Nematode Anticoagulant Protein C2 Research Articles

TF-FVIIa PAR2-β-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in β-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of β-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. These data provide insights into a TF-FVIIa signaling axis through PAR2-β-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

AbstractAntiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface–expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)–dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL–induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain–dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

Targeting myeloid cell coagulation signaling blocks MAP kinase/TGF-β1-driven fibrotic remodeling in ischemic heart failure.

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-β1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

Abstract 113: Novel Tissue Factor Inhibition For Thromboprophylaxis In COVID-19: Primary Results Of The ASPEN-COVID-19 Phase 2b Randomized Trial

Background: Tissue factor contributes to thrombo-inflammation in viral infection and may be a target in COVID-19. Recombinant nematode anticoagulant protein c2 (rNAPc2), a tissue factor inhibitor with anticoagulant, anti-inflammatory, and antiviral properties, has not been studied in COVID-19. Methods: In Phase 2b of ASPEN-COVID-19 (NCT04655586), an open-label blinded endpoint study, COVID-19 inpatients with elevated D-dimer are randomized 1:1:2 to higher or lower dose rNAPc2 given subcutaneously up to 5 days or local standard of care heparin. Primary efficacy and safety endpoints are D-dimer change (to Day 8 or discharge if earlier) and ISTH major bleeding. Recovery time is an exploratory outcome. Results: 160 patients were randomized at 24 sites in 3 countries. The average age was 56 years, 43% were female, 19% were Hispanic, and 21% were black. Baseline comorbidities included hypertension (51%), diabetes (35%), and smoking (35%); 11% were critically ill. On average, randomization occurred 10 days after COVID-19 symptom onset, and qualifying D-dimers were elevated 3.7-fold above the upper limit of normal. Median recovery time was 6 days (aggregate distribution shown in Figure). Enrollment has completed, database lock is anticipated Q1 2022, and final results will be ready for presentation at Vascular Discovery. Conclusion: ASPEN tests the hypothesis that compared to heparin, rNAPc2 reduces D-dimer in COVID-19. Results may help to provide a novel therapy for thromboprophylaxis in virus-related coagulopathies.

Rationale and design of a study to assess the safety and efficacy of rNAPc2 in COVID-19: the Phase 2b ASPEN-COVID-19 trial

BackgroundThe interaction between thrombosis and inflammation appears central to COVID-19-associated coagulopathy and likely contributes to poor outcomes. Tissue factor is a driver of disordered coagulation and inflammatory signaling in viral infections and is important for viral replication; therefore, tissue factor may be an important therapeutic target in COVID-19.Study DesignASPEN-COVID-19 (NCT04655586) is a randomized, prospective open-label blinded endpoint (PROBE), active comparator Phase 2b trial to evaluate the safety and efficacy of recombinant Nematode Anticoagulant Protein c2 (rNAPc2), a potent tissue factor inhibitor, in patients hospitalized with COVID-19 with elevated D-dimer levels. This report describes the design of the Phase 2b dose ranging and proof of concept study. Participants are randomly assigned, in a 1:1:2 ratio, to lower or higher dose rNAPc2 by subcutaneous injection on days 1, 3, and 5 or to heparin according to local standard of care; randomization is stratified by baseline D-dimer level (at 2X upper limit of normal). The primary efficacy endpoint for Phase 2b is proportional change in D-dimer concentration from baseline to Day 8 or day of discharge, whichever is earlier. The primary safety endpoint is major or non-major clinically relevant bleeding through Day 8. Phase 2b enrollment began in December 2020 and is projected to complete ∼160 participants by Q4 2021.ConclusionsASPEN-COVID-19 will provide important data on a novel therapeutic approach that may improve outcomes in hospitalized COVID-19 patients beyond available anticoagulants by targeting tissue factor, with potential effects on not only thrombosis but also inflammation and viral propagation.

Recombinant nematode anticoagulant protein c2 inhibits cell invasion by decreasing uPA expression in NSCLC cells.

