1 Ruth Martinez-Cruz, 1,2 Felix Cordoba, 1 Juan Carlos Mireles-Lopez, 2 Edgar Zenteno, 3 Eduardo Perez-Ortega, Margarito Martinez, and 1,3 Eduardo Perez Campos.
1 Unidad de Bioquimica e Inmunologia, Instituto Tecnologico, Oaxaca, 68,000 Mexico.
2 Departamento de Bioquimica, Facultad de Medicina, UNAM, 04510 Mexico.
3 Laboratorio de Patologia Clinica, Zaragoza 213, Oaxaca, Oax., C.P. 68,000, Mexico.
EFFECTS OF CONCANAVALIN A ON PROTEIN-C ACTIVITY
Life Sciences, Vol 64, N
10,pp. 879-885, 1999
Summary
Concanavalin A interacts specifically with the oligosaccharides from protein-C and modifies its anticoagulant activity. The lectin activates the protein-C activity in a dose dependent manner as demonstrated by in vitro and in vivo assays. Concanavalin A at low concentration (0.1 to 2 
g/mL) induces an increase on the catalytic activity of protein-C; at higher concentrations (5 to 20 g/mL), the catalytic activity returns to the baseline. The effect of concanavalin A was prevented by incubating the protein-C with (-methyl-mannoside or by treating the purified protein-C with (-mannosidase; furthermore, cleavage of mannosidic residues diminishes its catalytic activity. Our results indicate that the oligomannosidic portion of protein-C participates in the regulation of the catalytic activity of this protein.
Protein-C is a vitamin K-dependent glycoprotein that circulates as an active zymogen. This glycoprotein shows different biological activities such as anti-ischemic and anti-inflammatory activities (1, 2). Protein-C has been proved to protect against endotoxemia by inhibition of the production of macrophage-dependent cytocinins (3). This protein has been shown by inhibiting the macrophage-dependent proliferative response of T lymphocytes, and facilitates the invasion of the basal membrane by tumor cells (4). Severe acquired deficiency of protein-C during bacterial infections contributes to the pathogenesis of the thrombotic and necrotic lesions in diverse organs. Protein-C therapy has been proposed to be an excellent alternative to avoid mortality and thrombotic consequences due to bacterial infections (5).
Lectins have been proved to be useful tools in the study of the glycans in the coagulation factors, concanavalin A (Con A) is a lectin with specificity for oligomannosidic structures and has been used as a tool in the study of Factor I, Factor II, Factor V, and thrombin (6-11). In this paper, we show that the interaction of Con A with the oligosaccharides of protein C modifies its anticoagulant activity. These assays indicate the participation of oligosaccharide moieties of protein-C as regulatory determinants of its catalytic activity.
Materials and methods
Reagents and Human Plasma. The lectins Con A, and Lens culinaris agglutinin (LCA, specific for (-mannosidic residues), wheat germ (WGA, specific for N-acetyl-glucosamine), as well as all sugars and enzymes were from Sigma (Sigma Fine Chemicals, St Louis MO., USA). Con A-Sepharose 4B was obtained from Pharmacia (Pharmacia Biotech, Uppsala Sweden). Phaseolus coccineus var alubia (specific for lactosaminic type glycans) was purified by affinity chromatography on (1-acid glycoprotein as previously described (12). Plasma from human healthy donors and from BALB-c mice were collected in 3% citrate as anticoagulant and stored in liquid nitrogen until use. Protein-C-deficient plasma was obtained from Behringwerke AG, Germany.
Determination of protein-C activity. Protein-C activity was determined by a chromogenic method, the activator was a mixture of 2 mg/mL of venom from Agkistrodon contortrix contortrix in 25 mmol/L Hepes, 25 g/L polyethylene glycol, 30 mmol/L cesium chloride (pH 8.25). For the substrate, 3.3 mmol/L pyro-glutamic-proline-arginine-methoxy-nitroanilide acid (p-glu-pro-arg-MNA) (Berichrom, from Behring Werke AG Diagnostic, Marburg, Germany) was used.
Functional activity of protein-C. A mixture of 25 (L of the test sample and 25 (L of buffer was prepared in microwell plates (Nunc, Denmark). The resulting mixture was incubated at 37 C for 5 min and 25 (L of protein-C-deficient plasma was added and incubated at 37 C for 5 min. At this time 25 (L of 2 mg/mL Agkistrodon contortrix contortrix and 25 (L of thromboplastin reagent (Neothromtin from Behringwerke AG, Germany) were added and the mixture was incubated 230-240 seconds. Finally, the coagulation was activated with 25 (L of 25 mM CaCl2 and the coagulation time was measured with an ST4 coagulometer from Diagnostica Stago (Asničres, France).
