Targeting the host hemostatic system function in bacterialinfection for antimicrobial therapies

Yuanxi Xu • Haiqing Yu • Hongmin Sun

Published online: 31 December 2013

� Springer Science+Business Media New York 2013

Abstract The hemostatic system is an important player

in host’s response to infection. It has been shown that host

hemostatic factors as well as platelets, interact with various

proteins from bacteria and play important roles in host

defense against infections. This review summarizes studies

of function of host hemostatic system in host defense

against bacterial infections and efforts to target hemostatic

system interaction with pathogens to develop potential

antimicrobial therapies.

Keywords Infection � Hemostatic factor � Platelet �Antimicrobial therapy

Introduction

In recent years, antibiotic resistance has become a major

medical problem [1]. Due to inappropriate and irrational

use of antibiotics in human and farm animals, many

pathogens in the hospital are resistant to one of the drugs

commonly used to treat infections and many pathogens are

resistant to multiple antibiotics. There is an urgent need to

develop novel antimicrobial agents due to the emergence of

antibiotic resistant pathogens [1–4]. Interfering with the

host pathogen interaction will likely generate novel

therapeutic approaches supplementing the conventional

antibiotic therapy.

Host hemostatic system plays important roles in host

pathogen interaction in infectious diseases [5–10]. Infec-

tion by bacteria will cause inflammation and disruption of

host hemostatic system [9, 11]. Various members of host

hemostatic systems have been shown to interact with

pathogens or play roles in host response to pathogen

invasion [10]. It has been well established that a number of

bacterial pathogens produce either receptors or activators

of host plasminogen to exploit host fibrinolytic system to

facilitate bacterial invasion [5, 6, 10, 12]. Fibrinogen/fibrin

also plays important roles in host response to bacterial

infections and host inflammation [7]. At the site of infec-

tion, local fibrin deposition is a common feature. Fibri-

n(ogen) supports leukocyte adhesion and activation

through its interaction with the leukocyte integrin aMb2 [7,

13]. Mice with fibrin(ogen) defect in aMb2 binding

exhibited a major defect in the host inflammatory response

and were unable to clear bacteria when infected with

Staphylococcus aureus [14].

As a result, novel antimicrobial agents were developed

based on the knowledge of host hemostatic system inter-

action with pathogens against bacterial infections. In this

review, we will discuss examples utilizing the interactions

between host hemostatic system with pathogens to develop

novel antimicrobial therapies.

Agents targeting plasminogen pathogen interaction

Plasminogen is the central proteinase of the fibrinolytic

system. Plasminogen is cleaved by plasminogen activators

into plasmin to degrade the fibrin thrombus (Fig. 1). It has

been shown that host plasminogen is exploited by a number

Y. Xu � H. Yu � H. Sun

Department of Internal Medicine, University of Missouri

Hospital and Clinics, Columbia, MO, USA

H. Sun (&)

Division of Cardiovascular Medicine, University of Missouri,

CE306, Five Hospital Drive DC095.00, Columbia, MO 65212,

USA

e-mail: sunh@health.missouri.edu

123

J Thromb Thrombolysis (2014) 37:66–73

DOI 10.1007/s11239-013-0994-9

of bacterial pathogens to facilitate bacteria invasion [6, 10,

15–20]. The best known examples are the Pla protease and

streptokinase (SK). SK was used as the first thrombolytic

agent [21]. Yersinia pestis produces a protease Pla that is a

potent human plasminogen activator and a critical viru-

lence factor [19].

Group A streptococcus (GAS) (Streptococcus pyogenes)

produces SK that can activate human plasminogen. S. py-

ogenes is one of the most common human pathogens that

can cause a variety of human infections from tonsillitis,

scarlet fever and impetigo to life-threatening invasive

diseases, such as streptococcal toxic shock-like syndrome

and necrotizing fasciitis [22]. We have shown that the

interaction between SK and human plasminogen is critical

for GAS pathogenicity [23]. A transgenic mouse line was

established to produce human plasminogen. The human

plasminogen transgenic mice demonstrated significantly

increased susceptibility to GAS infection compared to their

wild-type sibling controls [23]. In order to further test the

roles of human plasminogen interaction with SK, the

human plasminogen transgenic mice were infected with a

GAS strain with inactivated SK gene. The increased sus-

ceptibility of transgenic mice to wild-type GAS was

essentially abolished by inactivating SK in the GAS, sup-

porting the critical roles of plasminogen SK interaction in

GAS pathogenicity [23].

