CROSSTALK BETWEEN COMPLEMENT AND COAGULATION: SHIGA TOXIN UPREGULATES TISSUE FACTOR GENE EXPRESSION IN ENDOTHELIAL CELLS IN

THE PRESENCE OF COMPLEMENT ACTIVATION

Presented by

Matthew Gerace

Completion Date:December 2013

Approved By:

Thomas Maresca, Biology DepartmentPatricia Wadsworth, Biology Department

Dr. Eric Grabowski, Massachusetts General Hospital

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Abstract

Title: Crosstalk Between Complement and Coagulation: Shiga Toxin Upregulates Tissue Factor Gene Expression in Endothelial Cells in the Presence of Complement Activation.

Author: Matthew GeraceThesis/Project Type: Independent Approved by: Thomas Maresca, Biology DepartmentApproved by: Dr. Eric Grabowski, Massachusetts General HospitalApproved by: Patricia Wadsworth, Biology Department, Commonwealth Honors College

It is well documented that the complement system and coagulation are closely related and that understanding the crosstalk and interplay between the two can have clinical implications in the context of diseases in which complement-coagulation interactions lead to the development of life-threatening complications. Hemolytic uremic syndrome (HUS) is one of those diseases and the pathophysiology of Shiga-toxin (Stx) related HUS is still unclear. We hypothesize that tissue factor expression on vascular endothelium is central to the disorder and is caused by both endothelial activation by inflammatory cytokines derived form the action of Stx and Stx induced complement activation which leads to further augmentation of tissue factor expression. We believe there is a clear relationship between activated complement pathways and the increase in prothrombotic events in the venous endothelium involved in HUS.

Abstract

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Title: Crosstalk Between Complement and Coagulation: Shiga Toxin Upregulates Tissue Factor Gene Expression in Endothelial Cells in the Presence of Complement Activation.

Author: Matthew GeraceThesis/Project Type: Independent Approved by: Thomas Maresca, Biology DepartmentApproved by: Dr. Eric Grabowski, Massachusetts General HospitalApproved by: Patricia Wadsworth, Biology Department, Commonwealth Honors College

It is well documented that the complement system and coagulation are closely related and that understanding the crosstalk and interplay between the two can have clinical implications in the context of diseases in which complement-coagulation interactions lead to the development of life-threatening complications. Hemolytic uremic syndrome (HUS) is one of those diseases and the pathophysiology of Shiga-toxin (Stx) related HUS is still unclear. We hypothesize that tissue factor expression on vascular endothelium is central to the disorder and is caused by both endothelial activation by inflammatory cytokines derived form the action of Stx and Stx induced complement activation which leads to further augmentation of tissue factor expression. We believe there is a clear relationship between activated complement pathways and the increase in prothrombotic events in the venous endothelium involved in HUS.

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Introduction and summary of previous research:

The pathophysiology of Shiga toxin-related hemolytic uremic syndrome(HUS) has yet to

be fully understood. Hemolytic uremic syndrome is described widely as a thrombotic

microangiopathy that can be clinically characterized by a low platelet count, non-immune

hemolytic anemia and acute renal failure. HUS is a disease that combines the effectors of the

complement system and coagulation together as partners in crime. We aim to uncover the

specific mechanisms in which endothelial cells become thrombogenic and how the complement

system interacts with these coagulation events to produce the overall pathophysiology of HUS.

“Approximately 10% of cases are categorized as atypical HUS (aHUS), a disorder that is mainly

associated with genetic or acquired defects in complement regulation” [1]. aHUS is usually

triggered by non-intestinal infections, pregnancy, cancer, and even use of drugs. Unfortunately,

the mortality rate for aHUS remains quite high where most progress to end stage renal failure.

Typical HUS, known as diarrhea-associated HUS (D+HUS) accounts for the majority of cases

and is very serious. Unlike aHUS, D+HUS arises from a gastrointestinal infection caused by

Shiga toxin-producing E. coli (STEC) [2]. It is not currently known why some children will

show a HUS phenotype upon infection with Shiga toxin producing E. coli while others will not.

However, it is thought that complement system dysregulation, differing strains of Stx and

enhanced expression of the receptor in which the toxin acts may play a role.

It is suspected that the damage caused to the endothelium by Shiga toxin is what

eventually leads to the microangiopathic condition along with disruptions with coagulation [2].

