POLYPHOSPHATE AND TISSUE FACTOR/FACTOR VIIA AS INITIATORS OF COAGULATION

BY

JOSHUA M. GAJSIEWICZ

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2016

Urbana, Illinois

Doctoral Committee:

Professor James H. Morrissey, Chair Professor Chad Rienstra Professor Emad Tajkhorshid Associate Professor Rutilio Fratti ABSTRACT

Protein-membrane interactions are a critical component of the coagulation cascade. For many coagulation factors, these interactions are mediated via γ-carboxyglutamate rich regions, or GLA domains, in a Ca2+-dependent manner. Nearly all studies of GLA domain-containing coagulation factors, and coagulation in general, have utilized supraphyisologic Ca2+ concentrations in order to fully occupy the 7 GLA domain metal binding sites (and thus maximize activity). Recent work has indicated that under physiologic conditions, 2-3 of the

GLA domain metal binding sites are actually occupied by Mg2+; furthermore, the concentration of Ca2+ required to maximize coagulation factor activity is reduced to near-physiologic levels in the presence of physiologic concentrations of Mg2+. Together, these results suggest an important role for Mg2+ in coagulation.

To investigate the role of Mg2+ further, I first demonstrated that tissue factor is required for the Mg2+-dependent rate enhancements and that tissue factor itself does not bind Mg2+. To determine which residues of TF mediate the effect of Mg2+, I performed alanine scanning mutagenesis and substrate activation experiments, identifying several key residues mediating these effects. Importantly, these residues reside in or near the putative substrate binding exosite, suggesting that Mg2+ occupation of GLA domain metal binding sites may mediate both protein- membrane and protein-protein interactions. Furthermore, in collaboration with the lab of Dr.

Ryan Bailey in the department of analytical chemistry at UIUC, we have probed the protein- membrane interactions of coagulation factors in a high-throughput manner utilizing photonic microring resonators. Additionally, we have investigated the binding of factor X to the tissue factor/factor VIIa complex assembled on Nanodiscs, leading to a deeper understanding of the effects of membrane composition and complex formation dynamics.

ii

The roles of anionic polymers such as inorganic polyphosphate and nucleic acids in both coagulation and inflammation have recently come to light. To better understand and delineate their effects, I performed a head-to-head comparison of the abilities of polyphosphate, DNA, and RNA to activate the contact pathway of coagulation, as well as to abrogate the anticoagulant activity of tissue factor pathway inhibitor (TFPI). Polyphosphate was a superior contact pathway activator on both a weight and molar basis, and was significantly more effective in abrogating

TFPI activity than DNA or RNA purified from biologic samples.

Both polyphosphate and nucleic acids are linear, anionic polymers; thus, it is possible that utilizing nucleic acid purification techniques on samples containing both nucleic acids and polyphosphate may result in co-purification of these polymers. To assess the purity of DNA or

RNA samples purified from biological sources, I examined the co-purification characteristics of polyphosphate with DNA or RNA using numerous commercially-available nucleic acid purification techniques. Polyphosphate readily co-purified with both DNA and RNA, though to a significantly greater extent with the former. In addition, I demonstrated that purified cell- derived DNA contained contaminant polyP using PAGE and staining with DAPI.

Because polyphosphate can co-purify with nucleic acids, investigations into the effects of

DNA and RNA on coagulation might be indirectly probing the effects of polyphosphate contamination within the purified nucleic acid sample. To investigate, I treated purified cellular

DNA with nucleases and polyphosphatases then assessed their ability to activate the contact pathway. Interestingly, treatment with either enzyme abrogated the procoagulant effect of purified DNA.

iii Though physiologic anionic polymers have recently been identified as activators of the contact pathway, their precise mode of action has not been elucidated. The contact pathway consists of 3 proteolytically active enzymes, each of which have been reported to bind –and active –in the presence of anionic polymers. Furthermore, an important cofactor, high molecular weight kininogen, also binds to anionic surfaces and has been demonstrated as important for mediating the activities of contact pathway proteins. Nonetheless, it is often overlooked when studying the contact pathway in vitro.

To investigate the reaction-by-reaction effects of physiologic polyanions, I performed a series of activity assays with purified proteins of the contact pathway. These assays were run in the presence of polyanions, and in some experiments HK. The results demonstrate the disparate effects of polyanions and HK, and highlight the importance of understanding contact pathway activation on a per-reaction level in addition to the pathway as a whole.

iv ACKNOWLEDGEMENTS

I would like to begin by thanking my thesis advisor, Dr. Jim Morrissey, for his excellent guidance and support during my tenure in his lab. Scientifically, I have learned a great deal through his mentorship and benefited immensely from his expertise and knowledge.