Nematode anticoagulant protein c2 (NAPc2) is an 85-residue polypeptide originally isolated from the hematophagous hookworm, Ancylostomacaninum. Several studies have shown that rNAPc2 inhibits the growth of primary and metastatic tumors in mice independently of its ability to initiate coagulation. We obtained bioactive recombinant rNAPc2 by splicing of the rNAPc2-intein-CBD fusion proteins expressed in E.coli ER2566. In the invitro assay, rNAPc2 obviously inhibited the invasive ability of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Furthermore, rNAPc2 suppressed tumor growth invivo by daily intraperitoneal injection of rNAPc2 in an NSCLC cell xenograft model of nude mice. Respectively, rNAPc2 downregulated the production of urokinase plasminogen activator (uPA) (P<0.05) and suppressed nuclear factor-κB (NF-κB) activity. We also identified that inhibition of NF-κB activity impaired cell invasion and reduced the uPA production in NSCLC cells. Meanwhile, NF-κB was found to directly bind to the uPA promoter invitro. These results demonstrated that rNAPc2 inhibits cell invasion at least in part through the downregulation of the NF-κB-dependent metastasis-related gene expression in NSCLC. Our results also suggest that uPA, a known metastasis-promoting gene, is indirectly regulated by rNAPc2 through NF-κB activation. These results indicate that rNAPc2 may be a potent agent for the prevention of NSCLC progression.

Descriptive Analysis of Ebola virus Proteins: Towards Development of Effective Therapeutics and Vaccines

Ebola virus (EBOV) is a non-segmented enveloped RNA virus. It has seven structural proteins and three non-structural proteins (∆-peptide, ssGP and sGP). Structural proteins include viral proteins (VP24, VP30, VP35, and VP40), glycoprotein(GP), nucleoprotein(NP) and RNA polymerase L. The functions of structural proteins range from replication, transcription and maintenance of the structural stability of EBOV. VP24 and VP35 are well known for suppressing host interferon (IFN) response systems. There are no approved treatments for Ebola virus disease (EVD). However, several virus vectored vaccines aimed at inducing B and T cell immunity against GP and NP are currently under development or undergoing clinical trials. Unlike virus vectored vaccines, virus like particle (VLP) based vaccines have been developed to elicit immunity against GP, NP, and VP40. They can also be engineered to include VP24 and VP35. Several compounds such as small interfering RNAs (siRNAs), phosphorodiamidate morpholino oligomers (PMOs), c3-NpcA, peptides, NSC629, E64, compound 3.47, compound 7, CA074, and Ca-c3 Ado that target different stages of the EBOV replication cycle have been found to be effective. Other compounds such as recombinant Review Article Munjita and Kwenda; BMRJ, 8(3): 457-479, 2015; Article no.BMRJ.2015.138 458 nematode anticoagulant protein c2 (rNAPc2) and Recombinant human activated protein C (rhAPC) have been designed to reduce coagulopathies seen in late stages of EVD. Cocktails of neutralising monoclonal antibodies are also effective against EBOV alone or in combination with IFNs. Therefore, this review discusses the structure and mechanism of action of EBOV proteins, current promising therapeutics and vaccines against EBOV, and different ways of improving their efficacy.

Ebola hemorrhagic fever: genetic biomarkers and vaccine development.

Ebola hemorrhagic fever: genetic biomarkers and vaccine development.

