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Staphylokinase Enzyme: An Overview of Structure, Function and Engineered

Forms

Article in Current Pharmaceutical Biotechnology · February 2018

DOI: 10.2174/1389201019666180209121323

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Current Pharmaceutical Biotechnology, 2017, 18, 000-000 1

REVIEW ARTICLE

Staphylokinase Enzyme: An Overview of Structure, Function and Engi-

neered Forms

Bahareh Vakili1, Navid Nezafat2, Manica Negahdaripour2,3, Maryam Yari1, Bijan Zare1 and
Younes Ghasemi1,2,3,*

1
Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of
Medical Sciences, Shiraz, Iran; 2Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shi-
raz, Iran; 3Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences,
Shiraz, Iran

Abstract: One of the most important causes of death in the modern lifestyle is acute ischemic stroke,
which is related to thrombosis in the blood vessels. Staphylokinase (SAK), a fibrinolytic agent, which
ARTICLE HISTORY is produced mainly by Staphylococcus aureus, is an indirect activator of plasminogen and belongs to
the third generation of fibrinolytic enzymes. Considering the very low level of production and immu-
Received: December 08, 2016
Revised: August 17, 2017 nogenicity concerns of natural SAK produced by Staphylococcus aureus, attempts have been made to
Accepted: January 06, 2018 produce recombinant SAKs with high production levels, more fibrinolytic activities and low immuno-
DOI: genicity. In this review, we summarized a number of expression systems based on recombinant DNA
10.2174/1389201019666180209121323 technology and protein-engineering approaches, which have been developed for the production of en-
gineered recombinant SAK molecules with higher fibrinolytic activities and lower antigenicity.

Keywords: Staphylokinase, Fibrinolytics, Recombinant, Thrombolysis, Immunogenicity, Plasminogen.

1. INTRODUCTION into direct and indirect activators. The direct activators have
serine protease activity and include reteplase, alteplase,
Cardiovascular diseases (CVDs) and specially stroke are
urokinase (u-PA), prourokinase (pro- u-PA), lanoteplase,
the most common causes of long-term physical disablement
tenecteplase, and APSAC (Sk-plasminogen activating com-
and the third most common cause of death in modern life- plex). Indirect activators are streptokinase and staphy-
style. One of the principle causes of CVDs is thrombosis that
lokinase, which do not have proteolytic action, and make a
occurs via formation of blood clots in blood vessels [1, 2].
1:1 stoichiometric complex with plasminogen and convert
Thrombosis is a highly complex process with many compo-
additional plasminogen to plasmin [8, 11]. Furthermore, in
nents [3]. In general, activation of the coagulation cas-
another method based on fibrin specificity, fibrinolytic en-
cade leads to the formation of thrombin that converts fi-
zymes are divided into 3 groups: first, second, and third gen-
brinogen to fibrin. The polymerized fibrin and other compo- eration. The first generation, streptokinase and urokinase, are
nents like red blood cells and platelets form a blood clot in a
systematic plasminogen activators, which are not selective
blood vessel. On the other hand, plasmin hydrolyzes the fi-
for blood clots and attach to both clot-bound and circulat-
brin clots and inhibits thrombosis [4, 5]. There are four
ing plasminogen. They have limited clinical usage, because
treatment strategies for thrombolytic therapy, including anti-
these enzymes produce considerable unwanted fibrino-
platelets, anticoagulants, surgeries, and recently, fibrinolytic
genolysis. The second generation, such as tissue plasmino-
enzymes [6-8]. Fibrinolytic enzymes change plasminogen gen activator (t-PA), is relatively selective for clot-bound
into plasmin, which is a natural fibrinolytic factor. In the
plasminogen. However, it still leads to the activation of cir-
clot, plasmin breaks the fibrinogen and fibrin, thereby, caus-
culating plasminogen. The third generation, staphylokinase
ing clot lysis [9, 10]. Fibrinolytic enzymes are classified
(SAK) and reteplase, are highly fibrin-specific and activate
based on different characteristics (Table 1). In the context of
plasminogen bound to fibrin without causing systemic plas-
plasminogen activation, fibrinolytic enzymes are categorized
minogen activation (Fig. 1) [12]. Finally, based on their ori-
gin, fibrinolytic agents are divided into two groups: autolo-
*Address correspondence to this author at the Department of Pharmaceuti- gous and heterologous agents. Autologous thrombolytic
cal Biotechnology, School of Pharmacy, Shiraz University of Medical Sci- agents such as t-PA and u-PA are non-immunogenic, as they
ences, P.O. Box 71345-1583, Shiraz, Iran; Tel/Fax: +98 7112426729; are derived from human body.
E-mail: [email protected]

1389-2010/17 $58.00+.00 © 2017 Bentham Science Publishers

2 Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 Vakili et al.

Table 1. Different categories of fibrinolytic drugs.

