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8/3/2019 ARTICULO ARBELAEZ

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Purification and Activation of Caprine and Canine plasminogen: Comparison with1

Human plasminogen2

3

4

Omaira Caas a, Alfonso Quijano Parra a, Lus Fernando Arbelez a*&5

6

aGrupo de investigacin en Qumica, Universidad de Pamplona, Pamplona, Colombia7

&Correspondence to Lus Fernando Arbelez Ramrez,[email protected]

9

10

11

Keywords, Plasminogen, Coagulation, Fibrinolysis, Plasmin12

13
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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ABSTRACT14

15

Objective. Unification of the purification and activation of the plasminogen of several16

species as human, caprine and canine. Materials and methods. In the purification17

procedure were used lysine-sepharose 4B and sephacel DEAE, for affinity and ion-18

exchange chromatographies respectivity. Results. Bands of 92 kDa corresponding to19

the native plasminogens were identified for the three species and their N-terminal20

sequences were determined to be EPLDDY, DPLDDY and XXLDDY for human,21

caprine and canine plasminogen respectively. Furthermore the degraded in vivo22

circulating plasminogens in the three species were purified and the N-terminal23

sequences determined to be KVYLSE, RITLL and RIYLS for the human, caprine and24

canine, respectively. Conclusion. Activation of the three plasminogens confirmed the25

formation of the typical electrophoretic bands for the human plasmin corresponding to26

the heavy A and the light B chains which also were identified for caprine and canine27

plasmin. This new purification methodology facilitates the comparisons and further28

elucidation of the fibrinolytic systems in mammals.29

30

31

32

33

.34

35

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RESUMEN39

40

Objectivo. Unificar la purificacin y activacin del plasmingeno de varias especies,41

como el humano, caprino y canino. Materiales y mtodos. En el procedimiento de42

purificacin fueron usadas lisina-sefarosa 4B y sefacel DEAE, para la cromatografa de43

afinidad y cambio inico respectivamente. Resultados. Bandas de 92 kDa fueron44

identificadas, correspondendientes a los plasmingenos nativos de las tres especies y las45

secuencias para el terminal-N fueron EPLDDY, DPLDDY y XXLDDY, para el46

humano, caprino y canino respectivamente, adicionalmente la degradacin del47

plasmingeno de las tres especies en la circulacin fue purificada y sus secuencias48

fueron determinadas, KVYLSE, RIT LL y RIYLS, para el humano, caprino y canino49

respectivamente. Conclusin. La activacin de los tres plasmingenos, confirman la50

formacin de bandas electroforticas tpicas de la plasmina humana, correspondientes a51

la cadenas pesada A y liviana B, las cuales tambin fueron identificadas para la52

plasmina caprina y canina. Este nuevo mtodo de purificacin facilita la comparacin53

del sistema fibrinoltico en mamferos.54

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56

57

58

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INTRODUCTION63

Haemostasis is a mechanism, which maintains blood in a fluid state within the64

vasculature.1 Coagulation of blood is mediated by cellular components and soluble65

plasma proteins in response to vascular injury. In the final step thrombin cleaves66

fibrinogen to generate fibrin monomers, which polymerize to form a chemically stable67

clot.68

The fibrinolytic (plasminogen/plasmin) system in the vasculature includes an inactive69

proenzyme, plasminogen (Pg), that can be converted to the active enzyme, plasmin70

(Pm), which degrades fibrin into soluble fibrin degradation products.2 Two71

immunologically distinct plasminogen activators (PA) have been identified: the tissue-72

type PA (t-PA) and the urokinase-type PA (u-PA). The t-PA mediated Pg activation is73

mainly involved in the dissolution of fibrin in the circulation. Inhibition of the74

fibrinolytic system may occur either at the level of the PA, by specific Pg activator75

inhibitors type 1 and 2 (PAI-1 and PAI-2), or at the level of Pm, mainly by 2-76

antiplasmin (2-AP).3,4 As most other plasma proteins, Pg is synthesized in the liver,77

and the human plasma concentration is approximately 2 M, 5 native Pg is a single-78

chain glycoprotein with glutamic acid as N-terminal for the human Pg, 6 and is therefore79

referred to as Glu-Pg. It is however easily degraded and autocatalytically cleaved by80

Pm, with release of a peptide of 8 kDa by cleavage at Lys76-Lys77 that converts Glu-Pg81

to Lys77-Pg.7 Calculations based on the primary sequence, corrected for82

carbohydrates,8,9 give molecular weights of 92 KDa and 82 KDa Da for Glu- and Lys-83