Tissue factor/factor VIIa pathway mediates coagulation activation in induced-heat stroke in the baboon

Excessive activation of coagulation, which can culminate in overt disseminated intravascular coagulation, is a prominent feature of heat stroke. However, neither the mechanism that initiates the coagulation activation nor its pathogenic role is known. We examined whether the tissue factor/factor VIIa complex initiates the coagulation activation in heat stroke and, if so, whether upstream inhibition of coagulation activation through its neutralization may minimize cellular injury and organ dysfunction. We also examined whether coagulation inhibition influences heat stroke-induced fibrinolytic and inflammatory responses. Randomized controlled study. Comparative Medicine Department, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. Baboons (Papio Hamadryas). Twelve anesthetized baboons assigned randomly to recombinant nematode anticoagulant protein c2, a powerful inhibitor of tissue factor/factor VIIa-dependent coagulation (n = 6), or a control group (n = 6) were heat-stressed in a prewarmed neonatal incubator at 44-47°C until systolic blood pressure fell <90 mm Hg, signaling the onset of severe heat stroke. Recombinant nematode anticoagulant protein c2 was administered as a single intravenous dose of 30 μg/kg body weight at onset of heat stroke. The control group received an equivalent volume of sterile saline intravenously. Heat stroke was associated with coagulation activation and fibrin formation as evidenced by the increased plasma thrombin-antithrombin complexes, endogenous thrombin potential, and D-dimer levels. Recombinant nematode anticoagulant protein c2 induced significant inhibition of thrombin generation and fibrin formation. Inhibition of coagulation in recombinant nematode anticoagulant protein c2-treated animals did not influence either fibrinolysis (assessed by tissue plasminogen activator, plasmin-α2-antiplasmin complexes, and plasminogen activator inhibitor) or the release of pro- and anti-inflammatory cytokines. No difference in markers of cell injury and organ dysfunction was observed between recombinant nematode anticoagulant protein c2-treated and control groups. Tissue factor/factor VIIa-dependent pathway initiates coagulation activation in induced-heat stroke in the baboon without an effect on fibrinolysis and inflammation. The findings suggest also that coagulation activation is not a prerequisite of cell injury and organ dysfunction.

RNAPc2 Inhibits Colorectal Cancer in Mice through Tissue Factor

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel antithrombotic activity. TF is highly expressed in human colorectal tumors, and levels are positively correlated with disease progression. To explore the therapeutic potential and mechanism of action of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 in several experimental colorectal cancer models in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion. Combining rNAPc2 with the cytotoxic agent 5-fluorouracil or bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody) resulted in additive growth inhibition and simultaneous reduction of microvessel density in HCT116 human colorectal tumor xenografts in nude mice. Furthermore, rNAPc2 potentiated CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 tumor cells. Long-term administration of rNAPc2 significantly suppressed spontaneous formation of intestinal tumors in Apc(Min/+) mice. Using a RNA interference approach, we showed that TF expression is necessary for rNAPc2-mediated inhibition of HCT116 human colorectal tumor xenograft growth in nude mice, indicating that the antitumor effect of rNAPc2 may be transduced through TF that is expressed on tumor cells. rNAPc2 is a potent anticancer agent when used in combination with chemotherapy or antiangiogenic therapy in mouse models of colorectal cancer, and TF positivity appears to be required for its activity.

RNAPc2, a Novel Inhibitor of Tissue Factor/Factor VIIa Complex, Inhibits Tumor Growth and Metastasis in Mouse Models of Colorectal Cancer.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastatic, anti-angiogenic, and anti-thrombotic activities. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with disease stage and inversely correlates with survival. To explore the therapeutic potential of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 efficacy in experimental colorectal cancers in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). Using CD31 and Ki67 immunohistochemisty, we found that rNAPc2 synergized with either 5-FU or bevacizumab in inhibiting microvessel density and tumor cell proliferation in HCT116 human colorectal tumor xenografts. Furthermore, rNAPc2 synergized with CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 human colorectal tumor cells. Long-term administration of rNAPc2 also significantly suppressed formation of intestinal adenomas and adenocarcinomas in ApcMin/+ mice. The dosing regimens of rNAPc2 used in these studies were well tolerated up to a three-month period by recipient mice without major hemorrhage or other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Marburg Virus Angola Infection of Rhesus Macaques: Pathogenesis and Treatment with Recombinant Nematode Anticoagulant Protein c2

