPI (rs5940) genes significantly affected the ETP-APC and/or the ETP+APC in FV Leiden carriers. Most of these SNPs have shown an association with VTE risk in conventional epidemiological studies, suggesting that the genetic dissection of thrombin generation leads to the detection of clinically relevant SNPs. In conclusion, we have identified several SNPs that modulate thrombin generation in FV Leiden heterozygotes. These SNPs may help explain the large variability in VTE risk observed among different FV Leiden carriers.

Superior in Vivo Hemostatic Properties of an Engineered Factor Va Variant for Hemophilia Mice

AbstractAbstract 17Approximately 1/3rd of hemophiliacs with inhibitors fail treatment with FVIIa-based bypassing agents. New intervention strategies are urgently needed. Activated FV (FVa) enhances thrombin formation by the prothrombinase complex, but is rapidly inactivated by activated protein C (APC). Mutation of the APC cleavage site, R506Q (FVLeiden), is prothrombotic and improves hemostasis in humans and mice with hemophilia. We therefore engineered FV-derivatives with additional mutations of the APC cleavage sites (Arg506/306/679Gln) and introduced a disulfide bond (Cys609-Cys1691) connecting the A2 and A3 domains (FV(A2-SS-A3)). Introduction of the disulfide bond prevents the dissociation of the A2 domain as the final irreversible step of inactivation. All FV mutants were activated with thrombin prior to experimentation. Procoagulant properties of FVa mutants were studied in vitro and in hemophilia mouse models. Mutation of all three APC cleavage sites (Arg506/306/679) and addition of the disulfide bond yielded “superFVa” that had 2- to 3-fold higher specific activity compared to recombinant human (rh)FVa, rhFVa(A2-SS-A3), rhFVaLeiden or rhFVaLeiden(A2-SS-A3) in purified prothrombinase assays and in thrombin generation assays in FV- and FVIII-deficient plasma. superFVa corrected the aPTT at 20-fold lower concentrations compared to rhFVa in FV-deficient plasma. superFVa was highly resistant to inactivation by APC in purified prothrombinase assays and in thrombin generation assays in FV-deficient plasma. While superFVa retained 90–100% activity in the presence of APC, FVa(A2-SS-A3) and FVaLeiden(A2-SS-A3) lost ∼90% and ∼40% of their activities, respectively. In FVIII-deficient mice, superFVa reduced bleeding significantly compared to rhFVa in a dose dependent fashion after tail clip (n=10–17 per group). Blood loss was determined separately for the first and second 10 min after tail clip to study the effects of FVa mutants on re-bleeding. FVa mutants were dosed at equal activities determined in prothrombinase assays (expressed in arbitrary units). At low doses of intravenous FVa mutants (40 units), mean blood loss during the first 10 min after clip in FVIII-deficient mice was significantly decreased following injection of superFVa compared to saline (6.5 μL/g vs. 15.5 μL/g; p=0.02), whereas blood loss following injection of rhFVa was not (16.7 μL/g). At high doses of FVa mutants (100 units), superFVa and rhFVa were effective in reducing blood loss in FVIII-deficient mice following intravenous injection. Mean blood loss with superFVa was further improved to 3.9 μl/g, and with rhFVa was 8.2 μl/g. Both results were significant compared to mean blood loss following saline injection (15.5 μL/g; p≤0.05). FVIII-deficient mice were considered protected at 10 min when blood loss was ≤ 9.9 μL/g, which is the mean plus 3 SDs derived from tail bleeding of wild-type BalbC mice. While only ∼45% of FVIII-deficient mice injected with saline or rhFVa (40 units) were protected, protection against bleeding increased to 83% with superFVa (40 units). When 100 units of FVa mutants were given, protection with both mutants (superFVa or FVa) was ≥ 90%. Of note, during the second 10 min and therefore during the combined 20 min of bleeding, only superFVa at high dose (100 units) effectively reduced bleeding. Mean blood loss in FVIII-deficient mice following intravenous superFVa was 11.7 μL/g during the 2nd 10 minutes of bleeding, compared to 20.2 μL/g with saline (p=0.02). Our results indicate that molecular engineering of FV, which combined two alterations for improving thrombin generation (increased specific activity and APC resistance), provided a FVa-mutant, superFVa, with unique procoagulant properties. In vivo, superFVa not only decreased initial bleeding, but also significantly decreased re-bleeding, indicating that superFVa efficiently improved clot formation and also enhanced clot stabilization. These results warrant more in depth characterization of the potential therapeutic benefits of superFVa as a novel bypassing strategy in hemophilia patients with inhibitors. Disclosures:No relevant conflicts of interest to declare.

