Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation.
Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T. M. (1993) J. Biol. Chem. 268, 5445-5449). A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin. Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids. Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology. The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin. A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins. Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation. This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets. Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes. Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor. The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo.
Keller
P M
PM
Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, Pennsylvania 19486.
Waxman
L
L
Arnold
B A
BA
Schultz
L D
LD
Condra
C
C
Connolly
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003094:Collagen / Q000494:pharmacology; D004247:DNA; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D013987:Ticks / Q000737:chemistry
D010975:Platelet Aggregation Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; C080268:moubatin; D003094:Collagen; D004247:DNA