Open access peer-reviewed chapter

Comparative Fibrinolysis

By Emma Beatriz Casanave and Juan Tentoni

Submitted: September 18th 2013Reviewed: November 4th 2013Published: May 7th 2014

DOI: 10.5772/57359

Downloaded: 1255

1. Introduction

Haemostasis prevents leaks or obstructions within the blood vessels following three interrelated sequences: formation of the haemostatic plug, platelet consolidation and dissolution of fibrin clot by the fibrinolytic system (Juhan-Vague and Hans 2003; Van Cott and Laposata 2001; Vasse 2008). Coagulation factors circulate in the blood as proenzymes until they are activated by vascular damage (Lane et al. 2005; Owens and Mackman 2010). These enzymes amplified and disseminated the sequence and then are stopped by natural inhibitors (Mulder et al. 2010; Middeldorp 2011) and the fibrinolytic system (Greenberg and Orthner 1999; Levi et al. 2012). Cellular phospholipids make the process much more efficient (Hoffman 2003; Gentry 2004; Rivera et al. 2009).Activated Factor XIIIa stabilizes the polymer (Sidelmann et al. 2000; Greenberg and Lai 2003; Muszbek et al. 2011). Plasminogen (Plg) is the key in thrombus lysis; and is synthesized in mammals principally by the liver (Stafford 1964; Degen 2001; Zhang et al. 2002; Zorio et al. 2008). Natural Plg activators are: tissue plasminogen activator (tPA) and urokinase (uPA) (Fleming and Melzig 2012); streptokinase (SK) acts as in an exogenous path (Sazonova et al. 2009). Free Plm is very active and degrades other proteins, such as complement, fibrinogen (Fbg), factors II, V and VIII or activates metallo-proteases involved in tissue remodeling by degradation of cellular matrix (Collen 2001; Parfyonova et al. 2002; Dewyer et al. 2007).The main inhibitors of Plm are the alpha2 plasmin inhibitor (α2PI) (Menoud et al. 1996; Fraser et al. 2011) and Plasminogen activator inhibitor type 1 (PAI-1) (Declerk et al. 1998; Vaughan 2005). Thrombin activatable fibrinolysis inhibitor (TAFI) is a link between the two systems, it is activated by thrombin generated during coagulation, and suppresses fibrinolysis (Marx 2004; Hilmayer et al. 2006; Milijic et al. 2010).

2. Selection of animal model in fibrinolysis, a challenge

There is a growing homology in the components of the fibrinolytic system along zoological evolution. Fibrinolysis is present in all vertebrates but invertebrates generally only have clumping of blood corpuscles (Withers 1992). Vertebrates factors involved in coagulation and fibrinolysis have evolved from common ancestral proteins and fibrinolytic ones seem to be related to digestive proteolytic enzymes used by rudimentary microorganisms to be released and disseminated, avoiding the host´s nonspecific defense and immunity response (Patthy 1990; Gladysheva et al, 2003; Opal and Esmon 2003; Levi et al. 2012).

Insects have rich sources of pharmacological active substances that may have medical value: The venom of Lonomia oblique caterpillar may induce a hemorrhagic syndrome in humans, and blood incoagulability in laboratory animals (Prezoto et al. 2002). Bee venom of Bombus ignites contains a Kunitz type serine protease inhibitor (Bi-KTI) that acts as an antifibrinolytic agent inhibiting plasmin (Choo et al 2012). In nature, there are many animals adapted to a diet of fresh blood, and they had to evolve mechanisms to control their host coagulation processes, to maintain the blood in a fluid state during intake and subsequent digestion (Tanaka-Azevedo et al 2010). A variety of coagulation inhibitors have been isolated from blood sucking animals such as ticks (Jacobs et al 1990; Waxman et al 1990), leeches (Sawyer 1986, 1991), hookworms (Cappello et al 1995) and bats (Gardell et al 1991).