With the principal route of infection being consumption of contaminated food and drinking

water, Shiga toxin-producing E. coli are a global health concern and were the main culprits in the

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unprecedented HUS outbreak in Germany in 2011 [1]. The Shiga toxin family belongs to a large

family of plant A:B toxins that includes cholera, pertussis and ricin [3]. Once infected with

STEC, E. coli release Shiga toxin into the gastrointestinal tract where it will cross the epithelium

and enter systemic circulation. The toxin accesses susceptible endothelial cells through binding

surface receptor globotriaosylceramide (Gb3). Once the toxin gains access to the endothelial cell,

it removes an adenine residue within eukaryotic 28S rRNA and inhibits protein translation [4].

Studies have shown that this Shiga toxin mediated inhibition has lead to endothelial damage and

apoptosis [4]. This endothelial damage leads these Shiga toxin activated cells to become

thrombogenic leading to fibrin rich clot formation through a mechanism which is believed to

involve the increased expression of cytokines such as TNF-alpha and clot promoting protein

tissue factor along with a decrease of its inhibitor, tissue factor pathway inhibitor (TFPI) [5]. As

this damage happens primarily in the fibrin-rich microvasculature of the renal glomeruli,

endothelial swelling or edema occurs along with an inflammatory process and narrowing of the

vessel lumen is observed. As the vessel walls become inflamed and swollen, the thrombogenic

endothelial cells form fibrin rich clots systemically in the micro vasculature which consume

circulating platelets producing a drop in platelet count. Hemolytic anemia results as red blood

cells being circulated in the endothelium are fragmented and broken up as they attempt to

squeeze through the fibrin-rich clots and thickening walls resulting in anemia. All of these events

together produce the symptoms associated with HUS including decreased glomerular filtration

rate, proteinuria and hypertension.

Equally important in unraveling the pathogenesis of HUS is understanding how the

immune system interacts with this procoagulent activity, specifically through the complement

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system. The complement system is a branch of the immune system that “complements” specific

immune cells to clear pathogens from an organism. Though the complement system is part of the

innate immune system it can aid the adaptive immune system. The complement system consists

of circulating plasma and membrane bound proteins. Oikonomopoulou 2012 describes

complement system activation as “the specific structural nature of initiators of the complement

pathways, activation does not readily occur unless a change in the microenvironment triggers an

appropriate surface pattern (pathogen or damage associated) and the subsequent binding of

complement factors”[6]. Endothelial injury due to Shiga toxin activation can activate

compliment and lead to inflammation cascades involving cytokines, leukocytes and interleukins

[7]. Complement activation proceeds by three different pathways, the classical, alternative and

mannose binding lectin (MBL) pathway. Each pathway behaves slightly differently with the

mannose binding lectin and the alternative pathways being most relevant to our studies with

HUS. The end game for each pathway is to eventually cause the potent complement protein C3

to go through hydrolysis by aid of enzyme C3 convertase and split into smaller complement

effector proteins C3b and C3a known as anaphylatoxins which eventually lead to the break down

of even more potent complement component C5 (Figure 1A,B). Anaphylatoxins are cytokine-

like polypeptides generated during complement system activation and released at the

inflammatory site [8]. The classical pathway works by supplementing the adaptive immune

system and is initiated by antigen-antibody and C-1 complexes to eventually lead to a pathogen

initiated cascade of reactions that lead to the hydrolysis of C3. The mannose binding lectin

pathway is initiated by the binding of either MBL or ficolin and other associated proteins to an

array of carbohydrate groups on the surface of a bacterial cell [9]. This pathway is just starting to

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be researched in relation to HUS and is at the forefront of complement research currently. Mainly

implicated in aHUS but also D+HUS is the alternative pathway which is constantly activated at

low levels and the most sensitive of all pathways. This pathway is not based on any pathogen

binding antibodies such as the other pathways aiding in its potency and sensitivity of being

constitutively active. The alternative pathway starts with C3 hydrolysis due to spontaneous

internal breakdown of an internal thioester bond [10]. The alternative pathway is the main

culprit in HUS because of its constant activation and sensitivity to being activated further by

damaged endothelium. Once endothelial cells are damaged by Shiga toxin they send out

cytokines and other immune effectors that up-regulate this innate mechanism of immune defense

which results is a huge systematic inflammatory response of the complement system.

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Figure 1. (A) From [35] shows each complement pathways converging in the eventual cleavage of C3 by C3 convertase to smaller more potent anaphylatoxins. (B) From [36] Shows the complement pathways with factor H regulatory protein of the alternative pathway and how each pathway aids in the formation of C5b-9 (Membrane attack complex).