Additionally, I cannot overstate the value I’ve gleaned from working with such an exceptional leader. Being a part of Jim’s lab was an incredibly rewarding and stimulating experience; I hope to one day emulate his leadership style and acumen so that I can foster a similarly exceptional and welcoming working environment.

Of course, I would also like to thank all of my coworkers, past and present, for their camaraderie and advice. Your support and friendship has truly enriched my Ph.D. experience. I’d like to thank my committee members for their input and intellectual discussions, as well as my collaborators in the Bailey, Rienstra, and Tajkhorshid Laboratories for their expertise and advice.

None of this would have been possible without the support of my friends and family.

Thank you to all of my friends, especially those at UIUC, who have made grad school fun and exciting. Thank you to my girlfriend, Sophie, who has provided me with endless support, motivation, and love. And last but certainly not least, a very special thanks to my parents, Gary and Cyndy. You nurtured my love for science from a young age and instilled in me the belief that

I could accomplish anything I set my mind to. I would not be where I am today without your unending love and support.

v TABLE OF CONTENTS

CHAPTER 1: Introduction to the Extrinsic and Intrinsic Pathways of Coagulation…………..1

CHAPTER 2: Mg2+ Effects on TF/FVIIa Activity ……………………………………………25

CHAPTER 3: Relative Contributions Of Nucleic Acids And PolyP To Coagulation………..40

CHAPTER 4: Characterization of Polyphosphate- and Ribonucleic Acid-Mediated Contact

Pathway Activation……………………………………………………………………………...55

FIGURES AND TABLES……………………………………………………………………..70

REFERENCES…………………………………………………………………………………99

vi CHAPTER 1: Introduction to the Extrinsic and Intrinsic Pathways of Coagulationa

PART 1: THE EXTRINSIC PATHWAY

Introduction

The extrinsic tenase complex, comprised of tissue factor (TF) and factor VIIa (FVIIa), is a two-subunit enzyme that initiates the coagulation cascade under most in vivo conditions

(Figure 1.1).1,2 The regulatory subunit of this complex, TF (also known as thromboplastin,

CD142, or coagulation factor III), is a cell-surface, transmembrane protein of the class II cytokine receptor family that is extensively expressed amongst adventitial and epithelial tissues; however, tissues exposed to the vessel lumen such as endothelial cells, platelets, and leukocytes constitutively express little or no TF.3-5 The enzymatic subunit is a plasma protein, FVIIa, that is a trypsin-like serine protease demonstrating homology with several other coagulation proteins, including its cognate substrates factors IX (FIX) and X (FX). Total FVII (active enzyme and zymogen) circulates in plasma at a concentration of approximately 10 nM; however, only about

1% is in the active form.6 Due to its poor enzymatic activity, free FVIIa in plasma largely escapes recognition by protease inhibitors and therefore circulates with a half-life of ~90 minutes.7,8 The extended half-life afforded to FVIIa may serve an important function, as low basal concentrations of pre-formed FVIIa may serve to “prime” the coagulation cascade for a rapid response to injury.6

a Part 1 of this chapter has been adapted in its entirety from a review article originally published online in the journal Seminars in Thrombosis and Hemostasis, and used in accordance with the publisher’s copyright privileges for publication in a dissertation thesis. Full Citation: J. M. Gajsiewicz and J. H. Morrissey (2015). "Structure-Function Relationship of the Interaction between Tissue Factor and Factor VIIa." Seminars in Thrombosis and Hemostasis 41(7): 682-690.

1 Under homeostatic conditions, TF and blood are physically separated, but vascular injury exposes plasma (which contains FVII and FVIIa) to a variety of TF-expressing cells. Once zymogen FVII binds to TF, it is rapidly converted to FVIIa by limited proteolysis, thereby generating the active TF-FVIIa complex. Formation of the TF-FVIIa complex greatly increases the enzymatic activity of FVIIa via allosteric interactions between TF and FVIIa, as revealed by about a 20- to 100-fold increase in the rate of hydrolysis of small, chromogenic peptidyl substrates (termed its amidolytic activity),9,10 and nearly a million-fold increase in the rate of activation of the macromolecular substrates, FIX and FX.11 Subsequently FIXa, in association with its regulatory subunit FVIIIa, further activates FX; FXa in turn complexes with its cofactor

FVa, to proteolytically cleave prothrombin to thrombin, leading to fibrin clot formation.