Streptokinase activates human plasminogen by forming

a SK-plasminogen complex. Furthermore, this complex can

also bind human fibrinogen to form a complex to capture

and activate plasma plasminogen on the surface of the

bacteria (Fig. 1) [5, 24, 25]. SK can hijack the host fibri-

nolytic system to penetrate through tissue barriers such as

local vascular thromboses that are formed during bacterial

infection. The local thrombotic barrier could wall off the

site of infection and prevent pathogen spread [5, 10, 26].

The hypothesis was further tested in mice with genetic

alterations in several coagulation factors [27]. Mice with

reduced thrombin generation such as mice with lower

plasma or platelet levels of coagulation factor V or defi-

ciency in fibrinogen demonstrated significantly increased

susceptibility to GAS infection, suggesting importance of

coagulation in host defense against bacterial infection [27].

It was also found that deactivating fibrinogen’s interaction

with the leukocyte integrin aMb2 also increased mice sus-

ceptibility to GAS infection, suggesting that fibrinogen’s

roles in inflammation was also important for host defense

against infections [27].

We hypothesized that interruption of this interaction of

host plasminogen with GAS by decreasing the production

of SK could lead to diminished virulence of GAS. A high

throughput screening was performed to screen small mol-

ecule libraries to identify compounds that can inhibit the

gene expression of SK [28]. A simple, growth-based, tur-

bidimetric, high throughput screening was designed to

search for low molecular weight compounds that inhibited

expression of the SK gene. A GAS strain was genetically

engineered to carry an extrachromosomal plasmid that had

a reporter gene (kanamycin resistance) under the control of

the SK gene promoter. The constitutively active SK gene

promoter enabled the GAS screening strain to grow under

kanamycin. A GAS strain with the same kanamycin

resistance gene driven by a different promoter was used as

counter screening strain. The GAS strains was then treated

with 55,000 compounds to identify lead compounds that

Fig. 1 Components of host

hemostasis system that were

explored as novel antimicrobial

therapeutic targets.

Antimicrobial agents were

developed to interfere with host

fibrinolysis system interaction

with bacteria. Synthetic peptide

was developed to block

activation of contact system to

mitigate infection. APC was

developed to treat sepsis.

Platelets act as antimicrobial

vehicle, secreting PMP

Targeting the host hemostatic system function 67

123

could inhibit the growth of the screening strain under

kanamycin selection, while not interfering with the counter

screening strain’s growth. These lead compounds could

inhibit the SK gene promoter specifically which led to

inhibition of growth of the screening strain under kana-

mycin. These lead compounds were then tested for their

effect on SK production in wild-type GAS [28]. A chem-

ical series of low molecule weight compounds were iden-

tified to be able to inhibit not only the SK gene expression,

but also the gene expression of a number of critical viru-

lence factors in GAS [28]. Virulence factors whose

expression patterns were changed included multiple adhe-

sins, antiphagocytic factors, and cytolytic toxins. Genes

involved in metabolism and energy production were also

affected in addition to virulence factors [28, 29].

The in vivo efficacy of the anti-virulence compounds

were further tested in the human plasminogen transgenic

mice. The lead compound protected mice against GAS

infection [28]. Further studies also demonstrated that ana-

logs of the lead compound could inhibit the gene expres-

sion of variety of S. aureus virulence factors and S. aureus

biofilm formation [30], suggesting that the compounds

inhibited function of an evolutionary conserved virulence

regulator [30]. Genes that are important for S. aureus

biofilm formation and structuring were down regulated as

well as a number of key virulence factors such as protein A

(SPA), Hla, PSMs and sspB [31–38]. Inhibition of these

critical virulence factors suggested the small compounds

can serve as anti-virulence agents [30]. As a result, the anti-

virulence compounds could also protect a host against S.

aureus infection. As a result, this chemical series of com-

pounds could have broad spectrum anti-virulence efficacy

against a number of important human pathogens.

