Functional Characterization of TAFI mutants Resistant to Activation by , Thrombin-

Thrombomodulin or

by

Mohammad Fahad Miah

A thesis submitted to the Department of Biochemistry in conformity with the requirement for the degree of Master of Science

Queen’s University Kingston, Ontario, Canada (January, 2009) Copyright © Mohammad Fahad Miah, 2009

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen

that acts as a molecular link between the and fibrinolytic cascades. TAFI can

be activated by thrombin and plasmin but the reaction is enhanced significantly when

thrombin is in a complex with the endothelial thrombomodulin (TM). The in

vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is

a thermally unstable that attenuates fibrinolysis by catalyzing the removal of

basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway,

however, is poorly defined and very little is known about the role of different activators

in regulating the TAFI pathway. In the present study, we have constructed and

characterized various TAFI mutants that are resistant to activation by specific activators.

Based on peptide sequence studies, these mutants were constructed by altering key amino

acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities

of all our mutants and found them to be similar to wild type TAFI. We have identified

that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or

thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI

mutants A93V and S94V were predicted to be resistant to activation by plasmin but this

was not observed. The triple mutant, DVV was not activated by any of the

aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to

evaluate the antifibrinolytic potential of our variants and were able to correlate their

effectiveness with their respective activation kinetics. In summary, we have developed

activation resistant TAFI variants that can potentially be used to explore the role of the

TAFI pathway in vivo.

ii Acknowledgements

First and foremost, I am forever grateful to Dr. Michael Boffa for giving me the

opportunity to work on this project. His help, advice, and support have been tremendous

and I have really appreciated him as my supervisor. I would also like to thank Dr.

Michael Nesheim and members of the Nesheim Lab for their advice and help on various issues.

I would also like to acknowledge the support that I have received from various

members of the Boffa/Koschinsky Lab, especially Diann Andrews. Her advice on various

technical issues has been much appreciated. I would also like to thank Susan Johnston for

her time and effort to keep my life in the lab in order.

Finally, I would like to thank my mom, for without her love, support, patience,

faith, and encouragement, I would never achieve such great heights.

iii Table of Contents

Abstract ii

Acknowledgements iii

Table of contents iv

List of Figures vi

List of Tables vii

Abbreviations viii

Chapter 1: Introduction 1

1.1 The coagulation and Fibrinolytic Cascades 1

1.2 Multifunctional Roles of Thrombin 7

1.3 Activation of TAFI 9

1.4 Inactivation of TAFI 11

1.5 Biological Role of TAFI: In Vitro Studies 14

1.6 Biological Role of TAFI: In Vivo Studies 16

1.7 Overall Hypothesis and Specific Objectives 20

Chapter 2: Methods and Materials 21

Chapter 3: Results 32

3.1 Construction and Expression of TAFI Variants 32

3.2 Time courses of thrombin-TM activation 34

3.3 Kinetics of Activation of TAFI variants by Thrombin 38

3.4 Kinetics of Activation of TAFI variants by Thrombin-TM 40

3.5 Kinetics of Activation of TAFI variants by Plasmin 42

3.6 Thermal Stabilities and Enzymatic Activities of TAFI variants 44

iv 3.7 Antifibrinolytic potential of TAFIa variants 46

Chapter 4: Discussion and Conclusion 49

4.1TAFI Pathway and Thrombolytic Therapy 49

4.2 The Kinetics of Thrombin Activation 50

4.3 The Kinetics of Plasmin Activation 55

4.4 The Kinetics of Thrombin-TM Activation 58

4.5 Thermal Stability 60

4.6 Antifibrinolytic Potential of the TAFI variants 61

4.7 Conclusion 62

References 65

v List of Figures

Figure 1-1: The balance between the coagulation and fibrinolytic cascades 2

Figure 1-2: The clotting cascade 4

Figure 1-3: Regulation of the fibrinolytic cascade 6

Figure 3-1: SDS-PAGE analysis of TAFI variants 34

Figure 3-2: Time course of TAFIwt activation by thrombin-TM 34

Figure 3-3: Time course of S94V activation by thrombin-TM 35

Figure 3-4: Time course of A93V activation by thrombin-TM 35

Figure 3-5: Time course of S90P activation by thrombin-TM 36

Figure 3-6: Time course of R92K activation by thrombin-TM 36

Figure 3-7: Time course of DVV activation by thrombin-TM 37

Figure 3-8: Time course of P91S activation by thrombin-TM 37

Figure 3-9: Activation of TAFI mutants by thrombin in the absence of TM 39

Figure 3-10: Activation of TAFI mutants by thrombin in the presence of TM 41

Figure 3-11: Activation of TAFI mutants by plasmin 43

Figure 3-12: Thermal stabilities of TAFI variants at 37 °C 45

Figure 3-13: The antifibrinolytic effect of activation-deficient TAFI mutants 48

vi List of Tables

Table 3-1: Recombinant TAFI variants used in this study 33

Table 3-2: Activation of TAFI mutants by thrombin in the absence of TM 38

Table 3-3: Activation of TAFI mutants by thrombin in the presence of TM 40

Table 3-4: Activation of TAFI mutants by plasmin 42

Table 3-5: Intrinsic stability of TAFIa variants 45

vii Abbreviations:

ε-ACA ε-aminocaproic acid

AAFR Anisylazoformylarginine

ABE1 Anion- 1

ABE2 Anion-binding site 2

APC Active protein C

ATIII Antithrombin III

CPN N

DIC Disseminated intravascular coagulation

EGF Epidermal growth factor

EPCR Endothelial protein C receptor

FDP Fibrin degradation product

GAG Glycosaminoglycans

GEMSA 2-guanidinoethylmercaptosuccinic acid

HBS/Tween HEPES-buffered saline/0.01% Tween detergent

Hipp-Arg Hippuryl-

II Prothrombin

IIa Thrombin kcat Turnover number kcat/KM Catalytic efficiency

KM Michaelis constant

MERGEPTA 2-mercaptomethyl-3-guanindinoethylthiopropannoic acid

viii PAI-1 Plasminogen activator inhibitor 1

PAR activated receptor

PC Protein C

PCI Protein C inhibitor

PCPS 75% Phosphatidyl choline: 25% Phosphatidyl serine

PF4 Platelet factor 4

PMSF Phenylmethanesulphonylfluoride

PPAck D-phenylalanylprolylarginyl chloromethylketone proCPB Procarboxypeptidase B proCPR Procarboxypeptidase R proCPU Procarboxypeptidase U

PSF Penicillin/streptomyocin/fungizone

PTCI Potato tuber carboxypeptidase inhibitor scu-PA Single chain plasminogen activator

SERPINS inhibitors

TAFI Thrombin activatable fibrinolysis inhibitor

TAFIa Active TAFI

TF Tissue factor

TFPI Tissue factor pathway inhibitor

TM Thrombomodulin

TME456 Thrombomodulin EGF 4th, 5th, 6th, domains t-PA Tissue plasminogen activator u-PA Urokinase plasminogen activator

ix VFKck Valylphenylalanyllysyl chloromethylketone

VWF von Willebrand Factor

x Chapter 1 Introduction

1.1 The Coagulation and Fibrinolytic Cascades

The balance between the activities of the coagulation and fibrinolytic cascades is crucial to the development of the haemostatic response at the site of injury, while maintaining blood in a fluid state elsewhere in the circulation. Injury to the vessel wall can initiate events mediated by both cellular components and soluble plasma proteins that lead to clot formation and dissolution [1]. Reactions of the coagulation cascade are responsible for the formation of the clot at the site of the injury while the activities of the fibrinolytic cascade can degrade the clot and remove it from circulation. The balance between the coagulation cascade and the fibrinolytic cascade is a tightly regulated process (Figure 1-1). Under normal conditions, the coagulation and fibrinolytic cascades are regulated such that the clot is contained temporally and spatially [2]. Imbalances within haemostasis can result in a tendency towards inappropriate clot formation that can lead to heart attacks and strokes or insufficient clot formation and protection, resulting in a tendency to bleed, as is observed in haemophilia.

The initial events of haemostasis are mediated by tissue components, plasma proteins, and receptors on platelets [3]. When the vascular wall is disrupted, flowing blood is exposed to the subendothelial matrix and the medial and adventitial layers of the vessel wall, where collagen and tissue factor (TF) associated with vessel wall can initiate platelet aggregation and the coagulation system. Platelet adhesion at the damaged site can be achieved through the plasma protein known as von Willebrand factor (VWF) [4].

1

Figure 1-1: The balance between the coagulation and fibrinolytic cascades. Thrombin generated by the coagulation cascade can catalyze the conversion of fibrinogen into fibrin. Plasmin generated by the fibrinolytic cascade can degrade fibrin into fibrin degradation products (FDP). Thrombin in a complex with its cofactor thrombomodulin can activate protein C (PC) and thrombin activatable fibrinolysis inhibitor (TAFI). Active protein C (APC) can downregulate thrombin generation by inactivating upstream coagulation factors. Active TAFI (TAFa) can downregulate plasmin generation by removing C-terminal from partially degraded fibrin. Thrombin generated by the coagulation cascade can regulate fibrinolysis through the activation of TAFI. (Figure adapted from Nesheim ME [5].)

2 VWF can bind to both collagen and platelet membrane glycoprotein Ib-V-IX

(GPIB-V-IX) thereby anchoring platelets at the site of injury. Platelets can also bind to

collagen through the glycoprotein Ia-IIa (GPIa-IIa), which can further stabilize platelet

adhesion at the damaged site [6]. Platelet adhesion is also accompanied by its activation,

which results in significant morphological changes that can expose negatively charged

phospholipids needed for the assembly of various coagulation enzyme complexes [7].

Morphological changes also induce platelets to release the contents of their α and dense

granules. α-granules contain a wide array of proteins that include platelet factor 4, PDGF,

EGF, fibrinogen, VWF whereas dense granules usually contain small molecules such as

ADP, calcium, and serotonin [7]. These molecules can activate nearby platelets, support

the initial reactions of haemostasis and promote contraction of the smooth muscle cells of

the vascular wall, thereby reducing the flow and loss of blood from the damaged site [7].

Activation of platelets also exposes the glycoprotein IIb-IIIa (GPIIb-IIIa) that can bind to

fibrinogen, VWF, fibronectin, which acts as a bridge between other activated platelets

and allow platelets to aggregate and form the platelet plug at the site of injury [8].

As the platelets are being recruited, TF located within the damaged vessel wall

can form a complex with the plasma protein factor VIIa (FVIIa) and Ca2+ to initiate the

coagulation cascade (Figure 1-2) [9]. It is not fully understood how factor VII (FVII) is initially activated but it is estimated that under normal conditions, plasma contains about

1% FVIIa and 99% FVII zymogen [10]. The TF-FVIIa-Ca2+ complex often referred to as

the “extrinsic Xase” that can initiate haemostasis by activating plasma factor IX (FIX)

and factor X (FX) into factor IXa (FIXa) and factorXa (FXa), which can feedback

3

Figure 1-2: Clotting Cascade. Damage to the endothelium can expose surface exposed tissue factor to the flowing blood. Tissue factor can interact with factor VIIa to convert factor IX into factor IXa and factor X into factor Xa. Factor IXa can also activate factor X into factor Xa. Factor Xa can convert factor II (prothrombin) into factor IIa (thrombin), which can catalyze the conversion of fibrinogen into fibrin. The tissue factor pathway inhibitor (TFPI) can regulate the amount of thrombin generated by forming inhibitory complexes with factor VIIa, tissue factor, and factor Xa. Factors VIII, V, and XI can be activated by thrombin and they can contribute to the positive feedback mechanism of thrombin generation. The thrombomodulin protein C protein S pathway can inactivate factors Va and VIIIa. Antithrombin III can inactivate factors XIa, IXa, Xa, IIa and these inhibitory reactions can be accelerated by heparin sulphate. Tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) can convert plasminogen into plasmin. Plasmin can degrade the fibrin clot that is deposited by the clotting cascade. (Figure adapted from Rosenberg AD [2].)

