Biochemical Characteristics And Physiological Functions Of Earthworm Fibrinolytic Isozyme III-1 & II From Eisenia Fetida

Thrombosis is one of the most common and devastating diseases, especially when happen in brain and heart. It could induce Vascular Dementia (VD). And it also led to the damage of brain vessels, Leukoaraiosis and the Alzheimer-like Disease (AD) histlogical change in brain. VD and AD are the two highest incidences of dementia diseases the entire world. Fibrinolytic enzymes are effectively in treating thrombosis, such as UK, SK, rt-PA, and SAK, have been studied as thrombolytic agents.Earthworm has been used as a traditional medicine to treat clotting diseases in eastern countries for centuries. In Compendium of Materia Medica edited by a Chinese traditional medical doctor Shizhen Li (1518-1593), earthworm was described as a kind of medicine prescribed for antipyretic and diuretic purposes in clinic. The dried earthworm prepared into powder, with a medicinal name: "Earth-dragon". So far, such a preparation is still used as a traditional folk remedy in China, Japan, Korea and other Asian countries. In 1878, Fredericq observed that an enzyme secreted from the alimentary tract of earthworm. In 1920, Keilin isolated a kind of earthworm and found several proteases contained in the worm that could degrade casein, gelatine and albumin. Tens years later, Mihara et al. separated a group of fibrinolytic enzymes (lumbrokinase, LK) from the extract of an earthworm Lumbricus rubellus. However, this group of enzymes is now commonly called earthworm fibrinolytic enzymes (EFEs). Recently pharmacological and clinical studies have focused on improving the efficacy and tolerability of EFE. If EFE were made into injection agent, it would be meaningful to treat AMI and other acute diseases involved in thrombosis.A fibrinolytic enzyme, EFE-III-1, is isolated and purified from earthworm (Eisenia fetida) by affinity chromotography. The molecular weight of EFE-III-1 estimated by SDS-PAGE and MS analysis is 34,000 and 29,557 Da, respectively.The N-terminal amino acid sequence is N-lle-Val-Gly-Gly-lle-Glu-Ala-Arg-, and the identity of EFE-lll-1 showed high homolog to those of trypsin-like enzymes. In substrates specificity and inhibition assays, seven substrates (BAEE, ATEE, Chromozyme -TH, -TRY, -ELA and -U) and four inhibitors (TLCK, TPCK, SBTI and Elastinal) were used to detect its enzymatic parameters. The Michaelis-Menten constant (Km) of EFE-lll-1 is much lower measured by BAEE (~1.7*1O"5 mol/L) than those for the Chromozym TH (~6.O10~5 mol/L), Chromozym TRY (-3.91 *10~5 mol/L) substrates, respectively. It indicates that the active site in EFE-lll-1 is more similar to the trypsin's substrate. Meanwhile, EFE-lll-1 has no activity using ATEE and Chromozym ELA as substrates.On the other hand, EFE-lll-1 is inhibited most strongly by soybean trypsin inhibitor (SBTI, a specific inhibitor of trypsin,), and weakly inhibited by TLCK, TPCK and elastinal, suggesting that EFE-III has a strong trypsin-like activity. It indicates further that EFE-lll-1 is a trypsin-like serine protease. EFE-lll-1 has a very strong fibrinolytic activity and maximum reactivity from pH 8.5-10 and 40-55°C measured by different substrates, it also shows EFE-lll-1 is a heat-resist and stable protease.Three methods are applied to assay EFE: light scattering, chromogenic substrate, and fibrin plate, and the activity of EFE-lll-1 is ~1.5><105 U/mg determined by light scattering method using standard LK as control. Assay of fibrin plate, the activity is 550 U/mg and 6.6x104 U/mg using standard UK and LK as control. These assays indicate that EFE-lll-1 has a strong fibrinolytic activity, and a strong amidolytic activity for various chromogenic substrates. EFE-lll-1 has been showed by fibrin plate that it could dissolve clotting through two pathways: (1) directly via hydrolyzing fibrin and fibriongen;and (2) indirectly via activating plasminogen to plasmin, then has the similar procedure to urokinase (UK).To investigate the mechanism of EFE-lll-1 involving anticoagulant steps, we use fibrinogen, plasminogen (PLg) and prothrombin, three important impact factors in blood, incubated with EFE-lll-1 to detect the degraded fragments. EFE-lll-1 can cleave all the three chains of fibrinogen, the Aa, B|3 and y chains. The cleavage sites are mainly at K53^K54 and K133^D134 in the Bp chain;K62M63 and K^S86 in the y chain. And EFE-lll-1 degrades fibrinogen mainly in the C-terminal region. And then, EFE-lll-1 was immobilized onto CDI (1,1'-Carboryl-diimidazole) activated Sepharose