Identification of an inhibitor of tissue-type plasminogen activator-mediated fibrinolysis in human neutrophils. A role for defensin.
An inhibitor of tissue-type plasminogen activator (tPA)-mediated and plasminogen-dependent fibrinolysis was isolated from human neutrophils. On a G-50 gel filtration column, the antifibrinolytic activity present in neutrophil homogenates comigrated with proteins of < 13 kDa. The inhibitory fraction had only a slight effect on urokinase with plasminogen- or plasmin-mediated fibrinolysis and no effect on urokinase- or plasmin-mediated cleavage of H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). The neutrophil-derived fraction inhibited tPA with plasminogen activity on S-2251 but not on H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288). The inhibition of tPA-mediated and plasminogen-dependent fibrinolysis or S-2251 cleavage showed a competitive pattern and could be relieved by increasing the concentration of plasminogen. The same fraction also inhibited binding of plasminogen to fibrin. Consecutive purification steps revealed that the molecular mass of the inhibitor was 1-5-kDa. Polylysine-Sepharose affinity chromatography indicated that the inhibitor is a protein of 4 kDa, migrating as one band on SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of this band showed the presence of two sequences, differing by one amino acid, which are identical to defensin I and II. Comparison of the sequences of plasminogen and defensin showed homology of defensin to the plasminogen kringles known to contain the lysine binding sites. The close structural similarity between defensin and plasminogen kringles and the ability of defensin to compete with plasminogen on binding to fibrin explain the ability of defensin to inhibit tPA-mediated, plasminogen-dependent fibrinolysis. These results suggest that the antifibrinolytic activity of defensin may have a biological function in preventing the spread of infection.
Higazi
A A
AA
Department of Clinical Biochemistry, Hadassah University Hospital, Jerusalem, Israel.
Barghouti
I I
II
Abu-Much
R
R
D000595:Amino Acid Sequence; D000933:Antifibrinolytic Agents / Q000302:isolation & purification* / Q000494:pharmacology; D001798:Blood Proteins / Q000302:isolation & purification* / Q000502:physiology*; D023082:Defensins; D005342:Fibrinolysis / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors*
D000933:Antifibrinolytic Agents; D001798:Blood Proteins; D023082:Defensins; D010959:Tissue Plasminogen Activator