4 amplify the system by activating FVII. As free , FIXa and FXa can convert FX

into FXa and prothrombin (FII) into thrombin (FIIa) respectively but these reactions are

highly inefficient. However, the small amount of FIIa that is generated can convert

circulating plasma factor V (FV), factor VIII (FVIII) and factor XI (FXI) into their

activated forms [11, 12, 13]. FVa acts as a cofactor with FXa and Ca2+ to form the

prothrombinase complex, which is highly efficient in converting FII into FIIa. Similarly,

FVIIIa acts as a cofactor with FIXa and Ca2+ to form the highly potent “intrinsic” Xase

complex, converting FX into FXa. FXIa can activate FIX, which can result in the

generation of more “intrinsic” Xase complex and a subsequent burst or amplification in

FIIa production [14, 15, 16]. Proteins of the tenase, prothrombinase, as well as FX and

prothrombin, can interact with the negatively charged phospholipid membrane through their gamma-carboxy glutamic acid residues (Gla domain). The assembly of enzymes, cofactors, and substrates on phospholipid surface can allow for efficient enzyme substrate interaction and catalysis as well as protection of these complexes from anticoagulants

[17]. The rate of thrombin generation by the coagulation cascade can be regulated at many different levels. Anticoagulants such as tissue factor pathway inhibitor (TFPI), protein C and protein S system, antithrombin III (ATIII) can restrict the formation of thrombin and the haemostatic plug [18].

Fibrinolysis is responsible for the dissolution of the fibrin clot at the site of injury.

Normal fibrinolytic response is necessary for proper wound healing, angiogenesis, and vessel recanalization after clot formation [19]. The fibrinolytic cascade can be initiated when endothelial cells release tissue-type plasminogen activator (t-PA) after stimulation by thrombin or urokinase plasminogen activator (u-PA) when perturbed. (Figure 1-3) [20,

5 Figure 1-3: Regulation of the Fibrinolytic Pathway. Plasminogen activator inhibitor 1 (PAI-1) can inhibit tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). Plasminogen and t-PA or u-PA can form a ternary complex with fibrin that can promote the formation of plasmin and subsequent lysis of the fibrin clot (Fibrin degradation products). Antiplasmin, either free or bound to fibrin, can inactivate plasmin. Activated FXIII can form isopeptide bonds between fibrin polymers to form cross-linked fibrin as well as bind antiplasmin in the fibrin clot. (Figure adapted from Kohler HP, Grant PJ [21].)

6 22]. In the presence of fibrin, which can act as a template to bind both t-PA and plasminogen, t-PA can efficiently convert plasminogen into plasmin, which can then cleave fibrinogen or fibrin into soluble fibrin degradation products (FDPs). As with the coagulation cascade, fibrinolysis is subject to multiple regulatory mechanisms. Serine protease inhibitors (SERPINS) such as α-2-antiplasmin can directly inhibit plasmin with

a rapid rate of association. Other SERPINS include plasminogen activator inhibitor-1

(PAI-1) that can inhibit both t-PA and u-PA. Plasmin can also upregulate its own

production through certain positive feedback mechanisms. Initial partial degradation of

fibrin by plasmin results in the appearance of carboxyl-terminal residues in fibrin,

as plasmin degrades fibrin by cleaving after certain lysines. These exposed carboxyl

terminal lysines act as ligands for the lysine binding sites of plasminogen and t-PA,

which can greatly enhance the efficiency of t-PA mediated activation of plasminogen

[21-26].

1.2 Multifunctional Roles of Thrombin

Thrombin is a multifunctional enzyme and the ultimate proteinase in the

coagulation cascade, acting specifically on a wide array of haemostatic substrates. Once

generated, thrombin can catalyze the removal of fibrino-peptides A and B from

fibrinogen which results in the formation of fibrin monomers and fibrin polymers that can

consolidate the initial haemostatic structure, the platelet plug, formed at the site of injury

[27]. Thrombin also catalyzes its own generation by activating FV, FVIII, and FXI and it

also stimulates platelet aggregation at the site of injury by cleaving membrane bound

protease-activated receptors (PARs 1, 3, 4) on platelets [27-29]. Thrombin is not only

7 responsible for the formation of the clot but it can also act to stabilize it in a number of

ways. Thrombin can activate plasma factor XIII (FXIIIa), which can catalyze the

formation of covalent isopeptide bonds between adjacent fibrin polymers thereby

crosslinking them and making fibrin polymers more resistant to lysis [30]. Activated

factor XIII can also cross link α-2-antiplasmin within the fibrin meshwork in order to regulate fibrinolysis [31]. The initial rate of thrombin formation can also affect the outcome of the clot architecture and consequently its stability. It has been demonstrated

that clots formed during high rates of thrombin formation tend to be less porous and more

resistant to lysis, whereas clots formed during low rates of thrombin formation are more

prone to lysis [32]. Furthermore, thrombin has been shown to protect the fibrin clot by

inactivating plasminogen activator known as single chain urokinase plasminogen

activator (scu-PA) [33].

Thrombin can escape from the fibrin meshwork and can form a high affinity

complex with the endothelial cell receptor thrombomodulin (TM) possibly located

adjacent to the thrombus [27]. In the presence of TM, thrombin loses its procoagulant

properties and gains anticoagulant and antifibrinolytic properties. Thrombin-TM complex can efficiently activate protein C [34]. Activated protein C (APC) in conjunction with its cofactor protein S can cleave and inactivate coagulation factors FVa and FVIIIa and downregulate thrombin generation. Protein C/S system and along with ATIII/heparin ensures that the procoagulant activities and deposition of fibrin are localized only at the site of injury. Thrombin escaping from the fibrin meshwork are rapidly mopped up by

either TM or ATIII [27].

8 A recent addition to the list of proteinase that can affect haemostasis is the plasma metallo-carboxypeptidase precursor termed thrombin activatable fibrinolysis inhibitor

(TAFI) also known in the literature as procarboxypeptidase U (proCPU) in reference to the protein being thermally unstable, procarboxypeptidase R (proCPR) for its preference for C-terminal ; and plasma procarboxypeptidase B (proCPB) for its close homology to pancreatic [35-38]. As the name suggests, Thrombin can activate TAFI either as a free enzyme or in a complex with TM [39]. The fibrinolytic enzyme plasmin can also activate TAFI. Activated TAFI (TAFIa) has been shown to interfere with the plasmin mediated positive feedback mechanism of plasmin generation, thereby attenuating fibrinolysis [40]. Furthermore, TAFI has been postulated to be a molecular link between the coagulation and fibrinolytic cascade since thrombin generated by the coagulation cascade can affect fibrinolysis through the activation of TAFI [5].

1.3 Activation of TAFI

The biochemical properties of TAFI have been extensively characterized [41-44].

Human TAFI consists of 401 amino acids that migrates with an apparent molecular weight of 60,000 (theoretical Mr ~ 46,000) on a SDS-PAGE gel due to the presence of N- linked glycosylation at four sites on the activation peptide [45]. The activation peptide consists of 92 amino acids and it shields substrates from accessing the . The catalytic domain consists of 309 amino acids, which contains residues thought to be important for binding zinc and substrate catalysis that are similar to pancreatic A1, A2, and B. The activation of TAFI occurs by proteolytic cleavage at the Arg92-Ala93 bond by thrombin or plasmin, which results in the dissociation of the

9 activation peptide and exposure of the active site. Activated TAFI (TAFIa) has been

shown to have a carboxypeptidase B like activity, catalyzing the removal of basic

carboxyl-terminal amino acids arginine or lysine from various peptides and proteins [35-

39].

Thrombin alone is a poor activator of TAFI. However, thrombin in a complex

with thrombomodulin can increase the efficiency of this reaction by almost 1250 fold

[39]. Unlike protein C activation by thrombin-TM, which can be further enhanced by

endothelial protein C receptor (EPCR), no such receptor has been found for the activation

of TAFI. As such, thrombin-TM, complex has been postulated to be the physiological

activator of TAFI in vivo. However, in vitro studies have demonstrated that the half

maximal inhibition of clot lysis time can be achieved at a TAFIa concentration of about 1

nM, which corresponded to less than 2% of the total plasma concentration of TAFI

zymogen [35, 46]. Therefore, it is reasonable to expect that activation of TAFI by thrombin alone can have significant impact on fibrinolysis in the presence of high

concentrations of thrombin generated by the feedback amplification system inherent of

the coagulation cascade. Indeed, an effect of TAFI on fibrinolysis has been demonstrated

in vitro in the absence of thrombomodulin, in a manner dependent on the presence of an

intact intrinsic pathway of coagulation [47, 48]. Moreover, an impaired intrinsic pathway,

which can be achieved using antibody that prevents activation of factor XI, can attenuate

the inhibitory effect of the TAFI pathway on thrombolysis [49].

The fibrinolytic enzyme, plasmin, can also activate TAFI by cleavage at Arg92

[50]. This reaction, however, can be accelerated in the presence of glycosaminoglycans

(GAG) such as heparin [51]. In vitro experiments have revealed that the catalytic

10 efficiency of thrombin-TM mediated cleavage is at least 10 fold greater than plasmin-

heparin [34, 51]. Plasmin-heparin mediated activation of TAFI can become significant

when injured sites on the blood vessel can expose significant amounts of

glycosaminoglycans from the extracellular matrix. Endothelium from these sites may also

be devoid of TM and therefore, plasmin-GAG mediated activation of TAFI becomes

important with respect to clot stability at these sites [51].

Taken together, the available data suggest that, in vivo, activation of TAFI could

be performed by thrombin, thrombin-TM, or plasmin. However, the relative significance

of the respective activators in vivo is unknown. Different vascular compartments differ in

their hemodynamic properties, in the phenotype of the endothelium, in the contribution of

platelets, and in the ratio of the volume of blood to the surface area of the endothelium

[52, 53]. Thus, it is likely that the contribution of the aforementioned activators in

regulating the TAFI pathway in vivo could be a function of the site of thrombosis and extent of activation of the coagulation or fibrinolytic cascades.

1.4 Inactivation of TAFIa

The intrinsic thermal instability of TAFIa has been described as the primary mode

of downregulation of the enzyme [54]. Fluorescence studies revealed that over time at

physiological temperatures, TAFIa undergoes two structural changes and it is the slower

of the two changes that has been associated with the inactivation of TAFIa. Furthermore,

thermodynamic interpretation of the data indicated that TAFIa underwent extensive

changes in its structural arrangements to become the inactive conformer. Large positive

enthalpy and entropy changes are implied by the data, which suggested that numerous

11 non-covalent bonds had to be broken and that inactive TAFIa adopted a less ordered possibly “unfolded” structure [54].

