Geetha Kassam

Characterization of the Heparin Binding Properties of Annexin II Tetramer

In this report, we have characterized the interaction of heparin with the Ca2+- and phospholipid-binding protein annexin II tetramer (AIIt). Analysis of the circular dichroism spectra demonstrated that the Ca2+-dependent binding of AIIt to heparin caused a large decrease in the α-helical content of AIIt from ∼44 to 31%, a small decrease in the β-sheet content from ∼27 to 24%, and an increase in the unordered structure from 20 to 29%. The binding of heparin also decreased the Ca2+ concentration required for a half-maximal conformational change in AIIt from 360 to 84 μm. AIIt bound to heparin with an apparentK d of 32 ± 6 nm (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). The binding of heparin to AIIt was specific as other sulfated polysaccharides did not elicit a conformational change in AIIt. A region of the p36 subunit of AIIt (Phe306–Ser313) was found to contain a Cardin-Weintraub consensus sequence for glycosaminoglycan recognition. A peptide to this region underwent a conformational change upon heparin binding. Other annexins contained the Cardin-Weintraub consensus sequence, but did not undergo a substantial conformational change upon heparin binding.

Role of Annexin II Tetramer in Plasminogen Activation

The enzymatic cascade triggered by activation of plasminogen has been implicated in a variety of normal and pathologic events, such as fibrinolysis, wound healing, tissue remodeling, embryogenesis, and the invasion and spread of transformed tumor cells. Recent data established that the Ca(2+)- and phospholipid-binding protein, annexin II heterotetramer (AIIt) binds tissue-type plasminogen activator (tPA), plasminogen, and plasmin, and dramatically stimulates the tPA-dependent conversion of plasminogen to plasmin in vitro. Interestingly, the binding of plasmin to AIIt can inhibit the activity of the enzyme, suggesting that plasmin bound to the cell surface is regulated by AIIt. The existing experimental evidence suggests that AIIt is the key physiological receptor for plasminogen on the extracellular surface of endothelial cells.

Regulation of plasmin-dependent fibrin clot lysis by annexin II heterotetramer

In a previous report we showed that plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if annexin II heterotetramer (AIIt) was present during fibrin polymer formation. Here, we show that AIIt inhibits fibrin clot lysis by stimulation of plasmin autodegradation, which results in a loss of plasmin activity. Furthermore, the C-terminal lysine residues of its p11 subunit play an essential role in the inhibition of fibrin clot lysis by AIIt. We also found that AIIt binds to fibrin with a K d of 436 nm and a stoichiometry of about 0.28 mol of AIIt/mol of fibrin monomer. The binding of AIIt to fibrin was not dependent on the C-terminal lysines of the p11 subunit. Furthermore, in the presence of plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen do not compete for identical sites on fibrin. Immunohistochemical identification of p36 and p11 subunits of AIIt in a pathological clot provides important evidence for its role as a physiological fibrinolytic regulator. These results suggest that AIIt may play a key role in the regulation of plasmin activity on the fibrin clot surface.