Anti-protein C antibodies and acquired protein C resistance in SLE: novel markers for thromboembolic events and disease activity?

OBJECTIVES: Risk factors for thromboembolism in SLE are poorly understood. We hypothesized a possible role for protein C, based on its dual activity in inflammation and haemostasis and on the evidence of an association between acquired activated protein C (APC) resistance (APCR) and high-avidity anti-protein C antibodies (anti-PC) with a severe thrombotic phenotype in venous thrombosis APS patients. METHODS: In a cross-sectional study of 156 SLE patients, the presence and avidity of IgG anti-PC was established by in house-ELISA, and APCR to exogenous recombinant human APC (rhAPC) and Protac (which activates endogenous protein C) was assessed by thrombin generation-based assays. Associations with aPL profile, thrombotic history and disease activity (BILAG and SLEDAI-2K) were also established. RESULTS: Anti-PC were detected in 54.5% of patients and APCR in 59%. Anti-PC positivity was associated with APCR to both rhAPC (P <0.0001) and Protac (P =0.0001). High-avidity anti-PC, detected in 26.3% of SLE patients, were associated with APCR in patients with thrombosis only (P <0.05), and with the development of thrombosis over time (range: 0-52 years; P =0.014). High-avidity anti-PC levels correlated with SLEDAI-2K (P =0.033) and total BILAG (P =0.019); SLEDAI-2K correlated inversely with APCR to Protac (P =0.004). CONCLUSION: Anti-PC occur in patients with SLE, independently of aPL profile, and are associated with APCR. High-avidity anti-PC are associated with thrombosis and with active disease and might prove a novel marker to monitor the risk of thrombosis and disease progression in SLE.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

The Immunoregulatory Activities of Activated Protein C in Inflammatory Disease.

Tissue injury prompts the initiation of host defense responses to limit blood loss, restrict pathogen entry, and promote repair. Biochemical and cellular pathways that lead to blood coagulation serve a fundamental role in generating a physical barrier at the wound site, but have also evolved to promote immune response to injury. Similarly, anticoagulant pathways that attenuate clot formation also regulate innate and adaptive immune responses. Of particular importance is activated protein C (APC) which serves as a principal regulator of thrombin generation, shapes the innate immune response to infection, and has been shown to contribute to the adaptive immune response in several preclinical models of autoimmune disease. APC controls blood coagulation by proteolytic degradation of procoagulant activated cofactors essential for fibrin clot development, but also cleaves multiple additional substrates and interacts with cell surface receptors to exert additional physiologically important roles. In this review, we focus on the molecular mechanisms utilized by APC to limit inflammation and, in particular, current understanding of the basis for APC anticoagulant and signaling activities. In particular, we provide an overview of established and emerging signaling pathways initiated by APC on endothelial cells, monocytes, neutrophils, dendritic cells, and T cells to control and regulate immune cell physiology. Finally, we consider the impact of APC activity in the context of both acute and chronic inflammatory disease, and the continuing efforts to harness the immunoregulatory properties of recombinant APC for therapeutic use.

Identification and characterization of CD4+ T cell epitopes on manganese transport protein C of Staphylococcus aureus.

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.

[Role of IgG plasma cells in the change of protein C system in ulcerative colitis].

The present study is designed to explore the role of plasma cells in the change of protein C system (PCS) in ulcerative colitis (UC). Dextran sulfate sodium (DSS, 4% in concentration) was used to induce mouse UC model. The plasma cells and the type of immune complex in colon were observed by immunofluorescence. The amount and type of plasma cells separated from colonic mucosal lamina propria were detected by flow cytometry using anti-CD54+CD38+ and IgA/M/G antibodies, respectively. After stimulation of macrophages by IgG type immune complex, TNF-α and IL-6 levels were evaluated by ELISA. After co-incubation of microvascular endothelial cells with TNF-α or IL-6, the expressions of endothelial protein C receptor (EPCR) and thrombomodulin (TM), and the activity of activated protein C (APC) were examined. As the results showed, the IgG type plasma cells infiltration and the quantity of IgG type immune complex were increased in DSS group in comparison with control group. After incubation with IgG type immune complex, the levels of TNF-α and IL-6 in the supernatant of macrophages were increased (P < 0.01) in a concentration-dependent manner. Meanwhile, after incubation with TNF-α or IL-6, the expressions of EPCR and TM in the microvascular endothelial cells were decreased (P < 0.05 or P < 0.01), while the activity of APC was reduced (P < 0.05 or P < 0.01). These results suggested that the quantity of IgG type plasma cells increases in UC and forms immune complexes, which affect the secretion of cytokines from macrophage, thereby affecting the function of endothelial cells and finally inhibiting PCS in UC. Therefore, plasma cell may be a novel target for the treatment of UC.