These studies demonstrated that it is feasible to develop

antimicrobial agents by targeting virulence factors and

their interactions with host hemostatic system. Further-

more, since these novel antimicrobial agents function

through a different pathway from antibiotics, they could

serve as viable alternative therapeutic solution to

antibiotics.

Agents targeting contact system pathogen interaction

The contact system is consisted of factor XII (FXII), factor

XI (FXI), plasma kallikrein (PK) and a co-factor, high-

molecular-weight kininogen (HK). When there is blood

vessel injury, the endothelium changes from an anti-

coagulant state to a pro-coagulant state. FXII can be auto-

catalytically activated, leading to activation of PK and FXI

by FXIIa. FXIa triggers the clotting cascade. PK will

cleave HK and release bradykinin (BK), leading to pro-

inflammatory reactions (Fig. 1) [8]. The BK peptide and

domain D5 of HK have antimicrobial activity [39]. Contact

system can be activated on the surfaces of pathogens such

as S. pyogenes, S. aureus and Salmonella, leading to pro-

cessing of HK and release of potent antimicrobial peptides

derived from HK [39]. When mice infected with S. pyog-

enes were treated with the FXII/kallikrein inhibitor to

inhibit the contact system activation, significantly more

bacteria were detected compared with control mice [39].

While activating contact system generates antimicrobial

peptides to the benefit of the host, systemic contact acti-

vation could lead to kinin-induced vascular leakage and

bleeding disorders to the detriment of host. A synthetic

peptide from a region of HK interacting with bacterial

surfaces was designed to block the activation of the contact

system (Fig. 1). The peptide was used to treat mice infected

with invasive GAS [40]. The peptide was able to protect

mice from lung damage. The peptide also significantly

prolonged survival time. When combined with antibiotic

clindamycin, the peptide also increased the survival rate of

infected mice [40]. The studies demonstrated that contact

system activation by bacterial pathogens can be either

beneficial or deleterious to the host. A massive activation of

contact system will result in pathological symptoms such as

coagulopathy in sepsis and septic shock. On the other hand,

contact system activation will generate antimicrobial pep-

tides and interact with alveolar macrophages to attract

neutrophils to eliminate invading pathogens [8]. Manipu-

lating the interaction of contact system with pathogens

could lead to discovery of novel antimicrobial approaches.

Proteins targeting inflammation and coagulation

pathway

Host innate immune system can detect and mount defen-

sive response against invading pathogens. The host pattern

recognition receptors (PRRs) will recognize pathogens and

activate caspase-1. Caspase-1 will then modulate inflam-

matory and host defense response by activating the proin-

flammatory cytokines, leading to variety of local and

systemic immune response including the induction of

fever, attraction of leukocytes to sites of infections and

activation of T helper cell responses [41]. The PRRs form

inflammasomes with caspase-1, ASC (apoptosis-associated

speck-like protein containing a CARD), and upstream

activator. The inflammasomes are key regulators of host

defense against pathogens [41, 42]. A number of pathogens

have evolved ways to either inhibit or evade inflamma-

somes [41, 42]. While the activation of inflammatory

reaction could eliminate or limit the pathogens, it could

also cause serious damage to the host such as tissue, neuron

damages and cell death, contributing to the virulence of

bacteria [41, 42].

68 Y. Xu et al.

123

Sepsis is the overwhelming host systemic inflammatory

response to infections. It has been well documented that

inflammation reaction and hemostatic abnormality are

interrelated [9, 43–45]. Host’s inflammatory response to

bacterial infections can induce tissue factor (TF) expres-

sion in monocyte, leading to activation of coagulation

system [46]. Thrombin generation can induce pro-inflam-

matory cytokine production in cultured monocytes and

endothelial cells [47]. It is believed that cross-talk of

inflammation and coagulation is a major mechanism to

control the host response to invading bacteria. Disruption

of this mechanism causes various syndromes of sepsis such

as disseminated intravascular coagulation (DIC) and mul-

tiple organ failure, resulting in high mortality in patents

[48].

