Absence Of Activated Protein C Research Articles

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. .

Assessment of acquired activated protein C resistance with the FibWave and comparison with the ETP-based APC resistance.

Activated protein C (APC) resistance is a major risk factor of venous thrombosis which may be acquired by hormonal therapy or other causes. The FibWave, a sensitive global clot-based assay design to analyze the coagulation kinetics in plasma, may be a good candidate to assess this prothrombotic state. This study aims to assess the suitability of the FibWave to differentiate the coagulation kinetics of women on oral contraceptives. Fifty-four healthy volunteers were divided into 5 groups: men [n=13], women not using hormonal contraception [n=12], women using second [n=12] or third generation [n=12] combined oral contraceptives, and women using progestin only contraceptive [n=5]. Patients with coagulation abnormalities were also assessed [n=8]. The APC resistance was assessed on the FibWave using exogenous APC or Protac, and on the Calibrated Automated Thrombogram using the ETP-based APC resistance assay. Either in presence or in absence of APC or Protac, the FibWave was able to detect a hypercoagulable state in plasma samples. All combined oral contraceptives showed a lower FW-Max1 , FW-Max2, and FW-Min2 percentage of inhibition and a lower FW-Ttpeak ratio than the other groups. The sensitivity of the FibWave was similar to the one of the ETP-based APC resistance assay. The FibWave is able to differentiate APC resistance levels observed in women on combined oral contraceptive. The FW-Max1 , FW-Max2, and to a lesser degree FW-Min2 were identified as the most sensitive parameters with a similar performance to the ETP-based APC resistance assay.

Identification and functional characterization of a novel F5 mutation (Ala512Val, FVBonn) associated with activated protein C resistance

Essentials Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. A novel missense mutation (Ala512Val - FVBonn ) was characterized in vitro and in silico. FVBonn is a new cause of APC resistance and venous thrombosis. FVBonn expresses additionally enhanced procoagulant activity in the absence of APC. Background Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. This phenotype is most commonly associated with the factor V Arg506Gln mutation (FV Leiden), which impairs the APC-mediated inactivation of both activated FV (FVa) and activated FVIII (FVIIIa). Objectives Here, we report the identification and characterization of a novel FV mutation (Ala512Val, FVBonn ) in six patients with APC resistance and venous thrombosis or recurrent abortions. Methods FVBonn was expressed in a recombinant system and compared with recombinant wild-type (WT) FV and FV Leiden in several functional assays. Results FVBonn conferred APC resistance to FV-depleted plasma, both in the activated partial thromboplastin time (APTT)-based test (APC sensitivity ratio [APCsr] of 1.98 for FVBonn versus 4.31 for WT FV and 1.59 for FV Leiden) and in the thrombin generation-based test (normalized APCsr of 5.41 for FVBonn versus 1.00 for WT FV and 8.99 for FV Leiden). The APC-mediated inactivation of FVaBonn was slower than that of WT FVa (mainly because of delayed cleavage at Arg506), but was greatly stimulated by protein S. The APC cofactor activity of FVBonn in FVIIIa inactivation was ~ 24% lower than that of WT FV. In line with these findings, an in silico analysis showed that the Ala512Val mutation is located in the same loop as the Arg506 APC cleavage site and might hamper its interaction with APC. Moreover, FVBonn was more procoagulant than WT FV and FV Leiden in the absence of APC, because of an increased activation rate and, possibly, an enhanced interaction with activated FX. Conclusions FVBonn induces hypercoagulability via a combination of increased activation/procoagulant activity, decreased susceptibility to APC-mediated inactivation, and slightly reduced APC cofactor activity.