Background. The procoagulant tissue factor (TF) is thought to play a role in the coagulation disorders that characterize filoviral infections. In this study, we evaluated the pathogenesis of lethal infection with the Angola strain of Marburg virus (MARV-Ang) in rhesus macaques and tested the efficacy of recombinant nematode anticoagulant protein c2 (rNAPc2), an inhibitor of TF/factor VIIa, as a potential treatment. Methods. Twelve rhesus macaques were challenged with a high dose (1000 pfu) of MARV-Ang. Six macaques were treated with rNAPc2, and 6 macaques served as control animals. Results. All 6 control animals succumbed to MARV-Ang challenge by day 8 (mean, 7.3 days), whereas 5 of 6 rNAPc2-treated animals died on day 9 and 1 rNAPc2-treated animal survived. The disease course for MARVAng infection appeared to progress more rapidly in rhesus macaques than has been previously reported for other strains of MARV. In contrast to Ebola virus (EBOV) infection in macaques, up-regulation of TF was not as striking, and deposition of fibrin was a less prominent pathologic feature of disease in these animals. Conclusions. These data show that the pathogenicity of MARV-Ang infection appears to be consistent with the apparent increased human virulence attributed to this strain. The apparent reduced efficacy of rNAPc2 against MARV-Ang infection, compared with its efficacy against EBOV infection, appears to be associated with differences in TF induction and fibrin deposition.

Recombinant Nematode Anticoagulant Protein c2 in non-ST Segment Elevation Acute Coronary Syndrome and Beyond

Patients with acute coronary syndromes (ACS) have high recurrent ischemic event rates despite management with current guideline-based therapies. Recombinant nematode anticoagulant protein (rNAP)c2 provides factor Xa-dependent inhibition of the tissue factor/factor VIIa complex acting proximally on the clotting cascade. It may be administered either intravenously or subcutaneously and has an elimination half-life of approximately 50-60 h. rNAPc2 reduces thrombin formation in patients undergoing elective percutaneous coronary interventions (PCI) and in patients with non-ST segment elevation ACS managed with an early invasive strategy, while bleeding rates are comparable with currently used anticoagulants. Patients receiving rNAPc2 undergoing emergent coronary artery bypass surgery within 96 h of dosing have increased rates of major bleeding. Some heparin coadministration may be necessary to avoid PCI-related thrombotic complications. Large-scale trials are needed to confirm these findings and to evaluate the impact of rNAPc2 on clinical events.

Recombinant Nematode Anticoagulant Protein c2 in Patients With Non–ST-Segment Elevation Acute Coronary Syndrome: The ANTHEM–TIMI-32 Trial

We sought to evaluate the safety and efficacy of recombinant nematode anticoagulant protein c2 (rNAPc2) in patients with non-ST-segment elevation acute coronary syndrome (nSTE-ACS). Recombinant NAPc2 is a potent inhibitor of the tissue factor/factor VIIa complex that has the potential to reduce ischemic complications mediated by thrombin generation. A total of 203 patients were randomized 4:1 to double-blinded intravenous rNAPc2 or placebo every 48 h for a total of 1 to 3 doses in 8 ascending panels (1.5 to 10 microg/kg). All patients received aspirin, unfractionated heparin (UFH), or enoxaparin and early catheterization; clopidogrel and glycoprotein IIb/IIIa blockers were encouraged. Two subsequent open-label panels evaluated 10 mug/kg rNAPc2 with half-dose UFH (n = 26) and no UFH (n = 26). The primary end point was the rate of major plus minor bleeding. Pharmacokinetics, pharmacodynamics, continuous electrocardiography, and clinical events were assessed. Recombinant NAPc2 did not significantly increase major plus minor bleeding (3.7% vs. 2.5%; p = NS) despite increasing the international normalized ratio in a dose-related fashion (trend p < or = 0.0001). Higher-dose rNAPc2 (> or =7.5 microg/kg) suppressed prothrombin fragment F1.2 generation compared with placebo and reduced ischemia by >50% compared to placebo and lower-dose rNAPc2. Thrombotic bailout requiring open-label anticoagulant occurred in 5 of 26 patients treated without UFH, but none in the half-dose UFH group (19% vs. 0%; p = 0.051). In patients with nSTE-ACS managed with standard antithrombotics and an early invasive approach, additional proximal inhibition of the coagulation cascade with rNAPc2 was well tolerated. rNAPc2 doses > or =7.5 microg/kg suppressed F1.2 and reduced ischemia, though some heparin may be necessary to avoid procedure-related thrombus formation. (Anticoagulation With rNAPc2 to Eliminate MACE/TIMI 32; http://www.clinicaltrial.gov/ct/show/NCT00116012?order=1; NCT00116012).

