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International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
57
Lumbrokinase – A Potent and Stable Fibrin–Specific Plasminogen
Activator
Mahendra Kumar Verma1, KK Pulicherla2
1Lecturer, Department of Biotechnology
2Professor & Head, Department of Biotechnology
RVR & J C College of Engineering, Chowdavaram, Guntur-522 019, Andhra
Pradesh, India
mahendra.sonal1983@gmail.com, pkkanth2006@gmail.com
Abstract
Cardio and cerebrocascular disorders lead to about 26 million deaths every year around
the world. Cardio and cerebrovaccular disorders have not only high mortality rate across
the globe but also leading to subsequent complication such as thrombolysis could favorably
influence the outcome of such life-threatening disorder as myocardial infarction,
cerebrovascular thrombosis, and venous thromboembolism. Our healthy system is capable to
overcome to these consequences but when there is imbalance of defensive and aggressive
factor in our system result come as blood clot in systemic circulation. Now role of
thrombolytic agents come in picture as artificial plasminogen activators that convert
plasminogen, an inactive form of plasmin to dissolve the clot by converting inactive
plasminogen in active plasmin. Plasmin dissolves the fibrin blood clot, but may also degrade
normal components of the hemostatic system which can further create another life
threatening consequence and death also so there is always need of such an agent who
specifically dissolved clot which are in circulation but not others.
Keywords: Thromboembolic disorders, plasminogen activators, Lumbrokinase,
Earthworm Fibrinolytic Enzyme
1. Introduction
A blood clot (thrombus) develops in the circulatory system can cause vascular blockage
leading to serious consequences including death. According to World Health Organization
(WHO) report year 2008-2010 numbers of deaths due to cardio and cerbrovascular disorder is
comparatively large than any other disorder or disease. A healthy homeostatic system
suppresses the development of blood clots in normal circulation, but reacts extensively in the
event of vascular injury to prevent blood loss. [1] The consequences of a failed hemostasis
include stroke, pulmonary embolism, deep vein thrombosis and acute myocardial infarction.
Pathologies involving a failure of hemostasis and the development of clot which requires
clinical intervention consisting of administration of artificial thrombolytic agents.
Lumbrokinase is one such agent. Other thrombolytic or fibrinolytic agents include
streptokinase, urokinase and the tissue type plasminogen activator (t-PA). [2]
A progressive research is going on to find out effective thrombolytic drugs with minimum
risk hence numerous trails have been conducted with the various thrombolytic agents but
could not active to target. Reason most of them get failed in certain aspect as specificity, side
effect or cost factor. Hence there is an urgent need of such an agent which can full fill all
International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
58
above criteria. A lot of research is going on for the hunt for potent thrombolytic agent. As a
result researcher has found lumbrokinase a potent fibrin specific thrombolytic agent to cure
the life threatening cardio and cerebrovascular disorder. Recombinant lumborkinase
formulations are commercially available in China and Korea since long time as capsule and in
other dosage form for the treatment of cardiac thromboembolic disorder [3]. The use of
lumborkinase in thrombolytic therapy holds great promise for becoming an important
therapeutic adjunct in the treatment of acute vascular occlusions, but such therapy has not
reached the stage for general clinical use at global level. Till date proper method of
administering thrombolytic agents has not yet been determined, and the processes that bring
about lysis of clots are incompletely understood [4]
This review will focus on the biochemical and thrombolytic properties of Lumborkinase,
an eukaryotic protein exist in six isoforms (isoenzyme) form tissues and intestinal fluid of
earthworm with the fibrin specific plasminogen activator that would appear to have potential
thrombolytic agent for treatment of cardio and cerebrovaccular disorder.
Blood Clot Formation
Blood Clot formation is a dynamic process which leads to clot formation and dissolution
simultaneously in a controlled fashion. Once clot formed has to be dissolve to make
homeostatic balance. In the normal blood circulation blood clotting factor runs in inactive
form and form clot when it needed and activates clot dissolving factor to cleanup.