Coagulation response in dogs with and without systemic inflammatory response syndrome – Preliminary results

The impact of systemic inflammatory response syndrome (SIRS) on all phases of coagulation is largely unknown in dogs.Fifty-six healthy dogs (controls) and 25 diseased dogs were included. Based on physical and hematological examination, dogs were classified as “no-SIRS” (n=7) or “SIRS” (n=18). Evaluated coagulation variables included platelets, coagulation times, fibrinogen, antithrombin (AT), FVIII, protein C, protein S, activated protein C (APC)-ratio, calculated from aPTT with and without presence of APC, and kaolin-activated thrombelastography (TEG).Overall, no-SIRS and SIRS were characterized by hypocoaguable state (P<0.001 compared to controls) i.e., prolonged coagulation times, decreased AT (median 59U/L and 89U/L versus 126U/L), and FVIII (median 19U/L and 70U/L versus 102U/L). In no-SIRS and SIRS, APC-ratio was significantly lower than in the controls (median 1.1 and 2.0 versus 2.5, P<0.01, P<0.001).Severe coagulopathies may be present in critically ill dogs without concurrent SIRS. APC-resistance is a frequent finding in severely diseased dogs.

TFPI-dependent activities of Protein S

Protein S is an essential anticoagulant protein that acts as a cofactor for full length tissue factor pathway inhibitor (TFPI) and activated protein C (APC) in the down regulation of coagulation. Protein S enhances APC-mediated inactivation of the coagulation factors Va and VIIIa, and it stimulates inhibition of factor (F)Xa by TFPI. Because TFPI is a tight binding, but slow inhibitor of FXa, the TFPI/protein S system fails to regulate FXa generation at high tissue factor/FVIIa concentrations. In this review, we explain how TFPI/protein S can regain its activity at high tissue factor concentrations in the presence of APC, resulting in an intertwinement of TFPI- and APC-cofactor activities of protein S, and making TFPI a major determinant of APC-anticoagulant activity in plasma.

Effect of Experimental Endotoxemia on Thrombelastography Parameters, Secondary and Tertiary Hemostasis in Dogs

Thrombelastography (TEG) and indicators of secondary and tertiary hemostasis might be altered in dogs with endotoxemia. Endotoxemia influences measures of coagulation in dogs. Ten healthy cross-bred dogs. Prospective laboratory study between controls (n = 5) receiving 0.9% saline IV and the study group (n = 5) treated with low-dose lipopolysaccharide (0.02 mg/kg IV). Physical examination and sampling for measurement of leukocytes, platelets, and coagulation variables were performed at time points 0, 1, 4, and 24 hours. Coagulation variables included kaolin-activated TEG, 1-stage prothrombin time (OSPT), activated partial thromboplastin time (aPTT), fibrinogen, factor VIII, antithrombin, protein C, protein S, activated protein C (APC)-ratio calculated from aPTT with and without presence of APC), and D-Dimers. Endotoxemia-induced clinical signs included lethargy (n = 5/5), diarrhea (n = 4/5), emesis (n = 4/5), and abdominal pain (2/5). After 1 hour there was severe leukopenia (2.5 ± 0.7 × 10(9)/L; mean ± SD, P < .0001) and a 2.2-fold increase in D-Dimers (0.81 ± 0.64 mg/L, P < .0001). After 4 hours there was hyperthermia (40.3 ± 0.4°C, P < .0001) and increases in OSPT (10.5 ± 2.7 seconds, P < .0001), aPTT (16.7 ± 5.2 seconds, P = 0.002). A significant decrease in fibrinogen (1.5 ± 1.0 g/L, P = 0.001), protein C (31 ± 33%, P < .0001), protein S (63 ± 47%, P < .0001), TEG α (58 ± 19, P = .007), and TEG maximal amplitude (50 ± 19 mm, P = .003) was seen compared with the controls. APC-ratio rose significantly (2.5 ± 0.2, P < .0001) without exceeding the reference interval (n = 4/5). D-Dimers are the earliest indicator for endotoxemia-associated coagulation abnormalities followed by decreased protein C concentration. APC-ratio and TEG were not good screening variables.