Widespread, constitutive expression of TF on cells surrounding organs, blood vessels, and skin serves to create a “hemostatic envelope” that initiates clotting upon vascular injury.3

An additional point of modulation of TF-FVIIa is via the phospholipid bilayer. In concert with allosteric activation of the active site of FVIIa upon binding to TF, formation of the extrinsic tenase on a suitably procoagulant phospholipid bilayer increases the rate of FIX or FX activation, in a Ca2+-dependent manner, an additional 1,000-fold.11 In fact, nearly all reactions of the coagulation cascade are reliant upon exposure of phosphatidyl-L-serine (PS) on membrane surfaces.12 The roughly million-fold overall increase in FX activation by the TF-FVIIa- phospholipid complex relative to free FVIIa is critical regulatory point for the coagulation cascade.13 The roles of TF-FVIIa in hemostasis, thrombosis and other biological processes are numerous, and the structural underpinnings comprising the foundation of their activities continue to be a point of focus and investigation.

2 Structure

Tissue Factor

TF, a 263 amino acid glycoprotein with a molecular weight of ~46kDa and member of the cytokine class II receptor family, is composed of three domains: a 219 amino acid N-terminal extracellular domain (residues 1-219); a 22 amino acid transmembrane domain (residues 220-

242); and a 21 amino acid cytoplasmic C-terminal tail (residues 242-263). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and one S-palmitoylation site at Cys245.

Removal of the cytoplasmic domain has no deleterious effects on TF coagulant activity. The TF transmembrane domain is composed of a single-spanning alpha-helix, the precise identity of which has been shown unimportant for TF procoagulant function;14 anchoring of a histidine- tagged extracellular domain of TF to the membrane using nickel-chelating lipids resulted in full restoration of procoagulant activity of TF.15 The extracellular domain of TF (sTF) is composed of two fibronectin type III domains, and is connected to the transmembrane domain through a six-amino acid linker. This linker likely exhibits sufficient flexibility to conformationally decouple the extracellular domain of TF from the transmembrane and cytoplasmic domains.14,16

The fibronectin type III domain structure, composed mainly of beta-strands connected by β- loops, is a member of the immunoglobulin-like family of protein folds and is conserved amongst a wide variety of extracellular proteins.17

The procoagulant activity of TF does not necessarily correlate with its levels of cell- surface expression. Much of the TF expressed on a cell surface is ‘encrypted’, and must first be

‘decrypted’ to participate fully in coagulation reactions. The process by which this occurs has yet to be fully explained, and is likely a combination of several mechanisms. One clear contributor is exposure of anionic phospholipids. Healthy cells actively sequester anionic phospholipids such as

3 PS to the inner leaflet of the plasma membrane,18,19 but following cellular damage, activation, or increased levels of cytosolic Ca2+ this bilayer asymmetry is lost, resulting in increased PS exposure on the outer leaflet which increases the specific activity of cell-surface TF-FVIIa complexes. PS exposure is well known to decrease the apparent Km for activation of FIX and FX, but additional mechanisms could include conformational rearrangement of TF or the TF-FVIIa complex and subsequent exposure of substrate binding sites.16,20 Expression levels of GRP78, a molecular chaperone protein, have also been shown to mediate TF procoagulant activity in a Ca2+- dependent manner and thus may also play a role in its decryption.21,22

A fascinating suggestion is that disulfide linkages play a role in TF encryption/decryption, and in particular, that the membrane-proximal cysteine pair in TF

(Cys186-Cys209) is an 'allosteric' disulfide that is subject to redox control, leading to TF encryption/decryption.23-25 Others have disputed this conclusion, however, and have proposed alternative explanations for the observed effects (reviewed in 26,27). Though the importance of TF disulfide bond formation towards its cofactor activity has yet to be resolved, FVIIa binding to TF is not dependent on the oxidation state of Cys186 and Cys209.23 A number of insightful and detailed reviews focus on controversies surrounding TF decryption are available.28-32

Factor VII/VIIa

The trypsin-like serine protease FVII (in the inactive precursor, or zymogen form) is a

~50KDa, single-chain polypeptide consisting of 406 residues, with an N-terminal γ- carboxyglutamate-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and