The structural basis for the intrinsic thermal stability of TAFIa has been inferred

from the crystal structures of TAFI [55, 56]. The crystal structures show that TAFIa

stability is related to the dynamics of the 55 residue segment (residues 296-350), which also includes the wall of the active site. In the zymogen form, this region is stabilized by

interactions with the activation peptide and the dynamics of this region can be markedly

increased when the activation peptide is dissociated from the TAFIa molecule. The

increased mobility of this region can drastically increase the plasticity of the entire TAFIa

molecule and consequently result in the complete unfolding of TAFIa and exposure of

the cryptic thrombin cleavage site at Arg302. In line with this view, the reversible TAFIa

inhibitor GEMSA, which has been previously shown to stabilize TAFIa, can reduce the

dynamic nature of this region once bound to the active site of TAFIa and stabilize the

entire molecule [55, 56].

Proteolytic cleavage has also been implicated in the inactivation of TAFIa and also for TAFI. For example, proteolytic cleavage and inactivation of TAFIa by thrombin

at Arg302 has been observed but it can only occur after thermal inactivation of TAFIa

[54, 57]. Whether this secondary mode of TAFIa inactivation has any physiological

relevance is not yet known. Interestingly, plasmin has also been shown to cleave TAFIa

at Arg302, Lys327, and Arg330 either before or after thermal inactivation of the enzyme,

although thermal inactivation of TAFIa definitely accelerates these cleavages. In

addition, it appears that plasmin, when present in large amounts, can cleave at Lys327

12 and Arg330, rendering a truncated form of TAFI that will not develop carboxypeptidase

activity following cleavage at Arg92 [50].

Although endogenous fast acting inhibitors of TAFIa have not been found in vivo,

exogenous inhibitors of TAFIa have been identified based on TAFIa active site sequence

homology with other carboxypeptidases [58]. These inhibitors have been described as reversible competitive inhibitors of TAFIa, which includes the lysine analog ε-

aminocaproic acid (ε-ACA) and arginine analog 2-mercaptomethyl-3-

guanindinoethylthiopropannoic acid (MERGEPTA) and 2-

guanidinoethylmercaptosuccinic acid (GEMSA). Potato tuber carboxypeptidase inhibitor

(PTCI), which is a globular peptide, can also inhibit TAFIa. The mechanism of inhibition

is expected to be similar to the inhibition of by PTCI [59].

An interesting phenomenon was observed when reversible competitive inhibitors

were used against TAFIa. Apart from inhibition of TAFIa, it was also noted that

saturating concentrations of these inhibitors were able to stabilize TAFIa at physiological

temperature [60]. Further experiments revealed the paradoxical finding that, depending

on the dose of the reversible inhibitor, fibrinolysis can either be promoted or inhibited

[61]. At high concentrations of the inhibitor, all of the TAFIa would be expected to be inhibited and therefore this would expect to promote fibrinolysis. On the other hand, when smaller amounts of the inhibitor are used, only a fraction of the TAFIa population would be bound by the inhibitor. The remainder would be allowed to exert their antifibrinolytic activity. However, as these uninhibited TAFa underwent thermal deactivation, equilibration between the two pools of TAFIa would force the release of more TAFIa from the inhibitor stabilized pool. In this way, not all the TAFIa is subjected

13 to thermal inactivation at one time. Since only a small amount of TAFIa is needed to exert antifibrinolytic effect, this method of inhibitor mediated gradual release of TAFIa can actually result in enhanced inhibition of fibrinolysis [61].

1.5 Biological Role of TAFI: In Vitro Studies

In vitro studies using clots made either from platelet poor plasma or purified components have shown that the TAFI pathway can inhibit fibrinolysis both in the presence and absence of thrombomodulin [35, 47, 48, 59]. In the absence of thrombomodulin, however, the intrinsic coagulation pathway must be fully functional for the TAFI pathway to exert its antifibrinolytic effect [49]. The antifibrinolytic activity of

TAFIa has been attributed to its ability to remove C-terminal lysines and arginines from peptides and proteins [40]. The removal of these basic amino acids by TAFIa interferes with the fibrinolytic activities of plasmin in a number of ways.

Firstly, removal of these basic amino acids can interfere with plasmin generation

[62]. Plasmin cleavage of fibrin can generate partially degraded fibrin with exposed lysines and arginines. In addition to fibrin, these exposed lysines and arginines can act as a template and provide extra binding sites for t-PA and plasminogen and enhance plasminogen activation. Furthermore, partially degraded fibrin also acts as a cofactor in the conversion of Glu-plasminogen to Lys-plasminogen by plasmin. Lys-plasminogen in turn is a much better substrate for t-PA. Hence, removal of these C-terminal lysines and arginines can attenuate the positive feedback steps in plasmin generation.

Secondly, the removal of the C-terminal lysines and arginines can decrease the protective effect of partially degraded fibrin against antiplasmin inhibition of plasmin

14 [63]. In the absence of fibrin or partially degraded fibrin, plasmin is inhibited by

antiplasmin very rapidly. However, this effect is reduced significantly in the presence of

fibrin or partially degraded fibrin with exposed lysines and arginines because they

provide a surface to which plasmin can bind and thereby prevent lysine mediated

interaction with antiplasmin. In the presence of TAFIa, however, this effect is decreased

and plasmin becomes more vulnerable to inhibition by antiplasmin.

Thirdly, TAFIa may be able to mediate its antifibrinolytic activity by preventing

the cooperative cleavage of fibrin [64,65]. It has been shown that complete digestion of

fibrin by plasmin is not necessary to solubilize the fibrin clot. Plasmin cleavage of fibrin

can occur on a few locations on the fibrin meshwork that can release FDPs as a family of

molecules of various molecular weights and solubilize the clot. Cleavage through the α-,

β- and γ-chains in the plasmin-sensitive coiled-coil domains of fibrin appears to be a cooperative process where carboxyl-terminal lysines generated by plasmin cleavage of one of the chains can attract additional plasmin molecules to accelerate digestion of the remaining chains. Presumably, TAFIa can prevent this by removing the newly-generated carboxyl-terminal lysines. However, TAFIa, by removing the C- terminal lysines and arginines, can prevent the accumulation of t-PA and plasmin and thereby forcing plasmin to be spread over the entire fibrin network to make more cleavages than necessary on the fibrin surface to completely dissolve the clot.

The removal of basic amino acids from partially degraded fibrin by TAFIa

constitutes the fundamental aspect of TAFIa’s antifibrinolytic activity. However with

regards to the fibrinolytic system, TAFIa has been shown to mediate its antifibrinolytic

activity by a threshold dependent effect [66]. This implied that as long TAFIa

15 concentration remained above certain threshold value, fibrinolysis would be inhibited or minimized. The time period TAFIa concentration remained above the threshold however depended on the initial TAFIa concentration and its half-life. TAFI variants that have greater stability, has been shown to exert greater antifibrinolytic effect [66]. Due to the intrinsic instability of TAFIa, the concentration of TAFIa would fall below the threshold value and fibrinolysis would resume again. Thus, TAFIa is not considered to be a true inhibitor of fibrinolysis but it simply acts to prolong the lifetime of the clot temporarily.

Apart from acting on partially degraded fibrin, TAFIa has also been shown to remove carboxyl-terminal arginines from vasoactive peptides such as bradykinin and anaphylatoxins C3a and C5a resulting in their inactivation [67, 68]. These substrates of

TAFIa implied that the coagulation pathway could potentially also affect the inflammatory response or the complement pathway through the activation of TAFI.

Interestingly, plasma also contains another carboxypeptidase termed carboxypeptidase N

(CPN), which is constitutively active, and stable that can also modify the aforementioned inflammatory peptides. With the discovery of TAFI, it appears that regulation of these peptides can also be achieved locally at the site of injury.

1.6 Biological Role of TAFI: In Vivo Studies

Evidence for a role of the TAFI pathway in arterial and venous thrombolysis has been demonstrated in various animal models. For example, inhibition of factor XI activity leading to decreased TAFI activation was shown to result in enhanced lysis of jugular vein rabbit clots [49, 69]. Furthermore, the administration of potato tuber carboxypeptidase inhibitor (PTCI), which specifically inhibits TAFIa, in conjunction

16 with t-PA, was shown to enhance both arterial and venous thrombolysis in vivo [49, 69,

70, 71]. To further investigate the roles of the TAFI pathway in vivo, mouse strain in which the TAFI gene has been inactivated by homologous recombination have been generated [72]. Curiously, TAFI knockout mice did not exhibit embryonic lethality.

Furthermore, these mice reproduced, developed and grew normally and did not exhibit any haemostatic abnormality although clots formed from these mice in vitro did lyse

more rapidly than normal. When presented with acute haemostatic challenges, such as

thrombin induced thromboembolism, endotoxin induced disseminated intravascular

coagulation (DIC), these TAFI knockout mice also performed in a similar manner as the

wild type strains [72].

An effect of the TAFI pathway, however, has been observed in mice in which the

plasminogen system has been compromised by hemizygosity [73]. These mice have half

the normal amount of plasminogen and partial deficiency of TAFI (plasminogen+/-,

TAFI+/-) or total deficiency of TAFI (plasminogen+/-, TAFI-/-) in these mice has been

shown to promote fibrinolysis compared to wild-type (plasminogen+/-, TAFI+/+) in a

pulmonary clot lysis model and increased thioglycollate induced leukocyte migration to

the peritoneum [73]. The role of TAFI in cell migration is postulated to depend on its

capacity to remove carboxyl terminal lysines, preventing plasminogen from binding to

cell surfaces as well as dampening plasmin generation as previously described. These

effects were hypothesized to hamper matrix degradation by plasmin, necessary for cell

migration [74]. In support of this view, it has also been reported that TAFI can promote

the downregulation of matrix proteolysis and tube formation in cultured microvascular

endothelial cells, thereby attenuating cell migration [75].

17 In a pulmonary embolism model, where batroxobin was used to initiate clot

formation, TAFI deficiency resulted in a lower retention of fibrin in the lungs, which

suggested increased rate of fibrinolysis, compared to wild type variants [76]. Finally,

TAFI deficiency was also shown to result in impaired wound healing in both cutaneous

and colonic anastomosis wound healing models. In this case, the absence of TAFI,

probably led to increased plasminogen activation and plasmin activity, which increased

degradation of the extravascular fibrin required for appropriate cell migration during

would healing [77].

Although TAFI can be activated by thrombin, plasmin, and thrombin-TM, their relative contribution in regulating the TAFI pathway in vivo is unknown. Interestingly,

regulation of haemostasis has been shown to depend on vascular compartment. For

example, it has been demonstrated that TM plays a significant role in the anticoagulant activities in the blood vessels of the lung, whereas in the blood vessels of the liver, it has

a very minor role [78]. Further studies have revealed that disrupting the anticoagulant

functions of TM in a mouse model resulted in the increased deposition of fibrin only in the lungs, heart, and spleen compared to wild type variants [79]. These findings strongly

suggest that haemostasis is most likely regulated in a tissue-specific manner.

Activation of TAFI and its antifibrinolytic effect in vivo, is also likely to be

sensitive to the dynamics of coagulation, the relative concentrations of various activators,

inhibitors, co-factors and the type of blood vessel in which haemostasis is initiated.

Although the full extent of these effects are not fully understood, preliminary in vitro

studies have revealed important clues. For example, the relative concentrations of TM

can be an important factor that can determine the extent of TAFI activation by thrombin.