Activated Protein C-Catalyzed Factor Va Inactivation Predominantly Contributes to the Downregulation of Coagulation Rather Than Factor VIIIa Inactivation

Activated protein C (APC) directly inactivates factor (F)Va and FVIIIa dependently of protein S (PS), and furthermore inactivates FVIIIa through cofactor function of FV. Any impairment in these inactivation pathways may increase thrombotic risk. A previous report described that the APC resistance (APCR) associated with FV-Leiden was mainly due to the impaired cofactor activity of FV (Castoldi et al. Blood 2004;103, 4173). We have recently reported that FV-Nara carrying W1920R mutation exhibited APCR caused by a loss of FVa susceptibility to APC and APC cofactor activity resulting in more serious thrombotic potential than FV-Leiden (Nogami et al. Blood 2014;123, 2420). The contribution of each inactivation pathway associated with FVIII(a) and/or FV(a) for the hemostatic regulation remains unclear, however. An ACE910, bispecific antibody (Ab) to FIXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII(a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In this study, to elucidate the contribution of each pathway, we investigated the regulatory mechanism(s) by APC utilizing ACE910. FXa generation with ACE910 (10μg/ml) or with FVIIIa (0.3 nM) in FVIII-deficient plasma (ΔFVIII) was evaluated by the addition of APC (0-0.5 nM), PS (5 nM), and FV (1 nM). FXa generation with FVIIIa was reduced by ~60% maximally in an APC dose-dependent manner, whilst that with ACE910 was unaffected, indicating that ACE910 itself was not susceptible to inactivation by APC through the cofactor activity of FV. Thrombin generation in ΔFVIII with ACE910 (0-30μg/ml) was enhanced in an ACE910 dose-dependent fashion and ~10-fold of peak thrombin (PeakTh) (442 ± 7.0 nM), equivalent to 77.5% of PeakTh in pool normal plasma (PNP), was observed with ACE910 (30μg/ml) compared to that without ACE910 (44.6 ± 5.0 nM). To investigate the effect of APC upon the hemostatic enhancement by ACE910 in ΔFVIII, thrombin generation in ΔFVIII mixed with ACE910 (30μg/ml) and APC (0-16 nM) was examined. PeakTh showed the APC-dependent reduction to 156 ± 22nM and the %inhibition was 64.7%. Subsequently, we evaluated the effects of APC on thrombin generation in PNP or in PNP with an anti-FVIII Ab (FVIIIAb) (16BU/ml) supplemented by ACE910. Of note, the %inhibition of PeakTh by APC in latter with ACE910 and FVIIIAb (64%) was greater than that in PNP alone (35%) by ~1.9-fold. To elucidate the contribution of APC-catalyzed inactivation of FV(a) to the hemostatic regulation, we evaluated the thrombin generation in FV-diluted plasma (FV-dil) consisting of FV-deficient plasma and FV (8nM) corresponded to ~25% of its physiological concentration mixed with or without ACE910 and FVIIIAb in the presence or absence of APC. The %inhibition of PeakTh by APC in FV-dil with ACE910 and FVIIIAb (46.5%), being reduced in comparison with that of PNP with both Abs, was greater than that in FV-dil alone (18.3%) by ~2.5-fold, indicating that the inhibition of thrombin generation by APC-catalyzed FV(a) inactivation was FV(a) dose-dependent but enhanced in the absence of FVIII(a). Furthermore, the effects on thrombin generation with APC (0-16 nM) in plasmas carrying FV-Leiden without or with FVIIIAb and ACE910 (30μg/ml) were examined. It was of surprise that PeakTh in latter with FVIIIAb and ACE910 reduced exponentially by ~40% in an APC dose-dependent manner, whilst APC showed any little effects upon thrombin generation in FV-Leiden plasma alone indicating APCR. The susceptibility index to APC calculated by the rate of inhibition with FVIIIAb and ACE910 was 0.174, greater than that with FV-Leiden plasma alone (0.085) by ~2-fold. These results strongly suggested that APC could regulate the hemostatic enhancement by inactivation of FV(a) even FV-Leiden in the absence of another substrate, FVIII(a). We conclude that APC potentiates its catalytic activity to FVa more in the absence of FVIII(a) than in the presence of FVIII(a) and this mechanism would play significant roles in the downregulation of coagulation as one of the breaking systems. DisclosuresYada:Chugai Pharmaceutica Co., Ltd: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Activation of Matrix Metalloproteinases 2, 9, and 13 by Activated Protein C in Human Osteoarthritic Cartilage Chondrocytes