Numerous studies and clinic trials have been performed

to study the effects of anti-inflammation and antithrom-

botic agents on sepsis outcomes. Tissue Factor Pathway

Inhibitor (TFPI) was shown to be able to protect baboons

from lethal intravenous Escherichia coli infusion [49]. Low

TF mice had reduced mortality compared with control mice

in an endotoxemia model [50]. Only recombinant activated

protein C (APC) demonstrated efficacy in clinic trials for

improving 28 day mortality rate of septic shock patients in

2001 [51]. However, drotrecogin alfa (activated) (recom-

binant human activated protein C) was withdrawn from the

market in October, 2011 [52]. A multicenter investigator-

led trial also found no evidence of benefit or harm of

recombinant human APC in adults with septic shock [53].

APC was demonstrated to have anti-inflammation and cy-

toprotective function in addition to anticoagulant function

(Fig. 1) [48, 54–59]. As a result, APC could mitigate the

inflammatory damages by infections. However, withdraw-

ing APC from market demonstrated the complicity of host

response to infections and difficulty to manipulate the

inflammation and coagulation reactions to the advantage of

the patients. As a result, much more effort is still needed to

mine the APC pathway for more effective and broad

spectrum drugs for sepsis.

APC was also shown to ameliorate Bacillus anthracis

lethal toxin (LT) induced lethality in rats [60]. B. an-

thracis is the causative agent of anthrax. LT induced

vascular collapse, vascular shock and coagulopathy, but

no strong inflammatory response [61], which was differ-

ent from the endotoxic shock in sepsis [60, 62]. Rats

injected with LT suffered acute lung injury and acute

respiratory distress syndrome associated with coagulopa-

thy. APC was used to treat coagulopathy in rats and

significantly improved the survival rate of LT-treated rats

[60]. The APC pathway has shown promises as a thera-

peutic target for infectious diseases, in spite of setback

with recombinant human APC.

Platelet as an antimicrobial vehicle

In addition to being a critical component of coagulation

system, platelet also plays important roles in host defense

against infections. Platelets are highly responsive to and

activated by agonists associated with vascular injury,

infection and inflammation. Platelets can interact with

bacteria pathogens either directly or indirectly. A number

of bacterial surface proteins can mediate platelet interac-

tion through interaction with host fibronectin, collagen and

fibrinogen as well as with platelet receptors [63–65]. The

roles of platelet pathogen interaction in pathogenesis of the

infection are still under debate.

While there is evidence that activated platelets can

internalize bacteria [66], the more intensive investigation

of antimicrobial function of platelet focuses on platelet

microbicidal proteins (PMP) which belong to antimicrobial

peptides (Fig. 1). Antimicrobial peptides (AMPs) are

peptides and small proteins with microbicidal activity.

They are parts of the primitive immunity that has been

identified in insects and other non-vertebrate organisms

and crucial for defense against pathogens [67]. Bacteria

and fungi also produce antimicrobial peptides and many of

them have been successfully developed into antibiotics

such as vancomycin and teicoplanin [67]. AMPs also play

critical roles in human immunity. Human tissues and cells

that are exposed to microbes can produce AMPs. Two

classes of the most important AMPs are defensins and

cathelicidins. They are mainly produced by epithelial cells

and neutrophils. AMPs generally have multiple functions

and act in synergy with other components of the innate and

adaptive immune system to defend host against pathogens

[67]. The discovery of PMPs further illustrated the roles of

hemostatic system in immunity. Activated platelet releases

PMPs from a granules (Fig. 1). A subset of PMPs are

conventional chemokines with microbicidal activity and

named as kinocidines [63]. The PMPs exist as native spe-

cies that are proteolytically processed into autonomous

functional domains by thrombin, platelet derived proteases

and proteases activated by tissue injury, phagocytes and

inflammation [63]. PMPs are derived from five lineages

including platelet factor 4 (PF4), platelet basic protein

(PBP) and its protein derivatives connective tissue-acti-

vating peptide 3 (CTAP-3) and neutrophil activating pep-

tide 3 (NAP-2), RANTES (released upon activation,

normal T cell expressed and secreted), thymosin-b-4 (T b-

4) and fibrinopeptides A and B (FP-A and FP-B) [63].