18 The TM concentration on endothelial cells has been shown to vary from 1 nM in large

arteries to 100 nM in capillary beds [80]. In vitro studies have demonstrated that higher

concentrations of TM inhibited TAFI activation because of increased activation of

protein C, thereby promoted a profibrinolytic state. Conversely, lower concentrations of

TM were shown to promote TAFI activation and an antifibrinolytic state [81]. Further

layers of complexity are provided by the differential relative distribution of TM and

EPCR across the endothelium. EPCR has been shown to enhance protein C activation by

thrombin-TM. Interestingly, EPCR expression on endothelial cells was shown to be the

opposite of TM, in that EPCR is relatively abundant on large vessels and negligible

quantities are present in small vessels. Therefore in the presence of EPCR, protein C

activation can be performed efficiently in the presence of lower concentrations of TM,

and shifting the balance towards a profibrinolytic state [82, 83].

Other factors that can affect overall TAFI activation are platelet factor 4 (PF4)

and protein C inhibitor (PCI). PF4 released by the α granules upon platelet activation can

enhance protein C activation and inhibit TAFI activation by thrombin-TM. However,

thrombin activation of TAFI in the presence of PF4 is unaffected [84, 85, 86]. Finally,

PCI, which is a plasma serine protease inhibitor, can inhibit anticoagulant factor APC,

and procoagulant factors thrombin and FXa. The inhibition of thrombin by PCI, however,

is enhanced when thrombin is in a complex with TM [87].

In summary, these examples highlight that the activation and regulation of

fibrinolysis by TAFI can depend on the complex interplay between various known and

unknown factors and further in vivo studies are needed to fully elucidate the

physiological role of the TAFI pathway.

19 1.7 Overall Hypothesis and Specific Objectives

To begin to understand and generate a comprehensive knowledge about the TAFI

pathway in vivo, TAFI mutants specifically impaired in activation with respect to thrombin, plasmin, and thrombin-thrombomodulin will be needed. It has been previously demonstrated that the endothelial cells of the vascular system display considerable

phenotypic variation to accommodate widely varying blood flow, pressure and the

distinct needs of individual tissues [88]. Thus, the regulation of the TAFI pathway will be

a function of its different activators and the endothelial cell heterogeneity within the

vasculature. Hence, these mutants will be used to study the overall hypothesis that

different activators of TAFI can regulate TAFI activation in different vascular

compartments. The aim of the current study is to develop TAFI mutants resistant to

activation by thrombin, plasmin, and thrombin-TM and perform their complete

biochemical characterization.

The specific objectives of the current study are as follows:

1. Construction and expression of various TAFI mutants.

2. Characterization of activation kinetics of the TAFI mutants with respect to their

activation by thrombin, thrombin-thrombomodulin, and plasmin.

3. Characterization of the enzymatic activity and stability (i.e. half-life) of each

enzyme.

4. Evaluation of the antifibrinolytic potential of each mutant using clot lysis assays.

20 Chapter 2 Methods & Materials

Materials

The synthetic carboxypeptidase substrate anisylazoformylarginine (AAFR) was

obtained from Bachem Americas Inc (Torrance, CA, USA). Hippuryl-L-arginine was obtained from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada) D- valylphenylalanyllysyl chloromethylketone (VFKck), and D-phenylalanylprolylarginyl chloromethylketone (PPAck) were purchased from Calbiochem (San Diego, CA, USA).

Amicon Ultra centrifugal devices were purchased from Millipore (Etobicoke, ON,

Canada). DNA restriction and modification enzymes were purchased from New England

Biolabs (Mississauga, ON, Canada) and Stratagene (La Jolla, CA, USA).

Oligonucleotides for mutagenesis were purchased from Cortec DNA Services

Laboratories, Inc. (Kingston, ON, Canada) or Integrated DNA Technologies, Inc.

(Coralville, IA, USA). Dulbecco’s modified Eagle’s medium/nutrient mixture F-12, Opti-

MEM, Trypsin-EDTA, penicillin/streptomyocin/fungizone (PSF) were obtained from

Gibco/Invitrogen (Burlington, ON, Canada). Newborn Calf Serum was obtained from

Sigma-Aldrich Canada Ltd (Oakville, ON, Canada). FuGENE 6 transfection reagent was from Roche Diagnostics (Laval, PQ, Canada). Methotrexate (Wyeth-Ayerst Canada Ltd,

Montreal, PQ, Canada) and recombinant t-PA (Alteplase) were purchased from Kingston

General Hospital Pharmacy. Thrombin, rabbit-lung TM and plasmin were obtained from

Haematologic Technologies (Essex Junction, VT, USA). Monoclonal antibody MA-T4E3 was coupled to CNBr Activated Sepharose 4B (GE Healthcare Life Sciences,

21 Mississauga, ON, Canada) according to manufacturer’s instructions (2-6 mg antibody/mL

resin). Synthetic 75% phosphatidyl choline: 25% phosphatidyl serine (PCPS) vesicles

were prepared as described previously [61].

Construction of TAFI Mutants

Mutagenesis was carried out using the QuikChange mutagenesis kit (Stratagene)

as outlined in the manufacturer’s protocol. In all cases, the template for the mutagenesis

was TAFI-pNUT, which contains human TAFI cDNA that has threonine at positions 147

and 325. TAFI variants with threonine at 325 have a half-life of about 8 minutes whereas

isoleucine at this position can increase half-life to about 15 minutes. The presence of the

mutations was verified by DNA sequence analysis. The mutagenesis was carried out by

Kyra Nabeta prior to my arrival in the laboratory.

Expression and Purification of Recombinant TAFI Variants

BHK cells were cultured in DMEM/F12 containing 5% (v/v) NCS and 1% (v/v)

PSF in a humidified incubator at 37°C in a 95% air/5% CO2 atmosphere. Expression

plasmids encoding the respective TAFI variants were transfected into the cells using

FuGENE 6 transfection reagent (10 μg plasmid DNA and 30 μL FuGENE per 100 mmm

plate of cells). After 24 h incubation with the transfection mixture, the cells were then

cultured in DMEM/F12 containing 5% (v/v) NCS, 1% (v/v) PSF, and 400 μM methotrexate for approximately 2 weeks, with the media changed every other day, for the selection of stable cell lines expressing the respective recombinant TAFI variants. For

22 recombinant TAFI production, stably expressing lines were cultured in triple flasks (500

cm2, Nunc, Roskilde, Denmark) in Opti-MEM containing 1% (v/v) PSF and 40 μM

ZnCl2. Conditioned medium was harvested at 48 hour intervals and replaced with fresh

medium. Harvested conditioned medium was centrifuged at 3000 × g for 10 minutes,

supplemented with Tris-HCl, pH 8.0 (to 5 mM), reduced glutathione (to 0.5 mM) and

phenylmethanesulphonylfluoride (PMSF) to (2 μM) and stored at -20°C.

To isolate the TAFI variants, usually 2 liters of conditioned, supplemented

medium was passed through a 0.22 μM filter and then over a 2-mL MA-T4E3-Sepharose

column at 4°C. The column was extensively washed with 20 mM HEPES pH 7.4

containing 150 mM NaCl and 0.01% (v/v) Tween 80 (HBS/Tween). TAFI was eluted with 0.2 M glycine pH 3 and 2-mL fractions were collected into tubes containing 1-mL aliquots of 1 M Tris-HCl, pH 8. Protein containing fractions were pooled and then were

concentrated and exchanged into HBS/Tween using Amicon centrifugal devices. Purified

TAFI was quantified by measurement of absorbance at 280 nm (ε1%, 280 = 26.4, Mr =

60,000), aliquoted and stored at -70°C.

Activation of TAFI and Determination of Thermal Stability

Purified recombinant TAFI variants (1 μM) were activated by incubation with thrombin (25 nM), TM (50 nM), and CaCl2 (5mM) in HBS/Tween at 24°C for 15 min.

The thrombin was then quenched by the addition of PPAck (to 1 μM) and the reactions

were then incubated in a 37°C water bath. At various times, 10 μL aliquots were removed

and mixed with 80 μl AAFR (120 μM final concentration) in a microtitre plate, and the

23 initial rates of substrate hydolysis were measured at 350 nm. The hydrolysis of AAFR by

TAFIa can be described by the following reaction:

Enzymatic removal of the anisylazoformyl moiety results in a decrease in the substrate

absorption at 350 nm and the generation of nitrogen and carbon dioxide gas.

The amount of TAFIa activity relative to the initial TAFIa activity was plotted as

a function of time, and the data were fit by non-linear regression to the equation for first-

order exponential decay (SigmaPlot):

-k.t [TAFIa] = [TAFIa]0.e (1)

Where [TAFIa]0 is the initial TAFIa concentration and [TAFIa] is the residual

concentration at various time intervals. The estimated first order decay constant k, which

was obtained from non-linear regression for each variant using SigmaPlot, was used to

determine the half-life of each mutant. The half life is defined as the time when [TAFIa]0

= 0.5*[TAFIa], therefore equation (1) can be rewritten as following:

24

-k.t 0.5*[TAFIa]0 = [TAFIa]0.e

0.5 = e-k.t

t = -ln(0.5)/k (2)

By substituting the decay constants into (2), the half-life of each variant was obtained.

Kinetics of Activation of TAFI Mutants by Thrombin or

Thrombin-TM

For activation by thrombin-TM, a range of concentrations of the TAFI variants

(0.032 – 1 μM) was incubated in the wells of microtiter plates (preconditioned overnight)

in HBS/Tween containing 1% (v/v) Tween in the presence of 5 mM CaCl2, 1 – 5 nM

thrombin, and 5 – 10 nM rabbit-lung TM in HBS/Tween for 10 – 20 minutes at 22°C. For

the TAFI variants S94V, A93V, S90P, and R92K, 1 nM thrombin and 5 nM TM was

incubated for 10 minutes at 22°C. For P91S and DVV, 5 nM thrombin and 10 nM TM

was incubated for 20 minutes at 22°C. Reactions were stopped by the addition of a

solution containing AAFR (120 μM final concentration) and PPAck (1 μM final

concentration) to a final volume of 200 μl. The absorbance was then monitored at 350 nm

to measure the initial rate of AAFR hydrolysis as described before. Depending on the

TAFI mutant, the zymogen shows about 0.5% to 2% of the activity of TAFIa towards the

AAFR substrate. Therefore, the proportion of the AAFR hydrolysis attributable to

remaining zymogen had to be corrected for using standard curves for the hydrolysis of

25 AAFR by TAFIa (wild-type) and TAFI (each of the respective mutants). For activation

by thrombin alone, the TAFI variants (0.05 – 0.5 μM) were incubated with thrombin (50 nM) for 20 min at 22°C, after which the reactions were stopped by the addition of a solution containing AAFR (120 μM final concentration) and PPAck (1 μM final concentration) to a final volume of 200 μl. The absorbance was then monitored at 350 nm to measure the initial rate of substrate hydrolysis. The amount of TAFIa formed was calculated using the following equation:

r = K1[TAFI] + K2[TAFIa] (3)

where r is the rate of hydrolysis of AAFR measured as change in absorbance over time

(dAbs/dt), K1 is the slope determined from the initial rate of AAFR hydrolysis versus the

TAFI zymogen concentration, and K2 is the slope determined from the initial rate of

AAFR hydrolysis versus various TAFIa concentrations. The TAFIa for K2 determination

was prepared by completely activating 1μM TAFI (wild type), which then was diluted to

prepare various known TAFIa concentrations. The total TAFI concentration in our

experimental assays can be written as [TAFI]0 = [TAFI] + [TAFIa], which can be used to

rearrange equation (3) into the following:

[TAFIa] = (r-K1[TAFI]0)/(K2 – K1) (4)

By knowing the initial input TAFI concentration for each assay [TAFI]o, the initial rate of hydrolysis r and the two constants K2 and K1, the amount of TAFIa formed can be

26 calculated using (4). The amount of TAFIa formed was then converted to rate of TAFIa

formation (moles of TAFIa formed per second per mole of thrombin) and the data were

fit to the Michaelis-Menten equation by non-linear regression analysis (Sigmaplot 10,

SPSS Inc., Chicago, IL) for thrombin-TM kinetics.