A B

Central to this complement system response are the anaphylatoxins C5a, C3a and C5b-9

which is known as the membrane attack complex. C5b-9 is a product of the alternative pathway

and a complex of complement anaphylatoxins C5b, C6, C7, C8 and C9 (C5b-9) which aggregate

on a cell surface to form a pore with the purposes of lysing the cell (Figure 2). Each one of these

anaphlytoxins produced by the hydrolysis of C3 are the main cross talk between the immune

system and coagulation systems. Binding of these anaphylatoxins to their receptors or integration

of the membrane attack complex onto the endothelial cell have been implicated in increased

inflammatory and prothrombotic responses along with decreased anti-coagulant factors [11]. Our

research aims to elucidate the mechanism in which endothelial cells become activated by Shiga

toxin to become prothrombotic and majorly how the complement system interacts with and up-

regulates coagulation in a HUS system.

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Figure 2. From [27] shows an annotated view of the classical and alternative pathways as well as the events that occur to eventually produce the membrane attack complex.

Figure 3. From [11] The coagulation cascade. The relevant extrinsic pathway demonstrates how tissue factor and other coagulation factors eventually lead to the conversion of prothrombin to thrombin and the formation of fibrin clots.

Previous research has aided to elucidate the mechanism for which endothelial cells

become activated by Shiga toxin or immune mediated events. Central to this prothrombotic

activation is the protein tissue factor (TF). Tissue factor is an important transmembrane

glycoprotein which is a major initiator of the extrinsic pathway of blood coagulation, as soon as

TF comes into contact with circulating factor VII the coagulation cascade is initiated [12] (Figure

3). “Tissue factor is constitutively expressed in sub-endothelial cell such as the vascular smooth

muscle cells leading to rapid initiation of coagulation when the vessel is damaged. Though

endothelial cells and monocytes don’t express TF under physiological conditions, TF is induced

in these cells in response to stimuli such as inflammation and vascular wounding” [13]. Having

TF constitutively expressed in vascular cells that have no direct contact with blood provides a

form of hemostatic protection that is initiated after vessel injury [14]. The main part TF plays in

this cascade is forming a TF-FVIIa complex with circulating factor VII and then proceeding to

activate factor X converting it to factor Xa. Factor Xa is involved in one of the most important

events in the coagulation cascade, “factor Xa catalyzes the conversion of prothrombin to

thrombin, thereby leading to fibrin formation, platelet activation and generation of a

thrombus” [13]. The coagulation cascade has regulatory proteins and molecules that promote

anticoagulation, such as tissue factor pathway inhibitor (TFPI), a protein that inhibits factor Xa.

The “expression of TF in different cells is regulated at many levels. Different components of

bacteria, proinflammatory cytokines, as well as oxidative, hypoxic or temperature stresses induce

TF expression in different cell types by activation of signaling pathways that involve binding of

transcription factors activator protein (AP)-1 and nuclear factor (NF)-kb” [12].

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Shiga toxin begins this activation by binding to the endothelial cells Gb3 receptor.

Nestoridi 2004 showed that cytokines such as TNF-alpha are necessary and potent effectors of

TF induction, showing that shiga-toxin alone was not enough to induce TF production without

the activation of cytokines and immune effectors. This research established a strong role for TF

in the pathophysiology of HUS and demonstrated a clear cross talk between the immune system

and coagulation. Further more it’s been shown that coagulation factors such as thrombin and Xa

may activate cell specific receptors on leukocytes, platelets, and endothelial cells by binding to

protease-activated receptors (PARs). PARs lead to down stream signaling that stimulates

transcription of proinflammatory cytokines [12]. Once Shiga toxin enters systemic circulation

inflammation begins with a cascade of immune effector production and chemotaxis such as the

up-regulation of cytokines which signal and regulate inflammatory processes. When cytokines

such as “TNF-alpha, interleukin-1-beta or CD40 bind to receptors a signal pathway is initiated

that is known to induce TF expression. TNF-alpha has been shown to activate a signaling

pathway that involves MAP kinases p38, p44/42 (ERK) and c-jun terminal NH2-kinase (JNK)

lead to the activation of transcription factors (NF)-kB and EGR-1” [13]. Also, unlike MAP

kinases or protein kinase C, PI3-kinase negatively regulates endothelial TF expression; as a

consequence, inhibition of PI3-kinase or it’s downstream mediators increases TF expression in

response to TNF-alpha, thrombin and vascular endothelial growth factor [14]. This pathway

represents a view of the pathophysiology of typical HUS caused by intestinal infection of Shiga

toxin producing E. coli, however atypical HUS is mostly caused by mutations of complement

regulators and not an enteric infection. This brought up the thought that the complement system

may also play a role in the pathophysiology of typical HUS. Orth 2009 showed strong evidence

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for a role of complement in typical HUS by analyzing complement regulatory proteins. Orth

found that Shiga toxin activated complement via the alternative pathway and was found to bind

to complement regulatory protein, activated factor H [9]. Factor H is a soluble glycoprotein that

circulates in human plasma that regulates the alternative pathway of complement making sure

that the complement system is directed towards pathogens and foreign substances and does not

damage host tissue. Factor H works by slowing the alternative pathway C3-convertase by

promoting dissociation of C3 convertase complexes and plays a role in inactivating C3b,

inhibiting the effect of complement on host cells. Factor H exerts it’s protective mechanism by

binding to glycosaminoglycans that are present on host cells only and not on pathogens. Shiga

toxin has been shown to bind to factor H causing a delay effect of its binding to cell surfaces and

cofactors resulting in enhanced complement mediated inflammation [9].

