JPH0564588A - Human protein c having short heavy chain and activated human protein c - Google Patents

Abstract

PURPOSE:To provide the subject protein C having improved stability, useful as an anticoagulant, etc., and having a structure obtained by removing several amino acids from a natural-type activated human protein C and having a heavy chain having an amino acid at a specific position on the heavy chain of the human protein C as the C-terminal amino acid. CONSTITUTION:A DNA fragment coding natural-type human protein C is prepared by conventional process. The fragment is integrated in a proper manifestation vector and introduced into a host to effect the transformation of the host. The transformant is cultured to obtain a recombinant protein C. A solution of the recombinant protein C is concentrated by ultrafiltration, dialyzed with 0.05M tris-HCI buffer solution (pH 7.5), added with a bovine thrombin solution, incubated at 37 deg.C for 3hr and purified by the successive cation-exchange column chromatography and gel-filtration to obtain a human protein C or activated human protein C having either one of the 239th to 246th amino acids on the heavy chain of a natural-type activated human protein C as the C-terminal amino acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to human protein C having a short heavy chain or activated human protein C. More specifically, it relates to short-chain human protein C or activated human protein C that can be used as an anticoagulant or a fibrinolysis promoter.

In this specification, the amino acid sequences are abbreviated by the method adopted by the IUPAC-IUB Biochemistry Committee (CBN), and the following abbreviations are used, for example.

Ala L-Alanine Arg L-Arginine Asn L-Asparagine Asp L-Aspartic acid Cys L-Cysteine Gln L-Glutamine Glu L-Glutamic acid Gly Glycine His L-Histidine Ile L-Isoleucine Leu L-Leucine Lys L-Lysine Met L-methionine Phe L-phenylalanine Pro L-broline Ser L-serine Thr L-threonine Trp L-tryptophan Tyr L-tyrosine Val L-valine

The sequence of DNA is abbreviated by the type of base contained in each deoxyribonucleotide constituting reed, and for example, the following abbreviations are used.

A adenine (representing deoxyadenylic acid) C cytosine (representing deoxycytidylic acid) G guanine (representing deoxyguanylic acid) T thymine (representing deoxythymidylate)

[0006]

BACKGROUND OF THE INVENTION Protein C is a type of plasma serine protease precursor that is activated on the surface of platelets and vascular endothelial cells by limited degradation by a complex of thrombin and its receptor, thrombomodulin. Activated protein C, which is a protease (hereinafter AP
C). APC exerts anticoagulant activity by selectively degrading activating factor 5 and activating factor 8 of the blood coagulation system. This activity is known to be enhanced by protein S. Also, AP
C is an inhibitor of tissue plasminogen activator
Cleavage of PAI-1 is believed to have fibrinolytic activity.

The amino acid sequence of human protein C has already been described in Proc. Natl. Acad. Sci. USA, 82 , 4673-467
7 (1985) and others, a light chain having a Gla domain and an epidermal growth factor (EGF) -like domain on the amino terminal side (molecular weight: about 21,000), a heavy chain consisting of an activated peptide and a catalytic domain (molecular weight: About 4
1,000) is a disulfide-bonded (double-stranded type).

Specifically, the natural human protein C is shown in FIG.
As shown in, the light chain consists of 155 amino acids from amino terminal Ala to carboxyl terminal Leu, and the heavy chain consists of 262 amino acids from amino terminal Asp to carboxyl terminal Pro. The chains are (H
₂ N-) light chain-Lys-Arg- is biosynthesized in the form of heavy chain (-COOH), and Lys-Arg is cleaved to form a double chain in the process of being secreted from cells. The light chain and the heavy chain are the Cys 141 from the amino terminus of the light chain and 120 from the amino terminus of the heavy chain.
It is connected to the second Cys by an SS bond.