In the case of certain poorly activated TAFI mutants, and for all variants in the

absence of TM, only the catalytic efficiency (kcat/Km) could be obtained. This was

obtained by modifying the Michaelis-Menten equation in the following manner:

V = kcat[E]T[S]/(KM + [S])

V/[E]T = kcat[S]/(KM + [S]) (5)

By assuming that Km>> [S], equation (5) can be rewritten as following:

V/[E]T = kcat[S]/KM (6)

By plotting V/[E]T, which is moles of TAFIa formed per second per mole of thrombin,

versus substrate concentration, the catalytic efficiency was obtained from the slope of the

graph.

Kinetics of Activation of TAFI Mutants by Plasmin

For activation kinetics by plasmin, TAFI mutants at a range of concentrations (7 –

112 nM) were incubated in a cuvette with 25 nM plasmin for 20 minutes at 22°C in a volume of 100 μL. The reaction mixture was quenched with a solution containing VFKck

27 (1 μM final concentration) and Hipp-Arg (1 mM final concentration) to a final volume of

200 μL. The absorbance was then measured at 260 nm to measure the initial rate of Hipp-

Arg hydrolysis. The change in absorbance of the substrate hipp-arg into hippuric acid is described by equation (7)

(dA/dt) (1/(ε.l)) = d[S]/dt (7)

where dA/dt is the change in absorbance, [S] is the initial concentration of hipp-arg, ε is

the extinction coefficient for the conversion of hipp-arg into hippuric acid and l is the

pathlength of the cuvette. The change in hippuryl-arginine can also be described by the following Michaelis-Menten equation:

d[S]/dt = kcat[TAFIa][S]/(KM + [S]) (8)

Where kcat is the turnover number and Km is the binding affinity of TAFIa for Hipp-arg.

Km, kcat, and ε have been previously defined [59]. By putting (7) into (8), and rearranging

to solve for [TAFIa], the following can be obtained:

[TAFIa] = (dA/dt)(1/(ε.l)){(KM + [S])/[S]}(1/kcat) (9)

The concentration of TAFIa was converted to moles of TAFIa formed per second. Rate data were modeled with the quadratic form of the Michaelis Menten equation using non- linear regression analysis (Sigmaplot 10, SPSS Inc., Chicago, IL). The quadratic form of

28 the Michaelis-Menten equation was used since the concentration of substrate was not negligible to the concentration of the enzyme. In other words, the concentration of free

substrate in the reaction cannot be approximated as the input substrate concentration as

previously done for regular Michaelis-Menten kinetics. The enzyme substrate interaction

and subsequent catalysis is characterized by equation (10), where the Michaelis constant

Km, is defined by (11).

(10)

KM = [E][S]/[ES] (11)

Where [E] and [S] are free plasmin and TAFI respectively. The total plasmin, [E]T can be written in terms of free plasmin, [E], and plasmin bound to TAFI, [ES].

[E]T = [E] + [ES] (12)

Similarly, the total TAFI, [S]T can be written in terms of free TAFI [S] and TAFI bound

to plasmin, [ES].

[S]T = [S] + [ES] (13)

By replacing the free plasmin and TAFI terms in equation (11) using (12) and (13) and

rearranging, the following can be obtained:

29 ([E]T – [ES])([S]T – [ES]) = KM[ES] (14)

2 [E]T[S]T – [ES]([S]T + [E]T) + ([ES]) = KM([ES]) (15)

2 ([ES]) – [ES](KM + [E]T + [S]T) + [E]T[S]T = 0 (16)

2 1/2 ([ES]) = 0.5(KM + [S]T + [E]T ± ((KM + [S]T + [E]T) – 4([E]T[S]T)) ) (17)

To resolve the ± ambiguity in the above equation, we can set [S]T = 0 in equation (17) for

which the [ES] would also be 0.

2 1/2 0 = 0.5(KM + [E]T ± ((Km + [E]T) ) (18)

0 = 0.5(KM + [E]T ± ((Km + [E]T)) (19)

For all KM and [E]T values, the condition in (19) is satisfied only when ± in (19) is replaced with negative sign. Therefore (17) can be written as:

2 1/2 ([ES]) = 0.5(KM + [S]T + [E]T - ((KM + [S]T + [E]T) – 4([E]T[S]T)) ) (20)

From equation (10), the rate of TAFIa formation can be written as:

V = kcat[ES] (21)

Therefore, by substituting equation (20) into (21), the quadratic form of the Michaelis-

Menten equation can be obtained:

30 2 1/2 V = kcat(0.5.(KM + [S]T + [E]T - ((Km + [S]T + [E]T) – 4([E]T[S]T)) )) (22)

TAFI-Deficient Plasma

To make TAFI deficient plasma, 50 mL of fresh-frozen citrated human plasma was thawed and passed over a 2-mL MA-T4E3-Sepharose column at room temperature.

The plasma was passed over the column repeatedly, and between each pass the column was washed with HBS/Tween. After each pass, TAFI was eluted from the column with

0.2 M glycine, pH 3. An aliquot of the plasma was then used for an in vitro clot lysis assay (in the absence of added TAFI) in the presence or absence of TM (see below).

When no difference was observed between the clot lysis times in the presence or absence of TM, the plasma was considered to be TAFI deficient.

Clot Lysis Assays

All clot lysis assays were performed in a final volume of 100 μl in microtitre plates at 37°C. TAFI-deficient plasma was diluted 1:3 in HBS/Tween and supplemented with different concentrations of the respective TAFI variants (0 – 200 nM), PCPS vesicles (20 μM) in the absence or presence of TM (10 nM) before clotting was initiated by the addition of these mixtures to the wells of microtitre plates containing small, separated aliquots of thrombin (5 nM, final), CaCl2 (10 mM, final), and tPA (2 nM, final). Clot lysis was monitored by the change in turbidity of each reaction at 410 nm in a

Spectramax Plus plate reader (Molecular Devices, Sunnyvale, CA) and the time to 50% lysis was determined graphically from the midpoint between maximum and minimum turbidities of the clots.

31 Chapter 3 Results

3.1 Construction and Expression of TAFI Variants

In order to construct TAFI variants (Table 3-1) that would be resistant to

activation by thrombin and plasmin, we have based our mutagenesis experiments on

previous peptide studies looking at ideal and non-ideal amino acids in the recognition

sites of thrombin and plasmin. It was found that proline at the P2 site was an absolute

requirement for thrombin substrates and bulky residues such as phenylalanine,

tryptophan, or tyrosine are preferred by plasmin at this site [89]. Therefore, we decided to

mutate the proline at this position in TAFI to serine (P91S variant) in the expectation that

the resultant variant would be resistant to cleavage by thrombin. However, the effect of

this mutation on plasmin cleavage couldn’t be predicted. Thrombin also has a strong

preference for arginine over lysine at the P1 position whereas plasmin does not

discriminate between either [90]. Therefore, we hypothesized that introducing this

substitution into TAFI (R92K variant) would yield a TAFI variant with reduced ability to

be activated by thrombin but activation by plasmin would be unaffected.

Compared to thrombin, plasmin is considered to be a much more promiscuous

enzyme, but peptide studies have revealed that plasmin does not favour valines at the P1’

and the P2’ positions and proline and aspartic acid at the P3 position [89, 90]. Although the substrate specificities at the P1’ and P2’ of thrombin have not been explored by these peptide studies, it was demonstrated that proline or aspartic acid at the P3 site was compatible with thrombin cleavage [89]. Thus, we replaced the alanine at the P1’

32 position, serine at the P2’ and P3 positions of TAFI to construct the A93V, S94V and

S90P variants, respectively. We also constructed a triple mutant containing aspartic acid at the P3 position and valines at the P1’ and P2’ positions (DVV variant) in the expectation that these mutants would be resistant to activation by plasmin.

All TAFI variants were expressed in BHK cells and purified to homogeneity from conditioned medium harvested from stably-expressing cell lines using immunoaffinity chromatography. We then proceeded to measure the thermal stability, the rate of activation by thrombin, thrombin-TM, and plasmin and finally evaluate the antifibrinolytic potential of each TAFI variant using in vitro clot lysis assays.

Table 3-1. Recombinant TAFI variants used in this study. Sequences are shown relative to the wild-type TAFI sequence (TAFIwt). Mutated amino acids are shown in boldface type. Variant P4 P3 P2 P1 P1’ P2’ P3’ P4’ TAFIwt V S P R A S A S P91S V S S R A S A S R92K V S P K A S A S A93V V S P R V S A S S94V V S P R A V A S DVV V D P R V V A S S90P V P P R A S A S

33

Figure 3-1: SDS-PAGE analysis of TAFI variants. Recombinant TAFI proteins were purified as described under “Methods & Materials”. 100 ng of each purified protein was resolved on a 12% polyacrylamide gel under reducing conditions. Protein bands were visualized by silver stain.

3.2 Time courses of thrombin-TM activation

Figure 3-2: Time course of TAFIwt activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

34

Figure 3-3: Time course of S94V activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

Figure 3-4: Time course of A93V activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

35

Figure 3-5: Time course of S90P activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

Figure 3-6: Time course of R92K activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

36

Figure 3-7: Time course of DVV activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

Figure 3-8: Time course of P91S activation by thrombin-TM. TAFIwt at a concentration of 1μM was activated at 22°C by thrombin (5 nM) and TM (10 nM). At times indicated below the gel, aliquots were removed and quenched with PPAck. The samples were resolved on a 12% polyacrylamide gel and stained with Coomassie Blue.

37

3.3 Kinetics of Activation of TAFI variants by Thrombin

TAFI is a poor substrate for thrombin, to the extent that high concentrations of

TAFI are needed to accurately estimate the KM and kcat of activation by thrombin.

However, such high concentrations were not afforded by our expression/purification system. With the available range of TAFI concentrations, we could only estimate the catalytic efficiency (kcat/KM) of TAFI activation. The data (Fig. 3-9 and Table 3-2) reveal that while A93V is comparable to wild-type TAFI as a substrate for thrombin, S94V is a considerably more effective substrate. As expected, neither P91S nor R92K were detectably activated by thrombin. Surprisingly, the S90P and DVV variants were also not detectably activated by thrombin.

Table 3-2. Activation of TAFI mutants by thrombin in the absence of TM. kcat/KM ratios were determined from the data in Fig. 3-1, and are presented ± the standard deviation of the estimate based on three experiments. N.M.: Not measurable. -1 -1 Variant kcat/Km (μM .s ) TAFIwt 0.0004 ± 0.00011 P91S N.M. R92K N.M A93V 0.0002 ± 0.00008 S94V 0.0011 ± 0.00015 DVV N.M. S90P N.M.