Fig: 1 A Cascade of Blood Plasma Protein Govern Clot Formation
The blood clot formation is defensive mechanism of our system which regulated by
various factors and by their cooperative work a blood clot is going to form. There are two
mechanism proposed earlier to describe entire mechanism of clot formation, Intrinsic and
Extrinsic which involves various blood clotting factor which run in a cascade way to achieve
the process. In short 13 factors which generally circulate in inactive form in blood get
activated with proper signal and activation of these factors is not random, work in cascade
way i.e. one is going to activate other in systemic way and finally result in soluble fibrinogen
into insoluble fibrin. After word activation of fibrin dissolving factor which cleanup the blood
clot. [5] Hence a healthy system maintains a homeostatic balance in between the defensive
and aggressive factor and their availability in the systemic circulation.
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Vol. 3, No. 2, June, 2011
59
2. Thrombolysis or Fibrinolysis
The fibrinolytic enzyme, plasmin, like most other proteases, is capable of acting upon a
number of different protein substrates in addition to its physiologic substrate fibrin like
proteinaceous clotting-factors like fibrinogen, proaccelerin, antihemophilic factor,
prothrombin and Christmas factor, and certain other blood proteins. But in the healthy blood
circulation plasmin exist with its inhibitors antiplasmin as Plasmin- Antiplasmin complex
which regulates the plasmin activity. Antiplasmin is 30 fold higher than plasmin in plasma
concentration act as defensive factor for breakdown of other plasma protein [6]
The complete physiology of the fibrin–clot formation is relatively well understood. A
blood clot or thrombus is consists of blood cells occluded in a matrix of the protein fibrin.
Thrombolysis or fibrinolysis is enzyme regulated process for dissolution of clot. In
mammalian circulation, the enzyme responsible for the fibrinolysis is plasmin which is a
trypsin-like serine protease [7]. In the presence of activator the fibrinolytically active plasmin
is produced from the inactive protein plasminogen which is present in systemic circulation.
The biochemical conversion of the inactive plasminogen to fibrinolytic plasmin involves a
limited proteolytic cleavage which is mediated by the various plasminogen activators [8].
Generally two plasminogen activators that occur naturally in blood are the tissue type (t-
PA) and the urokinase type (u-PA). The fibrinolytic activity in circulation is regulated by
inhibitors of plasminogen activators (e.g., plasminogen activator inhibitor-1, PAI-1, a fast-
acting inhibitor of t-PA and u-PA) and plasmin (e.g., a1-antiplasmin, a2 macroglobulin.
Recombinant forms of normal human plasminogen activators t-PA and u-PA are used in
clinical intervention. Another commonly used plasminogen activator is Lumbrokinase (l-PA),
an eukaryotic protein form earthworm that is having fibrin specific thrombolysis too [9]
Apart from Lumbrokinase, t-PA and u-PA do not have a direct fibrinolytic activity and
their therapeutic action is via the activation of blood plasminogen to the clot dissolving
plasmin. Thus, the plasminogen activating action of Lumbrokinase is fundamentally different
from the proteolytic activation brought about by t-PA and u-PA. So plasmin is always present
in systemic circulation but in the complex form with its inhibitor Antiplasmin. So it is
antiplasmin which protect other blood plasma protein form the degradation by plasmin [10]
Fig: 2 A Schematic Representation of Role of Antiplasmin (Plasmin Inhibitor) in
Clot Dissolution Mechanism.
International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
60
Current Treatment Regime for Thrombolytic Therapy:
Present scenario of thrombolytic therapy driven by recombinant Plasminogen Activators
or plasmin specific thromolyitc agents. Under the current clinical application streptokinase,
APSAC (Anisoylated plasminogen streptokinase activator complex- APSAC), Urokinase,
scu-PA. Tissue plasminogen activator has been used in last two decades are as t- PA, rt- PA
(altepalse), r- PA (reteplase), TNK-rt-PA. Current with the new advancement few third
generation agents are as n- PA (Lanoteplase), bat- PA (Desmodus rotundus), Bifunctional
molecules conjugating t-PA with monoclonal antibody [11].
Thrombolytic Agents Approved by the Food and Drug Administration
Following thrombolyitc agents has been approved by FDA for the thromolytic therapy
and are in clinical use with the better therapy.