Very little is known about the fibrinolytic system and its component concentrations in animals and the relevance of these models for human health is questioned due to many reasons: interspecies differences (Siller-Matula et al. 2008; Ralph and Brainard 2012), lack of reliable results (Vap et al. 2012), use of diagnostic equipment designed only for human care, inadequate relationship of test reagent to clotting factor concentration (Ravanat et al. 1995; Jagadeeswaran and Sheehan 1999; Kubalek et al. 2002, Münster et al. 2002; Gentry 2004; Weir-M et al. 2004). Also, anatomical features of the animal chosen can make it really difficult to obtain good quality blood samples (Saito et al. 1976; Meinkoth and Allison 2007). For example, vessel size and blood flow are important determinants of vascular function when mouse model is used for human research of aorta (Fay et al 2007).

3. Objective of this chapter

In this chapter we summarize the actual knowledge about fibrinolytic assays among different animal species and we compare these findings with healthy adult human beings.

4. Fibrinolytic parameters

A review of laboratory tests was conducted in a phylogenetic order: fish, amphibians, reptiles, birds and mammals. It was designed to assess the fibrinolytic system in its various stages: global (whole blood lysis time WBLT, whole blood diluted lysis time WDLT, euglobulin lysis time ELT), specific (Plg, PAI-1, tPA, α2PI and the thrombin-activatable fibrinolysis inhibitor TAFI) and degradation products generated from the degradation of fibrinogen / fibrin FDP, D Dimer DD, and Plm-α2PI, tPA-PAI-1, uPA-PAI-1 complexes (Blanco 2003; Urano and Suzuki 2011).

The results of these assays are summarized in Tables 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 (Tentoni et al, 2010).

In fishes the information is insufficient (Tables 1 and 2). WBLT is undetectable in lamprey and black fish, while lysis is fast in dog fish. The genes encoded for Plg and tPA were identified in the blowfish Fugu rubripes (Jiang and Doolittle 2003). Rats with diets based on fish oil decrease the fibrinolytic activity due to an increase of PAI-1 (Sano et al. 2003), whereas dietary supplementation with fish protein increases fibrinolysis by increasing tPA in blood (Murata et al. 2004).

In amphibians (Tables 1 to 3), the marine toad Bufo marinus and the tree frog Hyla caerula show spontaneous WBLT (Hackett and Lapage 1961, Hackett and Hann 1964), while it does not occur in the common frog Rana temporaria, leopard frog Rana pipiens or the clawed toad Xenopuslaevis(Table 2),but can be induced if possible inhibitors are removed, which suggests a large concentration of antifibrinolytic agents. The existence of a protein similar to Plg in Rana tigrina and Xenopus laevis is explained by the fibrinolysis produced after the addition of uPA (Srivastava et al. 1981).

There is no evidence of a fibrinolytic system in reptiles, lizards (Trachydosaurus rugosus rugosus, Tiliqua scincoides, Amphibolorus barbatus, Varanus acanthrus, Iguana iguana), turtles (Chelodina longicollis), crocodiles (Crocodylus porosus) or pitons (Liasis spp, Morelia spp) (Tables 1 and 10). A strong circulating antithrombin protects these vertebrates from intravascular thrombosis (Hackett and Hann 1964; Kubalek et al. 2002), however low concentrations of α2PI were detected in the circulation of the snake Bitis arietans using a chromogenic method (Table 10).

Snake venoms are mixtures of many peptides which affect the blood coagulation and fibrinolysis pathways such as Plg activators (Kini 2005; Miller et al 2009) and fibrinogen degradators (Meyer 2000). Recently a non hemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25, which has an efficient proteolytic action over fibrinogen (Sérgio et al 2011).

Birds are deficient in Factors XI and XII so the clotting times exceeding 70 minutes (Wartelle 1957; Soulier et al. 1959, Bigland 1964). Fibrinolysis can be activated with the saliva of the vampire Diaemus youngui (Cartwright and Hawkye 1969), but not with SK (Cliffton and Cannamela 1951). Plg concentration in quails is indetectable due the chromogenic assay is activated with SK instead of uPA. Vultures have the highest reported value DD concentration among the animals with reduced levels of Fbg and clotting factors, remaining a disseminated intravascular coagulation in man, with the advantage of being easily reversible (Weir-M et al. 2004).