Levels of activated protein C (APC) are elevated in the synovial fluid of patients with osteoarthritis (OA), and increased APC levels are correlated with the levels of active matrix metalloproteinase 2 (MMP-2). This study sought to investigate whether APC is a relevant protein for activation of MMPs in the degradation of human OA cartilage, and to elucidate its mechanisms of action. Human articular cartilage was cultured with or without interleukin-1α (IL-1α), in the presence or absence of APC or protein C, and an MMP or serine proteinase inhibitor. Aggrecan and collagen release and chondrocyte gene expression levels were quantified. Aggrecanase and MMP cleavage of aggrecan was examined with neoepitope-specific antibodies, and MMP activity was measured using gelatin zymography and fluorogenic peptide assay. In human OA cartilage, APC induced aggrecan and collagen release, whereas in non-OA cartilage, costimulation with IL-1α was required. Inhibition of MMP activity reduced APC-induced cartilage proteolysis, and MMP-induced aggrecanolysis was confirmed by Western blotting. In cultures with APC alone, the activity of MMPs 2, 9, and 13 was significantly increased in OA cartilage, although APC could not directly activate MMPs 2 or 9. Expression of MMP1, MMP2, MMP9, MMP13, TIMP1, and TIMP3 was not altered by APC in OA cartilage. Human OA chondrocytes expressed messenger RNA for protein C, endothelial protein C receptor, thrombomodulin, and protease-activated receptor 1, but these were unaltered or down-regulated by APC. The induction of MMP activation and cartilage degradation by APC was dependent on its serine protease activity. APC is a physiologically relevant activator of MMPs and cartilage breakdown in human OA. The effects of APC are dependent on its proteolytic activity and as-yet-undefined cell and/or cartilage matrix factors, and inhibition of this pathway may provide a novel therapeutic target to halt the progression of cartilage damage in OA.

Endogenous activated protein C limits cancer cell extravasation through sphingosine-1-phosphate receptor 1–mediated vascular endothelial barrier enhancement

Activated protein C (APC) has both anticoagulant activity and direct cell-signaling properties. APC has been reported to promote cancer cell migration/invasion and to inhibit apoptosis and therefore may exacerbate metastasis. Opposing these activities, APC signaling protects the vascular endothelial barrier through sphingosine-1-phosphate receptor-1 (S(1)P(1))activation, which may counteract cancer cell extravasation. Here, we provide evidence that endogenous APC limits cancer cell extravasation, with in vivo use of monoclonal antibodies against APC. The protective effect of endogenous APC depends on its signaling properties. The MAPC1591 antibody that only blocks anticoagulant activity of APC does not affect cancer cell extravasation as opposed to MPC1609 that blocks anticoagulant and signaling properties of APC. Combined administration of anti-APC antibodies and S(1)P(1) agonist (SEW2871) resulted in a similar number of pulmonary foci in mice in presence and absence of APC, indicating that the protective effect of APC depends on the S(1)P(1) pathway. Moreover, endogenous APC prevents cancer cell-induced vascular leakage as assessed by the Evans Blue Dye assay, and SEW2871 treatment reversed MPC1609-dependent vascular leakage. Finally, we show that cancer cells combined with MPC1609 treatment diminished endothelial VE-cadherin expression. In conclusion, endogenous APC limits cancer cell extravasation because of S(1)P(1)-mediated VE-cadherin-dependent vascular barrier enhancement.