· Streptokinase
· Urokinase
· Altepalse
· APSAC (Anisoylated plasminogen streptokinase activator complex- APSAC)
· Reteplase
· TNKase
All of listed above are the plasminogen activators, activating circulating inactive
plasminogen into active plasmin which degrade the fibrin clot. All of above listed drug
molecules are in clinical use for the thrombolytic therapy with numerous complication and
side effects. Reason for thrombolytic therapy complication most of these molecule are not
fibrin specific and with fibrin they also degrades other circulating plasma protein which
actually a healthy system needed for normal homeostasis. So still there is hunt of a drug
which will be fibrin specific and possibly Lumbrokinase will be ones of them as it activates
tissue plasminogen activators and also having fibrin specific thrombolysis.
Recombinant forms of normal human plasminogen activators t-PA and u-PA are used in
clinical intervention. Both t-PA and u-PA are trypsin-like serine proteases which activate
plasminogen directly and having the respective molecular weights of 70 and 55 kDa. Both
tPA and uPA are glycoprotein’s. u-PA is produced in the kidneys and secreted into the urine
[12] whereas tPA is produced by the vascular endothelial cells [13]. Tissue Plasminogen
Activator (t-PA) is the choice for therapeutic use is obtained mainly from cultures of
recombinant animal cells [14].
Because of its high affinity for the fibrin clot, t-PA activates the clot bound plasminogen
100-fold more effectively than the circulatory plasminogen. Consequently, t-PA is a poor
activator of circulatory plasminogen relative to the other available plasminogen activators.
But the cost point view t-PA is highly costly as its cloning and expression needs animal cell
line as being a eukaryotic protein also larger size is not possible in yeast and bacterial host
system due to inability to post translational modification of eukaryotic protein. Due to lack of
recognized plasminogen activators, attempts are underway to develop improved recombinant
variants of these compounds to achive better results [15].
Hunt for Ideal Thrombolytic Agent
There are numerous drugs and protein which were used for the treatment of cardio and
cerebrovascular disorder but results were not up to mark. So researcher are always in the hunt
of such an agent which can provide the medication up to level and also satisfy other
International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
61
parameter like cost factor. Here are some features listed below which supposed to
characteristic of thrombolytic agent. Characteristics of the Ideal Thrombolytic Agent:
· Rapid reperfusion.
· Easy Administration.
· Fibrin specific.
· Slow renal clearance
· Chemically stable
· Less first bypass
· Low incidence of systemic bleeding.
· Low incidence of intracranial hemorrhage.
· Resistant to plasminogen activator inhibitor-1 (PAI-1).
· Low reocclusion rate
· No effect on blood pressure.
· No antigenicity
· Reasonable cost
Most of thrombolytic agents what we are using now for the thrombolytic therapy are
unable to show all the characteristic. Majority of them are having limitation either for
substrate specificity and same time also dissolve necessary blot cloting protein and factor
which are essential for normal homeostasis. Hence it is triggers us to search the novel
molecule or refinement to the existing one for ideal drug molecule. Hence a lot of progressive
research is going on achieve the goal.
3. Lumborkinase
Historical Overview:
Earthworms are the key representatives of the soil fauna and are essential in maintaining
soil fertility through their burrowing, ingestion and excretion activities [16]. Around over than
8000 described species worldwide, existing everywhere but in Polar and arid climates [17].
They are increasingly recognized as indicators of agro ecosystem health and eco toxicological
sentinel species because they are constantly exposed to contaminants in soil. The following
earthworm species (e.g. Eisenia andrei, Eisenia fetida and Lumbricus terrestris) widely used
in standardized acute and reproduction toxicity tests belong to the Lumbricidae family
(phylum, Annelida; class, Clitellata; subclass Oligochaeta; order, Haplotaxida; super family,
Lumbricoidea; family, Lumbricidae.
The fibrinolytic enzymes in the earthworm, Lumbricus rubellus, were extracted and
named lumbrokinase a collective name for six fibrinolytic iso-enzyme proteins having
molecular weights of 25 to 32 KDa. [18] In the year 1992 a study on the crude extract of
earthworm shown to have potent thrombolytic effect. Lumbrokinase exist as is iso-enzyme in
the intestine and tissue fluid and intestinal fluid of earthworm. The reason why earthworm is
having such a potent protease as earthworm feed on the debris of plant and organic matter so
digest all these probably it produce Lumbrokinase a serine protease. As research progress LK
was isolated from other species of earthworm also and was having fibrinolytic activity.