Activation of the complement cascade has been shown to result in destruction of the

kidney and other organs, not only by direct lysis of cells but also via the release of chemotactic

anaphylatoxins C3a and C5a and the formation of the membrane attack complex C5b-9 [15].

Once the infection with Shiga toxin producing E. Coli is detected or host tissue damaged, the

complement system is activated. Usually sitting dormant as inactive zymogens, the complement

system comes alive amplifying the inflammatory effect of the immune response. These events

are usually associated with an increased propensity for blood clotting [16]. This interface

between the immune response and coagulation in a HUS model is what we are interested in.

With complement regulatory proteins such as factor H being inhibited by Shiga toxin,

complement activation, that is the enhanced cleavage of C3 and functionality of C3b which leads

to the formation of anaphylatoxins C3a,C5a and the membrane attack complex C5b-9 is

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enhanced greatly. This enhanced complement activation not only leads to uncontrolled systemic

inflammation but also to hypercoagulative states [17]. All of these anaphylatoxins have been

shown to aid in the genesis of prothrombotic states. When Shiga toxin binds to the Gb3 receptor

on endothelial cells or platelets it triggers increased expression in cell adhesion protein P-

selectin. P-selectin is found on activated endothelial cells and platelets, when inactive it is stored

in granules called the Weibel-palade bodies which become stimulated to increase levels of P-

selectin when Shiga toxin binds [18]. When P-selectin becomes over expressed it has the

capacity to amplify the activation of complement by acting as a C3b binding protein which

catalyzes the formation of the processes shown in (Figure 4.) resulting in increased generation of

C3a, C5a and the membrane attack complex [19]. Binding of C3a to it’s receptor C3aR has

shown to induce many prothrombotic events on its effector cell. C3a binding has been shown to

lead to increased P-selectin and von Willebrand expression which leads to increased thrombi

formation by increasing the cells ability to bind platelets. When platelets bind to these surfaces

they become active and release ADP and more Von Willebrand factor, leading to further

activation and thrombus formation [20]. “P-selectin is crucial in c-dependent thrombus formation

because functional blocking of P-selectin leads to a marked reduction of C3 accumulation and

limits thrombus formation on Stx-treated cells” [19]. C3a has also been shown to increase the

cleavage of thrombomodulin, a membrane protein responsible for aiding the conversion of

procoagulant thrombin to an anticoagulant enzyme. This C3a mediated decrease in

thrombomodulin also aids in the formation of a hypercoagulative state [19].

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Figure 4. From [19]. Shows the interpretation of how the endothelium could become activated by Shiga toxin and C3a. Shiga toxin binds to receptor Gb3, starting a cascade of cytokine release, P-selectin recruitment, thrombomodulin (TM) shedding and enhancement of formation of C3 convertase complexes.

Figure 5. From [10]. Demonstrates the view of a possible mechanism for how the coagulation cascade could effect complement. Factor IIa or thrombin, has been shown to aid in the breakdown of C3 and C5 to their anaphylatoxins [10]. This diagram also shows clearly which cell type binds each anphylatoxin and which types are known to induce tissue factor upon activation.

Even more potent than C3a are the anaphylatoxins formed from the cleavage of C5 which

contain C5a and the membrane attack complex C5b-9. C5a production is increased with the

expression of P-selectin as P-selectin increases the rate of production of the C3b complex that

aids in the cleavage of C5 (Figure 5). Increased levels of C5a have been associated with an array

of immune and hypercoagulative disorders such as sepsis and disseminated intravascular

coagulation (DIC) [21]. C5a binds to its receptor C5aR, these receptors are present on

neutrophils, monocytes, endothelial cells, mast cells, basophils and possibly more [10]. C5aR

belongs to the rhodopsin family of G-protein coupled receptors and is believed to be involved in

many signaling pathways which lead to increased inflammation and coagulation [8]. C5a is a

strong chemoattractant for granulocytes and shows strong chemotactic activity for monocytes