Thrombin generation and activated protein C resistance in the absence of factor V Leiden correlates with the risk of recurrent venous thromboembolism in women aged 18–65 years

Identification of patients at high risk of recurrence after a first event of venous thromboembolism (VTE) remains difficult. Resistance to activated protein C (APC) is a known risk factor for VTE, but data on the risk of recurrence is controversial. We wanted to investigate whether APC resistance in the absence of factor V Leiden, determined with global coagulation test such as the thrombin generation assay, could be used as a marker for increased risk of recurrent VTE among women 18-65 years old after a first event of VTE. In a cohort of 243 women with a first event of VTE, plasma was collected after discontinuation of anticoagulant treatment and the patients were followed up for 46 months (median). Thrombin generation was measured via calibrated automated thrombography, at 1 pM and 10 pM of tissue factor (TF). In women without factor V Leiden (n=117), samples were analysed in the absence and in the presence of APC. Increase in ETP (endogenous thrombin potential) and peak height analysed in the presence of APC correlated significantly with higher risk of recurrence. At 1 pM, peak height correlated with increased risk of recurrence. In conclusion, high thrombin generation in the presence of APC, in women after a first event of VTE is indicative for an increased risk of a recurrence. We also found that thrombin generation at low TF (1 pM) is correlated with the risk of recurrence. Our data suggest that APC resistance in the absence of factor V Leiden is a risk factor for recurrent VTE.

High Thrombin Generation Measured In the Presence of Activated Protein C Is Associated with An Increased Risk of Recurrence Among Women 18–64 Years After a First Event of VTE

AbstractAbstract 154 Background:Patients with venous thrombosis (VTE) have a high risk of a recurrence after discontinuation of anticoagulants. Both prolonged anticoagulant treatment with increased risk for bleeding and a recurrence reduces quality of life. In women younger than 65 years old the recurrence rate is relatively low. However, identification of the patients at risk of a recurrence remains difficult. Objective:The aim of our study was to investigate whether a global coagulation test such as thrombin generation can be used as a marker for the increased risk of a recurrence of VTE among women of 18–65 years old after a first event of deep venous thrombosis (DVT) or pulmonary embolism (PE). Methods:139 women with an objectively documented first event of DVT or PE were enrolled into the study at diagnosis. Plasma was collected at least 4 weeks after discontinuation of anticoagulant treatment. Patients with cancer or pregnancy within 3 months after the first event were excluded. All patients were followed up prospectively for median 47 months. Recurrences of VTE were objectively documented. No participants were taking oral estrogens treatment during the follow up. Thrombin generation was measured via calibrated automated thrombography (CAT), in the absence and in the presence of activated protein C (APC). The APC sensitivity ratio (APCsr) was calculated to estimate APC-resistance. All participants were screened for factor V Leiden and G20210A mutation. Results:The ETP and peak height in the presence of APC (ETP+APC, peak height+APC) were associated with a recurrence of VTE in a log-rank test. In a multivariate cox-regression patients with the ETP+APC > 169 nM*min had hazard ratios (HR) for recurrent VTE of 8.8 (p=0,008) compared to patients with the ETP+APC < 169 nM*min. Patients with the peak height+APC > 58 nM had HR of 7.2 (p=0,014). Patients with the APCsr > 3.32 had HR of 6,7 (p=0,006) for recurrence of VTE. Age, inherited thrombophilia (factor V Leiden, G20210A mutation) and BMI> 30 were included into the multivariate analysis. Conclusion: High thrombin generation in the presence of APC in women after a first event of VTE is indicative for an increased risk of a recurrence. Our data suggest that acquired APC resistance is a risk factor for recurrent VTE. Disclosures:No relevant conflicts of interest to declare.