Conversion from human protein C to activated human protein C is performed by activation from the 12th amino acid to the amino acid at the N-terminal of the heavy chain from the amino terminus (hereinafter referred to as the N-terminal) of the heavy chain of human protein C. This is done by removing the polypeptide. Therefore, the amino acid sequence of activated human protein C is the same as the amino acid sequence of human protein C except for all the amino acid sequence of human protein C from the 12th amino acid to the N-terminal amino acid of the heavy chain. However, the effect on the activity of the amino acid on the C-terminal side and the stability of the structure are not clear.

[0010]

[Object of the Invention] In order to produce activated human protein C (hereinafter abbreviated as APC) from human protein C (hereinafter abbreviated as PC), a suitable activator such as thrombin or thrombin-thrombomodulin complex is added to PC. It is necessary to act and remove the activating peptide from PC.

We have found that during the activation procedure, along with the activating peptide, other peptides derived from PC, especially those with the C-terminal peptide of the heavy chain removed from PC, have a short heavy chain. Nevertheless, they found that physiological activity still existed.

PC or APC from which the peptide containing the C-terminal amino acid, which is important for PC, has been removed is the one with the highest stability as it has already been subjected to the action of the enzyme.
Or it is APC.

[0013]

That is, the present invention basically has a structure of natural human protein C or activated human protein C, and the amino terminus of the heavy chain is 239 as calculated by natural activated human protein C. The present invention relates to a PC or APC having a heavy chain, which is characterized in that it is at the amino terminal of any of the 2nd to 246th amino acids.

The above primary structure of PC or APC is the most stable structure in PC or APC and
C has sufficient physiological activity.

The properties of PC include, for example, treatment of the protein of the present invention with a protein C activating enzyme so that it has anticoagulant activity, fibrinolytic activity, amidase activity and the like.

That is, the PC of the present invention is converted to the APC of the present invention by treating it with a protein C activating enzyme in the same manner as natural human protein C. Here, the protein C activating enzyme includes trobin and thrombin.
Examples thereof include thrombomodulin complex and snake venom.

Further, genetic engineering techniques can be used to obtain these APCs of the present invention. That is,
By deleting the peptide existing at the C-terminal of the heavy chain in advance, a stable structure can be obtained before activation.

It is also possible to directly express the APC of the present invention by deleting the activating peptide existing at the N-terminal of the heavy chain at the same time.

As described above, the APC activity is an anticoagulant activity that selectively decomposes activating factor 5 and activating factor 8 of the coagulation system, and an inhibitor of tissue plasminogen activator. Reacts with PAI-1 to PAI-
The fibrinolytic activity and the like resulting from deactivating 1 are mentioned.

In the present invention, the DNA sequence encoding the protein of the present invention means that when a plasmid containing it is introduced into a host and cultured, the protein having the properties of the present invention is expressed as a result. Any DNA sequence may be used, and a gene other than the structural gene (for example, an intron) may be interposed between the DNA sequences.

The DNA fragment encoding the protein of the present invention is
It is incorporated into an expression vector appropriately selected according to the host vector system. Specific examples of such expression vectors include the adenovirus major late promoter, SV40
A plasmid vector having an early promoter, SV40 late promoter, mouse metallothionein 1 / promoter, MMTV (mouse papilloma virus) promoter, RSV (Rous sarcoma virus) promoter, or a virus-derived vector such as Bovine papilloma virus is used.

The above-mentioned DNA fragment can be incorporated into such a vector by a method known per se, for example, Mi
ura O. et al., J. Clin. Invest. 83 , 1598-1604 (19
89) and the like.

On the other hand, the host animal cells may be human or non-human animal cells, for example, CHO, C127, BHK, Cos1, Cos7, LM, NIH3T3, 293, He.
la, etc., and CHO, BHK, 293 are preferable among them.

Transfection of these expression vectors into these cells can also be carried out by a method known per se well known in the art, for example, Span.
didos DA and Wilkie NM, Expression of Exogenous
DNA in Mammalian Cells, Ed. Hames BDan Higgins
SJ Transcription and Translation, IRL Press, Ox
It can be performed by the method described in the literature such as ford and ppl-48.