Fibrinolytic Agent Generation Fibrin-specific Plasminogen Activation Development Phase

Streptokinase (-) Indirect Approved

First
Urokinase (-) Direct Approved

t-PA (+) Direct Approved

Second
Prourokinase (-) Direct Approved

Alteplase (+) Direct Approved

Reteplase (+) Direct Approved

Tenecteplase (++) Direct Approved

Third
Lanoteplase (+) Direct Phase III clinical trial

APSAC (-) Direct Approved

Staphylokinase (+++) Indirect Phase III clinical trial

Fig. (1). Mechanism of fibrin-specific and fibrin-nonspecific agents in vessel lumen. Fibrin-specific agents only bind fibrin-bond plasmi-
nogen and convert it to plasmin, consequently lead to fibrin degradation and lysis the blood clotting. On the other hand, fibrin-nonspecific
agents bind circulating plasminogen which lead to fibrinogen degradation.

This group can be used several times for each patient. On non-human origin, they trigger an immune response. Thus,
the other hand, heterologous thrombolytic agents such as these agents should be used singly [13]. An ideal throm-
SAK and streptokinase come from bacteria, and due to the bolytic drug must have some principal features including:
Staphylokinase Enzyme: An Overview of Structure, Function and Engineered Forms Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 3

fibrin specificity, low or no reocclusion rate and systemic in only 34, 36, and 43 residues in mature SAK (Fig. 2) [34,
bleeding, resistance to plasminogen activator inhibitor- 35]. Due to the differences in three residues, they are differ-
1(PAI-1), non-antigenicity, and cost-effectiveness. These ent in thermostability; however, their potentials for activa-
days, there are several fibrinolytic drugs for treating throm- tion of plasminogen are similar (Table 2).
botic diseases that are different in pharmacodynamics and SakSTAR was selected for further studies for its throm-
pharmacokinetic features. Thrombolytic agents approved for
bolytic properties in humans, as it appeared to have a some-
clinical use are streptokinase (SK), urokinase (UK),
what higher temperature stability than other variants [36].
prourokinase (pUK), Tenecteplase (TNK), APSAC, rete-
SAK shows no significant homology with streptokinase,
plase, and t-PA. However, Common adverse effects of all
however, the mechanism of SAK activation is basically the
these thrombolytic drugs are bleeding complications related
same as streptokinase with little differences. Similar to strep-
to systemic fibrinogenolysis and lysis of normal hemostatic tokinase, SAK forms an equimolar complex (1:1) with plas-
plugs [14, 15]. Consequently, in recent years, research for
minogen. This complex generates an active site in plasmino-
new effective fibrinolytic drugs with less bleeding has been
gen, which converts plasminogen to plasmin. However, un-
developed. SAK has a high fibrin specificity and low bleed-
like the streptokinase-plasminogen complex, the staphy-
ing complications [16, 17]. However, SAK is a bacterial pro-
lokinase-plasmingen complex (Plg.Sak) is inactive and re-
tein of non-human origin, and triggers an immune response
quires staphylokinase-plasmin (Sak.Pli) complex for activa-
in patients [12, 18]. Although progress in recombinant DNA- tion [33, 37]. The generation of Sak.Pli complex is a rate-
based technology has enabled the synthesis of recombinant
limiting step, which is accelerated by Sak.Pli itself and de-
SAK molecules with reduced immunogenicities, currently,
layed by plasmin inhibitors like α2- antiplasmin (Fig. 3) [38].
different bioinformatics methods are used in various biologi-
It has been found that Arg719 in human plasminogen and
cal fields such as enzyme engineering to vaccine designing,
Met26 in SAK are very important for binding of these mole-
to lower the costs and improve the accuracy of empirical
cules to each other [39]. An amino acid substitution illustrated
investigations [19-24]. In this research, we attempt to review that Arg719 in human plasminogen has a substantial impact
a number of expression systems that have been designed for
on the formation of active staphylokinase:plasmin complex
large-scale production of recombinant SAK as well as engi-
[40]. In phase I trials, the half-life of SAK in plasma was 6.3
neered forms of enzyme, which have higher fibrinolytic ac-
minutes and its double-bolus administration was safe com-
tivity and lower antigenicity.
pared to a single bolus administration [36]. In comparison
with other fibrinolytic agents, SAK is significantly more fi-
2. STRUCTURE, FUNCTION AND PROPERTIES OF brin-specific, which is substantially important for its clinical
STAPHYLOKINASE
use. It was found that the fibrin-specificity of SAK is due to
An early study in 1948 showed that SAK, secreted by rapid inhibition of generated plasmin.staphylokinase complex
Staphylococcus aureus, has fibrinolytic properties [25, 26]. by α2-antiplasmin [41]. This rapid inhibition is needed for
A 163 amino acid precursor protein is encoded by the sak the attachment of lysine residues in the plasminogen. Inter-
gene whose first 27 residues encode a signal peptide. Finally, estingly, fibrin competes with these lysine residues in plas-
a mature protein of 136 amino acids is produced [27]. SAK minogen, thus the rate of inhibition is reduced more than
is produced mainly by Staphylococcus aureus after transfor- 100-fold at the fibrin clot [42]. The thrombolytic properties
mation by bacteriophages or after a lysogenic conversion. of SAK have been evaluated in several clinical studies (14,
The coding sequence is preceded upstream by a Shine- 41-43). A randomized trial with 100 patients that received
Dalgarno, and -10 and -35 prokaryotic promoter sequences. either SAK or recombinant t-PA (rt-PA), confirmed that
SAK belongs to the group of staphylococcal proteins, syn- SAK is significantly more fibrin-specific than rt-PA [43].
thesis of which takes place during the late exponential The therapeutic potential of SAK has been investigated
growth phase [28-30]. It is a single domain molecule made in the CAPTORS I and II trials by Armstrong et al. in 2000
from a single polypeptide chain without disulfide bonds [31]. and 2003, respectively (Collaborative Angiographic Patency
This protein does not contain cysteine, glutamine, and glyco- Trial of Recombinant Staphylokinase) [44, 45]. The ap-
sylation. It is made of two α-helices and eight β-sheets. SAK, proved efficacy/safety profile achieved with SAK was en-
such as streptokinase, is structurally similar to the members couraging [44]. Moreover, for improving therapeutic potency
of ß-grasp fold family proteins, according to SCOP protein of SAK, polyethylene glycol (PEG) was conjugated to SAK
classification [32]. The N-terminal region is shown to be that showed excellent thrombolysis in myocardial infarction
important for the activity of Staphylokinase [33]. The three [45].
natural variants, sakΦC, sak42D, and sakSTAR are different