38

Fig. 3-9. Activation of TAFI mutants by thrombin in the absence of TM. Different concentrations of wild-type TAFI (closed circles), S94V (open circles), and A93V (closed triangles) were incubated with 50 nM thrombin and 5 mM CalCl2 for 20 minutes at room temperature. The rate of activation was calculated and the slopes of the relationship between rate and the TAFI concentration were determined to estimate the catalytic efficiencies of activation. The estimated kcat/KM values are provided in Table 2, and correspond to the solid regression lines shown on the graph. Each data point represents the mean of three experiments. No activation was observed for S90P, P91S, R92K, and DVV.

39 3.4 Kinetics of Activation of TAFI variants by Thrombin-TM

The activation of TAFI by thrombin in the presence of thrombomodulin (TM) is

enhanced significantly (Table 3-3 and Figure 3-10). This is most likely due to the interactions between TM and thrombin that optimally position TAFI as a substrate for thrombin [45]. Unlike in the absence of thrombin, S94V was not appreciably more effective as a substrate for thrombin in the presence of TM. A93V was slightly less effective as a substrate. Interestingly, both S90P and R92K, neither of which were detectably activated by thrombin alone, were activated by thrombin-TM very effectively.

On the other hand, activation of P91S and DVV was barely detectable, with catalytic

efficiencies over two orders of magnitude smaller than for wild-type TAFI.

Table 3-3. Activation of TAFI mutants by thrombin in the presence of TM. Apparent kcat and KM values were determined from the data in Fig. 3-2, and are presented ± the standard deviation based on three experiments. N.M.: Not measurable. k /K (μM-1.s- Variant k (s-1) K (μM) cat(app) m(app) cat(app) m(app) 1) TAFIwt 0.315 ± 0.018 0.178 ± 0.030 1.77 P91S N.M. N.M. 0.0072 R92K 0.215 ± 0.011 0.229 ± 0.024 0.94 A93V 0.205 ± 0.013 0.169 ± 0.036 1.21 S94V 0.328 ± 0.020 0.232 ± 0.021 1.41 DVV N.M. N.M. 0.015 S90P 0.377 ± 0.028 0.162 ± 0.011 2.32

40

Fig. 3-10. Activation of TAFI mutants by thrombin in the presence of TM. Different concentrations of wild-type TAFI (closed circles), S94V (open circles), A93V (closed triangles), S90P (open triangles), and R92K (closed diamonds) were incubated with 1 nM thrombin, 5 nM TM, and 5 mM CaCl2 for 10 minutes at room temperature. The rates of activation were calculated for these mutants and the data were fit to Michaelis-Menten model of . DVV (closed squares) and P91S (open squares) were incubated with 5 nM thrombin, 10 nM TM and 5 mM CaCl2 for 20 minutes. For these last 2 variants, the data were fit to a linear model and the slope of the graph was used to obtain the catalytic efficiency. The estimated kcat(app) and Km(app) values are provided in Table 3 and correspond to the solid regression lines shown on the graph. The kinetic constants are considered “apparent” as they were determined at only one concentration of the cofactor TM. Each data point represents the mean of three experiments.

41 3.5 Kinetics of Activation of TAFI variants by Plasmin

The activation of TAFI by plasmin is more efficient than the thrombin catalyzed

reaction but not as efficient as the thrombin-TM reaction (Table 3-4 and Figure 3-11).

More specifically, TAFI activation by plasmin exhibits a lower KM but also a lower kcat

than the thrombin-TM reaction. Interestingly, the A93V and the S94V variants that were

designed to be resistant to activation by plasmin anticipated properties, as both of them

were as effective as substrates for plasmin as wild-type TAFI. However, the S90P and the

triple mutant DVV variants were only slowly activated by plasmin keeping in line with

previous predictions. The R92K variant was activated with similar kinetics to the wild

type variant and therefore plasmin did not prefer one basic amino acid over the other at

the P1 position as established previously [90]. Finally, the P91S variant, which was not

activated by thrombin or thrombin-TM, was activated by plasmin but the catalytic

efficiency was reduced to one third of the wild type TAFI.

Table 3-4. Activation of TAFI mutants by plasmin. kcat and Km values were determined from the data in Fig. 3-3, and are presented ± the standard deviation based on three experiments. -1 -1 -1 Variant kcat (s ) Km (μM) kcat/Km (μM .s ) TAFIwt 0.00042±0.00014 0.005±0.002 0.084 P91S 0.00020±0.00007 0.007±0.003 0.028 R92K 0.00039±0.00011 0.006±0.003 0.065 A93V 0.00043±0.00010 0.010±0.005 0.043 S94V 0.00030±0.00012 0.006±0.003 0.053 DVV 0.00004±0.00002 0.005±0.002 0.008 S90P 0.00008±0.00001 0.009±0.003 0.009

42

Fig. 3-11. Activation of TAFI mutants by plasmin. Different concentrations of wild- type TAFI (closed circles), S94V (open circles), A93V (closed triangles), S90P (open triangles), R92K (closed diamonds), P91S (open squares), and DVV (closed squares) were incubated with 25 nM plasmin for 20 minutes. The rates of activation (TAFIa/s) were calculated and the data were fit to the quadratic form of the Michaelis-Menten equation. The estimated kcat and Km values are provided in Table 4 and correspond to the solid regression lines shown on the graph. Each data point represents the mean of three experiments.

43 3.6 Thermal Stabilities and Enzymatic Activities of TAFI variants

Activated TAFI (TAFIa) is a highly unstable enzyme with a reported half life of about 8 or 15 min depending on the isoform and thermal instability of TAFIa has also been postulated to be the primary mechanism of TAFIa downregulation in vivo [56].

Furthermore, the half-life of TAFIa can significantly impact its antifibrinolytic effect

[67]. We determined the half-lives at 37°C of all variants following activation by thrombin-TM (Table 3-5 and Figure 3-12) and they were all very similar (within the range of the experimental error).

We were only able to quantitatively convert TAFI to TAFIa in the case of wild- type TAFI, A93V and S94V for the purpose of measuring the kinetics of substrate hydrolysis. Given that the S90P, S90D, P91S, and R92K substitutions would not be present in the TAFIa moiety, we would not expect the properties of TAFIa prepared from these variant to be different, as seen by the intrinsic stability measurements (which do not rely on quantitative conversion to TAFIa as the decay in activity is first-order). We also found that the wild-type TAFIa, A93V, and S94V displayed similar kinetics of hydrolysis of the substrates AAFR and Hipp-Arg. This indicated that the P1’ and P2’ substitutions do not affect the catalytic properties of TAFIa.

44

Table 3-5. Intrinsic stability of TAFIa variants. Decay constants estimated by the regression of the data in Fig.3-4 were used to calculate the half-lives of the TAFIa mutants. The data represent the mean of three experiments ± the standard deviation of the mean. Variant Half-life (minutes) TAFIwt 6.5 ± 1.2 P91S 6.1 ± 1.4 R92K 7.1 ± 1.3 A93V 6.5 ± 1.9 S94V 6.2 ± 1.4 DVV 5.8 ± 1.2 S90P 5.8 ± 1.1

Fig. 3-12. Thermal stabilities of TAFI variants at 37 °C. wild-type TAFI (closed circles), S94V (open circles), A93V (closed triangles), S90P (open triangles), R92K (closed diamonds), P91S (open squares), and DVV (closed squares) were activated by thrombin-TM and then incubated at 37°C. The residual TAFIa activity at different times was measured and the resultant data were fit by non-linear regression to the exponential decay function.

45 3.7 Antifibrinolytic potential of TAFIa variants

We measured the antifibrinolytic potential of each of the TAFIa variants using in

vitro clot lysis assays. These lysis assays were used in conjunction with our in vitro

kinetics data to identify the activators of TAFI during clot lysis. For these in vitro clot

lysis assays, 1/3 diluted TAFI-deficient plasma is supplemented with different

concentrations of purified TAFI variants and clotted in the presence of thrombin and

CaCl2, after which time t-PA stimulated lysis is measured. These experiments were

performed either in the absence or presence of TM, in which thrombin-TM has been

shown to very quickly activate virtually all the TAFI present in the clot. PCPS vesicles

were also included in these reactions in order to provide a consistent negatively charged surface for the production of thrombin by the coagulation cascade.

In the absence of TM, TAFIa has been shown to be generated in a biphasic

manner (see discussion). The amount of TAFIa formed is limited by the intrinsic

instability of the enzyme. In our system, the wild-type TAFI and A93V had similar

antifibrinolytic effects in the absence of TM while S94V was more effective, in keeping

with the kinetics of activation by thrombin (Fig 3-13). The R92K variant demonstrated

paradoxical behaviour in our lysis assays as it was able to prolong lysis times even

though our kinetic experiments showed that it is not detectably activated by thrombin.

None of S90P, P91S, or DVV had any antifibrinolytic effect. Of these, none are activated

appreciably by thrombin, but P91S is a reasonable substrate for plasmin. These findings

indicate that under these conditions, plasmin likely does not participate in TAFI

activation.

46 In the presence of thrombomodulin, all variants possessed some antifibrinolytic activity, with wild-type, S90P, R92K, A93V, and S94V having very similar profiles (Fig

4B). On the other hand, P91S and DVV, which were very poorly activated by thrombin-

TM, showed drastically reduced antifibrinolytic potency. The concentration of zymogen at which the inhibition of fibrinolysis was half maximal was almost 50 nM, over twenty- fold higher than the half-maximal concentrations of the other variants.

47

Fig. 3-13. The antifibrinolytic effect of activation-deficient TAFI mutants. TAFI deficient plasma (1/3 diluted) was mixed with wild-type TAFI (closed circles), S94V (open circles), A93V (closed triangles), S90P (open triangles), P91S (closed squares), DVV (open squares), or R92K (closed diamonds) at various concentrations and in the presence of 20 μM PCPS vesicles without TM (Panel A) or with 10 nM TM (Panel B). Clots were formed by adding the mixtures to microplate wells containing small separated aliquots of thrombin (6 nM; final) CaCl2 (5 mM; final) and t-PA (2 nM; final) The clots were incubated at 37°C and the turbidity of the clots monitored at 405 nm; the time to 50% clot lysis was measured and plotted as a function of the initial TAFI concentrations. Each data point represents the mean of two experiments.

48 Chapter 4 Discussion and Conclusion

4.1 TAFI Pathway and Thrombolytic Therapy

In vivo studies have demonstrated that TAFI can play a significant role in the regulation of the fibrinolytic pathway. Specific inhibitors of TAFIa, such has potato tuber carboxypeptidase inhibitor (PTCI), has been shown to enhance t-PA mediated thrombolysis in various animal models [49, 69-77]. Interestingly, TAFI deficient mice did not show any overt haemostatic or developmental abnormalities [73]. However, on a reduced plasminogen background, TAFI deficient mice compared with TAFI wildtype mice displayed enhanced fibrinolysis [74]. Antibody that specifically prevented TAFI activation by thrombin-TM, but not activation by thrombin alone or plasmin has been used previously to assess the role of the TAFI pathway in a baboon model [91]. The antibody prevented the consumption of TAFI following the administration of lethal dose of E. coli. Consequently, infusion of this antibody also accelerated fibrinogen consumption and the appearance of fibrin degradation product as would be expected since plasmin generation would be increased [91]. While, these findings highlight a role for thrombin-TM in TAFI activation in a sepsis model, the nature of the activator in other contexts, such as arterial thrombolysis, is yet to be defined.