Pg respectively.584

By cleavage of a single peptide bond (Arg560-Val561), Pg is activated to Pm.10 This85

cleavage is equivalent to the activation cleavage of other serine enzymes and the two86

chains are held together by two disulfide bonds.11

The full length cDNA of the human87


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Pg has been cloned and analysis of the DNA sequence revealed that Pg contains 79188

amino acids residues.1289

The Pg molecule contains lysine binding sites (LBS) and to these sites, lysine or90

lysine analogues, which carry both an amino group and a free carbonyl group bind,91

92

this group. One strong and five weak LBS were found,13 assuming that one of the low-93

affinity sites represents the active site, the other five sites corresponds with the five94

kringles domains in the Pg A-chain.11 The binding of Pg to fibrin, 2-AP, Histidine rich95

glycoprotein and thrombospondin is mediated via the kringles.14-1696

The Pg of three species has been isolated and studied, and the human Pg is until now the97

most studied. It has been purified by different methods and multiple molecular forms of98

human Pg have been identified and structurally analyzed.1799

The properties of Pg from other mammalian species, notably of cow,18 rabbit and100

sheep.19 Have been investigated up to now and compared with the human Pg. It is well101

established that Pg of different species differ in their behaviour towards streptokinase.102

Human, cat and monkey Pg are readily activated by catalytic amounts of streptokinase,103

whereas bovine, pig, sheep, rat and mouse Pg are not.20 The proenzymes from dog and104

rabbit require high concentrations of streptokinase for activation. Human Pg and Pm105

form a stoichiometric 1:1 complex with streptokinase.21106

In the present study we report the results of the a uniform method for the purification of107

the Pgs of three species out of which caprine Pg now is purified for the first time and108

the canine purification has been improved. A comparison of the concentrations of Pg in109

the plasma of these species and man has been performed, and the N-terminal sequences110

of these Pgs were determined and compared to those of human Pg.111

112


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MATERIALS AND METHODS113

Chemicals114

All buffer substances, salts and other chemicals employed were of the highest available115

purity. ACA, phenylmethanesulfonyl fluoride (PMSF), dimethylsulfoxide (DMSO),116

methanol, acetic acid were from Fluka, N, N- methylene-bis-acrylamide, ammonium117

persulfate, N,N,N,N-tetramethylethylenediamine, -mercaptoethanol, sodium dodecyl118

sulfate (SDS) were from Biorad, sodium chloride (NaCl) and sodium acetate from119

Riedel-de Han, di-sodium hydrogen phosphate dihydrate from Merck, and Lysine-120

Sepharose 4B, was supplied by Amersham Biosciences, diethylaminoethyl (DEAE),121

aprotinin were supplied by Sigma, chromogenic substrate for Pm Spectrozyme,122

Urokinase (UK), were supplied by American diagnostica Inc, Spectrolyze123

Plasminogen SK kit from Trinity Biotech, molecular weight markers employed: 180124

KDa (2-macroglobulin), 92 kDa (Glu-Pg), 66 kDa (-chain human fibrinogen), 52 kDa125

( -chain human fibrinogen), 46 kDa ( -chain human fibrinogen), and 23 kDa126

(Trypsin), molecular weight markers were supplied by the laboratorios de investigation127

en biomolculas from Pamplona University (Pamplona Colombia).128

Plasmas samples129

Both human and animal blood were drawn in bags containing citrate dextrose and130

adenine (PDA-1) as anticoagulant. Human Pg was obtained from fresh plasma from the131

Erasmo Meoz Hospital (CcutaColombia), analyzed before use and certified free of132

antigens of hepatitis, VIH, Chagas and another infectious diseases.133

Blood from the animals were obtained from the Experimental farm Villa Marina134

(Pamplona- University-Colombia). 200 ml plasma of each species were made 1mM135

PMSF (disolved in DMSO) and 730 IU/ml Aprotinin.136

Affinity Chromatography137


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All Pgs were purified by affinity chromatography on lysine-Sepharose 4B, according138

to the method of Deutsch and Mertz,22 using 35 ml of Lysine-Sepharose 4B, packed139

in a Column of 12 x 2.0 cm from Biorad, equilibrated with 3 column volumes of 0.1 M140

phosphate buffer containing 0.15 M of NaCl pH 7.3 (PBS) at a flow rate of 2 ml/min141

following by plasma sample application (200ml) and washed with the same buffer until142

the absorbance A280was 0.01. The bound Pgs were eluted with 100 ml of PBS143

containing 0.05M ACA and 2 ml fractions were collected. The concentration of Pg was144

determined at A280 using (1%)1cm = 1.68 as absorption coefficient.

