Purification of Protein C, a Natural Anticoagulant, from Human Plasma by Affinity Chromatography with Concanavalin A

E. Perez-Campos, F. Cordoba, E. Perez-Ortega, M. Martinez, and E. Zenteno
Biochemistry and Immunology Unit of the Technological Institute of Oaxaca.
Biochemistry and Experimental Medicine. School of Medicine of the National University Autonomous of Mexico. Mexico City.
Laboratory of Clinical Pathology. Zaragoza 213, Oaxaca. Oaxaca City. 68000, Mexico.

Purification of Protein C, a Natural Anticoagulant, from Human Plasma by Affinity Chromatography with Concanavalin A

PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, 26(3 & 4), 183-188 (1996)

ABSTRACT

We have developed a rapid, efficient, and inexpensive method to purify protein C from plasma using Concanavalin A conjugated to Sepharose. With this method we have obtained protein C purified to homogeneity, as measured in polyacrylamide gels. We discuss each of the steps of our method and give the results of the purification.

INTRODUCTION

Protein C is a vitamin K dependent glycoprotein which is activated by thrombin bound to thrombomodulin. Protein C, when combined with its cofactor Protein S, has anticoagulant activity, inactivating Factor Va and Factor VIII by limited proteolysis ^{1, 2}. Activated protein C also stimulates fibrinolysis by inactivating plasminogen activator-inhibitor. The relevance of protein C in the regulation of coagulation is the result of a newly discovered mechanism which helps to explain recurrent thrombosis associated with protein C resistance ^{4, 5}.

Until recently, most investigators have purified protein C by one or another of the following methods: barium citrate adsorption and elution; ammonium sulphate fractionation with or without DEAE-Sephadex chromatography; ^{6, 7, 8}. calcium-dependent or calcium-independent monoclonal antibodies; ^{9, 10}. dye-ligand chromatography ¹¹. and DEAE-Sephadex preparative polyacrilamide gel electrophoresis ¹². We have developed a new method to purify protein C which is as efficient as the above mentioned methods but has the advantage of being considerably less expensive to use on a routine basis. Our method of purification employs affinity chromatography with concanavalin A conjugated to Sepharose.

MATERIALS

The human plasma was donated by the local Red Cross. Concanavalin A-Sepharose was obtained from Vector Laboratories, Inc., Burlingame, CA, USA. Barium citrate was obtained from Spectrum Chemical Mfg. Corp, New Jersey, USA. Protein C assay kit was obtained from Behringwerke AG Diagnostic, Marburg, Germany. Protein C chromogenic assay from American Diagnostics Inc, Greenwich, CT, USA. S-2366 was purchased from Chromogenix, Möndal, Sweden. Ammonium sulphate, benzamidine, imidazole, Freud's Adjuvant, concanavalin A, North American copperhead venom (Agkistrodon contortrix contortrix), methyl alpha-D-mannopyranoside, and Sephacryl S-400 HR were purchased from Sigma Chemical Co. St. Louis, Mo, USA. Agarose, immunodiffusion grade, was purchased from The Binding Site, LTD, Birmingham, England. Biogel P-100, Acrylamide, Bis-acrylamide, ammonium persulfate and TEMED were purchased from Bio Rad, CA, USA. For the chromogenic assay we used a microstrep reader, Behring EL 301, from Behringwerke AG Diagnostic. Marburg. Germany. To measure the molecular weight we used an automatic chromatography reader Protec II, (Gilson, USA). For the electrophoresis in polyacrylamide gel, we used a mini Protean II (Bio Rad. California. USA). We used a densitometer Junior 24 (Helena, Saint-Leu La Forêt, France) to obtain the data shown in Figure 1.