The transformed cells thus obtained can be cultured by a conventional method under conditions suitable for each cell, and the desired protein of the present invention can be recovered from the culture solution.

The method for quantifying PC or APC of the present invention is disclosed in
It is possible to use the method shown in Japanese Patent Publication No. 61-283868. According to this method, it is also possible to measure only the protein having the activity of Gla-bearing human protein C and / or activated human protein C.

For purification of PC or APC of the present invention, a method for purifying natural human protein C including a barium adsorption method and an ion exchange chromatography method can be applied, but the conformation of the Gla domain by calcium ion is applied. It is particularly preferable to use affinity column chromatography using an anti-human protein C monoclonal antibody that recognizes changes (see Japanese Patent Laid-Open No. 64-85091). This method is excellent in that only PC or APC having Gla can be purified, and that a mild eluent called EDTA can be used.

The activity of the PC of the present invention was examined in advance by activating it with a snake venom (Protac, American Diagnostica) and cleaving the synthetic substrate (PCa, American Diagnostica). Since the PC and APC of the present invention have the same catalytic domain as the natural type, they have a synthetic substrate cleaving activity and thus have an anticoagulant activity, which is the original activity.
It can exhibit fibrinolytic activity.

[0029]

EXAMPLES In the examples of the present invention, the following methods were used. Preparation of recombinant PC (hereinafter abbreviated as rPC) rPC is a known gene recombination technique, that is, JP-A-64-
It was produced by the method described in Japanese Patent No. 85084.

Activation of PC (1) Activation reaction The rPC solution was concentrated to 2 mg / ml with an Amicon concentrator (Amicon, Grace Japan) equipped with an ultrafiltration membrane YM10, and 0.05M Tris / HCl pH 7.50. .1
It was dialyzed against 5M NaCl buffer. 2 mg / ml r above
To 1860 μl of PC solution, 69 μl of 2.7 mg / ml bovine thrombin solution (Mochida Pharmaceutical Co., Ltd.) and 500 mM Na ₂ EDT
1065 μl of a TBS solution containing A was added to make about 3 ml. After thoroughly mixing the solution, the mixed solution was incubated for 3 hours at 37 ° C. in a thermostat to activate rPC.

(2) Purification of APC A cation exchange column was used to purify APC by removing thrombin from the incubated mixed solution.

The above mixed solution containing APC was added to 3 ml of 0.
02M MES (2- (N-morpholino) ethan Sulfur
ic acid) / Tris pH 6.0, 0.05M Na
Diluted 2-fold by adding Cl buffer. The diluted solution was equilibrated with the same buffer solution and S of 1.5 cmφ × 4 cm
-It was passed through a Sepharose Fast Flow column (Pharmacia). After washing the column with the same buffer, the APC adsorbed on the column was eluted with a linear gradient of 0.05 to 1.0 M NaCl to pass through the fraction and 0.3 to 0.5 M N.
was included in the aCl fraction. Thrombin is about 0.6M
It was eluted with NaCl. APC and thrombin can be separated almost completely by this operation. APC
The combined fractions containing 30 ml of 0.02M MES /
Tris pH 6.0 buffer was added to reduce NaCl concentration.

Peptides released upon activation of PC
Amino acid sequence analysis of 100 μl of a mixed solution obtained by reacting PC and thrombin under the above conditions in the activation reaction of PC by thrombin
Was ultrafiltered using a centrifugal ultrafiltration device Centricon 10 (Amicon). Then, the free peptide contained in about 80 μl of the obtained filtrate was isolated under the following conditions. (Conditions) Column: Vydac C18 column (4.6 mm diameter x 250
mm length, 218TP54) Eluent A: 0.1% trifluoroacetic acid, 1 vol% acetonitrile aqueous solution Eluent B: 0.1% trifluoroacetic acid, 99.9 vol
% Acetonitrile Flow rate: 0.5 ml / min Column temperature: room temperature Detection: 215nm

All the detected peaks were collected, freeze-dried, and then acetonitrile / water = 1/1 (vol / vol) 100.
Re-dissolve in μl, and use 477A Protein Sequencer (A
When the amino acid sequence was analyzed by BI), peptides having the sequences shown in the following table were confirmed.