Association of common genetic variation in the protein C pathway genes with clinical outcomes in acute respiratory distress syndrome.

BACKGROUND: Altered plasma levels of protein C, thrombomodulin, and the endothelial protein C receptor are associated with poor clinical outcomes in patients with acute respiratory distress syndrome (ARDS). We hypothesized that common variants in these genes would be associated with mortality as well as ventilator-free and organ failure-free days in patients with ARDS. METHODS: We genotyped linkage disequilibrium-based tag single-nucleotide polymorphisms in the ProteinC, Thrombomodulin and Endothelial Protein C Reptor Genes among 320 self-identified white patients of European ancestry from the ARDS Network Fluid and Catheter Treatment Trial. We then tested their association with mortality as well as ventilator-free and organ-failure free days. RESULTS: The GG genotype of rs1042580 (p = 0.02) and CC genotype of rs3716123 (p = 0.002), both in the thrombomodulin gene, and GC/CC genotypes of rs9574 (p = 0.04) in the endothelial protein C receptor gene were independently associated with increased mortality. An additive effect on mortality (p < 0.001), ventilator-free days (p = 0.01), and organ failure-free days was observed with combinations of these high-risk genotypes. This association was independent of age, severity of illness, presence or absence of sepsis, and treatment allocation. CONCLUSIONS: Genetic variants in thrombomodulin and endothelial protein C receptor genes are additively associated with mortality in ARDS. These findings suggest that genetic differences may be at least partially responsible for the observed associations between dysregulated coagulation and poor outcomes in ARDS.

Anti-inflammatory effects of activated protein C on human dendritic cells.

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti-apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF-α and IL-6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL-12 and anti-inflammatory cytokine IL-10. Interestingly, treatment with 5 µg/mL, but not 25 µg/mL, of APC significantly enhanced production of IL-10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co-stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti-inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF-α and/or IL-6 production.

Inhibition of endogenous activated protein C attenuates experimental autoimmune encephalomyelitis by inducing myeloid-derived suppressor cells.

Activated protein C (PC) is an anticoagulant involved in the interactions between the coagulation and immune systems. Activated PC has broad anti-inflammatory effects that are mediated through its ability to modulate leukocyte function and confer vascular barrier protection. We investigated the influence of activated PC on the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. We modulated activated PC levels in the circulation during EAE induction through systemic administration of a mAb against PC/activated PC (anti-PC). We initially hypothesized that inhibition of activated PC may result in a heightened inflammatory environment, leading to increased EAE pathogenesis. Contrary to this hypothesis, mice treated with anti-PC Ab (anti-PC mice) exhibited attenuated EAE. Interestingly, despite reduced disease severity and minimal pathogenic conditions in the CNS, anti-PC mice exhibited considerable leukocyte infiltration in the brain, comparable to control mice with severe EAE. Furthermore, CD4(+) T cells were diminished in the periphery of anti-PC mice, whereas various CD11b(+) populations were increased, notably the myeloid-derived suppressor cells (MDSCs), a CD11b(+) subset characterized as potent T cell suppressors. MDSCs from anti-PC mice exhibited increased expression of T cell suppressive factors and effectively inhibited T cell proliferation. Overall, our findings show that activated PC inhibition affected EAE pathogenesis at multiple fronts, specifically increasing vascular barrier permeability, as evidenced by considerable leukocyte infiltration in the brain. Additionally, inhibition of activated PC modulated the functional responses of CD11b(+) cells, leading to the expansion and increased activation of MDSCs, which are suppressive to the CD4(+) T cells required for EAE progression, thereby resulting in attenuated EAE.