Several investigators purified and further characterized the fibrinolytic enzymes in L. rubellus
[19] and found the hydrolysis of the plasmogen-rich fibrin and plasmogen-free fibrin. The
fibrinolytic enzymes dissolve blood fibrin clots, which are important for clinical application
as chemotherapeutic agents [20].
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Vol. 3, No. 2, June, 2011
62
Isolation of Lumbokinase:
Lumbrokinase a collective name for six fibrinolytic iso-enzyme proteins having
molecular weights of 25 to 32 kDa. F1 – F6. The fibrinolytic activities of the all six isoforms
was found in the following order F6 > F2 > F5 > F3 > F1 > F4. (23) Hwan Cho et al 2003 Six
isoform of Lumbrokinase (LrPI- 0, LrP-I-1, LrP-I-2, LrP-II, LrP-III-1, and LrP-III-2) of
fibrinolytic enzymes were isolated from L. rubellus [3, 4, 23]. The molecular masses of the
isozymes measured by ion-spray mass spectrometry are 23,013; 24,196; 24,220; 24,664;
29,667; and 29,662, respectively. Usually in their peptide chains asparagine and aspartic acid
residues are comparative less lysine. May be this is the reason as Lumbrokinase have greater
stability in organic and inorganic solvent. They have a wide functional acidic range (pH 1.0–
11.0) and do not inactivate until 60◦C. The range of enzyme activity (LrP-II and LrP-III-1) is
maximally exhibited around pH 9.0 at 50◦C [21].
In the year 1988, Zhou and coworkers isolated least seven components with fibrinolytic
activity from earthworm E. fetida and found they are stable at pH 5.0– 9.0 and denaturated
below pH 2.6. The enzyme a member of serine protease and its molecule weight 45kDa. The
two constituting subunits (26k Da and 18kDa) with different fibrinolytic activities are bound
by hydrophobic interaction. Further isolation listed eight fibrinolytic enzymes as (Ef P-0-1,
Ef P-0-2, Ef P-I-1, Ef PI- 2, Ef P-II-1, Ef P-II-2, Ef P-III-1, and Ef P-III-2) through a stepwise-
purification procedure in 2007 [22].
Mechanism of Action:
The Lumborkinase (LK) group of proteolytic enzymes, extracted from the researched
species of earthworm, includes plasminogen activator and fibrin specific serine protease. The
mechanism to activate plasminogen in LK is similar to tissue plasminogen activator (t-PA)
from other sources, which makes it possible to show the thrombolytic activity only in the
presence of fibrin. The mechanisms of action of Lumborkinase include participation in the
activation of plasminogen, and direct activity on fibrin itself. Lumbrokinase primarily
proteolyzes fibrinogen and fibrin, hardly hydrolyzing other plasma proteins including
plasminogen and albumin. The enzymes lumbrokinase have very strong fibrinolytic activity,
are stable in a wide pH range, and show great stability against thermal inactivation and
degradation. Lumbrokinase are alkaline trypsin-like proteases that are greater than trypsins in
their stability and tolerance to organic solvents.
Fig: 3 Diagrammatic View of Lumbrokinase Mediated Thrombolysis. Hence
Lumbrokinase is not only plasminogen activators but also fibrin specific
thrombolytic agent.
International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
63
Lumbrokinase shows double thrombolytic effect and that is specific in one way it
activates plasminogen activator and start the normal cascade of our own system to dissolve
the blood clot and also it is having enormous affinity towards fibrin and specifically dissolved
clot.