[0035] _{NH 2 -Ser -His -Leu -COOH NH 2} -Arg -Ser -His -Leu -COOH ( SEQ ID NO: _{1) NH 2 -Lys -Arg -Ser -His} -Leu -COOH ( SEQ ID NO: 2) NH ₂ ―Asp ―Thr ―Glu ―Asp ―Gln ―Glu ―Asp
-Gln -Val -Asp-Pro -Arg -HOOH (SEQ ID NO: _{3) NH 2 -Ser -Trp -Ala -Pro} -COOH ( SEQ ID NO: 4)

From the amino acid sequence data of PC, these peptides were derived from the light chain C-terminal sequence, from the heavy chain N-terminal sequence (activation peptide), and from the heavy chain C-terminal sequence. Was confirmed.
Furthermore, these sequences are not autolysates of bovine thrombin, since the above sequences are not present in bovine thrombin.

Therefore, the C-terminal amino acid of the heavy chain of activated PC is present on the N-terminal side including amino acid: Lys of the 246th amino acid in FIG.

Solution of C-terminal sequence of H chain of activated protein C
Analysis 50mM Tris-0.15M NaCl-20mM
1 mg of purified rPC and 1 mg of activated rPC dissolved in 1 ml of EDTA (pH 7.4) buffer (weight is measured by absorbance at 280 nm using the following formula)
5 mg of dithiothreitol, 4 ml of 6M guanidine hydrochloride-0.5M Tris buffer (pH 8.
3) was added and the mixture was heated at 65 ° C. for 4 hr in a nitrogen atmosphere to reduce the disulfide bond connecting the heavy chain and the light chain, and separate the heavy chain and the light chain. Then add 2 to this solution
00 μl of 43 mg / ml monoiodoacetic acid aqueous solution (pH 4
.About.6) was added and the mixture was heated at 37.degree. C. for 30 minutes to carboxylate the SH group, thereby preventing re-oxidation of the SH group.
The solution thus obtained was added with 10 times 8M urea solution gradually, and the ultrafiltration membrane YM10 (Amicon) was added.
An ultrafiltration device (Amicon) (model number 8MC) equipped with was used for ultrafiltration to remove low-molecular compounds from the solution. In order to isolate the heavy chain from the solution containing the heavy chain and the light chain, reverse phase liquid chromatography was performed using a poly F column (manufactured by DuPont) under the following conditions.

Column: Poly F column (6.2 mmφ × 80 mm, DuPont) Eluent A: 0.1 M NH ₄ HCO ₃ (pH 8.0) Eluent B: Acetonitrile Flow rate: 0.5 ml / min Column temperature: room temperature Detection: 215nm

A peak containing a heavy chain (one in which a band having a molecular weight of 40,000 to 45,000 was confirmed by SDS-polyacrylamide gel electrophoresis) was lyophilized to obtain 50 mM Phosphate.
-1M urea buffer (pH 8.0) 200μl
And redissolved, added with 0.2 μg of endoproteinase Asp-N (Boehringer Mannheim), and 37 ° C.
After heating for 6 hrs, the N-terminal peptide bond of aspartic acid in the H chain was cleaved.

The peptides contained in this reaction solution were separated by reverse phase chromatography under the following conditions. (Conditions) Column: Vydac C18 column (4.6 mm diameter x 250
mm length, 218TP54) Eluent A: 0.1% trifluoroacetic acid, 1 vol% acetonitrile aqueous solution Eluent B: 0.1% trifluoroacetic acid, 99.9 vol
% Acetonitrile Flow rate: 0.5 ml / min Column temperature: room temperature Detection: 215nm

When all the detected peaks were collected and the sequence of peptides contained in each peak was analyzed,
The following sequence was confirmed as the sequence near the C-terminal of the heavy chain.

