Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo

摘要

Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light-scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high-affinity binding site for aegyptin, with a KD of approximately 5 n m. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat-denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO) and GFOGER with low affinity (o m range), which respectively represent glycoprotein VI and integrin l2o1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo. Structured digital abstract MINT-7299280, MINT-7299290: Collagen (uniprotkb: P02461) binds ( MI:0407) to Aegyptin (uniprotkb: O01949) by enzyme linked immunosorbent assay ( MI:0411) MINT-7298991, MINT-7299153, MINT-7299208: Collagen (uniprotkb: P02452) binds ( MI:0407) to Aegyptin (uniprotkb: O01949) by surface plasmon resonance ( MI:0107) MINT-7299266: Collagen (uniprotkb: P02452) binds ( MI:0407) to Aegyptin (uniprotkb: O01949) by fluorescence microscopy ( MI:0416) MINT-7299256: Collagen (uniprotkb: P02452) binds ( MI:0407) to Aegyptin (uniprotkb: O01949) by solid phase assay ( MI:0892)