and macrophages [22]. C5a binding has also been shown to cause oxidative burst in neutrophils,

enhance phagocytosis, reduce neutrophil apoptosis and function as a vasodilator [23,24]. Most

relevant to our research is that C5a has been shown to increase cytokine production and activate

coagulation pathways [24]. It has been demonstrated that C5a activates a number of pathways in

different cell types to produce an array of prothrombotic events. In monocytes the P38 MAPK

pathway and the ERK 1/2 pathway have been implicated in increasing activity of transcription

factor NF-kB leading to increased cytokine production and possibly tissue factor production but

that has yet to be delineated [7, 21]. In endothelial cells: P38, ERK 1/2 and other MAPK

pathways, p44 and p42 Erk kinases, and c-nh2 have been implicated [7, 25]. In neutrophils

similar pathways including protein kinase C (PKC) pathways are also involved [7]. There is a

need to clarify and objectify these pathways in each type of cell, a possible cross talk and their

ability to aid in prothrombotic events. C5a’s binding also has been shown to increase P-selectin

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expression further and the negativity of the membrane, which increases the affinity for platelets

to bind and become active [19]. Adding to this storm of inflammation and coagulation is the

binding of the membrane attack complex C5b-9, whether active or inactive has been shown to

cause very similar pro-inflammatory events as C5a and also a possible direct connection to tissue

factor expression [26]. C5b-9 can form on the endothelium and on platelets, it is not currently

known how C5b-9 localizes on the cell, it is known however to be associated with accelerated

platelet adhesion and activation as well as increased thrombin generation [27]. Thrombin is part

of the extrinsic coagulation cascade (Figure 3) and converts soluble fibinogen to insoluble fibrin

which results in clot formation. Thrombin actually further activates platelets by binding to PARs

on their membrane which leads to more aggregation and thrombin formation as the viscous cycle

continues [27]. C5b-9 like the other anaphylatoxins has shown to increase the expression of P-

selectin and E-selectin, both have very similar functions, but C5b-9 has been demonstrated to

induce tissue factor activity on the endothelium [25]. Though the binding of C5a and the

integration of C5b-9 into the membrane have been shown to induce tissue factor activity, it is not

known how C5b-9 localizes into the cell and what pathways function to lead to the increase in

tissue factor induced by C5b-9. Although some pathways have been identified for C5a little have

been tested with regards to HUS and shiga toxin binding, most studies are done using sepsis or

other inflammatory disorders as models. Our goal is to elucidate the many pathways with which

C5a and C5b-9 function, observe possible cross-talk and amplification signals, and assess the

expression of tissue factor in the presence of complement activation in a HUS model.

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Results and Current Research:

We hypothesize that tissue factor expression on vascular endothelium is integral to Shiga-

toxin related hemolytic uremic syndrome and is driven both by direct effects of Shiga toxin on

endothelium activated by cytokines derived from the action of Shiga toxin in the intestinal tract,

and by Shiga toxin induced complement activation which further augments tissue factor

expression. In context of complement we believe that induction of TF is dependent upon cell

signaling via the mechanisms of Shiga toxin driven increased C3 convertase activity which

results in C3b binding to upregulated endothelial P-selectin forming complexes to aid in

complement activation of endothelium via binding of C5a to its receptor, C5aR and formation of

C5b-9. First we demonstrated that Shiga toxin and TNF-alpha do lead to an increase in

functional TF by measuring TF activity through chromagenic assay (Figure 6.). We performed a

series of experiments (n=40) treating cultured human venous endothelial cell monolayers by

incubating them with TNF-alpha in hours 0-22 and at hours 18-22 10% plasma containing Stx-1

is added. We test for functional tissue factor using a chromogenic assay for factor Xa, since the

functionality of tissue factor depends on converting factor X to factor Xa. The elisa measures the

levels of Xa which are proportional to the level of functional tissue factor. Figure 6. Shows that

cell surface functional tissue factor was increased 3.24-fold (N = 40; p < 0.001) by treatment

with Stx-1 in combination with TNF-alpha, as opposed to TNF-alpha alone [5]. This shows

strong evidence that Shiga-toxin added to cytokine activated cells does induce increased

expression of tissue factor.

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To provide further evidence for the increase in TF expression per treatment of Stx and TNF-

alpha we looked at levels of tissue factor mRNA expression using real-time quantitative RT-PCR

in relation to our treatments (Figure 7). We found that fold changes increased 2.82 ± 0.92-fold

(N=13; p <0.0005) with TNFalpha alone vs. 1.25 ± 0.32-fold (N= 9; p =0.041) for Stx-1 alone

( Figure 7). TNFalpha plus Stx-1 yielded a 6.51 ± 3.48-fold increase (N=17; p <0.0005; Figure

7)[5]. This demonstrates further evidence that tissue factor is upregulated in cytokine activated

cells by Shiga toxin.