Protein S as Cofactor for TFPI

In the last decades evidence was obtained that protein S not only acts as cofactor of activated protein C (APC) in the downregulation of coagulation, but also expresses anticoagulant activity in the absence of APC. The search for the mechanism(s) underlying the APC-independent anticoagulant activity of protein S was hampered by the fact that protein S exhibited 2 seemingly identical anticoagulant activities in model systems and in plasma. Later it was shown that the anticoagulant activity of purified protein S in model systems was dependent on the concentration of phospholipid vesicles and was explained by low amounts of protein S multimers generated during purification that effectively inhibited phospholipid-dependent coagulation reactions via competition for phospholipid binding sites. Plasma does not contain multimers, and the anticoagulant activity of protein S in plasma was not affected by the phospholipid concentration but was dependent on the amount of tissue factor (TF) used for initiation of thrombin generation. This led to the discovery that protein S acts as cofactor of tissue factor pathway inhibitor (TFPI) which stimulates the inhibition of factor Xa by TFPI approximately 10-fold. The current review describes the background of the TFPI-cofactor activity of protein S as well as the rationale for the observation that the TFPI/protein S system particularly inhibits the TF pathway at low procoagulant stimuli.

In vitro Effects of Detergent Sclerosants on Antithrombotic Mechanisms

To investigate the in vitro effects of detergent sclerosants on antithrombotic pathways. Proteins C, S and antithrombin (AT) were assayed in normal plasma treated with increasing concentrations of sodium tetradecyl sulphate (STS) and polidocanol (POL). Activated protein C (APC) was investigated by mixing normal plasmas with sclerosants and testing with the activated partial thromboplastin time (APTT) and dilute Russell's viper venom time in the presence and absence of APC. The effect on factor Xa (FXa), heparin and enoxaparin was investigated using chromogenic anti-FXa and APTT methods. High concentration (>0.6%) STS significantly destroyed proteins C, S and AT whereas POL only caused a mild reduction in PC and AT and a moderate (60%) reduction in PS levels. STS potentiated the anticoagulant effect of APC while POL increased APC resistance. STS mimicked AT and demonstrated significant anti-Xa and anti-IIa activity. STS demonstrated a similar anticoagulant profile to heparin but was 1000x weaker. It also significantly potentiated the anticoagulant effect of heparin while POL had less effect. STS and POL demonstrated quite distinct and sometimes opposite effects on the antithrombotic mechanisms assayed. These effects were concentration-dependent and in general, STS had the greatest effect on antithrombotic proteins.

Differential Anticoagulant Effects of Protein S on Vascular Cells and Platelets.