METHODS

In order to prepare the protein C concentrate from human plasma, we followed four steps: 1) barium citrate adsorption and elution ^{8, 12}, 2) ammonium sulphate fractionation ^{8, 12}, 3) column chromatography with Biogel and 4) affinity chromatography with concanavalin A - Sepharose. The details of our procedure are as follows: plasma was anticoagulated with one-tenth volume of 0.11 M sodium citrate, and then frozen at -70 °C until used. To one liter of thawed plasma, we added 5 ml benzamidine-HCl 50 mM. All steps in the purification process were made at 4 °C. One-tenth volume of 1.0 M barium chloride was added slowly to plasma. The suspension was stirred gently at 4 °C for 24 hours and centrifuged at 6000 g for 10 min. The precipitate was washed with buffer A (0.1 M NaCl, 0.01 M BaCl₂, 1mM benzamidine-HCl and 0.02% NaN₃) and centrifuged again. The washing was repeated twice with buffer A, and the washed barium citrate precipitate was resuspended in 0.5 l of 40% saturated ammonium sulphate solution. One ml Benzamidine-HCl (1 mM) was added to the solution. The suspension was stirred at 4 °C overnight. The precipitate was then removed by centrifugation at 6000 g for 30 min, and the supernatant fraction was treated with saturated ammonium sulphate solution to 67% saturation for 24 hours. The precipitated material was collected by centrifugation for 30 min at 8000 g. The sediment was dissolved in 50 ml of 0.05 M sodium phosphate buffer, pH 6.0, containing 0.2 M NaCl and 1 mM benzamidine-HCl and dialysed at 4 °C overnight against 6 liters of 0.05 M sodium phosphate buffer pH 6.0, containing 0.2 M NaCl and 1 mM Benzamidine-HCl. The insoluble material produced during dialysis was removed by centrifugation. The clear supernatant solution was lyophilised, then redissolved in distilled water and passed through a chromatography column containing Biogel. The early fractions, which contained protein C, were then added to a Concanavalin A-Sepharose column with tris-imidazole buffer, pH 8.4, containing 1 mM CaCl₂ and 1 mM MnCl₂. The protein C was eluted from the column by adding methyl alpha-D-mannopyranoside. The latter has a higher affinity for concanavalin and displaces the protein C.

The method used to measure protein C employs the venom of Agkistrodon contortrix as activator and p-Glu-Pro-Arg-MNA as substrate ¹³. For the electrophoresis we used polyacrylamide which contained 7.5% acrylamide, 5% sucrose and 0.375 M tris- HCl ¹⁴. Antibodies to protein C were raised in adult rabbits using Freund's Adjuvant. To show precipitation lines, we used a method of inmmunodifusion with a gel containing 0.9% agarose, 1% Triton X-100, and glycine-phosphate buffer pH 7.4 ¹⁵.

RESULTS

The steps of purification of protein C are shown in Table I. It may be observed that our method gives a high degree of purification.

The protein C was, in fact, purified to homogeneity as shown by the densitometric tracings in Figure 1. The estimated molecular weight of the protein C using a column of Sephacryl S-400-HR with a buffer of 1 M NaCl, 0.05M lactose, pH 6.5, was 64 Kd. This molecular weight agrees well with the estimates of other investigators ⁷. In double immunodiffusion we observed a single line of precipitation between purified protein C and the antibody preparation obtained from rabbits (data not shown).

DISCUSSION

The purpose of this investigation was to obtain a rapid, efficient and inexpensive method to purify protein C, and we have accomplished that objective. In addition, we have shown that the protein C we have purified is homogeneous by several different assays, immunodiffusion, polyacrylamide gel electrophoresis, and a chromogenic assay with venom of Agkistrodon contortrix as activator and p-Glu-Pro-Arg-MNA as substrate.

When the eluate was applied to the concanavalin A column in the presence of Ca ²⁺ and Mn ²⁺, protein C was specifically bound to the column and could be eluted using 0.1 M methyl alpha-D-mannopyranoside. The purification in this latter step was approximately 100 fold. Often, when the protein C preparation was passed through the Concanavalin A column, we obtained a small but significant (not more than 10%) increase in total units. Apparently we purified the protein C away from some kind of inhibitor. As shown in Figure 1, the elution profile from the concanavalin A column showed a single protein peak which coincided with the protein C peak as determined by the chromogenic assay. When we analysed the eluate by Sephacryl S-400-HR column, the molecular weight was 64 Kd. In summary, the use of Concanavalin A- Shepharose allows a high degree of purification at minimal cost, of the important natural anticoagulant, protein C.

Table I

Purification of human protein C from plasma
Step	Volume (ml)	Concentration (U/ml)	Total units	Protein (mg/ml)	Specific activity	Purification
Plasma	930	41,200	38,316 x 10 ³	91.00	452	1.00
BaCl₂(1 M)	1,020	43,500	44,370 x 10 ³	29.00	1,500	3.31
((NH₄) ₂S0₄) Sat 67 %	330	21,900	7,227 x 10 ³	1.30	16,846	37.21
Con A-Sepharose	10	27,800	278 x 10 ³	0.48	57,916	127.92

Figure 1.
Polyacrylamide gel electrophoresis in nondenaturing sytem, with 0.375 M Tris-HCl, in A human protein C before Con -Sepharose. In B, after through Con A-Sepharose

ACKNOWLEDGEMENTS
We thank of Dr. Ramon Vasquez Lopez, Chief of the Blood Center of the Red Cross in Oaxaca, Mexico for his collaboration. We thank Ruth Martinez Cruz, and Juan Carlos Mireles Lopez for their valuable technical assistance. And we thank Dr. Robert B. Grant of Georgia State University, Atlanta, GA, for help with the writing of this paper.

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