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Figure 6. Shows factor Xa production for control, TNF-alpha, TNF-alpha + Stx and Stx-1 alone. The differences between Stx-1 +TNFalpha and TNF-alpha alone is significant (P < 0.001). The differences vs control with TNFalpha(P < 0.0001) and Stx-1 + TNFalpha (P < 0.0001) are also significant. *adopted from (grabowski reference). Bars represent the median.

Looking at sC5b-9 levels by treatment and concentration of Stx-1 we were able to see a clear

increase in sC5b-9 concentration (figure 8.) in the Stx-1 + TNFalpha treated cells over control

and Stx alone treated cells and an increase in complement concentration between 3pM Stx-1 and

10pM Stx-1 but did not increase further at 30pM Stx-1 (Figure 8). This provides supporting

evidence for the relation between activated complement and increased tissue factor expression

and we have reason to believe that augmented complement activation does lead to increased

tissue factor expression. As for the pathways involved and specific action of C5a further research

is needed and currently underway.

In order to determine which pathway was mainly involved in C5b-9 activation we did an

experiment to mediate only specific pathways functioning at specific times. We added 10pM

Stx-1 for 4 hours to culture medium with added 10% plasma or 10% serum, using a T12 plate in

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the absence of endothelial cells. In some wells we add no Stx-1 and no inhibitors, in other wells

we added 25 mM EDTA to block both classical and alternative pathways or 15mM EGTA plus

7.5mM Mgto block only the classical pathway. We measured amount of soluble C5b-9 in ng/ml

via ELISA (Quidel, San Diego, CA) in the endothelial cell supernatants. High concentrations

such as 1439 ng/ml of sC5b-9 were detected when 10% plasma supernatants were incubated with

Stx-1 in the presence of 15mM EGTA and 7.5mM Mg with comparable levels to the incubation

with the positive control with just Stx-1 at 1428 ng/ml, similar results were observed in 10%

serum. In contrast, incubation with Stx-1and EDTA showed only very low concentrations of

sC5b-9 at 313 ng/ml (Table 1.). The evidence presented here shows that blocking the classical

pathway results in nearly as much sC5b-9 formation as without blocking any complement

pathways supports that Stx-1 activates complement primarily through the alternative pathway.

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sC5b-9,ngml

10% plasma 10% plasma

Treatment no STX 10pM Stx-1

no inhibitor 780 1428

25 mM EDTA (Blocks both) 321 313

15 mM EGTA + 7.5 mM Mg (Blocks classical)

895 1439

Table 1. Levels of complement activation (ng/ml) in regards to alternative and classical pathways

Attempting to identify and objectify the pathways and crosstalk involved during

endothelial cell activation and interactions with complement to produce a thrombotic phenotype

is integral to our research. Figure 9 shows our current hypothesized diagram of the signaling

pathways involved in producing a thrombotic phenotype. Activation begins with the binding of

Stx, endothelial cell destruction, release of inflammatory cytokines and activation of the

complement system. the alternative pathway cascade begins with spontaneous hydrolysis of C3,

this change in confirmation allows the cleavage of plasma protein factor B into Ba and Bb. C3

and Bb bind together to form an unstable fluid phase C3 convertase, this unstable dimer becomes

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Figure 9. Our proposed pathway diagram from endothelial cell activation to prothrombotic phenotype in context of the alternative pathway of complement. Activation begins with the binding of Shiga toxin to Gb3 and increased levels of complement activation due to the inflammatory response of Stx binding.

stable and more functional with the help of P-selectin. As more C3 convertase forms, the more

C3b and Bb become available to form the C5 convertase, made up of C3b and Bb, which cleaves

C5 to form C5a and C5b-9. The prothrombotic phenotype is initiated upon Stx binding to a

degree (Figure 6), however as complement anaphylatoxins bind to their G-protein receptors,

kinase pathways become activated. C3a and C5a activate Akt (protein kinase B) which activates

the ERK1/2 pathway which activates transcription factor NFkB or it’s inhibitor EGR-1, NFkB

leads to expression of tissue factor [11]. It is not fully known how C5b-9 localizes on the

membrane but it’s suspected it localizes onto the lipid raft on the membrane [34]. C5b-9

integration, whether active or inactive, leads to activation of NFkb but has been demonstrated to

work through both a PKC pathway and an Akt pathway to activate the ERK1/2 pathway and then

NFkB. Also shown are known cytokine pathways that lead to tissue factor expression [13] and T-

PA mediated Thrombomodulin cleavage. T-PA is a protein that maintains thrombomodulin, an

anti-coagulant regulatory protein, and receives inhibitory signals with C5a and C5b-9 binding

which lead to the break down of thrombomodulin producing a procoagulant state. Increases in

tissue factor expression, thrombin and platelet adhesion creates a potent procoagulant state

derived from shiga toxin binding, cytokine activity and complement activation. Complement

activation and binding shows a clear theme of enhancing this procoagulent state in activated

endothelial cells.