Table 2. Different properties of three natural variants of staphylokinase.

Variant Number of Molecular Residues

Theoretical pI Instability Index Aliphatic Index
Name Amino Acids Weight (D) 34 36 43

SAK-STAR 136 15494.6 6.15 35.65 72.28 Ser Gly His

SAK-ΦC 136 15464.6 6.15 33.61 72.28 Gly Gly His

SAK-42D 136 15412.5 5.93 33.04 73.75 Gly Arg Arg

4 Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 Vakili et al.

Fig. (2). Protein sequence alignment of staphylokinase variants. Three natural variants of Staphylokinase has been characterized includ-
ing: SakSTAR, sakΦC and sak42D. SakSTAR has been cloned from the genomic DNA, while sakΦC and sak42D have been cloned from the
bacteriophages. These three variants are similar in plasminogen-activating potential, but differ in terms of stability. They have 136 amino
acides and are different in only three residues; 34, 36 and 43 in mature SAK:
1). SakSTAR (34; Ser 36; Gly 43; His)
2). sakΦC (34; Gly 36; Gly 43; His)
3). sak42D (34; Gly 36; Arg 43; Arg)

3. ADVANTAGES OF STAPHYLOKINASE IN COM- tokinase, SAK is active against both platelet-poor and plate-
PARISON WITH OTHER FIBRINOLYTIC AGENTS let-rich clots [28, 29]. SAK has a unique fibrin-dependent
mechanism of plasminogen activation compared to other
Staphylokinase has some distinct advantages over the
fibrinolytic drugs, which has led to a maintained interest in
other existing thrombolytic agents. Unlike all the throm-
research about its medical application.
bolytic drugs, SAK is less likely to cause bleeding complica-
tions. The SAK.plasmin complex neutralizes in blood plasma
4. NATURAL PRODUCTION OF STAPHYLOKINASE
compared to thrombus. Thus, SAK as a fibrinolytic drug,
does not cause the reduction of blood fibrinogen. It may Staphylokinase is naturally secreted by lysogenic Staphy-
yield a better potency if given at two doses daily [18]. Re- lococcus sp. strains or after transformation of these strains
combinant staphylokinase is more fibrin specific than rt-PA with bacteriophages [47]. However, industrial production of
[14]. Furthermore, it has been found that recombinant SAK Staphylococcus sp. is not cost-effective, because it grows
was equal in fibrinolytic activity with rt-PA, while signifi- very slowly in fermentation processes [31, 48]. Due to an
cantly more fibrin-selective [43]. In the presence of fibrin increasing demand for new fibrinolytic drugs, the overex-
clot, traces of plasmin are present (due to the physiological pression of recombinant SAK in new strains instead of natu-
plasminogen activation). These plasmin molecules are bound ral strains would be preferred.
to the clot via lysine binding site and therefore rapid inhibi-
tion by α2-antiplasmin is inhibited. Hence, SAK is concen- 5. DIFFERENT HOSTS FOR OVEREXPRESSION OF
trated near the clot and its activity increases near the clot. In RECOMBINANT STAPHYLOKINASE
the absence of fibrin clot, SAK-plasmin complex is quickly Considering the increasing incidences of coronary heart
inhibited by α2-antiplasmin. [46]. Furthermore, unlike strep- diseases along with the immense applicability of SAK as a
Staphylokinase Enzyme: An Overview of Structure, Function and Engineered Forms Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 5