22 Each preparation145

was concentrated, using a membrane of 10 kDa., to approximately 1mg/ml using an146

Amicon device (Millipore). The Pgs solutions were dialyzed over night at 4C, with147

0.06M Tris, 0.06M NaCl, 0.02M HCl pH 8.5. Buffer (A) in a dialysis tube of 25mm.148

Ion Exchange Chromatography149

All Pgs were further purified in a column of 5 cm x 0.25 cm from Biorad, packed with 4150

ml of DEAE Sepharose and equilibrated with buffer A. The sample was added and151

washed with buffer A until the A280 was 0.01 and the elution was performed by a152

linear gradient using buffer A and 0.07M Tris, 0.22M NaCl, 0.06M HCl pH 7.5 buffer153

(B), 3 ml fractions were collected at a flow rate of 1.5 ml/min. The concentration of Pgs154

were determined and the samples were concentrated as before and pelleted (dropping155

protein solution into liquid nitrogen) and stored at -80C until use.156

Determination of plasminogen concentration in plasma157

The concentration of Pg was determined using the Spectrolyze Plasminogen SK kit.158

The determination was made in both plasma and in each step of the purification.159

Electrophoretic analysis160

Gel electrophoresis was performed at denaturing (10 % SDS-PAGE) conditions161

according to Laemmli.23 Protein samples of 5 g were mixed with the sample buffer162


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SDS in a 1:1 (vol/vol) ratio. The proteins were allowed to react, before electrophoresis163

with SDS and -mercaptoethanol (10%) and were boiled for 5 min. at 100C. Proteins164

were visualized by staining with Coomassie Brilliant Blue R. A large range of markers165

(see materials and methods) were used as standards.166

Activation of Glu and Asp plasminogens167

To 1 mg of each Pg incubated at 37C, 6.72 l of UK was added to a final concentration168

of 739 IU/ml (activation solution). The reaction was followed spectrophotometrically169

at A405, using the Pm chromogenic substrate Spectrozyme as follows: To 8 test tubes 60170

l of substrate (0.3 mM), were added and 3 l of the activation solution was added to171

the substrate after 0, 1, 3, 6, 9, 15, 25 and 35 min. of incubation, after 12 seconds the172

reaction with substrate was stopped by addition of 10 l of 4M acetate pH 3.8. The173

development of color determined at A405 in each test tubes, was recorded.174

The activation solution was stopped by adding 100% glycerol to the final175

concentration of 25%, Glycerol. The solution was homogenized and stored at -20C176

until use.177

Determination of the plasmin concentration178

According to the substrate supplier, hydrolysis of the substrate with 10 mA (A405) at179

37C, corresponds to 1 nM Pm.180

To three test tubes 60 l substrate and 3 l of the activated solution were added and181

the tubes were incubated at 37C for 0, 1 and 2 min. After that time 10 l of stopping182

solution (4 M acetate pH 3.8), was added and the absorbance at A405 was determined.183

Protein sequence184

Sequence analysis for intact Glu-Pg and degraded Lys-Pg and corresponding proteins in185

the animals were performed: Twog of each Pg were diluted in 500 l of 0.1% acetic186

acid. To each solution membranes peaces of 3X3 mm of polyvinilidene fluoride187


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(PVDF) were added, moistened with 99.9% ethanol. The solutions were incubated at 2-188

8C for two days every 8 hours the solution were shaken for 3 min. The membrane189

peaces were dried followed by washes with 20% methanol.190

The N-terminal sequencing was kindly performed by Dr. Per-Ingvar Ohlsson at the191

University of Ume, Sweden using the Edman degradation methodology.24192

193

194


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RESULTS195

All Pgs display affinity for the Lysine-Sepharose matrix and the elution profile of all196

Pgs from the Lysine-Sepharose column, were the same for the three species as197

demonstrated in Figure 1, No significant contamination at washing or after elution was198

observed as can seen in Figure 1, fractions 0-10 and 30-35 respectively.199

All the species presented a very narrow peak of Pg in the plasma, both in the starting200

material as well as in the final step of purification as shown in the Table 1.201

The caprine Pg demonstrated the lowest concentration and the canine the highest Pg.202

These preparations were mixtures of all the physiologically known Pgs as Glu- and Lys-203