EGF2), and a C-terminal serine protease domain.33-36 Activation of FVII to FVIIa is

4 accomplished via specific proteolytic cleavage of the Ile153 -Arg152 bond in the short linker region between the EGF2 and protease domain, with the resultant light and heavy chains held together by a single disulfide bond (Cys135-Cys262). FVII has significant structural and sequence homology to coagulation factors IX, X, and protein C.37,38

FVIIa binds the phospholipid membrane in a Ca2+-dependent manner through its N- terminal GLA-domain. Containing 10 vitamin K-dependent, posttranslationally modified γ- carboxyglutamate (Gla) residues, GLA-domains coordinate 7-9 divalent metal ions such as Ca2+ and Mg2+, inducing conformational rearrangements that are requisite for interaction with membrane surfaces.39,40

Immediately C-terminal to the GLA domain is an aromatic stack and two epidermal growth factor (EGF) domains (EGF1 and EGF2). The aromatic stack connects the GLA to

EGF1 domain, which binds a single Ca2+ ion with moderately high affinity.41,42 Occupancy of this Ca2+-binding site increases FVIIa amidolytic activity and TF association.43 The FVIIa heavy chain comprises the trypsin-like protease domain, which is also homologous to other coagulation serine proteases such as FX, FIX, protein C, and prothrombin. The catalytic triad consists of

His193, Asp242 and Ser344, and binding of a single Ca2+ ion within the FVIIa protease domain is critical for catalytic activity.41,44 Additionally, proteolytic activation of FVII to FVIIa frees the newly formed amino terminus at Ile153 to fold back and insert into the activation pocket, forming a salt bridge with the carboxylate of Asp343 to generate the oxyanion hole.45 Formation of this salt bridge is critical for FVIIa activity; indeed, FVIIa with the mutation V154G is cleaved to the two-chain form normally and with wild-type macromolecular substrate affinity,46 but with significantly reduced ability of the resultant FVIIa to activate FX.46,47 Reduced N-terminal

5 hydrogen-deuterium exchange upon TF binding to FVIIa supports the hypothesis that the N- terminal Ile153 is not fully inserted into the activation pocket when free in solution.48

Additionally, unlike most other serine proteases, oxyanion hole formation in free FVIIa does not occur upon proteolytic activation, but instead upon substrate interaction.49 As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors,49 allowing it to circulate with a half-life of approximately 90 minutes.7,8 This is far longer than other coagulation enzymes such as FIXa, FXa and thrombin, whose plasma half-lives are on the order of seconds to minutes.50,51

Structure-Function Relationship

Association of TF with FVIIa allosterically activates the protease, creating what is essentially a dimeric enzyme in which TF is the regulatory subunit and FVIIa the catalytic subunit. The ability to cleave very small, tripeptidyl-amide substrates (amidolytic activity) of

FVIIa is increased approximately 50-fold upon binding of TF, with the largest change being

9,10 increased kcat, indicating that TF association induces conformational changes within the active site of the FVIIa protease domain.45,52-54 Additionally, pKa values of the catalytic triad are altered upon TF binding.55 It has been hypothesized that FVIIa in solution exists in an equilibrium between two states; one in which the heavy-chain N-terminus has inserted into the active site pocket, and one that is more zymogen-like with the N-terminus incorrectly or incompletely inserted. TF may preferentially bind FVIIa when it is in the catalytically active form,45 shifting the equilibrium towards the active (N-terminal buried) conformation. Hydrogen-deuterium exchange experiments coupled with mass spectrometry have demonstrated that several loop regions within the protease domain of FVIIa are stabilized upon binding to TF, through

6 rearrangement and strengthening of an extensive hydrogen bonding network.48,52 These structural changes are not limited to the protease domain but extend throughout most of

FVIIa,48 indicating widespread allosteric modulation of FVIIa by TF.

