Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the ␣(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and antibodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction.Type I and type III collagens are predominant vascular thrombogenic molecules. Platelet interaction with collagen is a complex phenomenon, involving several platelet membrane glycoproteins (1) and nonintegrin receptors (2), with different binding sites on the collagen molecule coming into play in a specific order during the sequential phases of platelet collagen interaction, i.e. platelet contact, spreading, and aggregation. GP 1 Ia/IIa (integrin ␣ 2 /␤ 1 , very late antigen-2) is a major collagen receptor. It binds to type I collagen via specific sequences within the ␣1(I)-CB3 fragment: residues 435-438 (DGEA) 2 (3) and residues 502-516 with an essential GER sequence (4), and within the ␣2(I)-CB4 peptide (5). Platelet GP Ia/IIa binds to the ␣1(III)-CB4 peptide on type III collagen and, more precisely, to a GGPP*GPR sequence (residues 522-528) (6). However, the role of GP Ia/IIa in platelet activation remains poorly understood, as it is not involved in the initial platelet contact (7) and it indirectly affects spleen tyrosine kinase phosphorylation during signal transduction (8). Spleen tyrosine kinase phosphorylation is induced by the binding of platelet GP VI to the GPP* sequences contained in the triple helical structure of collagen (9). Moreover, collagen-related peptides composed of a GPP* sequence repeats, are able to induce platelet aggregation via platelet GP VI (10). The platelet membrane glycoprotein GP IV (11) has been proposed as a collagen receptor, but its role seems to be restricted to type V collagen (12). Finally, Chiang et al. identified p65 as a specific type I collagen receptor that binds the ␣1(I)-CB5 fragment (13-15) and p47, which recognizes type III collagen (16).Previous studies from our laboratory showed that platelets bind to specific sequences of type I (17) and type III collagens (18 -20). In Particular, platelets react with an octapeptide in type III collagen that is located within the ␣1(III)-CB4 fragment (residues 49...

Toll-like receptor-4 (TLR4) pathways are major contributors to pathological inflammatory responses induced by tissue damage. NI-0101 is the first monoclonal antibody (mAb) blocking TLR4 signaling. This activity is independent of the ligand type and concentration, therefore, potentially blocking any TLR4 ligands. A phase I single ascending dose study was conducted in 73 healthy volunteers to evaluate NI-0101 tolerability, preliminary safety, pharmacokinetics (PKs), and pharmacodynamics (PDs), in absence and in presence of a systemic challenge with lipopolysaccharide (LPS), a TLR4 ligand. NI-0101 was well tolerated without safety concern. The PK profile was characterized by a half-life of ∼10 days at high concentrations and by a rapid elimination at low concentrations due to expected target-mediated drug disposition. NI-0101 prevented cytokine release following ex vivo and in vivo LPS administration and prevented the C-reactive protein (CRP) increase and the occurrence of flu-like symptoms expected following the in vivo administration of LPS.

ObjectivesAnti-citrullinated protein antibodies (ACPAs) form immune complexes with citrullinated proteins binding toll-like receptor (TLR) 4, which has been proposed as a mediator of rheumatoid arthritis (RA). NI-0101 is a first-in-class humanised monoclonal antibody blocking TLR4, as confirmed by inhibition of in vivo lipopolysaccharide-induced cytokine release in healthy volunteers. This study was design to confirm preclinical investigations supporting a biomarker-driven approach for treatment of patients with RA who present positive for these immune complexes.MethodsPlacebo-controlled, double-blind, randomised (2:1) trial of the tolerability and efficacy of NI-0101 (5 mg/kg, every 2 weeks for 12 weeks) versus placebo in ACPA-positive RA patients with inadequate response to methotrexate. Efficacy measures included Disease Activity Score (28-joint count) with C reactive protein (DAS28-CRP), European League Against Rheumatism (EULAR) good and moderate responses, and American College of Rheumatology (ACR) 20, ACR50 and ACR70 responses. Subgroup analyses defined on biomarkers were conducted. Pharmacokinetics, pharmacodynamics and safety were reported.Results90 patients were randomised (NI-0101 (61) and placebo (29)); 86 completed the study. No significant between-group difference was observed for any of the efficacy endpoints. Subgroup analyses using baseline parameters as covariants did not reveal any population responding to NI-0101. Treatment-emergent adverse events occurred in 51.7% of patients who received placebo versus 52.5% for NI-0101.ConclusionsWe demonstrate for the first time that in RA, a human immune-mediated inflammatory disease, blocking the TLR4 pathway alone does not improve disease parameters. Successful targeting of innate immune pathways in RA may require broader and/or earlier inhibitory approaches.