promoter and its own translation signals and was purified

using osmotic shock and CM-cellulose column chromatog-
raphy. SAK activity showed 60 times increase in the perip-
lasmic fraction [48]. This expression method has some prob-
lems, including proteolytic degradation of SAK and decrease
in the growth of recombinant E. coli host system. For im-
proving the level of SAK production, E. coli TG1 was trans-
formed with recombinant plasmids carrying the signal se-
quence of the sak42D and the sakSTAR gene that was re-
placed by an ATG start codon. SAK gene was expressed
under the control of two Shine-Dalgarno sequences and a tac
promoter. In this manner, SAK was 10% to 15% of intracel-
lular total protein. Moreover, r-SAK was purified from the
cytosol fractions with yields of 50% to 70% (200 mg/L of
fermentation broth) [50]. In 2008, an expression of pET32(a)
plasmid was constructed with the SAK gene and was trans-
formed to E. coli strain BL21(DE3). The expression of SAK
protein was increased to 35%. Subsequently, simple and
rapid chromatography was performed for the large-scale and
highly efficient purification of r-SAK from E. coli (>99 %)
[51]. In another study, SAK was expressed in E. coli BL21
in the presence of IPTG as the inducer, under the control of a
strong tac promoter. The expression level of recombinant r-
SAK was about 42% of the total cellular mass and 300 mg/L
of r-SAK was produced [52]. The highest expression level of
r-SAK expressed in E. coli system is 2.8 g/L of fermentation
broth, which was produced in BL21 (DE3) [53].

6.2. Bacillus subtilis

Secretory production of SAK has been investigated in
Bacillus subtilis in several studies. In addition to extracellu-
lar secretion of proteins by Bacillus subtilis, the non-
Fig. (3). Staphylokinase mechanisem. SAK forms an equimolar pathogenic nature of this system simplifies the purification
complex (1:1) with plasminogen, however unlike the streptokinase- process. In one of the earlier studies, a Bacillus subtilis
plasminogen complex, the staphylokinase-plasmingen complex strain, WB700, with a deficiency in seven extracellular pro-
(Plg.Sak) is inactive and it requires Staphylokinase-Plasmin teases was used. By a rapid three-step approach and the op-
(Sak.Pli) complex for activation [33]. The generation of plasmin. timization of fermentation conditions, 337 mg/L of SAK was
staphylokinase (Sak.Pli) complex is a rate-limiting step which is produced [54]. Moreover, a wprA-gene deficient Bacillus
accelerated by Pli.Sak itself and delayed by plasmin inhibitors (e.g., subtilis strain was used for the production of SAK. This
α2- antiplasmin). strain was transformed by an expression plasmid carrying a
SAK sequence fused to the signal sequence of subtilisin.
WprA is a subtilisin-type protease whose deletion enhanced
potential fibrinolytic treatment, attempts have been made to the stability of mature SAK. In this way, 181 mg/liter of
produce SAK by recombinant DNA technology in recent SAK was produced [55].
years. The objective is reaching high levels of SAK with
higher specificity and fibrinolytic activity. A number of host 7. EUKARYOTIC HOST
cells have been designed for the production of recombinant
SAK (r-SAK), including prokaryotic hosts (Escherichia coli 7.1. Yeast Expression Systems
and Bacillus subtilis) and eukaryotic hosts (Pichia pastoris
In recent years, some eukaryotes cells have been em-
and Hansenula polymorpha) (Table 3).
ployed as expression systems for overproduction of SAK,
including Pichia pastoris and Hansenula polymorpha. They
6. PROKARYOTIC HOSTS are methylotrophic yeasts that have the capability of post-
6.1. Escherichia Coli translational modifications, such as folding, glycosylation,
and disulfide bond formation, similar to higher eukaryotes.
E. coli was one of the first host cells for the production of However, the most important challenge by P. pastoris or H.
recombinant SAK molecule. In an early study, recombinant polymorpha is adding high-mannose oligosaccharides to the
SAK was expressed in E. coli JM109 with tac promoter and secreted heterologous proteins, which can be antigenic. To
ompA signal sequence, which produced 15mg/ml and overcome this problem, several glycosylation inhibitors as
5mg/ml into the periplasm and extracellular media, respec- well as humanized and genetically engineered yeast strains
tively [49]. SAK was also expressed in E. coli with λPR are being used [56].
6 Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 Vakili et al.

