Interference of neutrophil–platelet interaction by YC-1: A cGMP-dependent manner on heterotypic cell–cell interaction

Abstract

N-Formyl-Met-Leu-Phe (fMLP) activated neutrophils and then induced neutrophil–platelet complex formation in co-incubation condition. In addition, fMLP induce intracellular calcium mobilization in platelets, only when it is incubated along with neutrophils. This data established that fMLP-stimulated neutrophils activate platelets. 9E1, a monoclonal antibody of P-selectin, significantly blocks the formation of neutrophil–platelet complex induced by fMLP, indicating the involvement of P-selectin in the neutrophil–platelet complex formation. 3-(5′-hydroxymethyl-2′-furyl-1-benzylindazole (YC-1), an unique nitric oxide-independent activator of soluble guanylate cyclase, was evaluated for its effect on neutrophil–platelet complex. YC-1 inhibits fMLP-induced neutrophil–platelet complex formation in a concentration-dependent manner with an IC50 value of 15.3±3.5 μM. However, this effect of YC-1 is partially reversed by pre-treatment of 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ; 10 μM), which is a soluble guanylate cyclase inhibitor. Pre-treatment of either neutrophils or platelets with YC-1 (50 μM) prevent the fMLP-induced neutrophil–platelet complex formation, indicating that YC-1 could potentially exert its effects individually on either neutrophils or platelets alone. Cathepsin G released from fMLP-stimulated neutrophil activates the nearby platelets. YC-1 was also shown to inhibit this release of cathepsin G in a concentration-dependent manner. The IC50 value was 6.2±0.2 μM. This inhibitory effect of YC-1 on cathepsin G release is reversed by ODQ (10 μM) and a protein kinase G inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carbooxylic acid methyl ester (KT5835); 1 μM]. YC-1 inhibits cathepsin G-induced P-selectin expression on human platelet at the IC50 value of 32.5±2.6 μM. A further study showed that YC-1 inhibits fMLP-induced neutrophil–platelet complex formation in whole blood at the IC50 value of 35.8±8.1 μM in a concentration-dependent manner. According to these data, it was hypothesized that fMLP stimulates neutrophils to release cathepsin G, which subsequently activates the nearby platelets, creating neutrophil–platelet complexes. YC-1 inhibits fMLP-induced neutrophil from releasing cathepsin G via a cGMP-dependent pathway. This inhibitory effect of YC-1 on cathepsin G release is a major mechanism for affecting fMLP-induced neutrophil–platelet complex. YC-1's inhibition P-selectin expression on platelet may potentiate its effects. These inhibitory effects may contribute to the inhibition of neutrophil–platelet complex formation in whole blood.