Vilhardt and Lundin investigated whether earthworm fibrinolytic enzyme III-1 (EFE-III-
1) isolated from Lumbricus rubellus is capable of transporting into blood through intestinal
epithelium and keeping its biological function in circulation, they raised an antibody against
EFE-III-1. The immunological results showed that 10-15% of complete EFE-III-1 was
absorbed by the gastrointestinal epithelia [23]. The further evidence for intact absorption of
enzyme was concluded by immunohistochemistry approach and the presence in the intestinal
epithelial cells. The experiments were conducted on animal model, which concluded the
immunoreactive intact EFE-III-1 was found in serum or plasma after intraperitoneal injection
of rats. During the following study it was found 10% of the intact enzyme could transport
through the intestinal epithelium. The further confirmation for the maximum remaining
activity in blood was assayed around 60 min after the intraperitoneal injection [24].
Gene Structure of Lumbrokinase
As per the data form the GenBank there are eight cDNA clones of lumbrokinase are
available which are as (GenBank Accession Nos.; AY438622; U25644, AY178854;
AY187629; AF304199; AF433650; U25648; and U25643), the coding region of the
lumbrokinase cDNA is 852 bp in length and encode 283 amino acids, of which the first 36
amino acids consist of a signal peptide and the last 247 amino acids form the mature protein.
(25) The nucleotide sequence of each cDNA clone was analyzed and it was found that there
are 13 codons containing the “CG” motifs in the whole sequence, which are relatively rare for
mammalians and may not be efficiently translated after being transformed into mammalian
cells or tissue [26]. The c DNA of Eisenia foetida EFE-3 contained 859 nucleotides, with an
open reading frame starting from 112 to 853, encoding a polypeptide of 247 amino acid
residues [27] The extracellular enzyme lumbrokinase is isolated by various species of
earthworm but are having same mechanism for fibrinolytic action indirectly by activating the
circulatory plasminogen and direct on fibrin.
Protein Structure of Lumbrokinase
The protein sequence of various species of earthworm has been analyzed in order to have
complete proteomics information. The protein sequences of the isozymes from L. rubellus
and E. fetida have a lot of identical residues. The proteins have distinct predicted secondary
structures, for example, β-sheet, α-helix, turn, and coil. The sequence of a isoform of
lumbrokinase called as Earthworm fibrinolytic protease II (Ef P-II (EFEa)) is highly similar to
some related serine proteases with known structures [28-30] or other earthworm serine
proteases [31] the catalytic characterization of the earthworm protease is influenced directly
by their tertiary structures. The NMR and X ray study shows that Ef P-III- 1 (EFE-b) is a
trypsin-like protease with two chains (an N terminal, pyroglutamated light chain and an N-
glycosylated heavy chain) [32]. The structural features (Figure 4) probably endow Ef P-III-1
with high level of stability in resistance to heat, organic solvents, and proteases [33]. The
another isoform of Lumbrokianse Ef P-II is not only a chymotrypsin-like serine protease but
also has an essential S1 pocket of elastase.
International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
64
Recombinant Lumbrokinase
The isolation and purification of native lumbrokinase is form earthworm is tedious,
multistep and hence time consuming and even there will be chance for impurities of other
protein. So researcher has started cloning and expression of lumbrokinase gene into
prokaryote and eukaryote system form the different species of earthworm and majority of
them got success. In the year 2003 Hwan Cho, Eui Sung Choi and Hyung Hoan Lee
successfully cloned the lumbrokinase gene form Lumbricus rubellus into the bacterial system.
The complete c-DNA sequence of the lumbrokinase gene form Lumbricus rubellus was
amplified using an mRNA template, sequenced and expressed in E. coli cells [34].