CL-6B, it is found that the immobilized EFE-lll-1 degrades not only the Aa chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in theN-terminal region at the R19nIA/2o site.Human prothrombin was used to detect the characteristics of EFE-lll-1. Immobolized EFE-lll-1 degrades prothrombin at six hydrolytic sites: R|D58, RJT275 R|T288, R|N397, R|T159 and K|G478. The cleavage sites are alkaline amino acid residues (Lys-X, Arg-X) in C terminal, which shows the trypsin-like protease character of EFE-lll-1.EFE-lll-1 also degrades PLg at four hydrolytic sites: Lys77Mrg78, Arg 342^1^343, Ala444Mla445 and Arg557^lle558. The site Arg557-lle558 is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala444-Ala445 released mini-plasmin with secondary activity to hydrolyze fibrin. The results show that EFE-lll-1 has a broad specificity. This indicates that EFE-II functions in the anticoagulation system via several steps related to both coagulation and anticoagulation pathways: hydrolyzing fibrinogen/fibrin and activating PLg to produce plasmin and mini-plasmin.The relationship between the activity and conformation of EFE-lll-1 is investigated by using guanidine hydrochloride (GuHCI) denaturation. The changes of intrinsic fluorescence and activity are similar to those of trypsin as control. Kl quenching of the intrinsic fluorescence of EFE-lll-1 shows that unfolding of the molecule becomes distinguishable gradually. The results suggest that Trp residues may be near or at the exterior of the molecule.Meanwhile, we compared EFE-lll-1 with EFE-II obtained previously in this lab. The results show that EFE-lll-1 and EFE-II are the two different proteases, that is to say, EFE-lll-1 is a trypsin-like protease, and however EFE-II is both a trypsin-and an elastase-like protease.EFE-II recognized and reacted with the three substrates with different values, among which the Km of Chromozym ELA is the highest, about 27 times higher than that of Chromozym TH. This suggests that EFE-II acts strongly as a thrombin- and trypsin-like serine protease, as well as having relatively weak elastase-like function. . The inhibition of the enzyme by SBTI in the presence of Chromozym ELA (l50 = 5 nM) is less than in the presence of either Chromozym TH or Chromozym TRY. This suggests that EFE-II is a trypsin-like protease with low elastase activity. Nevertheless, the results show above all involve used small substrates, each with a single hydrolytic site between amino acid residue and 4-nitraniline. It is of interest whether EFE-II can recognize and cleave bothtrypsin- and elastase-like hydrolytic sites in a macromolecular protein, such as PLg orfibrinogen.EFE-II can cleave all the three chains of fibrinogen, the Aa, Bp and y chains. The cleavage sites are mainly at A^A49 and Y^Si43 in the Bp chain;A4/T83 in the y chain. And EFE-II degrades Aa chain of fibrinogen mainly in RnU/20 and V^E22, and most of the cleavage sites are located in the C-terminal region.EFE-II also degrades PLg at four hydrolytic sites: Lys77Mrg78, Arg342^Met343, Ala444Mla445 and Arg557^lle558. The site Arg557-lle558 is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala444-Ala445 released mini-plasmin with secondary activity to hydrolyze fibrin.Human a2-macroglobulin (a2M) inhibits most proteases;the effect of a2M has not been investigated on earthworm fibrinolytic enzymes (EFEs). SDS-PAGE showed that incubation of EFE-II with a2M resulted in formation of a subunit (-90 kDa), followed by formation of a high molecular weight complex (180-190 kDa).Resin coupled with m-aminophenylbornic acid was used to isolate a glycosylated component from homogenate of earthworm. The fraction showed a single band on SDS-PAGE with a molecular weight of 34,193 Da determined by mass spectroscopy. The N-terminal region is AQVCCPDI..., different from those of earthworm fibrinolytic enzymes reported previously. This glycosylated component showed a remarkable activity on digesting both Chromozym TH and fibrin, suggesting that it is a novel fibrinolytic enzyme.This study can provide more information for further research in choosing an earthworm fibrinolytic isozyme with higher fibrinolytic activity for the effective clinical medical agent in treating thrombosis. The results suggest that EFE-lll-1 is the isozyme with clearer enzymatic characteristics, higher fibrinolytic activity, stronger antigenic specificity and more stable molecular conformation. However, for the medical and clinical application, the relationship between its biochemical properties and physiological functions still need further research.