The WDLT in the Halichoerus grypus is lower than in humans (Table 3), suggesting the existence of an active fibrinolytic system.The Plg activity in Balaenoptera borealisis cannot be activated by SK but reacts against rabbit antibody antiPlg (Robinson et al. 1969).

FDP was undetectable in the Mirounga angustirostris elephant seal (Table 1 and 6).

Plg activators similar to tPA were discovered in the South American vampire bat´s Desmodus rotundus saliva (Verstraete 1995) and they all need fibrin as a cofactor (Schleuning et al. 1992).These activators do not degrade Fbg, or cause neuronal damage such as tPA does (Grandjean et al. 2004) and also have a prolonged plasma half-life (Zavalova et al. 2002).

In dogs (Tables 1, 3, 4, 5, 6, 7 and 10), except for the Plg when it is measured by activation with SK, the values of all the fibrinolytic assays are quite similar to the values in humans, as reported by Wohl et al. (1983).

In cats (Tables 1, 3, 5, 9 and 10) there is a marked difference in functional PAI-1 activity when compared to man, and its Plg cannot be activated with tPA but with uPA (Welles 1996).

In studied rodents, the fibrinolytic system is quite similar to that in humans, but Plg is poorly activated with SK; Plg, tPA, uPA and PAI-1 have been described in Mus musculus mouse (Tables 1, 7 and 8), the first two having high sequence homology with their human counterpart (Poplis and Castellino 2002). Interesting enough, PAI-1 deficient mice present a mild hyperfibrinolytic state in adulthood, whereas Plg deficiency predisposes to severe thrombosis (Eitzman et al. 2000; Mackman 2005). The main inhibitors of fibrinolysis in mice are α2PI and TAFI (Marx et al. 2000). In rodent capybara Hydrochaeris hydrochaeris (Tables 1 and 5), Plg cannot be activated even with 500 U/mL of SK (Leitao et al, 2000).

Rat (Tables 1, 3, 4, 5, 7, 8, 9 and 10), guinea pigs (Tables 4, 5 and 10) and rabbits (Tables 1, 3, 4, 5, 7, 9 y 10) are the most employed animal models in fibrinolytic research.

Plg cannot be activated with SK in cattle (Tables 1, 5, 6 and 10), pigs (Tables 1, 5, 7 and 10) and sheep (Tables 5, 7 and 10), (Cliffton and Cannamella 1953; Korninger and Colleen 1981; Wohl et al 1983; Zhang et al 2012). Horses (Tables 1, 5, 6, 7, 9 and 10) have higher levels of functional PAI-1 and α2PI when compared to humans (Barton et al. 1998). The fibrinolytic activity in llama is similar to that of horses and other domestic species (Morin et al 1995).

In armadillos Chaetophractus villosus our research group found prolonged WBLT and WDLT with PAI-1 functional activity four times greater than in man; this high concentration of inhibitor can be successfully removed with the ELT technique, despite the anticoagulant used (citrate/oxalate). The α2PI concentration is similar to that measured in humans. DD was undetectable in the immunological test (Tentoni et al., 2008). Nevertheless we found FXIII activity in this mammal, with a range from 32 to 78 percent (%) in relation to the calibration curve obtained with a pool of healthy humans platelets poor plasma using Berichrom chromogenix assay (Dade Behring). The fibrin plug was resistant to urea 5M for more than 36 hours; its coagulation factors depend on the vitamin K cycle because the oral administration of 0.28 mg/kg/day of acenocumarol increased baseline values of Prothrombin time PT (p<0.01) and activated Partial Thromboplastin time aPTT (p<0.05). When PTT-LA reagent is used in aPTT assays in armadillos, the typical shortened values of this specie (20 seconds) increases (26-30 seconds) (Tentoni et al., unpublished), as observed in pigs by Velik-Salchner et al. (2006).

