Comparative Fibrinolysis

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 (Staf‐ ford 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); streptoki‐ nase (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 activata‐ ble 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).


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

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;).
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(Sawyer , 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:

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.

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).
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).
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 (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.    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. 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. A Mirounga angustirostris; b Tursiops truncatus (n: 12); < less than. A Papio papio; b (n: 30); nd: not detectable; < less than; > more than. 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

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;. 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.