Discussion and Implications for Future research

It is well established that complement activation and other immune effectors are directly

correlated with physiological prothrombotic changes. The immune system and coagulation

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system have long since had evolutionary ties and are much more closely related than most may

think. Understanding the interplay between complement and coagulation has fundamental

clinical implications in the context of inflammatory diseases in which complement-coagulation

interactions contribute to the development of life-threatening complications. Currently, we have

shown support of our hypothesis and past research that tissue factor expression is increased by

shiga toxin binding (Figure 6). Also, we have shown support for that complement (C5b-9)

activation is associated with higher levels of tissue factor expression (Figure 7 and 8). In one

study of a rat model of sepsis researchers were able to show that in vitro C5a and C5b-9 induce

tissue factor expression on endothelial cells and monocytes, furthermore the assembly of C5b-9

on the surface of platelets has been shown to stimulate prothrombinase activity [26] .C5b-9 may

also have ties to regulating anti-coagulative properties because “C5b-9 contains a carboxyl-

terminal lysine similar to other proteins that bind and facilitate activation of plasminogen

(PG)” [27]. This research is interesting because most of the time there is only procoagulant

aspects of complement activation but knowing that C5b-9 has this lysine which can facilitate a

process to clear out fibrin clots may account for why low levels of complement activation doesn't

result in systemic procoagulant activities. It may mean a certain threshold of complement activity

must be reached before we see phenotypes such as the microangiopathy in HUS, also this may be

a reason why higher concentrated 30pM Stx-1 in Figure Y2 didn't show further increase, maybe

this “threshold” for complement activation was reached and anti-coagulative mechanisms were

more predominate promoting fibrinolysis after a certain degree of activation. For concurrent and

future research we intend to measure levels of C3a, C5a and more C5b-9 along with P-selectin

levels with and without neutralizing antibodies to C3, C5 [29] and mannose binding lectin. We

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will assess the relative importance of each putative mechanism using specific complement

neutralizing antibodies, and PKC and MAPKs by using specific inhibitors of ERK, p38, and c-

nh2, along with assessment of the activated transcription factors P47 and NF-kB. The use of

tissue culture incorporating human plasma will permit the presence of clotting factors, absent in

serum, which can become activated by complement. Lastly, we will investigate the mechanisms

of complement activation and enhanced procoagulant TF in a humanized MBL mouse model.

Another interesting aspect about the implication of complement activation in the context of HUS

is the mannose binding lectin pathway. This pathway has just begun to see research with regards

to HUS and preliminary work is seeing some interesting results. It turns out close to 50% of the

population is deficient in mannose binding lectin [30], the main component of the pathway, this

could relate back to the inflammatory storm involving complement in different inflammatory

disorders [30]. It could be that many of these cases of sepsis, HUS, and DIC could be due to over

active complement for those who do have MBL vs those who do not. For example, someone who

is lacking MBL and experiencing a complement mediated inflammatory storm may fare better

than someone who has MBL circulating in their blood. These findings will support therapeutic

approaches to typical HUS which can incorporate inhibitors of tissue factor induction pathways,

complement activation or both. Currently available for treatment of aHUS is Eculizumab, a

humanized monoclonal antibody against complement protein C5, inhibits activation of the

terminal complement pathway. Eculizumab has proven effective, especially when administered

early [29]. Another drug, 3F8 which is an anti-MBL monoclonal antibody has shown even more

promising results and in our studies blocking about 26% more tissue factor procoagulent activity

than eculizumab (E. Grabowski progress report 2013). It is apparent that complement plays an

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important role in the pathophysiology of HUS and has a clear association with the prothrombotic

activity that is such a detrimental manifestation of the disease. As we continue to uncover more

about the association between the complement system and coagulation we will discover more

effective treatments.