Background: Protein S is a vitamin K-dependent plasma protein and involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor (TFPI). The anticoagulant properties of protein S have been thoroughly characterized by in vitro methods. However, fewer studies focus on protein S function on vascular cells. These studies are limited to model systems employing purified coagulation factors. The aim of this study was to investigate the role of protein S in plasma that is in contact with natural cell membranes, including endothelial cells, smooth muscle cells and platelets.Method: We employed thrombography to evaluate protein S function in 50 % v/v recalcified citrated plasma in the presence of washed platelets, cultured umbilical vein endothelial cells or cultured umbilical artery smooth muscle cells. Since we aimed at a comparison between different cellular membranes, micro-particle free plasma was used. As a reference, we also examined synthetic phospholipid membranes composed of phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine in a 2/6/2 molar ratio. Thrombin activity was measured employing the fluorogenic substrate z-Gly-Gly- Arg-AMC. Protein S activity was probed with CLB-PS13, an antibody directed against the protein S Gla-domain. The APC-independent activity of protein S was assayed in the presence of an inhibitory antibody against protein C. In studies employing phospholipids, thrombin generation was triggered with relipidated tissue factor (TF). Expression of TF on endothelial cells was induced during a 6-hour preincubation with PMA.Results: In the presence of CLB-PS13, the APC-independent activity of protein S became apparent as an increase in peak height in the thrombogram. Lag time, time to peak and area under the curve remained essentially unaffected. Peak height was increased two-fold when examining phospholipids at standard conditions (4 μM lipids and 1 pM TF). This increase in peak height by CLB-PS13 was concentration dependent and complete at 10 μg/ml IgG. Increasing the TF concentration from 1 to 5 pM resulted in loss of the APC-independent activity of protein S on these membranes. APC cofactor activity was assessed in the presence of APC. Addition of APC resulted in inhibition of the thrombin formation on phospholipids with an IC50 of 0.4 nM. CLB-PS13 completely abolished this decrease in thrombin generation up to 50 nM APC, irrespective whether 1 or 5 pM TF was present. Our results are compatible with the view that at high procoagulant stimuli the TFPI-cofactor activity of protein S is abolished. Furthermore, these results show that in plasma APC is completely dependent on protein S. Protein S activity on platelets was studied in the presence of 1 pM TF. As for synthetic lipid membranes and in the absence of APC, CLB-PS13 increased the peak height in the thrombogram. APC inhibited platelet mediated thrombin generation (IC50 = 19 nM), and this inhibition was completely abolished by CLB-PS13. These observations suggest that platelets support both APC-dependent and APC-independent anticoagulant activities of protein S. Thrombography on TF-expressing endothelial cells and smooth muscle cells revealed massive thrombin generation that could not be enhanced with CLB-PS13, indicating that protein S does not contribute to regulation of thrombin formation under these conditions. The APC-dependent activity of protein S became apparent as an abolished inhibition by APC in the presence of CLB-PS13. However, APC proved relatively inefficient in inhibiting thrombin generation on TF-expressing vascular cells (IC50 > 50 nM). Inhibition of TF restored the APC-independent protein S activity.Conclusion: Our results indicate that, in plasma, vascular cells and platelets are able to support the APC-dependent as well as the APC-independent anticoagulant activities of protein S. The APC-independent activity on vascular cells is abolished upon increasing TF expression, while the APC-dependent activity of protein S is limited by the relatively low anticoagulant activity of APC on these cell surfaces. We conclude that protein S activity on cells require relatively high levels of APC or low expression of TF.