Activated protein C inhibits pancreatic islet inflammation, stimulates T regulatory cells, and prevents diabetes in non-obese diabetic (NOD) mice.

Activated protein C (aPC) is a natural anticoagulant with strong cyto-protective and anti-inflammatory properties. aPC inhibits pancreatic inflammation and preserves functional islets after intraportal transplantation in mice. Whether aPC prevents the onset or development of type 1 diabetes (T1D) is unknown. In this study, when human recombinant aPC was delivered intraperitoneally, twice weekly for 10 weeks (from week 6 to 15) to non-obese diabetic (NOD) mice, a model for T1D, the incidence of diabetes was reduced from 70% (saline control) to 7.6% by 26 weeks of age. Islets of aPC-treated mice exhibited markedly increased expression of insulin, aPC/protein C, endothelial protein C receptor, and matrix metalloproteinase (MMP)-2 when examined by immunostaining. The insulitis score in aPC-treated mice was 50% less than that in control mice. T regulatory cells (Tregs) in the spleen, pancreatic islets, and pancreatic lymph nodes were increased 37, 53, and 59%, respectively, in NOD mice following aPC treatment. These Tregs had potent suppressor function and, after adoptive transfer, delayed diabetes onset in NOD.severe combined immunodeficiency mice. The culture of NOD mouse spleen cells with aPC reduced the secretion of inflammatory cytokines interleukin (IL)-1ß and interferon-γ but increased IL-2 and transforming growth factor-ß1, two cytokines required for Treg differentiation. In summary, our results indicate that aPC prevents T1D in the NOD mouse. The aPC mechanism of action is complex, involving induction of Treg differentiation, inhibition of inflammation, and possibly direct cyto-protective effects on ß cells.

Protein C and acute inflammation: a clinical and biological perspective.

The protein C system plays an active role in modulating severe systemic inflammatory processes such as sepsis, trauma, and acute respiratory distress syndrome (ARDS) via its anticoagulant and anti-inflammatory properties. Plasma levels of activated protein C (aPC) are lower than normal in acute inflammation in humans, except early after severe trauma when high plasma levels of aPC may play a mechanistic role in the development of posttraumatic coagulopathy. Thus, following positive results of preclinical studies, a clinical trial (PROWESS) with high continuous doses of recombinant human aPC given for 4 days demonstrated a survival benefit in patients with severe sepsis. This result was not confirmed by subsequent clinical trials, including the recently published PROWESS-SHOCK trial in patients with septic shock and a phase II trial with patients with nonseptic ARDS. A possible explanation for the major difference in outcome between PROWESS and PROWESS-SHOCK trials is that lung-protective ventilation was used for the patients included in the recent PROWESS-SHOCK, but not in the original PROWESS trial. Since up to 75% of sepsis originates from the lung, aPC treatment may not have added enough to the beneficial effect of lung-protective ventilation to show lower mortality. Thus whether aPC will continue to be used to modulate the acute inflammatory response in humans remains uncertain. Because recombinant human aPC has been withdrawn from the market, a better understanding of the complex interactions between coagulation and inflammation is needed before considering the development of new drugs that modulate both coagulation and acute inflammation in humans.

Antibody SPC-54 provides acute in vivo blockage of the murine protein C system.