Anticoagulants and Their Reversal

In an exciting era with many alternatives to the old anticoagulants heparin and warfarin emerging on the scene, awareness of the possibility to reverse their effect is mandatory. In this review, the traditional antidotes for warfarin (vitamin K, plasma, and prothrombin complex concentrate) and for heparin (protamine) are described together with the newer alternatives (recombinant activated factor VII, concatameric peptides, and recombinant platelet factor 4). For some of the newer anticoagulants, possible antidotes have been identified, whereas other alternatives have been discarded. There is a very limited experience of deamino-d-arginine vasopressin or a von Willebrand factor VIII concentrate to counteract hirudin. The small direct thrombin inhibitors may be reversed with activated prothrombin complex concentrate but not with recombinant activated factor VII, whereas the latter agent appears to be effective against the pentasaccharides and the recombinant nematode anticoagulant protein C2. Additional options that may become available in the future are also discussed briefly.

RNAPc2, a Novel Inhibitor of Tissue Factor/Factor VIIa Complex, Suppresses Lung Metastasis and Synergizes with 5-FU or Bevacizumab in Mouse Models of Colorectal Cancer.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastasis, anti-angiogenesis, and anti-thrombosis activities. rNAPc2 has been previously shown to inhibit both the primary growth and metastasis of murine B16 melanoma and Lewis lung carcinoma in mice. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with the progression of malignancy. To explore the therapeutic potential of rNAPc2 during tumor growth and progression, we tested rNAPc2 efficacy in experimental colorectal cancer in mice. Both primary and metastatic colorectal tumor models were used in the current study and rNAPc2 was given to mice via daily intraperitoneal injections. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). The doses and dosing regimens of rNAPc2 used in these murine models of colorectal cancer were well tolerated by recipient mice without major complications of hemorrhage or any other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Endotoxaemia induces resistance to activated protein C in healthy humans

Systemic inflammation activates the tissue factor/factor VIIa complex (TF/FVIIa), leading to a procoagulant state, which may be enhanced by impairment of physiological anticoagulant pathways, such as the protein C system. Besides impaired protein C activation, resistance to activated protein C (APC) may occur. We studied the effect of endotoxemia on APC resistance, analysed its determinants and evaluated the effect of TF/FVIIa inhibition on endotoxin-induced APC resistance. Sixteen healthy male volunteers participated in the study, eight receiving endotoxin alone and eight receiving the combination of endotoxin and recombinant Nematode Anticoagulant Protein c2 (rNAPc2), a potent inhibitor of TF/FVIIa. Parameters of coagulation were subsequently studied. The sensitivity to APC was determined by two tests: a test based on the endogenous thrombin potential and a test based on the activated partial thromboplastin time. In response to endotoxemia, both tests detected a transient APC resistance that was predominantly mediated by an increase in factor VIII and was not influenced by TF/FVIIa inhibition. In vitro tests confirmed that an increase in factor VIII induced APC resistance, as measured by both tests. This finding suggests that APC resistance might play a role in the procoagulant state occurring during human endotoxemia.