Table 3. Different hosts for Overexpression of recombinant staphylokinase.

Organism Vector Promoter Yield Ref.

Strain 15mg/ml (periplasm media)

pKK-ompA tac [49]
JM109 5mg/ml (extracellular media)

E. coli Shine-Dalgarno sequences in tan-

TG1 pMEX602 200 mg/L (fermentation broth) [50]
dem and a tac promoter
BL21 (DE3) pESak tac 300 mg/L [52]
BL21 (DE3) pET21a 2.8 g/L (fermentation broth) [53]
337 mg/L (Fed-batch fermentation)
WB700 pSAKBQ P43 [54]
B. subtilis 140 mg/L (shake-flask)
wprA-deficient WB600 pSAK704 Subtilisin 181 mg/liter [55]
P. pastoris pPICZαA AOX1 1 g/L [58]
+
H. polymorpha pFPMTMFα MOX, FMD ≥ 1 g/L [59]

In one of the first studies, the recombinant Pichia carry- fusion of SAK and hirudin was a Y-shaped molecule (HE-
ing multiple copies of the sak gene produced a high-level (1 SAKK). This new variant is composed of engineered coiled-
g/l) of extracellular glycosylated r-SAK with insignificant coil sequences. In comparison with wild-type SAK, this fu-
plasminogen activation activity. Addition of tunicamycin as sion molecule had higher-level thrombolytic activity, which
a glycosylation inhibitor during the induction phase led to targeted thrombin-rich fibrin clots. Moreover, the reforma-
the expression of a non-glycosylated, highly active r-SAK. A tion of clots decreased during fibrinolysis reaction [60].
homogenous r-SAK of 95% purity was produced with two
simple steps of ion-exchange chromatography [57]. Recently,
SAK was inserted into the expression vector pPICZαA and
expressed under the control of the AOX1 promoter in
P. pastoris GS115. Non-glycosylated SAK was purified us-
ing ProBondNi2+-affinity chromatography [58]. In 2012,
large scale (80 L) production of non-glycosylated recombi-
nant SAK-2 (rSAK-2) was performed in H. polymorpha
through optimizing the fermentation process. Substitution of
some residues in N-acetyl glycosylation motif of SAK, high
yields of r-SAK were achieved, which were biologically
active (≥ 1g rSAK-2 per liter) [59].

8. DIFFERENT TYPES OF CHIMERIC STAPHYLOK-

INSE WITH ANTITHROMBOTIC AND ANTIPLATE-
LET ACTIVITIES
In order to combine the fibrinolytic activities of SAK
with the advantages of antithrombotic and antiplatelet activi-
ties, SAK has been infused with some antithrombotic and
antiplatelet agents, such as hirudin and RGD, respectively.

9. RECOMBINANT HIRUDIN-SAK
In order to introduce antithrombin activity to SAK, it has
been infused with hirudin, which is an antithrombotic agent
isolated from the salivary glands of the leech, Hirudo medi-
cinalis [60] (Fig. 4). In one approach, fusion SAK to hirudin
Fig. (4). Hirudin and RGD as antithrombin and antiplatelet
variant was perfomed (SAK-HV) using a thrombin-binding
agents. Hirudin binds to thrombin and acts as a strong inhibitor of
motif composed of 12 residues from hirudin. Then, the fu-
thrombin such as antithrombin III activity. RGD is an antiplatelets
sion protein was expressed in E. coli BL21 (DE3) strain. In
agent which can recognize the glycoprotein IIb/IIIa (GPIIb/IIIa)
vitro measurement of the thrombin-binding activity revealed
receptor on the membrane of platelets. The binding of surface gly-
that SAK-HV could bind to thrombin, in contrast to wild-
coprotein GPIIb/IIIa to fibrinogen mediates platelet aggregation.
type SAK which could not bind to thrombin. It means that
RGD peptide can prevent fibrinogen binding to GPIIb/IIIa on acti-
SAK-HV has more thrombolytic and anticoagulation activi-
vated platelets, therefore inhibiting platelets aggregation.
ties in comparison with the wild-type SAK [47]. Another
Staphylokinase Enzyme: An Overview of Structure, Function and Engineered Forms Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 7