The most potent isoform of Lumbrokinase for fibrinolysis F6 protease gene consisted of
pro- and mature sequences by gene sequence analysis, and the protease was translated and
modified into active mature polypeptide by N-terminal amino acid sequence analysis of the
F6 protease. The gene structure of F6 protease consisted of the 44 residues from methionine-1
to lysine-44 which is a pro-region and the mature polypeptide which is 239 amino acid
residues and one stop codon; 720 bp) which started from isoleucine-45 and continued to the
terminal residue. The F6 protease gene was expressed in E. coli system as a pro-mature
sequence and mature sequence produced inclusion bodies. After the expression into E coli
system these inclusion bodies were given orally to rats, where thrombus was already
generated a drastic reduction in size approximately 60% versus controls. Further these
inclusion bodies have shown hemolytic activity when solubilized in pepsin or trypsin
solutions. Consequently it was concluded the F6 protease has hemolytic activity, and that it is
composed of pro- and mature regions. [35]
Zhe-rong Xu and their team constructed and expressed the mature peptide of LK PI239 in
Escherichia coli; we amplified and optimized the gene of LK which was then cloned into the
prokaryotic expression vector pET-22b. The engineered construct LK (rLK) was expressed as
inclusion bodies and we have developed a purification process of rLK from these inclusion
bodies. Further urea concentration strategy was applied to the rLK renaturation process in
order to have mature protein. The purified and renatured rLK was successfully analyzed in
the animal model and has shown sufficient thromolysis, which again confirms LK as
therapeutic agent for thrombotic-associated diseases. [37].
Being a eukaryotic protein it has always problem in the expression in the prokaryotic
system as the prokaryotic system lacks post translational modification and protein folding
machinery. In this order many Lumbrokinase genes have been cloned, expressed and
sequenced but the expression of gene has not achieved up to optima due unknown reason or
codon bias. The coding region of the mature lumbrokinase c-DNA is 852 bp in length and
which encodes 283 amino acids, of which the first 36 amino acids consist of a signal peptide
(pro-peptide) and further last 247 amino acids form the mature protein. The sequence of each
cDNA clone was completely analyzed and was found that there are 13 codons containing the
“CG” motifs in the whole sequence, which are relatively rare for mammalians and may not be
efficiently translated after being transformed into mammalian cells or tissue. Hence those
codon were optimized in order to have optimum expression [38].
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The Position and Optimized Codon for Better Expression of Lumbrokinase:
S. No.
Codon No. Position
Original Codon
Optimized Codon
Amino Acids
1.
8
TCG
TCA
SER
2.
11
GCG
GCA
ALA
3.
57
CCG
CCA
PRO
4.
63
CGA
AGA
ARG
5.
80
CGT
AGT
ARG
6.
109
GCG
GCT
ALA
7.
113
CGT
AGG
ARG
8.
166
CGT
AGA
ARG
9.
188
CGA
AGA
ARG
10.
191
ACG
ACT
THR
11.
216
GCG
GCT
ALA
12.
217
TCG
TCA
SER
13.
267
CGC
AGG
ARG
To achieve better expression and folding of mature Lumbrokinase protein researcher has
started cloning and expression in the eukaryotic system. During the lumbrokinase PI239 is
expression in Escherichia coli, it possesses an extra cysteine residue and is produced as an
inclusion body. Hence a renaturation procedure is necessary to obtain a mature protein. In
addition, because E. coli is a prokaryote system it is unable to perform many eukaryotic
posttranslational modifications such as proteolytic processing, folding, and glycosylation etc.
The consequence the recombinant lumbrokinase expressed into E. coli system has low
enzymatic activity even after the denaturation and renaturation procedure. As a result there is
need of an eukaryotic system where expression and posttranslational modification will be
possible. (39) As a eukaryotic system Pichia pastoris has been developed into a highly
successful system for the expression of heterologous genes. Majority of fibrinolytic enzymes,
including streptokinase and urokinase plasminogen activator, have been expressed in P.
pastoris. Most of them use GS115 as the expression host and pPIC9K as the expression
vector. Recently, an effective system for expression of lumbrokinase (F-III-2) was developed
by using P. pastoris GS115 as the host strain and pPICZ_-A as the expression vector pPICZ_-
A is easier than pPIC9K to manipulate, and selection for the vector in both P. pastoris and E.
coli is based a single small, dominant selectable marker that confers Zeocin resistance [40].
With the successfully complete set of processes for the screening, expression, and
fermentation of lumbrokinase in yeast was established. Further with the high density
fermentation of engineered yeasts was performed for the first time high density fermentation
of yeasts engineered to express lumbrokinase was performed, and a preliminary investigated
of the conditions needed for recombinant lumbrokinase expression by high density
fermentation was conducted.