Understanding how the TAFI pathway is regulated may contribute to the development of novel throbolytic therapies. Current methods primarily rely on heparin to potentiate antithrombin III mediated arterial reperfusion after infarction but the success rate of this treatment has been shown to be only around 50% [92]. Part of the problem

49 lies with antithrombin III and heparin’s inability to inhibit thrombin that is bound to the

fibrin clot. It has been demonstrated that thrombin bound to clot is protected from inhibition and can stimulate and amplify its own production through the activation of platelets, factors V, VIII, and XI [93, 94]. Novel thrombin inhibitors that are more potent have been developed but they carry the risk of inhibiting thrombin distal to the target thrombus leading to anticoagulant state elsewhere in the body and subsequent bleeding complications [95]. In contrast, manipulation of the TAFI pathway, through our understanding of how each of its activators play a role in different thrombotic diseases might lead to the development of enhanced thrombolytic protocols.

In the present study, we have developed TAFI variants that can be used to explore

the TAFI pathway in vivo. By mutating selected residues surrounding the scissile bond, we have developed TAFI variants that can be specifically activated by plasmin (P91S

variant), thrombin-TM (S90P variant), or thrombin-TM and plasmin (R92K variant).

Certain mutants, however, failed to meet our expectations. The TAFI variants A93V and

S94V were hypothesized to be resistant to activation by plasmin but this was not

observed. These findings have implications for our understanding of how protease

specificity is determined.

4.2 The Kinetics of Thrombin Activation

The results we obtained from measuring the rate of activation of the TAFI

variants by thrombin can be rationalized in terms of the structural information that is

available on thrombin. Thrombin is a trypsin like member of the chymotrypsin family of

serine . Unlike trypsin, thrombin possesses large insertion loops that border and

50 shape the canyon like active site cleft. Particularly, the “60” and 149” insertion loops can

block access to wide range of substrates or inhibitors to the catalytic site thereby

contributing to the narrow substrate specificity of thrombin. Thrombin also has a Na+ binding site that can govern its conformation and subsequent procoagulant and anticoagulant activities [27-29].

Thrombin possesses a highly uneven charge distribution on its surface resulting in the formation of what is known as the “anion-binding site 1” (ABE1) and “anion-binding site 2” (ABE2). ABE1 was initially characterized as the fibrinogen binding site whereas

ABE2 preferentially binds to heparin [27-29, 96]. With increasing number of studies characterizing thrombin substrate interactions, it is becoming clear that substrates and co- factors often utilize one or both exosites in addition to the catalytic site for optimal substrate recognition.

The crystal structure of thrombin in a complex with its inhibitor D-Phe-Pro-Arg- chloromethylketone (PPAck) has been solved previously which explained how peptide substrates interact with thrombin [97]. Arginine is specifically preferred at the P1 site as it can form critical hydrogen bonds within the S1 pocket. The P2 position or the corresponding S2 site on thrombin is delimited by the rigid hydrophobic 60 loop which protrudes into the active site and only allows small hydrophobic residues, such as proline to be fit into the S2 cavity. The hydrophobic S4 cavity of thrombin, which is also known as the aryl binding site, generally favours hydrophobic or aromatic residues. Finally, the

P3 site is considered to have a negligible specificity since the side chain of this residue was shown to point away from the cleft [27-29].

51 Recently, crystal structures for human and bovine TAFI have been published [55,

56]. The structure revealed that the scissile arginine-alanine bond for TAFI activation is

located on a surface-exposed loop. It is also apparent from this structure as to why TAFI

is such a poor substrate for thrombin and plasmin. The Arg92 side chain does not project

out into the solvent, but appears packed against the site of the catalytic domain, anchored

between the aromatic side chains of Tyr97 and Phe113. The basic end of the Arg92 side

chain is in close proximity to the side chain carboxylate of Asp106 (implying the presence of a salt bridge) and the side chain hydroxyl of Ser109. Thus, the mobility of

Arg92 may be restricted, which might limit the activatability of TAFI. This may be an inherent feature of the protein designed to prevent excessive activation of TAFI by either

thrombin or plasmin.

Our results show that the TAFI variants R92K and P91S are not activated by

thrombin or their activation is below the detection limit of our substrate hydrolysis assay.

The lack of activation of the R92K variant is expected. Crystal structure of thrombin with

PPAck has shown that the S1 pocket of thrombin, in contrast to trypsin, is slightly more

hydrophobic. Water molecule inside thrombin’s S1 pocket can lower their free energy if

they receive a favourable hydrogen bond from the guanidinium group of arginine and this

indeed allows thrombin to better accommodate the arginine side chain at the S1 pocket.

In contrast, lysine side chain fits less tightly into the S1 pocket and also cannot provide

hydrogen bond to the water molecule in the S1 pocket, making lysine unfavourable at the

P1 position [98]. Studies have shown that thrombin can cleave peptides with lysine at the

P1 site but the rate of cleavage is significantly decreased [98].

52 The inability of thrombin to activate the P91S variant can be argued on the basis that serine at the P2 position will not be favoured at the small hydrophobic S2 pocket.

But the structures of TAFI may also offer an alternate explanation. As alluded earlier, the structures revealed that the region surrounding the scissile bond is located on a surface exposed loop with proline at the apex. Therefore it is also possible that substitution of this proline by any other residue could disrupt the “kink” and thereby offset the activation loop relative to the thrombin binding pockets so that it is no longer in

an optimal position for catalysis.

The S90P variant was also not activated by thrombin. Previous structural and

substrate analysis of thrombin pointed to negligible specificity at the P3 site [29, 89]. In

our case, it is plausible that the lack of activation of the S90P variant may be due to the

presence of tandem prolines (i.e. prolines at 90th and 91st residues) in the S90P variant.

As previously mentioned for the P91S variant, it is possible that the tandem prolines may have resulted in an altered geometry of the activation loop. In a study by Schneider et al.,

this serine was also replaced by an alanine [99]. The S90A variant was activated by

thrombin with similar kinetics to TAFI wild type, which is in line with previous peptide

studies showing minimal specificity at this position.

The inactivatability of the DVV variant by thrombin was interesting.

Hydrophobic residues at the P1’ and P2’ are favoured as demonstrated by the activation

of the A93V and S94V variants. However, closer inspection of the protein C activation

sequence, which is cleaved inefficiently by thrombin, revealed that it is very similar to

that of the DVV variant. Protein C has an aspartic acid at the P3 position and

hydrophobic residues, leucine and isolecine at the P1’ and P2’ sites respectively [27]. The

53 protein C activation sequence have been previously cloned by Schneider et al. into the

activation loop of TAFI, where it was demonstrated that thrombin alone was unable to

cleave this sequence in the context of TAFI [99]. This was in line with previous findings

which suggested that aspartic acids at the P3 and P3’ positions of protein C led to

unfavourable interactions between Glu-192 and Glu-39 of thrombin, respectively [100].

Thus, the inactivatability of the DVV variant could be attributed to the presence of the aspartic acid at the P3 position. However, although single valines by themselves at the

P1’ and P2’ did not have any effect, the potential effect of the tandem valines cannot be ignored. We are currently constructing two additional mutants, S90D and a mutant with just the tandem valines to resolve this ambiguity.

The S94V variant was consistently activated more efficiently by thrombin than

the wild type variant. Substrate specificity of thrombin for the prime sites (i.e. P1’, P2’,

etc.) have not been determined but structural analysis of the crystal structure of thrombin revealed that S2’ site has a strong preference for hydrophobic residues [27-29]. In fact natural substrate sequences of thrombin generally tend to have hydrophobic residues at the P2’ site. Hence, it could be argued that replacing the serine with valine led to more favourable thrombin-TAFI interactions. The activation kinetics of A93V variant by thrombin was comparable to that of the wild type. The structure of thrombin with antithrombin III showed that the side chain at the P1’ site is adjacent to the hydrophobic

60 insertion loop and points away from the main cleft [27-29]. Interestingly, inspection of

natural substrate sequences reveal a strong preference for polar (i.e. serine, threonine)

residues at the P1’ position, suggesting possible solvent interaction.

54 4.3 The Kinetics of Plasmin Activation

The fibrinolytic enzyme plasmin is responsible for removing the blood clot from circulation and restoring blood flow. Plasmin is derived from its precursor, plasminogen, which is a single chain glycoprotein containing five kringle domains. The kringle

domains, some of which contains “lysine binding sites” mediate specific binding of plasminogen to fibrin and the interaction between plasmin and antiplasmin [101]. All

plasminogen activators convert plasminogen to plasmin by cleavage of a single Arg561-

Val562 bond. The resulting two chain plasmin molecule comprises of a heavy chain

containing the five kringle domains and a light chain containing the [101].

The substrate specificity of plasmin is considered to be less stringent and different

than its closely related homologues such as t-PA and urokinase-type plasminogen

activator (u-PA) [90]. As with thrombin, the major determinant of P1 basic residue

specificity of plasmin lies with Asp189 deep within the S1 pocket. However, unlike

thrombin, our results showed that plasmin did not exhibit any preference for either

arginine or lysine at the P1 position, which was consistent with previous peptide studies

[89, 90] and crystal structures of plasmin, which showed that the specificity of the S1 pocket is similar to trypsin’s [101, 102]

Unlike other members of the chymotrypsin like serine proteases, plasmin has a six

residue deletion within its so-called “90-loop” which is involved in substrate interaction.

The deletion eliminates the “99-beta-hairpin” present in most chymotrypsin like serine

proteases and creates the “99-shunt”, which is characterized by substantial enlargement

of the S2 pocket of plasmin compared to other serine proteases. The enlarged pocket can

expose the catalytic triad to solvent rendering the catalytic machinery inactive unless the

55 exposed cavity is filled. Therefore, at the P2 site, plasmin shows a strong preference for

bulky residues such as tyrosine and phenylalanine [90, 101, 102].

The presence of proline at the P2 site likely already made wild type TAFI a poor

substrate for plasmin to begin with as explained above and this was further exacerbated

when proline was replaced with serine (P91S variant). In fact, replacing proline at the P2

site with serine reduced the catalytic efficiency of activation of TAFI by 70%. From inspection of the catalytic constant (kcat) and the binding affinity (KM) of activation

kinetics of the P91S variant, it can be seen that the KM remains relatively unchanged

whereas there was a marked decrease in the kcat. This suggests that serine at the P2 site

most likely interfered with the catalytic machinery of plasmin. It is probable that too

much solvent was able to access the active site and therefore reduced the nucleophilicity

of the catalytic serine.

The TAFI variant S90P was significantly impaired in activation by plasmin,

keeping in line with previous peptide prediction [89]. However, from a structural

perspective it is not clear why this mutant failed to be activated. One possibility would be

the tandem prolines in the activation loop as mentioned previously. The structure of

plasmin with its optimal substrate Lys-Thr-Phe-Lys has shown that the side chain of the

P3 residue pointed away from the enzyme into the bulk solvent [89]. Therefore, just like

P3 specificity of thrombin, one would expect minimal specificity at this site.

