Microthrombomodulin. Residues 310-486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation.
Thrombomodulin is the endothelial cell cofactor for thrombin-catalyzed activation of protein C. Recently, we isolated a 10-kDa thrombin binding fragment, CB3, from the epidermal growth factor precursor homology domain (epidermal growth factor (EGF)-like regions) of thrombomodulin (Kurasawa, S., Stearns, D. J., Jackson, K.W., and Esmon, C.T. (1988) J. Biol. Chem. 263, 5993-5996). The CB3 fragment did not, however, support protein C activation. A 29-kDa fragment, called CB23, has now been isolated and corresponds to residues 310-486 in the EGF-like region of thrombomodulin. The CB23 fragment bound thrombin and accelerated thrombin-catalyzed protein C activation. With two separate preparations of CB23, the Km for protein C was 1.6 and 1.9 microM and the Kd for thrombin was 8.9 and 13.2 nM. The carboxyl terminus of CB23 and CB3 was identified by isolation and sequence analysis of a tryptic peptide from CB3. The sequence of this peptide corresponded to Asn457-Ser486, indicating that the carboxyl terminus of these fragments is 6 residues beyond the sixth EGF-like region of thrombomodulin. In addition, although CB3 cannot accelerate protein C activation, CB3 did inhibit the rate of thrombin-catalyzed fibrinopeptide release from fibrinogen. Thus, like native thrombomodulin, CB3 will alter thrombin's substrate specificity, but protein C activation requires additional information all of which can be provided by other regions of the EGF-like domain.
Stearns
D J
DJ
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Kurosawa
S
S
Esmon
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003488:Cyanogen Bromide; D004815:Epidermal Growth Factor; D005340:Fibrinogen / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D011486:Protein C / Q000378:metabolism*; D011498:Protein Precursors; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000494:pharmacology*; D018179:Receptors, Thrombin; D012689:Sequence Homology, Nucleic Acid; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin
D009844:Oligosaccharides; D010446:Peptide Fragments; D011486:Protein C; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor; D005340:Fibrinogen; D014357:Trypsin; D013917:Thrombin; D003488:Cyanogen Bromide