Novel function of human ADAM15 disintegrin-like domain and its derivatives in platelet aggregation.
BACKGROUND
A Disintegrin and Metalloproteinase (ADAM) proteins are a family of multifunctional proteins containing disintegrin and metalloproteinase domains that perform both adhesive and proteolytic functions in cell-cell and cell-matrix interactions. ADAM15 is unique among these proteins in having an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain. This motif is known to interact with the integrin alphaIIbbeta3 on platelets.


METHODS
We cloned and expressed the human ADAM15 disintegrin-like domain and its derivatives in Pichia pastoris, and purified them by chromatographic fractionation. We then characterized the integrin binding specificities and their antiplatelet activities of the proteins. Antiplatelet function was assessed by inhibition of platelet adhesion and aggregation.


RESULTS
The yeast-expressed ADAM15 disintegrin-like domains were able to inhibit the binding of alphaIIbbeta3 as well as alphavbeta3 to its biological ligands in a dose-dependent manner. Remarkably, mutation of the three residues proximal to the RGD tripeptide sequence, RPTRGD sequence to NWKRGD, increased its affinity for alphaIIbbeta3. The NWK mutant had a much greater inhibitory action on human platelet aggregation than the original ADAM15 disintegrin-like domain.


CONCLUSIONS
The structural context of the RGD tripeptide sequence in the disintegrin domain determines the specificity and affinity of the protein for its binding partners. The human ADAM15 disintegrin-like domain may provide useful information for developing an antithrombotic agent.
Jeon
Ok-Hee
OH
Department of Biochemistry, College of Science, Yonsei University, Seoul, Republic of Korea.
Kim
Dongbum
D
Choi
Yong-Jun
YJ
Kim
Seung-Hee
SH
Choi
Won-Seok
WS
Kim
Doo-Sik
DS
D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000187:drug effects / Q000502:physiology; D020869:Gene Expression Profiling; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D009842:Oligopeptides / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000187:drug effects / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D051722:ADAM Proteins; C098660:ADAM15 protein, human