Summary
The complement inhibitor S protein, which is identical to the adhesive protein vitronectin,
functions as heparin-neutralizing factor by protecting thrombin as well as factor
Xa against fast inactivation by antithrombin III. The interference of S protein with
glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated
in these studies. S protein significantly counteracted the anticoagulant activity
of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of
0.3 μg/ml heparin, 0.5 μg/ml pentosan polysulfate, or 2 μg/ml dermatan sulfate, S
protein induced a concentrationdependent
reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted
in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin),
or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan
sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor
II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and
carboxymethylation of the protein increased the neutralizing activity of S protein
towards heparin and pentosan polysulfate. The results of these functional experiments
correlated well with the demonstration of direct binding of S protein to both polysaccharides
but not to
dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective
in neutralizing other dermatan sulfate compounds with varying degree of sulfation,
a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding
domain of S protein, counteracted their anticoagulant activity. Independent on the
polysaccharide used, S protein was found incorporated within a ternary complex with
thrombin and heparin cofactor II during the inhibition reaction as judged by crossed
immunoelectrophoresis, ultracentrifugation as well as ELISA analysis, emphazising
the function of S protein as scavenger protein for enzyme-inhibitor complexes of the
coagulation system. These findings demonstrate the role of S protein as effective
neutralising plasma protein of the anticoagulant activity of various glycosaminoglycans
also with respect to heparin cofactor II. Although the glycosaminoglycan-binding domain
of S protein readily neutralized different dermatan sulfate compounds, physiological
modulation of heparin cofactor-II-dependent
inhibition of thrombin by native S protein appears to be restricted to the vascular
compartments, where other glycosaminoglycans than dermatan sulfate appear to be operative.
Key words Heparin cofactor II - S protein (vitronectin) - Glycosaminogly-can neutralization
- Thrombin inhibition - Ternary complex
