Safety and efficacy of an anti-human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models.

Rebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

Blocking human protein C anticoagulant activity improves clotting defects of hemophilia mice expressing human protein C.

Hemophilia A and B are hereditary coagulation defects resulting in unstable blood clotting and recurrent bleeding. Current factor replacement therapies have major limitations such as the short half-life of the factors and development of inhibitors. Alternative approaches to rebalance the hemostasis by inhibiting the anticoagulant pathways have recently gained considerable interest. In this study, we tested the therapeutic potential of a monoclonal antibody, HAPC1573, that selectively blocks the anticoagulant activity of human activated protein C (APC). We generated F8-/- or F9-/- hemophilia mice expressing human protein C by genetically replacing the murine Proc gene with the human PROC. The resulting PROC+/+;F8-/- or PROC+/+;F9-/- mice had bleeding characteristics similar to their corresponding F8-/- or F9-/- mice. Pretreating the PROC+/+;F8-/- mice with HAPC1573 shortened the tail bleeding time. HAPC1573 pretreatment significantly reduced mortality and alleviated joint swelling, similar to those treated with either FVIII or FIX, of either PROC+/+;F8-/- or PROC+/+;F9-/- mice in a needle puncture-induced knee-joint bleeding model. Additionally, we found that HAPC1573 significantly improved the thrombin generation of PROC+/+;F8-/- mice but not F8-/- mice, indicating that HAPC1573 enhanced the coagulant activity of hemophilia mice by modulating human APC in vivo. We further documented that HAPC1573 inhibited the APC anticoagulant activity to improve the clotting time of human plasma deficient of FVIII, FIX, FXI, FVII, VWF, FV, or FX. These results demonstrate that selectively blocking the anticoagulant activity of human APC may be an effective therapeutic and/or prophylactic approach for bleeding disorders lacking FVIII, FIX, or other clotting factors.

Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice.

Thrombin-mediated proteolysis is central to hemostatic function but also plays a prominent role in multiple disease processes. The proteolytic conversion of fII to α-thrombin (fIIa) by the prothrombinase complex occurs through 2 parallel pathways: (1) the inactive intermediate, prethrombin; or (2) the proteolytically active intermediate, meizothrombin (fIIa(MZ)). FIIa(MZ) has distinct catalytic properties relative to fIIa, including diminished fibrinogen cleavage and increased protein C activation. Thus, fII activation may differentially influence hemostasis and disease depending on the pathway of activation. To determine the in vivo physiologic and pathologic consequences of restricting thrombin generation to fIIa(MZ), mutations were introduced into the endogenous fII gene, resulting in expression of prothrombin carrying 3 amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIa(MZ) Homozygous fII(MZ) mice are viable, express fII levels comparable with fII(WT) mice, and have reproductive success. Although in vitro studies revealed delayed generation of fIIa(MZ) enzyme activity, platelet aggregation by fII(MZ) is similar to fII(WT) Consistent with prior analyses of human fIIa(MZ), significant prolongation of clotting times was observed for fII(MZ) plasma. Adult fII(MZ) animals displayed significantly compromised hemostasis in tail bleeding assays, but did not demonstrate overt bleeding. More notably, fII(MZ) mice had 2 significant phenotypic advantages over fII(WT) animals: protection from occlusive thrombosis after arterial injury and markedly diminished metastatic potential in a setting of experimental tumor metastasis to the lung. Thus, these novel animals will provide a valuable tool to assess the role of both fIIa and fIIa(MZ) in vivo.

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.

Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury.

We previously reported that extracellular histones are major mediators of death in sepsis. Infusion of extracellular histones leads to increased cytokine levels. Histones activate TLR2 and TLR4 in a process that is enhanced by binding to DNA. Activation of TLR4 is responsible for the histone-dependent increase in cytokine levels. To study the impact of histone release on pathology we used two models: a Con A-triggered activation of T cells to mimic sterile inflammation, and acetaminophen to model drug-induced tissue toxicity. Histones were released in both models and anti-histone Abs were protective. TLR2- or TLR4-null mice were also protected. These studies imply that histone release contributes to death in inflammatory injury and in chemical-induced cellular injury, both of which are mediated in part through the TLRs.

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.

The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A.

Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl3 vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

The link between vascular features and thrombosis.

The mechanisms of vascular control of thrombotic events remain unclear. The vasculature possesses essential anticoagulant factors that regulate coagulation. Because the endothelium-to-blood ratios are much higher in the microcirculation, it is likely that stasis contributes to thrombotic risk, due in large part to failure to rapidly access the microcirculation and to gain access to this highly anticoagulant environment. Inflammation can potentiate thrombosis in part through downregulation of the vascular anticoagulants, a process that appears to be exacerbated in aging, a well-known risk factor for thrombosis. Surgery and trauma, two major risk factors for thrombosis, result in the release of a variety of cellular components that trigger coagulation through separate mechanisms.

Extracellular histones are major mediators of death in sepsis.

Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

Thrombomodulin mutations in atypical hemolytic-uremic syndrome.

BACKGROUND: The hemolytic-uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin-producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic-uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic-uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic-uremic syndrome. METHODS: We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic-uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic-uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells. RESULTS: Of 152 patients with atypical hemolytic-uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I-mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic-uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement. CONCLUSIONS: Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic-uremic syndrome.

Effects of membrane and soluble EPCR on the hemostatic balance and endotoxemia in mice.