4. Recent Advancement of Lumbrokinase:
Crystallization
Even the complete three Dimensional structural information of lumbrokinase is not
available. In order to have the information of lumbrokinase protein molecule researcher has
started crystallization of lumbrokinase. Hence the initial crystallization studies for
lumbrokinase were carried out using the hanging-drop vapour-diffusion method following the
sparse-matrix approach (44) (Jancarik & Kim, 1991). Microcrystals were obtained in a system
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66
containing 1.0 M (NH4)2SO4 as precipitant, 2.5% PEG 400 as additive and 0.05 M HEPES
buffer pH 7.2 at room temperature. After enormous optimized crystallization conditions, the
temperature was adjusted to 288 K and the initial drop solution was adjusted to contain 5 mg
mlÿ1 protein, 0.6 M (NH4)2SO4, 2.5%(v/v) PEG 400 and 0.05 M MOPS buffer pH 7.2, with
the reservoir solution containing 2.0 M (NH4)2SO4, 5.0%(v/v) PEG 400 and 0.10 M MOPS
buffer pH 7.2 [41].
F0F1-ATPase Micro- mixer
To archive the maximum enzyme activity and efficiency researcher has invent new
technology for better utilization of lumbrokinase for the treatment of cerebro and
cardiovascular disorders. F0F1-ATPase is a key enzyme in the biological world and one of
the most ubiquitous proteins in nature . The Enzyme ATP synthase an universal enzyme that
synthesizes ATP, the fuel that powers most of the cellular processes. The enzyme consists of
two parts; the F1 motor generates a mechanical torque using the hydrolysis energy of ATP
and the F0 motor generates a rotary torque in the opposite direction using a transmembrane
proton motive force. In the following design these two motors are connected by flexible
coupling, and each motor can be reversed: the F0 motor can drive the F1 motor to synthesize
ATP, and the F1 motor can drive the F0 motor to pump protons Ning Tao et al have
developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer
was constructed from arrays of chromatophore-embedded F0F1-ATPases, where the d-free
F1 part acted as a rotator to mix solutions, and the F0 part was driven by light. The
microscopic studies indicated that the micro-mixer did not touch directly on the fibrin labeled
with FITC. The force generated by the F0F1-ATPases motor directs drug movement in the
solution and accelerated the fibrinolysis process. The study strongly suggests that the micro-
mixers generated a nanomechanical force which accelerated the fibrinolysis process in the
presence of lower concentrations of lumbrokinase [42].
Immobilization and Chemical Modification
Immobilization and chemical modifications study has been performed to reuse the
enzyme in-vivo system which will minimize frequency of dosing Lumbrokinase was
immobilized on the surface of polyurethane using maleic anhydride methyl vinyl copolymer
as an enzyme carrier [43]. So Immobilized LK polyurethane surface has been shown highly
antithrombogenic activity and can reduce surface induced thrombus. Immobilized LK surface
may minimize platelet adhesion and activation by preventing fibrinogen from adsorption or
by altering the conformation of adsorbed fibrinogen at an early stage of blood contact [44].
Further in order to improve the efficacy and tolerance of thrombolytic agents, its
necessary to improve the specificity of the Lumbrokianse on fibrin to avoid the side effects
and enhance the resistance to plasminogen activator inhibitor to elongate the half-life. To
improve the stability of Lumbrokinase chemical modification has been used to stabilize the
native structure of the earthworm protease and decrease the antigenicity during
administration. The stabilization of the Lumbrokianse was done by chemical modification of
the enzyme with 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide and phenylglyoxal to
protect the activity from the autolytic inactivation [45].
The strongest fibrinolytic Lumbrokinase has been modified chemically with fragmented
human serum albumin (MW, 10,000–30,000). After the chemical modification enzyme lost
the antigenicity of the native enzyme. The Lumbrokinase is a non hemorrhagic protein and
does not induce platelet aggregation. The Lumbrokinase has been shown the potent
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proteolytic activity for fibrin and fibrinogen than that of human plasmin. The Lumbrokianse
easily solubilizes in vivo fibrin clots (thrombi) of whole blood induced by thrombin in a rat’s
vena cava.