Covalent antithrombin-heparin effect on thrombin-thrombomodulin and activated protein C reaction with factor V/Va

Thrombomodulin (TM), which variably contains a chondroitin sulfate (+/-CS), forms an anticoagulant complex with thrombin (IIa). IIa-TM(+/-CS) converts protein C (PC) into activated PC (APC), which then inactivates activated factors V (FVa) and VIII (FVIIIa). This reduces prothrombinase and tenase complexes that generate IIa. Heparin (H) increases the rate of IIa-TM inhibition by antithrombin (AT) and enhances FV cleavage by APC. Our novel covalent AT-H (ATH) product, has superior anticoagulant activity compared to AT + unfractionated H (UFH). We studied mechanisms by which ATH versus AT + UFH inhibits IIa-TM(+/-CS), and ATH influences on APC cleavage of FV/FVa compared to UFH. Findings would determine how these reactions moderate ATH's overall effects as an anticoagulant. Discontinuous second order rate inhibition assays of IIa-TM(+/-CS) inhibition by AT + UFH or ATH were performed in presence or absence of human umbilical vein endothelial cells (HUVECs). FV/FVa cleavage by APC in the presence of UFH or ATH was analysed by Western blots. ATH increased IIa-TM(+/-CS) inhibition to a greater degree than AT + UFH, both on plastic and HUVEC surfaces. Unlike UFH, ATH did not accelerate FV cleavage by APC, but ATH did enhance FVa cleavage relative to UFH. Increased IIa-TM inhibition by ATH downregulates PC activation. However, ATH does accelerate downstream inactivation of FVa, which increases its potency for IIa generation inhibition compared to UFH. This trend holds true in the presence of APC's cofactor, protein S. Overall, ATH may have a balanced function towards inhibiting or accelerating PC pathway activities.

Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function

Activated protein C (APC) is an anticoagulant, approved as a treatment for severe sepsis, that can prevent apoptosis, inflammation, and vascular leakage. The aim of this study was to investigate whether APC protects endothelial barrier function through the angiopoietin (Ang)/Tie2 axis. APC significantly up-regulated gene and protein expression of Tie2 and Ang1 in a dose (0.01-10 microg/ml)- and time (0.5-24 h)-dependent manner in human umbilical vein endothelial cells (HUVECs). Interestingly, it markedly inhibited Ang2 with an IC(50) of approximately 0.1 microg/ml. HUVEC permeability, measured using Evans blue dye transfer, was significantly reduced in the presence of APC, and, in concordance, the tight junction associated protein zona occludens (ZO)-1 was up-regulated and localized peripherally around cells, compared with controls. Smooth muscle cell migration toward APC-stimulated HUVECs was elevated compared with unstimulated cells. Blocking antibodies and small interfering (si) RNA treatment, compared with isotype (IgG) or scrambled siRNA controls, showed that APC requires 3 receptors, the endothelial protein C receptor, protease-activated receptor 1, and Tie2 to perform all these barrier stabilization functions. In summary, this study demonstrates that APC has novel effects on the Ang/Tie2 axis, which enhance endothelial barrier function and are likely to contribute to its therapeutic effect in sepsis and other diseases associated with vascular leakage.-Minhas, N., Xue, M., Fukudome, K., Jackson, C. J. Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function.