Recent studies have shown that endothelial protein C receptor (EPCR) polymorphisms and soluble EPCR levels are associated with thrombotic diseases. It is unknown whether membrane EPCR (mEPCR) heterozygosity and/or physiologically elevated sEPCR levels directly impact the hemostatic balance and the outcome of endotoxemia. In these studies, thrombin infusion experiments revealed that EPCR heterozygosity (Procr+/-) impaired protein C activation by approximately 30%. Infusion of factor Xa with phospholipid demonstrated that the Procr+/- genotype increased the coagulant response relative to wild-type mice. Challenge of the Procr+/- mice with lipopolysaccharide (LPS) did not significantly exaggerate their response compared with wild-type mice. We also generated mice in which one allele of full-length EPCR was replaced by sEPCR (Procrs/+). Compared with Procr+/- mice, Procrs/+ mice had 5-fold higher sEPCR and similar mEPCR levels. Procr+/- and Procrs/+ mice generated similar levels of activated protein C (APC) upon thrombin infusion. They also exhibited a similar coagulant response upon factor Xa/phospholipid infusion. Only supraphysiologic levels of sEPCR could influence protein C activation and exaggerate the coagulant response. In conclusion, mEPCR, but not physiologically elevated sEPCR, regulated protein C activation. Procr heterozygosity results in a mild increase of thrombosis tendency and little influence on the response to endotoxin.

Extraembryonic expression of EPCR is essential for embryonic viability.

The endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex. Deletion of the EPCR gene (Procr) in mice leads to embryonic lethality before embryonic day 10 (E10.0). EPCR is detected in the giant trophoblast cells at the feto-maternal boundary from E7.5 and weakly in embryonic aortic endothelial cells from E13.5, suggesting that extraembryonic EPCR expression may be essential for embryonic viability. Using conditional knock-out strategies, we demonstrate that Procr-deficient embryos with EPCR expression on placenta giant trophoblasts can be carried to term and then develop normally. Conversely, EPCR expression in the embryo, without expression in the giant trophoblast cells, does not rescue the mice. In genetically modified mice with low tissue factor activity, Procr deficiency is not lethal to the embryo. As adults, Procr-deficient mice generate more thrombin and activate less protein C in response to procoagulant stimuli. Spontaneous thrombin formation in the deficient animals increases with age. These findings show that extraembryonic EPCR expression is critical for embryo development.

Inhibition of APC anticoagulant activity on oxidized phospholipid by anti-{beta}2-glycoprotein I monoclonal antibodies.

Activated protein C (APC) anticoagulant activity and the ability to be inhibited by auto-antibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. beta(2)-glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti-beta(2)-GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on beta(2)-GPI were examined. Each inhibited the PE-, phospholipid oxidation-dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti-beta(2)-GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required beta(2)-GPI. Surprisingly, although the mAbs did increase beta(2)-GPI affinity for membranes, PE and oxidation had little influence on the affinity of the beta(2)-GPI antibody complex for the membrane vesicles. We conclude that antibodies to beta(2)-GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes.

PAR1 cleavage and signaling in response to activated protein C and thrombin.

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

Disruption of the endothelial cell protein C receptor gene in mice causes placental thrombosis and early embryonic lethality.

The endothelial cell protein C receptor (EPCR) is a type 1 transmembrane protein found primarily on endothelium that binds both protein C and activated protein C with similar affinity. EPCR augments the activation of protein C by the thrombin-thrombomodulin complex. To determine the physiological importance of EPCR, we generated EPCR-deficient mice by homologous targeting in embryonic stem cells. Genotyping of progeny obtained from EPCR(+/-) interbreeding indicated that EPCR(-/-) embryos died on or before embryonic day 10.5 (E10.5). Reverse transcriptase-PCR confirmed the absence of EPCR mRNA in EPCR(-/-) embryos. EPCR(-/-) embryos removed from extra-embryonic membranes and tissues at day E7.5 and cultured in vitro developed beyond E10.5, suggesting a role for EPCR in the normal function of the placenta and/or at the materno-embryonic interface. Immunohistochemistry revealed the lack of EPCR in trophoblast giant cells of EPCR(-/-) embryos. These cells, which normally express EPCR, are in direct contact with the maternal circulation and its clotting factors. In EPCR(-/-) embryos, greatly increased fibrin deposition was detected around these cells. To prevent this fibrin deposition, EPCR(+/-)-crossed female mice received a daily subcutaneous injection of enoxaparin through pregnancy. Although some EPCR(-/-) embryos were rescued from midgestational lethality, this regimen yielded no EPCR(-/-) pups. We conclude that EPCR is essential for normal embryonic development. Moreover, EPCR plays a key role in preventing thrombosis at the maternal-embryonic interface.

The crystal structure of the endothelial protein C receptor and a bound phospholipid.

The endothelial cell protein C receptor (EPCR) shares approximately 20% sequence identity with the major histocompatibility complex class 1/CD1 family of molecules, accelerates the thrombin-thrombomodulin-dependent generation of activated protein C, a natural anticoagulant, binds to activated neutrophils, and can undergo translocation from the plasma membrane to the nucleus. Blocking protein C/activated protein C binding to the receptor inhibits not only protein C activation but the ability of the host to respond appropriately to bacterial challenge, exacerbating both the coagulant and inflammatory responses. To understand how EPCR accomplishes these multiple tasks, we solved the crystal structure of EPCR alone and in complex with the phospholipid binding domain of protein C. The structures were strikingly similar to CD1d. A tightly bound phospholipid resides in the groove typically involved in antigen presentation. The protein C binding site is outside this conserved groove and is distal from the membrane-spanning domain. Extraction of the lipid resulted in loss of protein C binding, which could be restored by lipid reconstitution. CD1d augments the immune response by presenting glycolipid antigens. The EPCR structure is a model for how CD1d binds lipids and further suggests additional potential functions for EPCR in immune regulation, possibly including the anti-phospholipid syndrome.