These cationic peptides could disrupt the cell membrane of

bacteria to achieve bactericidal effects [68]. PMPs have

been shown to have bactericidal activity against multiple

pathogens. Variable bactericidal activity was demonstrated

by PMPs against E. coli and S. aureus [69]. PF4 and its

Targeting the host hemostatic system function 69

123

derivatives also demonstrated bactericidal activity against

S. aureus and S. typhimurium [70]. Two synthetic peptides

designed with structure–activity attributes characteristic of

PMPs were tested for their therapeutic potential in an

ex vivo model. E coli was mixed with complex fluid bi-

omatrices consisted for either whole human blood or

plasma and treated with the PMP synthetic mimetic pep-

tides. Both peptides exhibited potent antimicrobial activi-

ties in biomatrices [71]. Platelets also secret human b-

defensins (hBD). b-defensins are primarily expressed by

epithelial cells. Platelets release hBD-1 when stimulated by

a-toxin, a S. aureus toxin to inhibit growth of S. aureus

[72]. Platelet a-granules also secret complement C3,

complement C4 precursor and C1 inhibitor of the com-

plement system [73–75]. Platelets a-granules also contain

factor H that can regulate the activity of the C3 convertase

C3bBb [76]. However, the impact of platelets on comple-

ment activation and regulation is still unknown [74].

There have been studies to show that platelet rich

plasma (PRP) could have beneficial effects in clinic set-

tings including infections and sepsis. Human platelet con-

centrates has been reported to have antimicrobial activity

against bacteria such as S. aureus, E. coli and Klebsiella

pneumonia [77–80]. PRP without leukocytes inhibited the

growth of Enterococcus faecalis, Streptococcus agalactiae

and Streptococcus oralis [81]. It was also shown that

platelets activated by thrombin could significantly inhibit

the proliferation of S. aureus in an in vitro infective

endocarditis vegetation model [82].

However, the mechanism of the bactericidal effect of the

PRP is not fully understood. Burnouf et al. [80] suggested

that the antimicrobial activity was carried by plasma

components, rather than platelets or white blood cells.

Nevertheless, PRP has been used in clinic to help wound

healing. Autologous platelet-rich plasma enriched in

growth factors and antimicrobial proteins, known also as

platelet–leukocyte rich plasma was applied to induce

healing processes of an infected high-energy soft tissue

injury and demonstrated significant antimicrobial effect

[83]. PRP was used to prevent implant associated infec-

tions. Leukocyte- and platelet-rich plasma gel (L-PRP gel)

exhibited antimicrobial efficacy in vivo in a rabbit osteo-

myelitis model [84]. PRP also demonstrated efficacy in

treating of a chronic femoral osteomyelitis case [85].

Conclusion

Due to the rising prevalence of antibiotic resistance among

human pathogens, there is an increasing emphasis on

developing novel antimicrobial agents. The novel antimi-

crobial approaches described above could lead to alterna-

tive therapies to complement current antibiotics.

The roles of host hemostatic system in pathogenesis of

infections are still relatively understudied. The studies

described here demonstrated that host hemostatic system is

a critical player in host response to infections. Under-

standing the mechanism of host hemostatic system inter-

actions with pathogens could provide us with valuable

information to design innovative medical treatments to

combat infectious agents.

Some of the approaches target the pathogen host inter-

actions to diminish the pathogenicity of the pathogens [28,

30, 40]. Some of the approaches try to modulate host

response to infections [51]. Utilizing host’s own antimi-

crobial defense components such as platelets and its anti-

microbial peptides could also open new avenues to develop

novel therapies. These novel antimicrobial agents all

operate through independent pathways from the conven-

tional antibiotics. They can thus work in synergy with

antibiotics to potentially extend the shelf life of current

antibiotics [5]. Significant hurdles still exist to translate the

positive results on the bench top to the bedside. The

complicity of host’s interactions with and responses to

pathogens makes efforts to develop novel effective anti-

microbial therapies daunting tasks. Nevertheless, the

urgency of antibiotic resistance merits greater effort to

exploit hemostatic system to search for novel antimicrobial

approaches.

Acknowledgments The works of the author is supported by Grants

(P01HL573461) from the National Institute of Health. We would also

like to thank all our colleagues on the works discussed in the review.

We apologize to all colleagues whose works could not be cited due to

space limitations.

Conflict of interests The authors states no conflict of interests.

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