Purification of protein-C. Protein-C obtained from plasma of healthy human donors was purified by affinity chromatography on Con A coupled to Sepharose 4B, in a column of 3 mL containing 30 mg of coupled lectin. The column was equilibrated in PBS (0.05 M sodium phosphate buffer, 0.15 M NaCl, and 1 mM CaCl2 and MgCl2, pH 7.4), 12 mg protein in 1 mL of serum was applied to the column. Non-retained material was eluted with PBS until optical density was below 0.01 and protein-C was eluted from the column by adding 0.2 M (-methyl-mannopyranose. Fractions of 400 l were collected and protein-C was measured with the method previously described (13). Active protein C fractions were collected, dialyzed against water and freeze dried until use. The purified protein-C (1 mg) was incubated for 16 h at 37 C with 1 U almond (-mannosidase in 50 mM sodium citrate, 0.15 M NaCl pH 4.5, the reaction was stopped by increasing the pH to 7.4 (14). Mannosidase treated protein-C was passed through the column containing Con A and 100% of the protein-C deposed was not retained. Protein-C activity was tested as indicated.
Effect of Con A on protein-C in BALB-c murine coagulation. Female BALB-c mice, average weight 34.8
3 g, were obtained from the Pharmacology Department of UNAM, Mexico. The effect on protein-C activity of alubia, WGA, LCA, and Con A was tested by injecting each lectin intraperitoneally to groups of five mice, at different concentrations (0.01 to 30 g) in a total volume of 0.1 mL, control assays were performed in animals injected with Ringer solution. Blood samples were obtained at different time intervals (from 0 to 40 min) from treated mice by cardiac puncture in a one-tenth volume of 0.11 M sodium citrate containing 1 g/L benzamidine, mixed and centrifuged at 1500 x g for 10 min. Partial thromboplastine time (PTT), thromboplastine time (PT), thrombine time (TT time), and protein-C were determined in all plasma samples. Standard procedures were used for these assays. Protein-C activity was measured in a chromogenic assay as described above.
Results
Effect of Con A on protein-C activity. By adding Con A to human plasma with 100% protein-C activity, in chromogenic assays, using Agkistrodon contortrix contortrix as activator and p-glu-pro-arg-MNA as substrate, we identified an increase of protein-C activity at concentrations of 0.5 to 2 (g lectin/ml plasma. At higher concentrations (over 5 g/mL) of Con A, the catalytic activity returns to the baseline (Figure 1). The effect of Con A was eliminated by incubating the lectin previously with 0.2 M (-methyl-mannopyranose. Similar results on activation of protein-C activity with this lectin were obtained when purified protein-C was added to protein-C deficient plasma. Neither bovine serum albumin as control nor other lectins such as Lens culinaris lectin, wheat germ lectin, and alubia lectin modified the activity of protein-C. Chronometric assays revealed that Con A induced an increase in coagulation times (Figure 2).
Fig 1.
Effect of Con A on the activity of protein-C in a chromogenic assay. Normal plasma (
). Deficient protein C plasma, supplemented with purified protein C (
). Normal plasma containing 100 mM (-methyl-mannopyranose (
). Agkistrodon contortrix contortrix was used as activator and p-glu-pro-arg-MNA as substrate. Results were obtained at 405 nm. No significant effect on protein-C activity was observed in the presence of Lens culinaris (LCA), wheat germ or alubia lectins; protein-C activity remained unaltered even at lectin concentrations of 20 g/mL.
Fig 2.
Effect of Con A on the activity of protein-C in vitro (chronometric assay). Assays were performed in an ST4 coagulometer. Human plasma (), protein-C-deficient plasma supplemented with purified protein-C (), were incubated with Con A at different concentrations, at 37 C for 240 sec, then with Agkistrodon contortrix contortrix and thromboplastin for 240 sec; finally CaCl2 was added. Clot-formation time was measured. An anticoagulant effect was obtained with an increase in the concentration of Con A. Control experiments were performed with Con A incubated previously with 0.2 M (-methyl-mannopyranose ().
Modulation of protein-C activity by Con A in vivo. In vivo experiments in the presence of Con A, revealed an increase in the activity of protein-C in those animals who received low doses of Con A, optimal stimulatory effect was obtained at 10 g/mL (Fig. 3); when we increased the concentration of Con A, protein-C activity returned to base line. At concentrations over 50g/mL of Con A, signs of respiratory failure and ataxic movements were observed. Thrombin time (TT) and partial thromboplastin time (PTT) were also measured. In healthy BALB-c mice the results were 24 3.7, and 25 2.5 sec, respectively. When Con A was administered at low doses (0.1 to 2 g) the values obtained were TT 34 1 sec and PTT 543.2. When Con A was administered at doses over 50 g/mL, the plasma becomes uncoagulable even after 4 min, indicating that Con A induces defects in the intrinsic routes and fibrin formation (Figure 4). This effect was optimal for Con A 5 min after injection, although it could be observed even 3 min after injection (not shown).
