Physicochemical characterization of human S-protein and its function in the blood coagulation system

Abstract

S-protein, the main inhibitor of the assembly of the membrane attack complex of complement, was isolated from human plasma by a simple purification procedure, which includes barium citrate adsorption, ammonium sulphate precipitation, chromatography on DEAE-Sephacel and Blue Sepharose and gel filtration on Sephacryl S-200. The homogeneous protein (sedimentation coefficient 4.6 S) was obtained in approx. 5% yield relative to its concentration in plasma, which was found to be 0.3-0.5 mg/ml. The final product did not cross-react with antisera against complement proteins or other proteinase inhibitors of human plasma. On polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, S-protein migrated as a single-chain band with an apparent Mr of 74000 under non-reducing conditions and as a doublet of Mr 78000 and 65000 upon reduction. In plasma or serum S-protein also existed in two forms of corresponding Mr values, as was evidenced by an immunoblot enzyme-linked immunosorbent assay technique. S-protein was found to be an acidic glycoprotein with 10% (W/W) carbohydrate content and several isoelectric points in the range pH 4.75-5.25, and it contained one free thiol group per molecule of protein. The functional properties of S-protein in the complement system were demonstrated by its ability to inhibit complement-dependent cell lysis in a concentration-dependent manner (Ki 0.6 microM) and by its incorporation into the nascent SC5b-7 complex. A new function for S-protein could be revealed in the blood coagulation system. The slow progressive inhibition of thrombin by antithrombin III was not affected by S-protein, whereas the purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex. The acceleration of this inhibition reaction by heparin was counteracted by S-protein, indicating the ability of S-protein to neutralize heparin activity.

Fibroblasts Spread on Immobilized Fibrin Monomer by Mobilizing a β1-class Integrin, Together with a Vitronectin Receptor αvβ3 on Their Surface

“…Protein concentrations were determined spectrophotometrically by using absorption coefficients (A 1 cm 1% at 280 nm) of 15.1 for fibrinogen (11), 9.0 for vitronectin (19), and 13 for fibronectin (20).…”

Determination of the vitronectin binding site on plasminogen activator inhibitor 1 (PAI‐1)

“…Thrombin was radiolabeled with IlZ5 using the Iodogen method, resulting in a specific radioactivity of 1.6@/pg protein. Vitronectin was purified from human plasma to apparent homogeneity [22]. The protein concentration of the purified preparation was determined, assuming an extinction coefficient at 280 nm of E,, = 13.0 and a molecular weight of 75,000 [23].…”

“…It is conceivable that the physiologically relevant configuration of PAI-in plasma [9,10], in the subendothelial matrix [l l] and in releasates of platelets [12], is as a complex with its carrier protein vitronectin.…”

Incorporation of Vitronectin into Fibrin Clots

“…Vitronectin also regulates cell adhesion and pericellular proteolysis on surfaces of cells and extracellular matrices (1)(2)(3)(4)(5). Like fibrinogen, vitronectin is found in plasma at micromolar concentrations (6), and is stored in megakaryocyte and platelet ␣-granules (7)(8)(9). In plasma, vitronectin circulates as a native, monomeric form that is a mixture of 72-kDa single-chain and two-chain disulfide-linked species (10 -12).…”

“…Levels of the oligomeric forms of vitronectin in serum relative to plasma also increase; indicating the process of coagulation alters vitronectin structure and function (10 -12, 14). The altered, oligomeric form of vitronectin is generated, at least in part, by interactions with other plasma proteins such as thrombin-antithrombin complexes (11, 12, 14, 15, and complement C5b-9 complexes (11, 12, 16).A portion of vitronectin in plasma (17,18), and in platelets is complexed with type 1 plasminogen activator inhibitor (PAI-1) 1 (8,9,19), an interaction that induces the formation of higher order complexes (4,5,20,21) and influences the structure and function of both PAI-1 and vitronectin. Thus, when bound to vitronectin, PAI-1 is stabilized in its active conformation (22, 23), and thrombin is more readily inactivated by PAI-1 (24).…”