Pg in the human and the corresponding animal Pgs.204

The eluted Pgs were analysed by SDS-PAGE using a protein marker as indicated in205

materials and method and a band of 92 kDa was identified in the three species as206

demonstrated in Figure 2 lane 2 for the human Pg, lane 3 for canine Pg and lane 4 for207

caprine Pg. All these bands were at the same level that the band corresponding to the208

Glu-Pg in the marker lane 1.209

Physiologically and particularly in human plasma different types of Pg as Glu-and Lys-210

Pgs have been detected. These Pgs were separated by ion exchange chromatography as211

shown in Figure 3, in which peak 1 was identified as caprine Asp-Pg, not retain on the212

ion exchange column, while peak 2 is a Pg of the caprine with lower molecular weight213

as the identified Lys-Pg in human. All Pg for these three species presented the same214

chromatogram as demonstrated in Peak 1 and 2 Figure 3. The electrophoretical analysis215

of the separation of the different Pgs from the three species are shown in Figure 4. Lane216

2 presents the caprine Arg-Pg bound in the ion exchange chromatography column,217

(Figure 3 peak 2), lane 3 is the canine XX-Pg and lane 4 is the human Lys-Pg. The N218

terminal sequence were determined for both the intact Pg and the degraded Pg of the219


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three species, i.e. for the human Glu-Pg EPLDDY, for the canine Pg XXLDDY and220

for the caprine Pg DPLDDY. Furthermore the human Lys-Pg N-terminal was221

determined to KVYLSE and the corresponding sequence for the caprine RITLL and for222

the canine RIYLS, summarized in Table 2.223

The intact Pgs Glu- for human and Asp- for the animals were activated by UK to Pm224

and the concentration of the Pm found in the activation solution were 2.71 M for225

human Glu-Pg, 3.68 M for canine X-Pg and 6.30 M for caprine As-Pg, these values226

must be compared to the maximal theoretical values of 10 M. The human Glu-Pg227

presented the lowest activation, followed by the canine, and the caprine Asp-Pg was the228

most activated Pg of the three species, as summarized in Table 3. The successful229

activation of the three Pgs is visualized by the appearence of the typical A and B chains230

as shown in Figure 5. Lane 1 is the marker (see materials and methods), lane 2 is Glu-231

Pg, lane 3 is Glu-Pm, lane 4 is canine X-Pm, lane 5 is caprine Asp-Pm.232

233


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DISCUSSION234

This new purification procedure demonstrates its usefulness for different Pgs of several235

species. Human intact Pg and its degradation products in plasma have been purified as236

Glu-Pg and Lys-Pg, respectively,6 confirmed in this study by the sequence237

determination of the two separated human Pgs. They have been used as control samples238

for the corresponding Pg in the animals, the human Glu-Pg corresponds in the animals239

to Asp-Pg, as determined from the N-terminal sequence of the undegraded Pg in the240

animals and the human Lys-Pg corresponds to Arg-Pg in the animals. This is in good241

agreement with the results of the determination of both intact Pg and degrade Pg242

sequence for others species.18243

This new procedure also demonstrates that the Pg concentration in plasma of the three244

species are close despite the species differences. The purification of canine Pg early has245

been performed by different methods all of them very complicated, with more than 15246

steps and no separation of the different degraded Pgs in the canine plasma.25 In this247

study the method was improved by using only two steps i.e. the Lysine-Sepharose and248

the ion exchange chromatography and different physiologically active canine Pg were249

separated. The two first amino acids of the N-terminal of intact canine Pg, were difficult250

to identify. No apparent reason was detected, despite several determinations of the N-251

terminal, giving the same results. Maybe these amino acids are cleaved by Pm or other252

proteinasesin vivo in the dog plasma. But in all animals species known, the N-terminal253

sequence has been determined to Asp as primary amino acid.254

Purification of caprine Pg was performed for the first time and this Pg displays the same255

behavior as the human and canine Pgs during the purification steps. The determination256

of the concentration of Pg in the plasma for the three species indicated that they had257

very close amount of Pg in the plasma and the undegraded Glu-Pg for human and Asp-258


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Pg for the animal species. Their N-terminal sequence had in common the sequence259

