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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
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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
36
37
38
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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
55
56
57
58
59
60
61
62
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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
283
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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
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483484
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485