5. Clinical Application of Lumbrokinase
A Potent Fibrinolytic Agent
The Lumbrokianse an Earthworm Protease as a Fibrinolytic Agent has been used in China
Korea and Japan. The formation of thrombus in the blood causes many complications such as
stroke and myocardial infarction. Many of native and recombinant proteolytic enzymes have
been used as the thrombolytic agents including urokinase (UK), streptokinase, recombinant
tissue-type plasminogen activator, staphylokinase, and recombinant prourokinase [46]. Many
of them has shown good result but they also have some limitations such as fast clearance,
lack of resistance to reocclusion, bleeding complications, and other adverse effects.
The earthworm protease has dual functions in the fibrinolysis and plasminogen activation,
distinct from those enzymes (UK, tissue-type plasminogen activator, etc.) [47-50]. Therefore
have been used to treat the thrombosis. As Lumbrokianse during orally experiments both in
animals and clinics show significant fibrinolytic efficacy. Further more distinct amelioration
is observed in the treatment of blood high viscosity syndrome and thrombocytosis [51]. In
addition, the LK are quite stable under the long-term storage at room temperature [52], in the
form of oral dosage form. So for Lumbrokianse have been used in as oral dosage to prevent
and treat clotting diseases, such as myocardial infarction and cerebral thrombus [53].
Protection of Cerebral Ischemia:
Lumbrokinase has been a suitable drug candidate for ischemic complication. The anti-
ischemic activity of LK is due to its anti-platelet activity which raises c-AMP level and
lowers the calcium release from calcium stores, the anti-thrombosis action due to inhibiting of
ICAM-1 expression, and the anti-apoptotic effect due to the activation of JAK1/STAT1
pathway. At the molecular level Intercellular adhesion molecule-1 (ICAM-1) and Janus
Kinase1/Signal Transducers and Activators of Transcription1 (JAK1/STAT1) pathway in
protecting brain against ischemic injury by anti-thrombosis and anti-apoptosis [54]
Assistant to Implantation
During and after artificial organ transplantation small thrombus is usually formed on the
surface of the graft which may further leads to the more complication as graft rejection. Even
after great advancement in medicine and transplantation to improve the blood compatibility to
biomaterial. Even though the results, so far, are not satisfactory. During 1994, Lumbrokinase
was immobilized on the surface of polyurethane using maleic anhydride methyl vinyl ether
copolymer as an enzyme carrier [55]. So Immobilized LK polyurethane surface has been
shown highly antithrombogenic activity and can reduce surface induced thrombus.
Immobilized LK surface may minimize platelet adhesion and activation by preventing
fibrinogen from adsorption or by altering the conformation of adsorbed fibrinogen at an early
stage of blood contact [56].
International Journal of Bio-Science and Bio-Technology
Vol. 3, No. 2, June, 2011
68
6. Discussion and Conclusion:
Lumbrokinase are becoming more popular now a day in the medical Science for the
thrombolytic therapy. Being a eukaryotic protein it has even more scope in the treatment of
cardio and cerebrovascular disorder. As advancement in recombinant technology
Lumbokinase has been produced in bacterial and eukaryotic system in order to minimize
drawback of native Lumbokinase. Further attempt has been made for more effective drug by
alteration at gene level, designing suitable host system for enhance expression of recombinant
enzyme, Immobilization for the reuse of enzyme and chemical modification to reduce
antigenecity with the enhanced activity. In fact always there is progressive research is going
on to minimize the complication of thrombolytic therapy. In future it will be suitable drug
candidate for the thrombolytic therapy.
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Authors
Mahendra Kumar Verma is working as Lecturer at Department of
Biotechnology, R.V.R. & J.C. College of Engineering, Andhra Pradesh,
India. He is B.Pharma & M.Tech in Biotechnology and currently
pursuing PhD as part time. He teaches Molecular Biology Genetic
Engineering and Pharmaceutical Biotechnology for undergraduate
students.
KK Pulicherla is an Associate Professor and Head of the Department
at R.V.R. & J.C. College of Engineering, Andhra Pradesh, India. He
researches and teaches in several areas of Genomics, Proteomics, as well
as Bioinformatics. As a young researcher he has published 7 papers in
Journals and Magazines. He attended and presented 3 papers in Various
National and international conferences. He also has a patent to his
credits.