Materials and methods:

Cell culture procedure

We use three to five umbilical cords ( courtesy of the MGH Labor and Delivery Service

via the MGH Surgical Pathology service) under IRB protocol for discarded human tissue to

produce primary endothelial cell cultures. We extract endothelial cells by cell culture procedures

outlines in [13] that involve infusing collagenase to break down the venous walls, then

mechanically stimulating the umbilical cord with finally infusing media through the cord into a

collection tube to collect cells. The cells are then spun down in a centrifuge and plated in tissue

culture T-25’s coated with fibronectin. cultured monolayers were studied as passage 3 or 4 cells

on pressed and polished polyvinyl chloride for immunofluorescence (T6s), chromagenic

determinations of factor Xa generation (T12’s), qPCR (T75’s) or as T75s for subsequent

passaging of cells on T6, T12’s and T75s as passage 4 cells.

Reagents used for culture of human umbilical venous endothelial cells (HUVECs)

Medium 199 with Earle’s BSS, L-glutamine and 25 mM HEPES buffer (m199), L-

glutamine, penicillin/steptomycin, fetal bovine serum (FBS) and endothelial cell growth factor

21

(ECGF). Sterile culture plasticware, fibronectin, collagenase (type 2; 210 U/ml), Trypsin

inhibitor (5000units/ml), trypsin-EDTA, and porcine heparin.

Experimental conditions and complement measurements

After 5 or 6 days of culture, HUVEC monolayers were exposed to several conditions to

examine response to inflmatory mediators and to Stx-1. A) control: complete media with no

additives; B) TNF-alpha: tumor necrosis factor alpha alone for 22 hours; C) Stx-1 alone for

hours 18-22; D) Stx-1+TNF-alpha: combination of TNF-alpha for 22 hours with addition of

Stx-1 for hours 18-22. For complement studies, we used experimentally (described above)

treated cell supernatants to be tested for soluble C5b-9 levels (ng/ml) via compliment ELISA

(Quidel, San Diego, CA).

RNA extraction, preparation of cDNA and quantitative real-time PCR

RNA was extracted using the guanidinium thiocyanate-phenol-chloroform isolation

method [13]. Reverse transcription Polymerase Chain Reaction (RT-PCR) was performed to

prepare cDNA using an Eppendorf Realplex thermal cycler (Eppendorf NA, Hauppauge, NY),

cycles consisted of 2 hours at 37 degrees Celsius, followed by 10 minutes at 95 degrees.

Prepared cDNA was stored at -20 degrees Celsius.

Measurement of TF mRNA expression

Tissue factor (TF) expression was measured by real-time quantitative FT-PCR using a

Mastercycler EP. The reactions contained 5ul cDNA, 1.2 uM TF

22

(5'TGTGGCAGCATATAATTTAACTTGGAA-3' and 5'ACCCGTGCCAAGTACGTCTG-3').

The PCR protocol specified 3 min at 95 degrees celsius, followed by 40 cycles of 15 sec at 95

degrees, 45 sec at 54 degrees, and 45 sec at 72 degrees celcius. Samples were analyzed in

triplicate and normalized to GAPDH. Fold-changes in mRNAs ere calculated using the

comparative ct method [28], with fold-changes for a given set of HUVECs derived from the

mean of triplicate sample values.

Chromagenic assay for factor Xa

Functional activity of tissue factor was measured as the production rate of activated

Factor X, in units of fmol/cm2/min, using an elisa calibrated using purified factor Xa. Since

functional tissue factor converts factor X to factor Xa, levels of Xa are a direct measure of

functional tissue factor. Specificity of activity for tissue factor was confirmed in control

experiments by including vs not including (hours 22-23) 30 or 100nM of the previously

described monoclonal antibody directed against human tissue factor.

Statistical methods

Experimental fold changes in mRNA reported as mean ± standard deviation, N denotes

number of separate experiments for a given treatment. Values for procoagulant TF (factor Xa

production) on the other hand, were non-normally distributed. Data for levels of procoagulent TF

are expressed as median and interquartile levels. Statistical analysis was performed using Stata

software (Stata Corp., Collefe Station, TX), version 11.0 for windows. T-test differences between

mRNA fold-changes we performed a log transformation of the fold changes and then carried out

23

a one-way ANOVA. In the case of levels of procoagulent TF, we first carried out a log

transformation of these levels and then carried a one-way repeated measures ANOVA.

Differences were considered significant if the probability (p) value was less than 0.05 (*), 0.01

(**), or 0.001 (***).

Acknowledgments:

All of this work was done as part of three summers spent as a student in the laboratory of Dr.

Eric Grabowski, and under the mentorship of Dr. Grabowski and his colleague, Dr. Rafail

Kushak. The work presented here is part of a larger overall effort by Drs. Grabowski and Kushak

to uncover interactions between complement activation and procoagulent tissue factor activity in

the pathophysiology of epidemic hemolytic uremic syndrome. In addition, I wish to acknowledge

the expert help of Mr. Bohan Liu of the Grabowski Laboratory.

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