Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo.
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 K(D) of approximately 5 nM. 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)(10) and GFOGER with low affinity (microM range), which respectively represent glycoprotein VI and integrin alpha2beta1 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.
Calvo
Eric
E
Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases/NIH, Bethesda, MD 20852, USA.
Tokumasu
Fuyuki
F
Mizurini
Daniella M
DM
McPhie
Peter
P
Narum
David L
DL
Ribeiro
José Marcos C
JM
Monteiro
Robson Q
RQ
Francischetti
Ivo M B
IM
D000330:Aedes / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D002341:Carotid Artery Thrombosis / Q000517:prevention & control*; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology; D006909:Hydroxyproline / Q000737:chemistry; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D038982:Integrin alpha2beta1 / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D014841:von Willebrand Factor / Q000378:metabolism*
D018076:DNA, Complementary; D005343:Fibrinolytic Agents; D019476:Insect Proteins; D038982:Integrin alpha2beta1; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D012471:Salivary Proteins and Peptides; C098341:platelet membrane glycoprotein VI; D014841:von Willebrand Factor; D003094:Collagen; D006909:Hydroxyproline