10. RECOMBINANT RGD-SAK lergenic than streptokinase. However, the majority of pa-
tients develop a neutralizing antibody after Wild-type SAK
It has been found that after a thrombolytic therapy, acti-
administration, because it is a heterologous protein [69, 70].
vated platelets play an important role in rethrombosis by
However, levels of anti-SAK antibodies in the general popu-
platelet aggregation [61]. Arg–Gly–Asp (RGD) peptide is an
lation are lower than those of anti-streptokinase antibodies
antiplatelet agent that can recognize the glycoprotein IIb/IIIa [46]. Nowadays, both B- and T-cell epitopes of SAK have
(GPIIb/IIIa) receptor on the platelet membrane. Research
been identified [71-73] (Fig. 5). Many efforts have been
illustrated that the binding of the surface glycoprotein
done for reducing the immunogenicity of SAK (Table 4).
GPIIb/IIIa to fibrinogen mediates platelet aggregation. RGD
Wild-type SAK contains three nonoverlapping B-cell epi-
peptide can prevent fibrinogen binding to GPIIb/IIIa on acti-
topes including K35, E38, E80, and D82 in epitope 1 and
vated platelets, therefore inhibiting platelets aggregation [62]
K74, E75, and R77 in epitope 3. The other epitope may be
(Fig. 4). A novel SAK variant (RGD-SAK) was designated located adjacent to the dimer interface [73, 74]. Moreover,
by using site-directed mutagenesis with substitution of K35
SAK has five T-cell epitope. Among these five epitopes, C3
with Arg to constitute an RGD motif. The engineered RGD-
(amino acids 71–87) is the most prominent immunoreactive
SAK was structurally similar to SAK, while its affinity with
region and could induce proliferation of T-cell [75]. Recent
platelets was much higher than SAK. Moreover, this new
mutagenesis studies have been revealed that modification of
bifunctional variant inhibited ADP-induced platelet aggrega-
these epitopes leads to lower immunological properties of
tion [63, 64]. SAK without significant loss of its thrombolytic potency
compared to wild-type Sak [76, 77]. Early studies to reduce
11. MULTIFUNCTIONAL SAK VARIANTS the immunogenicity of SAK focused on the deletion of B-
cell epitopes [77]. For instance, two less-immunogenic vari-
In order to improve the antiplatelet and anti-thrombin ants namely SakSTAR.M38 (K35, E38, K74, E75 and R77
activities of r-SAK, several multifunctional r-SAK have been substitution with A) and SakSTAR.M89 (K74, E75, R77,
constructed. A multifunctional SAK was designed by com- E80 and D82 substitution with A) were identified and con-
bining SAK molecule with RGD, the Kringle 2 domain (K2) sidered in rabbits and baboons. These two SAK variants
of t-PA, containing a fibrin-specific binding site and hirudin showed significantly less circulating IgG than wild-type
(SAK-RGD-K2-Hir). This new SAK molecule was signifi- SAK [70, 74]. Interstingly, another variant with only a single
cantly a more potent and faster thrombolytic agent in com-
parison with the standard r-SAK, while its antithrombin ac-
tivity was similar to hirudin alone [65]. Another multifunc-
tional fusion protein based SAK, SRH (SAK-RGD-hirudin),
was expressed as a soluble form in the osmotically inducible
E. coli GJ1158 and purified with a yield of 0.27 g/L. The
antithrombin activity and platelet binding activity of SRH
were significantly higher than SAK [66]. To overcome reoc-
clusion, a multifunctional Y-shaped molecule r-SAK (HE-
SAKK) has been constructed using engineered coiled-coil
sequences, which dramatically minimized clot reformation
and showed powerful thrombolytic properties compared to
other r-SAK [62]. A fast-acting engineered SAK (SAKM3-
L-K1) including Kringle-1 domain, as a fibrin-targeting do-
main, from human plasminogen and the C-terminal end of
SAK with a linker sequence was designed with a substituting
Thr-30 with alanine in a glycosylation site, which led to a
non-glycosylated biologically active fusion protein and was
expressed in P. pastoris. SAKM3-L-K1 showed a reduction
in clot lysis time [67]. PLATSAK (Platelet-Anti-thrombin-
Staphylokinase) was a multifunctional SAK designed by
three inhibitory regions, including (1) RGD, (2) a part of
fibrinopeptide A, a thrombin inhibitor, and (3) a C- terminal
tail of hirudin. PLATSAK significantly elongated activated
thromboplastin and thrombin time and repressed amidolytic
activity of thrombin. However, due to the unfavorable three
dimensional structure, PLATSAK didnot inhibit platelet ag-
Fig. (5). Staphylokinase structure (Uniprot code: P68802). SAK
gregation substantially [68].
in a single polypeptide chain without disulfide bridges which con-
sist of eight ß-sheet and a central α-helix. Wild-type SAK contains
12. STAPHYLOKINASE VARIANT WITH REDUCED
three nonoverlapping B-cell epitopes including K35, E38, E80 and
IMMUNOGENICITY
D82 in epitope 1 (magenta) and K74, E75 and R77 in epitope 3
Current clinical research illustrates that the allergic reac- (red). The other epitope may be located in the vicinity of the dim-
tions to SAK in patients are rare. Vanderschueren et al. mer interface. SAK has five T-cell epitope of SAK epitopes. Of
found that staphylokinase administration did not induce al- these T-cell epitopes C3 (amino acids 71–87) is the most prominent
lergic reactions[18]. Furthermore, staphylokinase is less al- immunoreactive region (yellow).
8 Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 Vakili et al.