LDDY, while the two first amino acids for the canine Pg could no be determined. The260

difference between the human and caprine Pg was only one amino acid. Apparently the261

three species showed very similar degradation products of Pg in plasma.262

The activation of the Glu-Pg and Asp-Pg of the three species by UK demonstrated that263

the activation to Pm of the three species generated the same bands, identified in the264

human case as chains A and B.26 Furthermore caprine Pg was activated to 63% (6.3 M265

of 10 M) while the canine and human Pg were activated to only 36.8% and 27.1%266

respectively. These last results indicate that the Pg/Pm system (fibrinolytic system) is267

activated differently in these species. In several publications from our laboratories we268

demonstrated that the Pg/Pm of the canine 27, equine 27, bovine27, and bufaline 28, had269

higher affinity for substrates made for human Pg/Pli.270

This probably indicated that the fibrinolytic system in animals recognition preferentially271

the blood clots formed in the vasculature than the human. These results are in good272

agreement with all the studies of the human coagulation and fibrinolytic system, which273

indicate man as being the most susceptible species to thrombotic diseases. 29-32274

Furthermore molecular abnormal Pgs have been detected with different mutation within275

the Pg molecule which predispose these patients for thrombosis.33 Pg deficiency also276

has been demonstrated to be related to different human health problems.34-37277

With no doubt, Pg of many species can be purified by this method, faciliting278

comparison of the fibrinolytic system between species, opening the possibility to279

identify Pgs in species which more efficiently degrades blood clots, this Pg can be use280

in clinical determination of parameters of the fibrinolytic system, that cause281

cardiovascular problem in human.282

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ACKNOWLEDGMENTS284

We thank Dr. Per-Ingvar Ohlsson for performing the sequence analysis, Erasmo Meoz285

Hospital for supplied the human plasma samples, Dr. Carlos Mario Duque for the286

animals plasma samples and the Medical Faculty of the University of Ume in Sweden.287

288


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FIGURE LEGENDS410

Figure 1. Elution profile of the Pgs of the three species from lysine-Sepharose column.411

Human Pg ( ), Canine Pg ( ), and caprine Pg ( ).412

413

Figure 2. Denaturating (10%) SDS PAGE of Pgs from three species eluted from lysine-414

Sepharose: lane 1 Molecular Weight Markers (see materials and methods), lane 2415

Human Pg, lane 3 Canine Pg, and lane 4 caprine Pg.416

417

Figure 3. Chromatogram of the separation of different physiologically caprine Pgs in418

DEAESepharose column. Peak 1 Asp-Pg, Peak 2 Arg-Pg eluted as indicated in419

material and methods.420

421

Figure 4. Denaturating (10%) SDS-PAGE of the degraded Pgs in plasma separated by422

ion exchanged chromatography, lane 1 Molecular Weight Markers (see materials and423

methods), lane 2 caprine Arg-Pg, lane 3 human Lys-Pg, lane 4 canine XX-Pg.424

425

Figure 5. Activation of human and animals Pg by UK. Lane 1 is the Molecular Weight426

Markers (see materials and methods), lane 2 is Glu-Pg, lane 3 is Glu-Pm, lane 4 X-Pm427

for canine and lane 5 Asp-Pm for caprine.428

429


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Table 1. Yield of Pgs Human (H), Canine (C) and Caprine (Ca) from plasma and from430

different purification steps, as determine by Spectrolyze Plasminogen SK kit.431

432

433

Procedure Steps V/ml Amount ofPg (mg)

Pg recovered(%)

FoldPurification

1. Startingmaterial

2. Lysine-Sepharose

Chomatography

3. Ion-exchangeChomatography

HCCa

HCCa

HCCa

200200200

131510

6118

283226

232522

192219

100100100

928992

767879

111

333530

353733

434

435


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Table 2. Amino acid sequences of the N-terminals of the undegrade and degrade Pgs,436

determined using Edman degradation methodology.437

438

Plasminogen N-terminal sequence

Undegrade (Glu, Asp, X)

N-terminal sequence

Degrade (Lys, Arg, X)

Human EPLDDY KVYLSE

Caprine DPLDDY RITLL

Canine XXLDDY RIYLS

439


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Table 3. Activation of the undegraded Plg (Glu-Pg and Asp-Pg) in the three species440

with human UK using a chromogenic substrate.441

442

Plasminogen (1 mg) PmM Theorical

Human 2.71 10 M

Canine 3.68 10 M

Caprine 6.30 10 M

443

444


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Figure 1445

446

447

0

0,5

1

1,5

2

2,5

3

3,5

0 5 10 15 20 25 30 35

Fractions Number

Absorbance280nm

Human

Canine

Caprine


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Figure 2.448

449

450

451


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Figure 3452

453

454

455


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Figure 4.456

457

458

459


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Figure 5.460

461

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467

468

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483484


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485

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