SpeciesFbg (mg/dL)Author
human188 - 381Williams et al, 2005
armadilloa211 - 333Casanave et al, 2006
armadillo258 - 380Tentoni et al, 2008
whaleb147Saito et al, 1976
iguanac420 - 440Kubalek et al, 2002
catd50 - 165O´Rourke et al, 1982
cat150 - 400Herring and McMichael, 2012
eaglee80 - 160García-Montijano et al, 2002
frogf590 - 990Coppo et al, 2005
dolphing269 - 417Tibbs et al, 2005
mouseh200 - 260Tsakiris et al, 1999
dog141 - 227Mischke et al, 2000
dog179. - 329Machida et al, 2010
dog150 - 400Herring and McMichael, 2012
rat168 - 192Honda et al, 2008
japanese quaili140 - 260Belleville et al, 1982
pigj181 - 534Velik-Salchner et al, 2006
pig130 - 170Schöchl et al, 2011
rabbitk257 - 286Marval et al, 1992
cowl125 - 697Heuwieser et al, 1989
sheep178 - 215Wilhelmi et al, 2012
horsem78 - 156Barton et al, 1998
monkeyn119 - 239Suzuki et al, 1977
elephant sealo50 - 162Gulland et al, 1996
capybarap124Leitâo et al, 1999
ostrichq172 - 356Frost et al, 1999
caimanr430 - 1500Arocha-Piñango et al, 1982.
marine fishs220 - 280Pavlidis et al, 1999
asian elephantt412 - 510Gentry et al, 1996
vultureu< 20Weir-M et al, 2004
llamav140 - 300Morin et al, 1995

Table 1.

Fibrinogen (Fbg) concentration values in different vertebrates

A Chaetophractus villosus (n:20); a´ (n:24); b Balaenoptera borealis (n:1); c Iguana iguana (n:26); d (n:21); e Aquila adalberti (n:12); f Rana catesbeiana (n:302); g Tursiops truncatus (n:17); h Mus musculus; i Coturnix coturnix japonica ; j(n:80); k New Zealand rabbits (n:102); l (n:90); m foals (n:53); n Macaca fuscata (n:52); o Mirounga angustirostris (n:19); p Hydrochaeris hydrochaeris (n:2); q Struthio camelus (n:30); r Caiman crocodilus; s Dentex dentex; t Elephas maximus; u Coragyps atratus (n:2); v (n: 46 adult females); < less than.


SpeciesWBLT (hours)Author
human"/> 24Conard, 1976
lampreyandHawkey, 1971
black fishbndHawkey, 1971
common frogcndBlofield, 1965
leopard frogdnd
clawed toadend
domestic birdsndNiewiarowski & Latallo, 1959
dogfishf2 – 4Hawkey, 1971
Doolittle & Surgernor, 1962
japanese quailg"/> 72Belleville et al, 1982
armadillo"/> 72Tentoni et al, 2008

Table 2.

Whole blood lysis time (WBLT) values in different vertebrates

a Petromyzon marinus; b Tautoga onitis; c Rana temporaria; d Rana pipiens; e Xenopus lavéis; f Mustelus canis; g Coturnix coturnix japonica (n:10 adult males); nd: not detectable; > more than.


SpeciesWDLT (hours)Author
human> 20Fearnley et al, 1957
tiger froga> 48Srivastava et al, 1981
sealb5.9 – 8.5Lohman et al, 1998
dogc> 20Hedlin et al, 1972
ratd> 20
rabbite> 20
rabbitf> 30Hassett et al, 1986
catgndWelles et al, 1994
armadillo> 72Tentoni et al, 2008

Table 3.

Whole blood diluted lysis time (WDLT) values in different vertebrates

nd: not detectable; a Rana tigrina (n:6) measured at 4 and 37ºC; at 22ºC WDLT range was 31.5-45.3 hours; b Halichoerus grypus (n:2, both females), before immersion; c (n:3); d (n:6); e (n:4); f New Zealand male rabbits (n:4); g (n:15); > more than.