NH ₂ ―Asp ―Lys ―Glu ―Ala ―Pro
―Gln ―Lys ―Ser ―Trp ―Ala ―Pro ―COOH (SEQ ID NO: 5) NH ₂ ―Asp ―Trp ―Ile ―His ―Gly ―His ―Ile
-Arg-COOH (SEQ ID NO: 6)

Is derived from the C-terminal peptide of the heavy chain (amino acids Nos. 240 to 250), and is derived from the peptide bound at its N-terminal (amino acids Nos. 232 to 239).
). Although these peptide fragments were detected in both rPC and activated rPC, the peptide-to-peptide molar ratio in rPC (measured by amino acid composition analysis)
Was 100, the value was 21 in the activated rPC.

The amount of peptide was almost the same in both rPC and activated rPC. Therefore, in activated rPC, amino acid No.
There are H chains with either Arg of 239 to Lys of No. 246.

[Sequence list]

SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence Arg Ser His Leu 1

SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence Lys Ars Ser His Leu 15

SEQ ID NO: 3 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Asp Thr Glu Asp Gln Glu Asp Gln Val Asp Pro Arg 1 5 10

SEQ ID NO: 4 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence Ser Trp Ala Pro 1

SEQ ID NO: 5 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence Asp Lys Glu Ala Pro Gln Lys Ser Trp Ala Pro 1 5 10

SEQ ID NO: 6 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment sequence Asp Trp Ile His Gly His IleArg 15

[Brief description of drawings]

FIG. 1 shows the structure of native activated human protein C in a form in which a prepro leader sequence at the N-terminus of the light chain and an activation peptide are added.

[Procedure amendment]

[Submission date] November 10, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

Specifically, the natural human protein C is shown in FIG.
As shown in, the light chain consists of 155 amino acids from amino terminal Ala to carboxyl terminal Leu, and the heavy chain consists of 262 amino acids from amino terminal Asp to carboxyl terminal Pro. The chains are (H
₂ N-) light chain-Lys-Arg- is biosynthesized in the form of heavy chain (-COOH), and Lys-Arg is cleaved to form a double chain in the process of being secreted from cells. The light chain and the heavy chain are the Cys 141 from the amino terminus of the light chain and 120 from the amino terminus of the heavy chain.
It is connected to the second Cys by an SS bond. Also,
The Gla domain contains nine calcium-binding amino acids,
Including γ-carboxyglutamic acid (Gla) residue
Is known.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction content]

The PC or APC from which the peptide containing the C-terminal amino acid of the heavy chain of PC has been removed has already been subjected to the action of the enzyme, and therefore has the highest stability of PC.
Or it is APC.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

The above mixed solution containing APC was added to 3 ml of 0.
02M MES (2- (N-morpholino) ethan Sulfur
ic acid) / Tris pH 6.0, 0.05M Na
Diluted 2-fold by adding Cl buffer. The diluted solution was equilibrated with the same buffer solution and S of 1.5 cmφ × 4 cm
-It was passed through a Sepharose Fast Flow column (Pharmacia). After washing the column with the same buffer, the APC adsorbed on the column was eluted with a linear gradient of 0.05 to 1.0 M NaCl, and APC was passed through the fraction and 0.3 to 0.
It was included in the 5M NaCl fraction. Thrombin was eluted at about 0.6M NaCl. APC and thrombin can be separated almost completely by this operation. Fractions containing APC are combined and about 30 ml of 0.02M
MES / Tris pH 6.0 buffer was added to reduce NaCl concentration.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0033

[Correction method] Change

[Correction content]

Peptides released upon activation of PC
Amino acid sequence analysis of 100 μl of a mixed solution obtained by reacting PC and thrombin under the above conditions in the activation reaction of PC by thrombin
Was ultrafiltered at 4 ° C. using a centrifugal ultrafiltration device Centricon 10 (Amicon). Then about 8 filtrates obtained
The free peptide contained in 0 μl is a high-performance liquid under the following conditions.
It was isolated by chromatography (HPLC) . (Conditions) Column: Vydac C18 column (4.6 mm diameter x 250
mm length, 218TP54) Eluent A: 0.1% trifluoroacetic acid, 1 vol% acetonitrile aqueous solution Eluent B: 0.1% trifluoroacetic acid, 99.9 vol
% Acetonitrile Flow rate: 0.5 ml / min Column temperature: room temperature Detection: 215nm