Table 4. Different properties of SAK variants with reduced immunoactivity.

Sequence Molecular Instability Total Number of

SAK Variants Theoretical pI Ref.
Length Weight Index Charge Residues

34.13 (Asp + Glu): 23 Uniprot

wt-Sak 163 18490.2 6.75
Stable (Arg + Lys): 23 P68802
SakSTAR; code SY155)
(K35A, E65Q, K74R, D82A, 35.08 (Asp + Glu): 20
163 18227.9 6.74 [76]
S84A, T90A, E99D, T101S, Stable (Arg + Lys): 20
E108A, K109A, K130T, K135R)
34.13 (Asp + Glu): 22
SakSTAR(R77A,E80A) 163 18347.1 6.75
stable (Arg + Lys): 22
35.31 (Asp + Glu): 21
SakSTAR (R77A, E80A, D82A) 163 18303.1 7.78 [71]
stable (Arg + Lys): 22

SakSTAR (K74Q, R77S, E80S, 38.01 (Asp + Glu): 21

163 18351.0 6.75
D82A) stable (Arg + Lys): 21
34.13 (Asp + Glu): 22
Sak(E80A) 163 18432.2 7.78
stable (Arg + Lys): 23
[72]
34.13 (Asp + Glu): 22
Sak(E80S) 163 18448.2 7.78
stable (Arg + Lys): 23
SakSTAR (code SY 161)
K35A, E65Q, K74R, E80A, 35.28 (Asp + Glu): 19
163 18185.9 7.82 [77]
D82A, T90A, E99D, T101S, stable (Arg + Lys): 20
E108A, K109A, K130T, K135R

substitution of K74 with A in epitope 3 showed intact spe- and prolonged half-life without reduction in the fibrin-
cific activity, while immunololigal responces decreased specificity. Because of these favorable properties, this vari-
markedly [75]. Recently, the elimination of T-cell epitopes ant has been selected for more clinical development [82, 83]
has presented a new approach to lessen the immunogenicity (Fig. 6).
properties of Sak [78-80]. In a rational drug design, SAK
variants, with 2 to 4 amino acid substitutions in the region
CONCLUSION
71-87, were designed and evaluated by both functional as-
says and computer modeling. These variants were no longer In parallel to the growing developments of fibrinolytic
recognized by specific T-cells, and consequently didnot drugs in treating CVDs, a continuing demand for less anti-
show new SAK-variant specific immune response. Further- genic and cost-effetctive fibrinolytic drugs has risen. While a
more, there was no substantial impact on thrombolytic prop- wide range of such drugs have been identified, they have
erties of Sak, probably because the modified regions are out- some common side effects, such as bleeding and limited fi-
side plasmin binding and plasminogen activation domains brin-specificity. To overcome these drawbacks, attempts for
[78]. In another study, a recombinant SAK based on mutage- improving thrombolytic drugs, which are especially en-
nesis of Glu80 residue was rationally designed. In wild-type zymes, through increasing fibrin specificity as well as reduc-
SAK, Glu80 is not only an important T-cell epitope, but also ing their antigenicity, are needed. SAK, a member of third
an essential residue of the B-cell epitope. Two Sak mutants, generation of fibrinolytic drugs, is not only a highly fibrin-
including Sak (E80A) and Sak (E80S), were expressed in E. selective agent, but also more resistant to inhibition by
coli DH5a and showed significant decrease in the level of plasma inhibitors in thrombolytic therapy. Furthermore,
specific anti-Sak IgG antibodies, as well as a slightly higher SAK is comparably active in the unretracted and platelet rich
fibrinolytic activity compared to the wild-type SAK [79]. In clots.
an effort to reduce SAK immunogenicity, 350 SakSTAR
variants were constructed. From these 350 variants, Sak- Efforts have been undertaken to develop expression sys-
STAR (code SY155) with 12 amino acid substitution (K35A, tems for producing biologically active SAK in large quanti-
E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, ties for biochemical studies and clinical uses. Promising im-
K109A, K130T, K135R) showed markedly fewer antibody provements have been achieved in both prokaryotic and eu-
attachment, but an intact thrombolytic potency [81]. Another karyotic hosts for overexpression of rSAK. While E. coli is
SakSTAR variant (code SY161-P5), with 12 amino acid sub- still the preferred expression host for r-SAK production, but
stitutions (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, high-level production (1 g/l) of r-SAK has been achieved
T101S, E108A, K109A, K130T, K135R), and with a muta- using yeast cells. Researches showed that the optimization of
tion in position 3 (Ser to Cys) illustrated reduced antigenicity fermentation conditions has a substantial impact on the final
Staphylokinase Enzyme: An Overview of Structure, Function and Engineered Forms Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 9