SpeciesELT (minutes)Author
human"/> 120Kowalski et al, 1959
armadilloa15.4 – 45.6Bermúdez, 2003
armadillo 24.5 - 93Tentoni et al, 2008
tiger frogbndSrivastava et al, 1981
japanese quailcndBelleville et al, 1982
dog21 - 109Hedlin et al, 1972
guinea pigd< 90Kaspareit et al, 1988
rabbit270 - 450Hassett et al, 1986
monkeye240Suzuki et al, 1977
vulturefndWeir-M et al, 2004
rat105 - 145Groza et al, 1988

Table 4.

Euglobulin lysis time (ELT) values in different vertebrates

a Chaetophractus villosus using citrated plasma (n:20, 10 females and 10 males); a´ using oxalated plasma; b Rana tigrina (n:6); c Coturnix coturnix japonica (n:10 young males); d Cavia porcellus (n:45); e Macaca fuscata; f Coragyps atratus (n:2); nd: not detectable; > more than; < less than.


SpeciesPlg (%)Author
human80 - 120Perkins, 1999
japanese quaila0Belleville et al, 1982
dog102 - 115 #Lanevschi et al, 1996b
dog3,2 - 4,4Karges et al, 1994
cat50 - 200O´Rourke et al, 1982
cat94 - 122Karges et al, 1994
rat6 - 14
guinea pigb0.4 – 6.1
rabbit2
rabbit147 - 217 #Marval et al, 1992
rabbit84 - 108 #Hassett et al, 1986
sheep0.7 – 1.5Karges et al, 1994
cow0
monkeyc24 - 39
monkeyd164 #Suzuki et al, 1977
capybarae0Leitâo et al, 2000
pig2.1 – 5.2Karges et al, 1994
pig0Hahn et al, 1996
horsef66.5 – 98.1Barton et al, 1998
whaleg112 #Saito et al, 1976
armadillo28 - 40Tentoni et al, 2008

Table 5.

Plasminogen (Plg) activity values in different vertebrates

Results are expressed as percent for Plg activity in relation to the calibration curve obtained with a pool of healthy humans platelets poor plasma, using a chromogenix assay after activation with SK.


a Coturnix coturnix japonica (n:10 young males); b Cavia porcellus; c Macaca fascicularis; d Macaca fuscata; e Hydrochaeris hydrochaeris, it was impossible to activate its Plg with 500 U/mL of SK; f neonatal foals, Plg calibration curve was performed using equine pooled plasma; g Balaenoptera borealis (n:1); # Plg measured using uPAas activator.


SpeciesFDP (μg/mL)Author
human< 10Amiral et al, 1990
dog< 5Boisvert et al, 2001
dog< 5Stokol, 2003
dog< 5Griffin et al, 2003
dog0 – 1.18Machida et al, 2010
dog< 10Herring & McMichael, 2012
cat< 10Herring & McMichael, 2012
horse5.5 – 10.9Barton et al, 1998
horse< 10Stokol et al, 2005
elephant seala0Gulland et al, 1996
dolphinb< 10Tibbs et al, 2005
cow< 5Irmak & Turgut, 2005
armadillo0 - 10Tentoni et al, 2008

Table 6.

Fibrin fibrinogen degradation products (FDP) concentration values in different mamals

A Mirounga angustirostris; b Tursiops truncatus (n: 12); < less than.


SpeciesDD (μg/mL)Author
human< 0.50Estève et al, 1996
dog0.08 – 0.39Stokol et al, 2000b
dog0.02 – 0.28Caldin et al, 2000
dog< 0.25Nelson, 2005
dog< 0.25Herring & McMichael, 2012
cat< 0.25Herring & McMichael, 2012
rat0.18Asakura et al, 2002
rat< 0.02Ravanant et al, 1995
hen< 0.02
rabbit< 0.02
sheep< 0.02
monkeya< 0.05
mouse< 0.02
mouse0Tsakiris et al, 1999
pig0Roussi et al, 1996
pig< 0.01Schöchl et al, 2011
horseb0.46 – 0.92Monreal et al, 2000
horse0 – 0.91Machida et al, 2010
horse< 0.50Stokol et al, 2005
ostrich0.25Frost et al, 1999
vulture"/> 1Weir-M et al, 2004
armadillondTentoni et al, 2008
dolphin< 0.50Tibbs et al, 2005

Table 7.