Multiple protective effects of pharmacological activated protein C (APC) are reported in several organ pathologies. To help evaluate the endogenous murine PC system, we characterized a rat monoclonal anti-mouse PC antibody, SPC-54, which inhibited the amidolytic and anticoagulant activities of murine APC by>95%. SPC-54 blocked active site titration of purified APC using the active site titrant, biotinylated FPR-chloromethylketone, showing that SPC-54 blocks access to APC's active site to inhibit all enzymatic activity. A single injection of SPC-54 (10mg/kg) neutralized circulating PC in mice for at least 7days, and immunoblotting and immuno-precipitation with protein G-agarose confirmed that SPC-54 in vivo was bound to PC in plasma. Pre-infusion of SPC-54 in tissue factor-induced murine acute thromboembolism experiments caused a major decrease in mean survival time compared to controls (7min vs. 42.5min, P=0.0016). SPC-54 decreased lung perfusion in this model by 54% when monitored by vascular perfusion methodologies using infrared fluorescence of Evans blue dye. In LD50 endotoxemia murine models, SPC-54 infused at 7hr after endotoxin administration increased mortality from 42% to 100% (P<0.001). In summary, monoclonal antibody SPC-54 ablates in vitro and in vivo APC protective functions and enzymatic activity. The ability of SPC-54 to block the endogenous PC/APC system provides a powerful tool to understand better the role of the endogenous PC system in murine injury models and in cell bioassays and also to neutralize the enzymatic activities of murine APC in any assay system.

Activated protein C N-linked glycans modulate cytoprotective signaling function on endothelial cells.

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporine-induced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma ß-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.

Application of low dose of FK506 in pancreas transplantation model in Wistar-SD rats.

OBJECTIVE: To probe into application of low dose of FK506(Tacrolimus) in pancreas transplantation. METHODS: Effects of low-dose FK506 (Tacrolimus) in pancreas transplantation with examination of ELISA Electron microscopy and TUNEL by method of random control were studied. RESULTS: Blood glucose concentration in control group is higher than that in treated group A (FK506) and treated group B (CsA) 7 days after transplantation (p < 0.05). Serum C-peptide and insulin concentrations in control group are less than that in treated group A (FK506) and treated group B (CsA) 7 days after transplantation (p < 0.05). Blood glucose, serum C-peptide and insulin concentrations are same as that in control group, group A (FK506) and group B (CsA) (p > 0.05).There are more apoptotic nuclei in control group than that in treated group A (FK506) and treated group B (CsA) (p < 0.05). There is no significant difference between group A (FK506) and treated group B (CsA) in sum of apoptotic nuclei (p > 0.05). There is no significant difference among treated group A (FK506) and treated group B (CsA) in electron microscopy fields. CONCLUSION: Low-dose FK506 applied in pancreas transplantation could not only be effective for immunosuppressive, but also be safe for islet cells of pancreas.

Impact of endogenous protein C on pulmonary coagulation and injury during lethal H1N1 influenza in mice.

Influenza accounts for 5-10% of community-acquired pneumonia cases, and is a major cause of mortality. Sterile and bacterial lung injury are associated with procoagulant and inflammatory derangements in the lungs and down-regulation of the protein C (PC) pathway has been correlated with disease severity and mortality in severe bacterial pneumonia and sepsis. In addition, during lethal influenza pneumonia, pulmonary and systemic coagulation are activated, which can be attenuated by the administration of recombinant activated (A) PC. We here determined the role of endogenous PC in lethal H1N1 influenza A infection. Male C57BL/6 mice pretreated with an inhibitory monoclonal antibody directed against murine PC or a control antibody were intranasally infected with a lethal dose of a mouse-adapted H1N1 influenza A strain. Mice were killed at 48 or 96 hours after infection, after which lungs and bronchoalveolar lavage fluid were harvested, or observed for up to 9 days. Anti-PC antibody treatment aggravated pulmonary activation of coagulation as compared with control antibody treatment, as reflected by increased lung concentrations of thrombin-antithrombin complexes and fibrin degradation products, as well as intravascular thrombus formation. Anti-PC antibody treatment aggravated lung histopathology, but lowered bronchoalveolar neutrophil influx and total protein levels, and delayed mortality. In conclusion, endogenous PC has strong effects on the host response to lethal influenza A infection, inhibiting pulmonary coagulopathy and inflammation on the one hand, but facilitating neutrophil influx and protein leak and accelerating mortality on the other hand.

Multiple receptor-mediated functions of activated protein C.