Les nouveaux antithrombotiques

Current antithrombotic agents include anticoagulants (unfractionated and low-molecular-weight heparin, and antivitamin K) and platelet aggregation inhibitors (aspirin, ticlopidine, clopidogrel). Two areas are under particular investigation: specific inhibition, direct or indirect, of factor Xa and factor IIa. Pentasaccharide, an indirect anti-Xa, has proved effective in curing deep-vein thrombosis and more effective than enoxaparin for prophylactic treatment after orthopedic surgery. Administered in a single subcutaneous injection daily, it has no risk of thrombocytopenia; laboratory surveillance is based on anti-Xa activity. Hirudin and melagatran act by direct thrombin inhibition. Unlike hirudin (which requires monitoring of active coagulation time or ecarin clotting time), melagatran requires no laboratory monitoring. It is not associated with an increased risk of hemorrhage. But there is no true antidote at this time. If its efficacy is confirmed, ximelagatran, the orally active prodrug of melagatran, may facilitate the long-term treatment now reserved for antivitamin K. Three antagonists of the tissue factor-factor VIIa complex are also under development: rNAPc2 (Recombinant Nematode Anticoagulant Protein C2), ASIS (Active Site Inhibitor Factor Seven) and recombinant TFPI (Tissue Factor Pathway Inhibitor). Antiplatelet drugs are the reference antithrombotic agents for the prevention and treatment of arterial thrombosis. Aspirin remains in first place (75 to 300 mg/d) but the modest superiority of the thienopyridines (clopidogrel and ticlopidine) is established. Hemogram monitoring is no longer necessary for clopidogrel. Use of aspirin + a thienopyridine after placement of a coronary stent has been validated. Laboratory monitoring of antiplatelet treatments has not been codified.

Inhibition of the tissue factor/factor VIIa pathway does not influence the inflammatory or antibacterial response to abdominal sepsis induced by Escherichia coli in mice.

Anticoagulants have gained increasing attention for the treatment of sepsis. Inhibition of the tissue factor (TF)/factor (F) VIIa pathway has been shown to attenuate the activation of coagulation and to prevent death in a primate model of sepsis caused by gram-negative bacteria. To determine the role of the TF/FVIIa complex in the host response to peritonitis, mice received an intraperitoneal injection of live Escherichia coli with or without concurrent treatment with recombinant nematode anticoagulant protein c2 (rNAPc2), a selective inhibitor of the TF/FVIIa pathway. Peritonitis was associated with an increase in the expression of TF at the tissue level and activation of coagulation, as reflected by elevated levels of thrombin-antithrombin complexes and by increased fibrin(ogen) deposition in the liver and lungs. rNAPc2 strongly attenuated this procoagulant response but did not influence the inflammatory response (histopathology, leukocyte recruitment to the peritoneal cavity, and cytokine and chemokine levels). Moreover, rNAPc2 did not alter bacterial outgrowth locally or dissemination of the infection, and survival was not different between rNAPc2-treated mice and control mice. These data suggest that TF/FVIIa activity contributes to the activation of coagulation during E. coli peritonitis but does not play a role in the inflammatory response or antibacterial host defense.

Recombinant nematode anticoagulant protein c2, an inhibitor of tissue factor/factor VIIa, attenuates coagulation and the interleukin‐10 response in human endotoxemia

The tissue factor-factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc2. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc2 was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc2 was administered subcutaneously. rNAPc2 elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of prothrombin time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc2 completely blocked the endotoxin-induced thrombin generation, as measured by plasma prothrombin fragment F1+2. The endotoxin-induced effect on fibrinolytic parameters such as plasmin-antiplasmin complexes and plasminogen activator inhibitor type 1 was not affected by rNAPc2. The administration of rNAPc2 attenuated the endotoxin-induced rise in interleukin (IL)-10, without affecting the rise in other cytokines. In conclusion, rNAPc2 is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc2 completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc2 attenuated the endotoxin-induced rise in IL-10, without affecting the rises in other cytokines.