D Dimer (DD) concentration values in different vertebrates.

A Papio papio; b (n: 30); nd: not detectable; < less than; > more than.


SpeciesPAI-1 immunologic (ng/mL)Author
human4 – 43Declerck et al, 1988
mousea1.3 – 2.5Tsakiris et al, 1999
mouse1 – 2Matsuo et al, 2007
pig0Roussi et al, 1996
pigb5.6 – 9.0Schöchl et al, 2011
armadillo1.0 – 2.2Tentoni et al, 2008
rat3.9Nieuwenhuys et al, 1998

Table 8.

Immunological Plasminogen activator inhibitor type 1 (PAI-1) concentration in different mammals

A Mus musculus (n: 160); b measured with Porcine PAI-1 Activity Assay


SpeciesPAI-1functional (U/mL)Author
human< 10Van Cott & Laposata, 2001
cat0Welles, 1996
rabbit0.06 – 0.16Hassett et al, 1986
horse19.6 – 42.2Barton et al, 1998
armadillo24.8 – 37.7Tentoni et al, 2008
rat1.0Nobukata et al, 2000
rat4.9 – 7.4Emeis et al, 1992

Table 9.

Functional Plasminogen activator inhibitor type 1 (PAI-1) concentration in different mammals

Results are expressed as units of PAI-1 present in plasma in relation to the calibration curve obtained with a commercial standard when using immunological test; < less than


Speciesα2PI (%)Author
Human70 - 130Teger-Nilsson et al, 1977
japanese quaila65 - 85Belleville et al, 1982
ostrichb115.6Frost et al, 1999
hen109.4
snakec10
Sheep68.8
whaled50Saito et al, 1976
dog96 - 103Lanevschi et al, 1996b
dog92 - 94Karges et al, 1994
cat70 - 86
rat118 - 138
guinea pige91 - 101
sheep90 - 109
pig63 - 104
monkeyf82 - 99
rabbit91 - 108
rabbit66 - 92Hassett et al, 1986
pig87 - 127Hahn et al, 1996
rat120Nobukata et al, 2000
horse154 - 240Barton et al, 1998
cow80 - 94Daugschies et al, 1998
armadillo72 - 101Tentoni et al, 2008

Table 10.

alpha2 plasmin inhibitor activity (α2PI) in different vertebrates

Results are expressed as percent for α2PI activity in relation to the calibration curve obtained with a pool of healthy humans platelets poor plasma, using a chromogenix assay after activation with an excess of Plm.


a Coturnix coturnix japonica; b Struthio camelus; c Bitis arietans; d Balaenoptera borealis (n:1); e Cavia porcellus; f Macaca fascicularis.


5. Conclusions

The information summarized in this chapter helps the choice of appropriate animal experimental models for studying fibrinolysis and the correct extrapolation of animal results toward humans. Previous work from our laboratory, has identified the choice of the armadillo as an animal model because it adapts well to captivity conditions, endures repeated blood sampling, shows excellent tolerance to cardiac puncture and recovers quickly from anaesthesia (Bermúdez et al. 2004; Casanave et al. 2005; 2006). Chaetophractus villosus has a hypercoagulable and hypofibrinolytic profile (Tentoni et al., 2008) as pigs, which are frequently used as an animal model in human research. Finally, the study of animals’ haemostatic mechanisms is important in the field of zoology, for the advancement of scientific knowledge and in biomedicine, helping to select a suitable experimental animal model.

Acknowledgments

This work was supported by Secretaría General de Ciencia y Tecnología, Universidad Nacional del Sur (SGCyT-UNS), Project 24/B152 and by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), PICTR 74/02, Argentina.

© 2014 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Emma Beatriz Casanave and Juan Tentoni (May 7th 2014). Comparative Fibrinolysis, Fibrinolysis and Thrombolysis, Krasimir Kolev, IntechOpen, DOI: 10.5772/57359. Available from:

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