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction content]

All the detected peaks were collected, freeze-dried, and then acetonitrile / water = 1/1 (vol / vol) 100.
re-dissolved in μl, 477A protein sequencer (A
When the amino acid sequence was analyzed by Pride Biosystems , peptides having the sequences shown in the following table were confirmed.

[Procedure Amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction content]

Solution of C-terminal sequence of H chain of activated protein C
Analysis 50mM Tris-0.15M NaCl-20mM
1 mg of purified rPC and 1 mg of activated rPC dissolved in 1 ml of a buffer solution of EDTA (pH 7.4) (weight is the following formula {“PC, activity
Weight of denatured PC (mg / ml) "=" Absorption at 280 nm
Degrees "÷ solution of 5mg of measurement) using a 1.4} dithiothreitol, 4 ml of 6M guanidine hydrochloride-0.5 M T
By adding ris buffer (pH 8.3) and heating at 65 ° C. for 4 hours in a nitrogen atmosphere, the disulfide bond connecting the heavy chain and the light chain was reduced, and the heavy chain and the light chain were separated. Next, 200 µl of 43 mg / ml monoiodoacetic acid aqueous solution (pH 4 to 6) was added to this solution, and the mixture was heated at 37 ° C for 30 minutes to carboxylate the SH group, thereby preventing re-oxidation of the SH group. The solution thus obtained is ultrafiltered with an ultrafiltration device (Amicon) (model number 8MC) equipped with an ultrafiltration membrane YM10 (Amicon) while gradually adding 10 times 8M urea solution. , The low molecular weight compound was removed from the solution. In order to isolate the heavy chain from the solution containing the heavy chain and the light chain, reverse phase liquid chromatography was performed using a poly F column (manufactured by DuPont) under the following conditions.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Item name to be corrected] 0040

[Correction method] Change

[Correction content]

A peak containing a heavy chain (one in which a band having a molecular weight of 40,000 to 45,000 was confirmed by SDS-polyacrylamide gel electrophoresis) was lyophilized to obtain 50 mM Phosphate.
200 μl of -1 M urea buffer (pH 8.0) was added and redissolved, and 0.2 μg of endoproteinase A was added.
sp-N (Boehringer Mannheim) was added, and the mixture was heated at 37 ° C for 6 hours to cleave the N-terminal peptide bond of aspartic acid in the H chain.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0044

[Correction method] Change

[Correction content]

Is derived from the C-terminal peptide of the heavy chain (amino acids Nos. 240 to 250), and is derived from the peptide bound at its N-terminal (amino acids Nos. 232 to 239).
). These peptide fragments were detected in both rPC and activated rPC, but the molar ratio of peptide to peptide in rPC ( the number of moles of peptide divided by the peptide)
The number of activated rPC was 21, when the number of moles of the protein was measured by amino acid composition analysis) and the value was 100.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Seiichi Yokoyama, 4-3-2 Asahigaoka, Hino-shi, Tokyo Inside Teijin Limited Tokyo Research Center (72) Inventor, Yataro Ichikawa 4--3, Asahigaoka, Hino-shi, Tokyo Teijin Limited Tokyo Research Center

Claims (3)

[Claims] 1. A human protein C or an activated human protein C having a heavy chain whose C-terminal is any of amino acids 239 to 246 of the heavy chain in natural activated human protein C. 2. The human protein C according to claim 1, which has a light chain having a C-terminal of any of amino acids 141 to 155 of the light chain of natural activated human protein C.
Or activated human protein C. 3. The human protein C according to claim 1, which has a light chain whose C-terminal is any of amino acids 149 to 155 of the light chain in natural activated human protein C.
Or activated human protein C.