Interestingly, peptide studies have shown that proline at this site only can also hamper

substrate cleavage by plasmin, which was the rationale for constructing the S90P mutant

in the first place [89]. Though our ultimate goal of creating a TAFI variant that couldn’t

be activated by plasmin was accomplished, the exact structural details behind its

56 inactivatability remain elusive. Moreover, we have yet to construct a variant of TAFI that

is resistant to plasmin cleavage yet that exhibits normal activation by thrombin or

thrombin-thrombomodulin.

Like the S90P variant, the DVV variant exhibited resistance to activation by

plasmin. But once again, the mechanistic details regarding its inactivatability are not

clear. Aspartic acid at the P3 site was shown to be unfavourable for plasmin [89]. The

tandem valines by themselves were also shown to be unfavourable in a separate peptide

study [90]. The presence of the tandem valines makes the activation sequence of TAFI

similar to plasminogen’s activation sequence (KKSPGRVVGGSVAH), which is not

cleaved by plasmin. This is an important regulatory feature of the plasminogen pathway.

Throughout evolution, plasmin has been postulated to have undergone strong selection

against cleavage of its own activation sequence. This "negative selection", however, is

different than "positive selection", where enzymes gradually develop restricted substrate

specificity, catalyzing specific substrates relative to related molecules. In contrast,

plasmin, through negative selection, has evolved to catalyze many related substrates

while leaving plasminogen intact. This allows plasmin to regulate many different

physiological processes or substrates rather than specific proteases evolving through positive selection to regulate these processes. Negative selectivity also ensures that plasmin does not activate plasminogen itself, safeguarding the fibrinolytic system from deregulated activity while maintaining the integrity of the fibrinolytic cascade [90].

Interestingly, t-PA and u-PA, which are close homologs of plasmin, cannot also

cleave plasminogen derived sequences efficiently even though they are the physiological activators of plasminogen. These enzymes, however, have developed productive

57 secondary enzyme-substrate interactions through exosite interactions that can overcome

suboptimal interactions surrounding the scissile bond to achieve efficient catalysis [90].

Single valines at the P1’ and P2’ positions (i.e. A93V and S94V) only caused

mild impairment in activation kinetics. The A93V and S94V variants were hypothesized

to be resistant to cleavage by plasmin but our studies show that the effect is not as

dramatic as it was demonstrated through peptide studies [90]. A possible explanation is

that the context that these residues are in can affect protein substrate recognition. In other words, peptides used to probe subsite interactions may not account for restriction of conformational freedom once the same residues are placed in the context of a protein.

4.4 The Kinetics of Thrombin-Thrombomodulin Activation

Thrombomodulin is a single chain glycoprotein that is primarily expressed on the

surface of endothelial cells that line the blood vessels. Thrombomodulin has five distinct

regions: the N-terminal lectin like domain, six consecutive EGF (epidermal growth

factor) like repeats, a proteoglycan like serine threonine rich region that is mostly o-

glycosylated, a single transmembrane helix, and finally C-terminal cytoplasmic tail [103,

104]. Crystal structures of thrombin in a complex with a thrombomodulin fragment have

been solved previously [105]. This structure revealed that thrombomodulin primarily

interacts with thrombin via the exosite 1 motif of thrombin. Once thrombomodulin is

bound to thrombin, it cannot cleave procoagulant substrates such as fibrinogen but

becomes an anticoagulant enzyme by activating protein C, which can cleave factor V,

and VIII and downregulate the coagulation cascade.

58 The binding of thrombomodulin to thrombin has been shown to enhance the activation of TAFI by almost 1250 fold [35]. The exact mechanism behind this enhancement is not fully understood and the structural basis for the TM mediated

enhancement of TAFI cleavage by thrombin has yet to be fully established. However, it

has been determined that residues surrounding the scissile bond R92-A93 or more

specifically the P6 to P3’ residues of TAFI do not affect TM dependence of TAFI

activation [99]. This finding is in accordance with the crystal structure of thrombin with

the 4th, 5th, and 6th EGF-like domains (TME456) of TM. This structure revealed that

binding of the TME456 fragment did not change the active site conformation of

thrombin, which suggested that thrombin-TM catalysis was not mediated by allosteric modification of the active site.

It has also been determined that 4th, 5th, and the 6th and 13 residues comprising

the 3rd disulfide loop of the 3rd EGF-like domains of thrombomodulin were absolutely

necessary for TAFI activation. This contrasts with the other major substrate of thrombin- thrombomodulin, protein C, which requires only the 4th, 5th, and 6th EGF-like domain in

TM [106]. Taken together, it is apparent that thrombomodulin acts as a template to maximize the efficiency of the interaction between thrombin and its substrates, either

TAFI or protein C. Thrombomodulin binds to thrombin in an identical fashion for activation of both substrates, but TM also makes contacts with the substrates themselves, in part through different TM domains.

In our study, thrombin did not detectably activate S90P, P91S, R92K, and DVV

but thrombin-TM was able to activate S90P and R92K with catalytic efficiencies that

were comparable to the wild-type TAFI. Activation of P91S and DVV by thrombin-TM

59 was barely detectable. These findings indicate that TM can allow thrombin to overcome some non-ideal thrombin cleavage sites but not others. Unfortunately, we were not able to detect activation by thrombin of any of these four variants, even at elevated thrombin concentrations, and so we cannot at this time rationalize why S90P and R92K are good substrates for thrombin-TM.

4.5 Thermal Stability

Activated TAFI (TAFIa) is a highly unstable enzyme with a half-life that is dependent on the TAFI isoform. There have been four reported wild-type TAFI isoforms:

TAFI-(Thr147/Thr325), TAFI-(Ala147/Thr325), TAFI-(Thr147/Ile325), TAFI-

(Ala147/Ile325) [27]. The four variants are activated in a similar manner by thrombin and thrombin/TM. However, isoleucine at the position 325 has been shown to extend the half life of TAFI from 8 to 15 minutes at 37 C, whereas alanine or threonine at 147 has no known effect [107]. In our present study, we have constructed all TAFI variants using the wild-type TAFI with threonine at 147 and 325 as template. All of our mutants exhibited half-lives that were comparable to the wild-type TAFI which suggested that our mutagenesis experiments did not drastically alter the overall structure nor the catalytic activity of our variants.

A recent study on the role of glycosylation of TAFI has reported that mutations in the activation peptide can affect the catalytic properties of TAFIa through an interaction between the activation peptide and TAFIa after TAFI activation [108]. However, our thermal stability experiments with the TAFI mutants suggest that this may not be the case. The similar intrinsic stabilities of our TAFI variants, as seen from these assays,

60 indicate that mutations in the activation peptide did not affect the enzymatic activity, in

keeping with a recent study by Marx and coworkers [109].

4.6 Antifibrinolytic Potential of the TAFI variants

The antifibrinolytic potential of our TAFI variants were measured using in vitro

clot lysis assays in the presence or absence of TM at 37 C. In the absence of TM, the

TAFI variants S94V and A93V prolonged lysis times to a similar extent as wild type

TAFI, whereas P91S and DVV variants did not prolong lysis times. The antifibrinolytic

potential of these variants also correlated very well, with one exception, with their

activation kinetics by thrombin. For example, the S94V variant was activated more

efficiently by thrombin than the wild type variant and this was also reflected in the clot

lysis assays as S94V prolonged lysis times better than the TAFIwt when there was no TM

present. The only exception was the R92K variant, which prolonged lysis times in the

absence of TM even though this variant was not detectably activated by thrombin. One

possible explanation for this discrepancy between the activation kinetics and clot lysis

assays is that activation kinetics were measured at room temperature (23 °C) whereas the

clot lysis assays were performed at 37 °C. It might be possible that at higher

temperatures, the mobility of the scissile bond is less restricted, which might promote

enhanced activatability of TAFI. To investigate this matter, we are currently studying the

activation kinetics of our variants at 37 °C.

In our in vitro assays, it is apparent that only thrombin and thrombin-TM but not plasmin, were the primary activators of TAFI. This was evident from the inability of the

P91S variant to inhibit fibrinolysis in the absence of TM. The P91S variant was poorly, if

61 at all, activated by thrombin but it was a reasonable substrate for plasmin. Therefore, plasmin under these conditions did not play any role in activation of TAFI. Interestingly, it has been previously demonstrated that the activation of TAFI occurred in two phases, where the first activation was mediated by thrombin prior to lysis and the second activation was mediated by plasmin after lysis [110]. The significance of the second activation of TAFI by plasmin is not fully understood but it may become important in situations where there is reduced fibrinolytic activity and therefore a longer lysis time.

The P91S variant can be used to explore this scenario.

We have also noticed that several of the variants that are poorly activated by thrombin such as the S90P, P91S, and DVV variants do not inhibit fibrinolysis in the absence of thrombomodulin even at elevated zymogen concentration. This finding contradicts recently published results that implicate the zymogen activity of TAFI to contribute to the inhibition of fibrinolysis [111]. Other independent studies have also cast doubt on the functional significance of the zymogen activity of TAFI [112].

4.7 Conclusion

The present study can be seen as a stepping-stone in an attempt to understand the regulation of TAFI pathway in vivo. To date, it is not yet fully understood how each of the activators of TAFI, identified via in vitro studies, affect TAFI activation in an in vivo setting. In vitro clot lysis assays are only an approximation and do not take into account other factors such as anatomical, hemodynamic, and phenotypic variables that can influence TAFI activation. For example, the effective concentration of thrombomodulin, which can determine the balance between anticoagulant and antifibrinolytic states [81]

62 will depend not only on the diameter of the blood vessel, but also the vascular site, as the expression of thrombomodulin has been shown to vary in different areas of the vasculature [2]. Thus, it could be hypothesized, for example, that the thrombin-TM complexes can play a significant role in TAFI activation in the microcirculation than in large arteries and veins, because the local concentration of thrombomodulin would be much higher in smaller vessels. The S90P variant, which can only be activated by the thrombin-TM complex, can be used to explore this hypothesis.

Thrombin activation of TAFI may predominate in arterial clots, which are platelet rich, because the presence of activated platelets supports the activity of the intrinsic pathway of coagulation while inhibiting fibrinolysis through the release of plasminogen activator inhibitor-1 (PAI-1) [113]. In this case, the R92K variant, which cannot be activated by thrombin, can become a useful tool in assessing the role of thrombin activation of TAFI in arterial thrombolysis.

The fibrinolytic enzyme plasmin may also play a role in regulating TAFI activation at sites where injuries to the endothelium can expose significant amounts of vessel wall glycosaminoglycans [51]. As these sites may also be devoid of thrombomodulin, plasmin mediated activation of TAFI and stabilization of the haemostatic clot may become significant. The P91S variant, which is a reasonable substrate for plasmin but not for thrombin or thrombin-TM, can be used to assess whether plasmin mediated activation of TAFI play any role in such scenario.

Plasmin activation of TAFI may also play a role in platelet-poor, venous clots, where thrombus resolution requires prolonged activity of the fibrinolytic system [114].

63 The P91S variant, which can only be activated by plasmin, can be used to assess whether

plasmin mediated activation of TAFI play any role in venous thrombolysis.

In conclusion, we have constructed and fully characterized various activation

resistant TAFI mutants. These mutant versions of TAFI reported here would constitute useful tools for examining the regulation of the TAFI pathway in different contexts. We have constructed and characterized variants that are specifically activated by plasmin

(P91S), thrombin-TM (S90P), or thrombin-TM and plasmin (R92K). Furthermore, by constructing these various mutants, we were able to gain interesting insights into how thrombin, thrombin-TM, and plasmin can interact with their substrates.

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