The central effector protease of the protein C pathway, activated protein C (APC), interacts with the endothelial cell protein C receptor, with protease activated receptors (PAR), the apolipoprotein E2 receptor, and integrins to exert multiple effects on haemostasis and immune cell function. Such receptor interactions modify the activation of PC and determine the biological response to endogenous and therapeutically administered APC. This review summarizes the current knowledge about interactions of APC with cell surface-associated receptors, novel substrates such as histones and tissue factor pathway inhibitor, and their implications for the biologic function of APC in the control of coagulation and inflammation.

Antiphospholipid antibodies and the protein C pathway.

Haemostasis is a delicate balance between procoagulant and anticoagulant processes. In the human body usually anticoagulant mechanisms prevail over procoagulant mechanisms, thereby preventing a prothrombotic state. The antiphospholipid syndrome is an example in which this balance is shifted to a more prothrombotic state due to the presence of antiphospholipid antibodies. One of the most extensively proposed pathogenic mechanisms within the antiphospholipid syndrome is the inhibition of protein C by antiphospholipid antibodies. Antiphospholipid antibodies have been described to have different actions on the protein C pathway, for example decreasing protein C and/or S plasma levels, inducing increased resistance against activated protein C and lowering thrombin levels (resulting in an impaired protein C activation). This review briefly discusses the actions of protein C in human body but mainly focuses on the effects of antiphospholipid antibodies on the protein C pathway that have been described in literature.

Levels of C-reactive protein and protein C in periodontitis patients with and without cardiovascular disease.

Since periodontitis is a chronic and inflammatory disease, a number of hypotheses have proposed that it has an etiological or modulating role in cardiovascular disease (CVD). This study aimed to ascertain the changes in the plasma levels of C-reactive protein (CRP) and protein C (PC), a natural anticoagulant also having an anti-inflammatory effect, in patients who have mild-to-severe periodontitis with or without CVD. The test group consisted of 26 patients with CVD and chronic periodontitis and the control group consisted of 26 patients with chronic periodontitis and no systemic disease. In both groups Community Periodontal Index of Treatment Needs scores were recorded and blood samples were collected. CRP levels were significantly high and PC activity was significantly low in the test group compared to the control group (p < 0.001). There was a negative correlation between tooth loss and PC and between CRP and PC. How PC is affected by the inflammatory events and its association with CRP is an active area of investigation.

Laboratory tests for protein C deficiency.

Hereditary protein C deficiency is a hypercoagulable state associated with an increased risk for venous thrombosis. The recommended initial test for protein C is an activity (functional) assay, which may be clotting time based or chromogenic. The advantages and disadvantages of the various testing options are presented. The causes of acquired protein C deficiency are much more common than hereditary deficiency. Therefore, this article describes the appropriate steps to take when protein C activity is low, to confirm or exclude a hereditary deficiency. The causes of falsely normal results are also described, including lupus anticoagulants and direct thrombin inhibitors.

Cerebral palsy in siblings caused by compound heterozygous mutations in the gene encoding protein C.

We report two sisters with extensive bilateral periventricular haemorrhagic infarction (PVHI) causing cerebral palsy (CP). The older sister presented at 20 months with cortical visual blindness, spastic diplegia, and purpura fulminans. The younger sister presented aged 3 days old with apnoeas and multifocal seizures. She subsequently had global developmental delay, cortical visual blindness, spastic quadriplegia, epilepsy, and purpura fulminans at age 2 years. Neuroimaging of both siblings showed bilateral PVHI consistent with bilateral cerebral intramedullary venous thrombosis occurring at under 28 weeks' gestation for the older sister and around time of birth for the younger sister. At latest follow-up, the older sister (13y) has spastic diplegia at Gross Motor Function Classification System (GMFCS) level II, and the younger sister (10y) has spastic quadriplegia at GMFCS level IV. Both sisters showed partial quantitative reduction in plasma protein C antigen and severe qualitative reduction in plasma protein C anticoagulant activity. They were heterozygous for two independent mutations in the protein C gene (PROC). There was no other risk factor for CP. To our knowledge, this is the first family reported with compound heterozygous PROC mutations as the likely genetic cause of familial CP. This report adds to the list of known monogenic causes of CP.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.