Fig. (6). Comparison of wt-SAK and some SAK variants with reduced immunogenicity. Sequence alignment of reviewed structures of
wt-SAK (Uniprot code: P68802) with seven different SAK variants with reduced immunogenicity. They all contain 163 amino acid residues
and including: SakSTAR; code SY155 (K35A, E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), Sak-
STAR (R77A, E80A), SakSTAR (R77A,E80A,D82A), SakSTAR(K74Q,R77S,E80S,D82A), Sak(E80A), Sak(E80S), SakSTAR (code SY
161) K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R.

yield of r-SAK. They also found that fed-batch fermentation going researches to achieve new recombinant SAKs with
led to the production of more r-SAK than batch culture at the low immunogenicity and high fibrinolytic activity.
large scale. Several studies have presented successful con-
structions of the chimeric SAKs, in which a thrombus- LIST OF ABBREVIATIONS
specific peptide, such as RGD or hirudin was combined with
SAK = Staphylokinase
SAK to enhance the thrombolytic specificity. The results
obtained from these engineered SAK variants in animal CVDs = Cardiovascular Diseases
models were encouraging. Moreover, neutralizing antibodies u-PA = Urokinase
against SAK were demonstrated in the majority of the pa- pro- u-PA = Prourokinase
tients. Thus, reducing the immunogenicity of SAK is the
most important challenge in the thrombolytic therapy. In APSAC = Sk-plasminogen Activating Complex
light of protein engineering approches, the immunogenicity t-PA = Tissue Plasminogen Activator
of SAK protein can be reduced by rational modification of PAI-1 = Plasminogen Activator Inhibitor-1
both B- and T-cell epitopes. Successful results from different
SK = Streptokinase
studies revealed that substitution of immunodominant resi-
dues by suitable amino acids is a promising method to mod- UK = Urokinase
ify B- and T-cell epitopes in the wild-type SAK, in order to pUK = Prourokinase
reduce the immunogenicity of SAK for further clinical uses TNK = Tenecteplase
in human.
Plg.Sak = Staphylokinase-plasmingen
In summary, many efforts have been undertaken to pro- Sak.Pli = Staphylokinase-plasmin
duce high levels of biologically active recombinant SAK
with reduced immunogenicity. However, there are still on- rt-PA = Recombinant t-PA
10 Current Pharmaceutical Biotechnology, 2017, Vol. 18, No. 13 Vakili et al.

CAPTORS = Collaborative Angiographic Patency Trial of front-loaded alteplase in acute myocardial infarction. Am Heart J
Recombinant Staphylokinase 1997, 134, 213-219.
[19] Nezafat, N.; Ghasemi, Y.; Javadi, G.; Khoshnoud, M. J.; Omidinia,
r-SAK = Recombinant SAK E., A novel multi-epitope peptide vaccine against cancer: an in
silico approach. J. Theor. Biol. 2014, 349, 121-134.
RGD = Arg–Gly–Asp [20] Zamani, M.; Nezafat, N.; Negahdaripour, M.; Dabbagh, F.;
K2 = Kringle 2 Domain Ghasemi, Y., In silico evaluation of different signal peptides for the
secretory production of human growth hormone in E. coli. INT J
PLATSAK = Platelet-Anti-thrombin-Staphylokinase PEPT RES THER 2015, 21 (3), 261-268.
[21] Gholami, A.; Shahin, S.; Mohkam, M.; Nezafat, N.; Ghasemi, Y.,
Cloning, characterization and bioinformatics analysis of novel cy-
CONSENT FOR PUBLICATION tosine deaminase from Escherichia coli AGH09. INT J PEPT RES
THER 2015, 21 (3), 365-374.
Not applicable. [22] Farhadi, T.; Nezafat, N.; Ghasemi, Y.; Karimi, Z.; Hemmati, S.;
Erfani, N., Designing of complex multi-epitope peptide vaccine
CONFLICT OF INTEREST based on omps of Klebsiella pneumoniae: an in silico approach.
INT J PEPT RES THER 2015, 21 (3), 325-341.
The authors declare no conflict of interest, financial or [23] Hajighahramani, N.; Nezafat, N.; Eslami, M.; Negahdaripour, M.;
otherwise. Rahmatabadi, S. S.; Ghasemi, Y., Immunoinformatics analysis and
in silico designing of a novel multi-epitope peptide vaccine against
Staphylococcus aureus. Infection, Genetics and Evolution 2017, 48,
ACKNOWLEDGEMENTS 83-94.
[24] Negahdaripour, M.; Nezafat, N.; Ghasemi, Y., A panoramic review
This study was supported by a Grant from Shiraz Univer- and in silico analysis of IL-11 structure and function. Cytokine &
sity of Medical Sciences, Shiraz, Iran. Growth Factor Reviews 2016, 32, 41-61.
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PMID: 29424308

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