Abstract
Background Details of the molecular interaction between tissue-type plasminogen activator (tPA)
and plasminogen activator inhibitor type-1 (PAI-1) remain unknown.
Methods and Results Three distinct forms of high-molecular-weight complexes are demonstrated. Two of
the forms were detected by mass spectrometry. The high molecular mass detected by
MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry)
was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA
(65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367
Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass:
3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed
by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single
band was observed. However, after treatment by SDS and Triton X-100, two distinct
forms of the complex with different mobilities were shown by SDS-PAGE. The higher
molecular weight band demonstrated specific tPA activity on fibrin autography, whereas
the lower molecular weight band did not. Peptide sequence analysis of these two bands,
however, unexpectedly revealed the existence of the C-terminal cleavage peptide in
both bands and its amount was less in the upper band. In the upper band, the sequences
corresponding to the regions at the interface between two molecules in its Michaelis
intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl–enzyme
complexes, wherein only the upper band liberated free tPA under the conditions employed.
Conclusion These data suggest that under physiological conditions a fraction of the tPA–PAI-1
population exists as nonacylated–enzyme inhibitor complex.
Keywords
tissue-type plasminogen activator (tPA) - plasminogen activator inhibitor type 1 (PAI-1)
- SERPIN - serine protease - fibrinolysis