A Single Amino Acid Substitution (I18V) in the Gla Domain of Activated Protein C Markedly Impairs Ability of PE and GlyCer to Enhance Anticoagulant Activity

Phosphatidylethanolamine (PE) and glucosylceramide (GlyCer) are cell surface lipids that preferentially enhance anticoagulant, rather than procoagulant pathways. In particular, both PE and GlyCer enhance the anticoagulant activity of activated protein C (APC). Previous studies have indicated that specific APC Gla domain residues may mediate APC interaction with PE and GlyCer. To investigate whether specific APC residues mediate PE and GlyCer enhanced APC anticoagulant activity, we expressed a series of APC variants in which APC Gla domain residues not shared with the human prothrombin Gla domain were substituted with their prothrombin amino acid equivalent. The anticoagulant activity of each APC Gla domain variant was assessed in a tissue factor-initiated thrombin generation assay containing phospholipid vesicles of differing composition (80% PC/20% PS); or PC/PS/PE (60%/20%/20%); or PC/PS/GlyCer (60%/20%/20%). For each of these lipid mixtures, thrombin generation (endogenous thrombin potential, ETP) was not significantly different in the absence of APC. In the presence of PC/PS vesicles, APC reduced thrombin generation by 63±3% at the highest APC concentration tested (6nM). However, APC impairment of thrombin generation was enhanced 3-fold in the presence of PC/PS/PE compared with vesicles containing PC/PS alone, and in the presence of PC/PS/GlyCer was enhanced 4.3-fold. Enhancement of anticoagulant function by PE and GlyCer was similar for the majority of the APC Gla domain variants tested. Interestingly, one APC variant (APC-I18V) exhibited similar anticoagulant activity to that of wild type APC with PC/PS vesicles, but was not enhanced by the presence of PE- or GlyCer-containing vesicles. Phospholipid vesicles containing PE or GlyCer have been previously described to enhance protein S cofactor enhancement of APC. Therefore, to further characterize APC-I18V, we assessed the ability of wild type APC and APC-I18V to be enhanced by protein S in the presence of PC/PS, PC/PS/PE or PC/PS/GlyCer using a protein S-sensitive thrombin generation assay. In the presence of PC/PS, increasing protein S concentration in protein S-deficient plasma resulted in an APC-mediated slow decrease in thrombin generation, irrespective of whether wild type or APC-I18V was used (IC50 for protein S-mediated APC inhibition of thrombin generation with PC/PS, 130nM). However, in the presence of PC/PS/PE or PC/PS/GlyCer, thrombin generation was impaired by wild type APC at 3–4-fold lower protein S concentration than that observed when PC/PS vesicles alone were used (IC50, PC/PS/PE=31.5nM and PC/PS/GlyCer=37.5nM). APC-I18V, however, did not exhibit a similarly increased sensitivity to protein S in the presence of PE or GlyCer, as the anticoagulant activity of this variant was the same as when only PC/PS was included. To investigate whether the loss of specific neutral lipid enhancement in APC-I18V affected its ability to initiate cytoprotective signaling via EPCR-PAR-1 on endothelial cells, the capacity of APC-I18V to inhibit thrombin-induced endothelial cell barrier permeability was assessed. When cells were pre-treated with either wild type APC or APC-I18V, there was a significant enhancement in barrier integrity and attenuation of thrombin-induced permeability (P<0.05), demonstrating that loss of PE/GlyCer enhancement of APC anticoagulant activity does not adversely affect EPCR binding and EPCR/PAR-1 cytoprotective signaling. Collectively, these results suggest PE and GlyCer enhancement of APC anticoagulant activity is mediated by increased sensitivity to protein S, and that Ile-18 in the APC Gla domain is critical for mediating APC-specific functional enhancement by PE/GlyCer.

Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera

AbstractWe used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2V617F mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (FII), factor V (FV), and neutrophil elastase were measured in plasma; CD11b was measured on neutrophils. Compared with normal controls, patients had a lower endogenous thrombin potential in the absence of APC but had a higher endogenous thrombin potential in the presence of APC, showing the occurrence of APC resistance. The nAPCsr increased in JAK2V617F carriers compared with noncarriers and was highest in JAK2V617F homozygous patients. FII, FV, free PS, and TFPI levels were reduced in patients, mainly in JAK2V617F carriers. Multiple regression analysis indicated the low free PS level as major determinant of the increased nAPCsr. Elastase was increased in patients and inversely correlated with free PS. In conclusion, these data indicate the occurrence of acquired APC resistance in ET and PV patients, probably because of a reduction in free PS levels. The APC-resistant phenotype is influenced by the JAK2V617F mutational load.

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

In Vivo Anti-Thrombotic Potency of Engineered Activated Protein C Variants.

Activated protein C (APC) has both anticoagulant activity via inactivation of factors Va and VIIIa and cytoprotective activities on cells that include anti-apoptotic and anti-inflammatory activities, alterations of gene expression profiles and protection of endothelial barrier function. The relative importance of APC's anticoagulant activity vs. APC's direct cytoprotective effects on cells for reduction of mortality in severe sepsis patients and protective effects in animal injury models is not entirely clear. In this current study, genetically engineered APC variants with different activity spectra were tested for in vivo anti-thrombotic potency. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191-193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, ASH 2006, J Exper Med , 2007). In contrast to 5A-APC, mutation of E149 to A in APC increased anticoagulant activity in clotting assays while diminishing cytoprotective effects on cells. Murine APC variants, E149A-APC and 5A-APC (KKK192-194AAA + RR230/231AA) were used to determine in vivo anti-thrombotic potency in an acute carotid artery thrombosis model in mice, using FeCl3-induced injury. Under the conditions employed, first occlusion occurred within 3.5 min (mean: 171 sec; range 150-200 sec) in the absence of APC. Murine wild type (wt)-APC effectively delayed time to first occlusion in a dose-dependent manner (0 to 1.8 mg/kg wt-APC; mean: 561 sec; range 400-960 sec). The E149A-APC variant exhibited potent in vivo anti-thrombotic activity (1.8 mg/kg; mean: 1020 sec; range 540- >1600 sec) and was superior to wt-APC as evident by the absence of appreciable occlusion in 2/6 E149A-APC vs. 0/6 wt-APC treated animals. Thus E149A-APC was hyperactive in plasma clotting assays as well as hyperactive in an acute FeCl3-induced arterial thrombosis model. To test the hypothesis that an increased protein S cofactor activity contributed to its enhanced anticoagulant activity, E149A-APC anticoagulant activity was tested in normal and protein S deficient plasma. Compared to wt-APC, E149A-APC showed 3-fold increased anticoagulant activity in normal plasma but not in protein S deficient plasma. In studies with purified proteins, protein S concentrations required for half-maximal stimulation of factor Va inactivation by E149A-APC were 3-fold lower compared to wt-APC, whereas factor Va inactivation rates were indistinguishable in the absence of protein S. These data support our hypothesis that increased protein S cofactor activity is, at least partially, responsible for the observed hyper anticoagulant and anti-thrombotic potency in vitro and in vivo. In contrast to E149A-APC, 5A-APC was severely deficient in anti-thrombotic activity in vivo. Even at concentrations up to 8 mg/kg, 5A-APC (mean: 245 sec; range 172-300 sec) failed to delay significantly time to first occlusion compared to no APC. These data highlight important distinctions between structural requirements for APC's anticoagulant, anti-thrombotic and cytoprotective functions. Engineered APC variants with differentially altered activities (e.g. cytoprotective vs. anticoagulant) may lead to safer or better therapeutic APC variants for a variety of indications including sepsis, ischemic stroke or other pathologies.

Coagulation factors and the protein C system as determinants of thrombin generation in a normal population.

Thrombin generation is a powerful tool to probe overall plasma coagulability. To determine which plasma factors influence the various parameters of the thrombin generation curve, for example lag time, peak height and endogenous thrombin potential (ETP), under different experimental conditions. Plasma levels of coagulation factors and inhibitors, as well as thrombin generation at 1 pm tissue factor (TF) +/- thrombomodulin (TM) and at 13.6 pm TF +/- activated protein C (APC), were determined in plasma from 140 healthy individuals. Data were analysed by multiple regression models. Thrombin generation increased with age and was higher in females than in males. Under all conditions, the lag time was mainly dependent on the levels of free tissue factor pathway inhibitor (TFPI), free protein S (PS), factor VII (FVII), FIX and fibrinogen. The major determinants of thrombin generation (ETP and peak height) at 1 pm TF were fibrinogen, FXII (despite inhibition of contact activation), free TFPI and antithrombin (AT), both in the absence and in the presence of TM. Thrombin generation in the presence of TM was also dependent on protein C levels. At 13.6 pm TF, thrombin generation was determined by prothrombin, AT, fibrinogen, free TFPI and FV levels in the absence of APC, and by free TFPI, free PS and FX levels in the presence of APC. The lag time, ETP and peak height of thrombin generation depend on the levels of multiple coagulation factors and inhibitors. The specific assay determinants vary with the experimental conditions.

Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

Activated protein C (APC) inactivates factor Va (fVa) by proteolytically cleaving fVa heavy chain at Arg(506), Arg(306), and Arg(679). Factor Xa (fXa) protects fVa from inactivation by APC. To test the hypothesis that fXa and APC share overlapping fVa binding sites, 15 amino acid-overlapping peptides representing the heavy chain (residues 1-709) of fVa were screened for inhibition of fVa inactivation by APC. As reported, VP311-325, a peptide comprising residues 311-325 in fVa, dose-dependently and potently inhibited fVa-dependent prothrombin activation by fXa in the absence of APC. This peptide also inhibited the inactivation of fVa by APC, suggesting that this region of fVa interacts with APC. The peptide inhibited the APC-dependent cleavage of both Arg(506) and Arg(306) because inhibition was observed with plasma-derived fVa and recombinant R506Q and RR306/679QQ fVa. VP311-325 altered the fluorescence emission of dansyl-active site-labeled APC(i) but not a dansyl-active site-labeled thrombin control, showing that the peptide binds to APC(i). This peptide also inhibited the resonance energy transfer between membrane-bound fluorescein-labeled fVa (donor) and rhodamine-active site-labeled S360C-APC (acceptor). These data suggest that peptide VP311-325 represents both an APC and fXa binding region in fVa.

