A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site.
We have isolated a fragment (approximately equal to 10 kDa) of thrombomodulin containing the fifth and sixth epidermal growth factor (EGF)-like regions which retains thrombin binding capacity. The amino-terminal sequence of a 50-kDa active fragment of thrombomodulin derived from elastase proteolysis begins 11 residues before the first EGF-like structure of native thrombomodulin. Subsequent digestion with cyanogen bromide yields a 10-kDa thrombin binding fragment. The amino-terminal sequence of this fragment starts at the fifth EGF-like structure (Phe407). The amino acid composition suggests that this fragment contains the fifth and sixth EGF-like structures with a total of approximately 77 residues. This fragment lacks cofactor activity, but acts as a competitive inhibitor for protein C activation (Ki = 8.6 +/- 1.4 nM). We propose that the fifth and sixth EGF-like structures contain the thrombin binding site of thrombomodulin.
Kurosawa
S
S
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Stearns
D J
DJ
Jackson
K W
KW
Esmon
C T
CT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D003488:Cyanogen Bromide / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000032:analysis*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*
D000596:Amino Acids; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D004815:Epidermal Growth Factor; D013917:Thrombin; D003488:Cyanogen Bromide