Fig 3.
Effect of Con A (), LCA () and alubia () and WGA () on the activity of protein-C in BALB-c mice. Lectins were injected intravenously at different concentrations, and protein C activity was measured by a chromogenic method 5 min after injection. Results represent the mean of 5 mice and the corresponding SE.
Fig 4.
Effect of Con A on activated partial thromboplastin time (), and TT thrombin time () in BALB-c mice. The lectin was injected intravenously in the tail. Protein-C was tested in plasma obtained by intracardiac puncture, and measured using the chromogenic method. Each point represents the mean SE of 5 mice. Con A concentration equal or above 50 g/mL renders the plasma uncoagulable.
Discussion
Blood clotting involves many proteins that act harmoniously in response to vascular injury to produce a fibrin plug; however, unregulated clotting results in occlusion of blood vessels and thrombosis. Regulation of the balance between the pro-coagulant and coagulation mechanisms is extremely important for survival. In this study we demonstrated that Con A modifies the proteolytic activity of protein-C. This effect is mediated by specific recognition of saccharidic structures on protein-C. As many coagulation factors, protein-C is a glycoprotein, and as reported previously its oligosaccharidic portion is N-glycosidically bound (15).
The effect of Con A on protein-C activity was evaluated in human plasma and confirmed with the purified protein-C; in both cases, the effect of the lectin was abolished by addition of its specific inhibitor and by cleaving, specifically with almond (-mannosidase, the mannosidic branches of the purified protein-C. Although there are no reports indicating the specific role of glycans in protein-C activity, elimination of mannose residues does not modify its catalytic activity, and Con A does not exert any effect on this enzymatically-treated protein-C, thus our results indicate that the complex protein-C-Con A, at low doses, renders a more stable conformation of the protein to interact with its specific substrate. Lectins as LCA, WGA, or alubia do not modify the protein C activity, neither in the plasma nor in the purified protein-C. Con A recognizes the trimannosyl core of oligomannosidic structures by specific interaction with the man(1,6 branch, rather than with the man (1,3 (16); the presence of a bisecting GlcNAc group on the ß-man is well tolerated by the lectin. Although LCA recognizes oligomannosidic structures, it seems that an (-1,6-linked fucose residue is an important determinant for the binding of the oligosaccharidic structure (16). WGA interacts only with bisected hybrid type N-glycans and to a lesser extent with bisected biantennary N-acetyl-lactosamine type glycans (16). Alubia interacts with bisected tri- and tetra-antennary N-acetyllactosamine type glycans (12). Our results confirm that protein-C contains oligomannosidic glycan structures, which are able to interact with Con A.
In vivo experiments showed that Con A at concentrations equal or above 50 g per animal, induce massive bleeding and death, probably by inducing disseminated coagulation. The BALB-c mouse was selected as the experimental organism because previous works have shown that the catalytic regions of protein-C in the mouse are homologous to human protein-C. (17). Protein C is the zymogen of the key anticoagulant enzyme, activated protein C. The thrombin-thrombomodulin complex on endothelial cell surfaces (18) activates this enzyme. Activated protein C forms complexes with protein S and inactivates the coagulation factors Va and VIIIa. Protein-C, activated protein C, and protein S bind specifically to cell surfaces such as endothelial cells (19); thus it is possible that Con A inhibits the activation of protein-C by interacting with specific sites of the protein-C where thrombin exerts its catalytic effect, as demonstrated with other coagulation factors (12). As demonstrated by in vitro assays, low doses of Con A (0.1 to 2 g), administered intravenously, induce activation of protein-C, thus confirming that Con A, when it interacts with specific glycans on protein-C, induces conformational modifications that favor its catalytic effect. Similar concentrations of the lectin increased the activity of protein-C and the partial thromboplastin time (PTT), which forms part of the intrinsic also increased 100% above the normal time. Con A has no effect on the extrinsic route of coagulation since preliminary results have indicated that prothrombin time was unaltered by the presence of Con A. In summary, we have shown that Con A is a useful tool to study the physiology of protein-C. Furthermore, the results strongly suggest that oligomannosidic structures on protein-C play an important role in the conformation of the protein and, very probably, in the interaction with other molecules or specific receptors on cells and endothelium.
Acknowledgments
This work was supported in part by Universidad Nacional Autonoma de Mexico (PADEP 012305), and CONACyT, Mexico (3931-M and SIBEJ S-7). Authors wish to thank Robert B. Grant, Ph.D., from Georgia State University, Atlanta, GA for fruitful comments.
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Corresponding author: Zaragoza 213, Oaxaca, Oax. Mexico, CP 68000, Fax: + 52 (951) 61140, eduardo@laboratorio.com.mx
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