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia.

Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non-carriers with comparable prothrombin levels. Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non-carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver-synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non-carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels. Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC-resistant than individuals with comparable prothrombin levels because of other causes.

Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.

Cholesterol enhances phospholipid‐dependent activated protein C anticoagulant activity

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities.

Inhibition of thrombin generation by protein S at low procoagulant stimuli: implications for maintenance of the hemostatic balance

The activated protein C (APC)-independent anticoagulant activity of protein S on tissue factor-induced thrombin generation was quantified in plasma. In absence of APC, protein S significantly decreased the endogenous thrombin potential (ETP) in a concentration-dependent manner. The APC-independent anticoagulant activity of protein S in plasma was not affected by phospholipid concentrations but strongly depended on tissue factor concentrations: protein S inhibited the ETP from 6% at 140 pM tissue factor to 74% at 1.4 pM tissue factor. Plasma with both 60% protein S and 140% prothrombin showed an ETP of 240% compared to normal plasma, suggesting an APC-independent protective role of protein S in the development of thrombosis as a result of protein S deficiency and the prothrombin-G20210A mutation. At high tissue-factor concentrations, protein S hardly expressed APC-independent anticoagulant activity but exerted potent APC-cofactor activity when thrombomodulin or APC were added to plasma. Neutralization of protein S under these conditions resulted in a 20-fold reduction of the anticoagulant activity of APC. The present study shows that protein S effectively regulates coagulation at 2 levels: at low procoagulant stimuli, protein S maintains the hemostatic balance by directly inhibiting thrombin formation, and at high procoagulant stimuli, protein S restores the hemostatic balance via its APC-cofactor activity.

Stabilization of monocyte chemoattractant protein-1-mRNA by activated protein C

The activated protein C (APC) pathway has been suggested to be a common link between coagulation and inflammation. APC may function to restore hemostasis via modulation of cytokine expression. We investigated the effect of APC on the endothelial expression of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is controlled by the activation of central proinflammatory transcription factors, such as nuclear factor-kappa B (NF-kappaB). We found that human APC (2.5-10 micro g/ml) upregulated the amount of MCP-1-mRNA in human umbilical vein endothelial cells (HUVEC) and caused a time- and dose-dependent increase in MCP-1 protein production (p <0.001 for APC 2.5 micro g/ml at 4 up to 24 h). In this cell culture model MCP-1 induced an improvement of cell migration and wound repair after injury to endothelial monolayers. After stimulation of MCP-1-mRNA-transcription with TNF-alpha (0.1-1 ng/ml), HUVEC's were washed and an inhibitor of gene transcription, Actinomycin D (1 micro g/ml), was added in the presence or absence of APC. HUVEC's receiving APC contained more MCP-1-mRNA than controls after one hour and up to eight hours suggesting an inhibitory effect of APC on MCP-1-mRNA degradation (with APC: 753 +/- 56 atto mol of MCP-1-mRNA per ml of cell lysate vs. 263 +/- 60 atto mol/ml without APC at t =4 h; p <0.001). Electrophoretic mobility shift assays revealed that APC attenuated NF-kappaB DNA-binding capacity implying that NF-kappaB may not be involved in the upregulatory effect of APC on MCP-1 production. The ability of APC to upregulate the production of MCP-1, most likely by increasing the stability of MCP-1-mRNA rather than by transcriptional activation via NF-B, identifies a novel immunomodulatory pathway, by which APC may control the local inflammatory reaction, thereby initiating wound repair and modulating the extent of endothelial injury.