Active Site Positioning

In vivo enzymatic activity of the TF-FVIIa complex occurs exclusively on the phospholipid membrane surface, and is dependent upon the interaction of FVIIa, FIX and FX with the membrane through their membrane-binding GLA-domains. Fluorescence resonance energy transfer (FRET) experiments indicate that free FVIIa adopts a stable, extended structure when bound to the membrane, with its active site positioned ~80 Å above the membrane surface.56 This distance is in good agreement with those seen for the homologous proteins FIXa57 and FXa58. Upon FVIIa binding to TF, the FVIIa active site is repositioned ~6 Å closer to the membrane, a modulation that may aid in proper alignment of the FVIIa catalytic triad with the target substrate cleavage site.56 In comparable FRET experiments using GLA-domainless FVIIa, the active site was still positioned a similar distance above the membrane, demonstrating that TF is able to fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions.59 Further, experiments using multiple approaches have shown that TF supports full

FVIIa proteolytic activity as long as the TF extracellular domain is tethered in some way to the membrane surface, while the exact nature of this membrane tether is essentially irrelevant.14,15,60

In contrast, raising the active site of FVIIa greater than 80 Å above the membrane surface using

TF/P-selectin chimeras greatly reduced the ability of the TF-FVIIa complex to activate FX, but did not diminish TF-FVIIa amidolytic activity. This indicates that TF-mediated positioning of

7 the FVIIa active site above the membrane surface is important for its activity towards cognate substrates.61

Molecular dynamics (MD) simulations of TF-FVIIa in the presence of membrane surfaces indicate TF reduces FVIIa flexibility. Intra-domain dynamics (Cα RMSD) are small compared to inter-domain flexibility, suggesting TF interaction significantly rigidifies FVIIa domains relative to each other.16

Spatial Stabilization

Free FVIIa is an inherently dynamic molecule, with MD simulations, fluorescence anisotropy, and hydrogen-deuterium exchange data indicating intra- and inter- domain flexibility.16,52,62-64 A major component of TF allosteric modulation of FVIIa activity is the stabilization and reduced flexibility of FVIIa upon TF binding. In the protease domain, stabilization of the 170-loop located near the TF interaction site appears to be important for

FVIIa amidolytic activity.63,64 Replacement of the loop with that of a similar but truncated loop from trypsin results in an increase in FVIIa amidolytic activity even in the absence of TF, suggesting that stabilization of the 170-loop plays a significant role in TF-mediated allosteric activation of FVIIa.65 Additionally, hydrogen exchange experiments and MD simulations show that TF binding aids the insertion of Ile153 into the activation pocket of FVIIa and stabilizes its structure, even after removal of the N-terminal insertion.52,63 Specific TF and FVIIa residues have been identified that contribute to stabilization of FVIIa within the TF-FVIIa complex.

Alanine scanning mutagenesis studies66 and crystallography data41 demonstrated that Met306 in

FVIIa plays a pivotal role in TF interactions, restricting the flexibility of FVIIa’s 170-loop upon

8 FVIIa-TF complex formation. The crystal structure of free FVIIa,67 in which the 170-loop and precluding α-helix (containing Met306) are more disordered, also supports this idea.

TF-Substrate Interactions

In addition to the role of TF in allosterically activating FVIIa, binding interactions between the ‘exosite’ region of TF and macromolecular substrates are also implicated in TF-

FVIIa catalytic activity. The known physiologic substrates of TF-FVIIa are FVII, FIX, FX and certain protease activated receptors (PARs). TF mutational analysis has identified a number of residues that, when mutated, support full FVIIa amidolytic activity towards small peptidyl substrates but are deficient in their ability to support macromolecular substrate (FVII, FIX, FX) activation.68-70 Several crystal structures have shown disorder in the TF loop region at residues

159-165,41,49 and residues in or adjacent to this flexible loop have been shown to be especially critical for proteolytic activity of the TF-FVIIa complex, thereby defining the proposed substrate-binding exosite region of TF that is quite distant from the FVIIa active site.68,69

Interestingly, mutation of Gly164 of TF to a marginally more bulky alanine significantly impairs

TF-FVIIa proteolytic activity, suggesting the flexibility afforded by glycine is critical for macromolecular substrate recognition.68,70

TF residues Lys165 and Lys166 have also been demonstrated to be important for substrate recognition and binding; mutation of either of these residues to alanine results in a significant decrease in the cofactor function of TF.68,69,71,72 However, TF with mutations at K165A and

K166A activated GLA-domainless FX at rates comparable to that of the wild-type TF, and utilization of GLA-domainless FVIIa greatly muted the effects of these TF mutations on FX activation.69 Crystal structures have indicated that Lys165 and Lys166 face away from each other,

9 with Lys165 pointing towards FVIIa in most TF-FVIIa structures, and Lys166 pointing into the substrate binding exosite region.41,73 Putative salt bridge formation between Lys165 of TF and

Gla35 of FVIIa would support the notion that TF interaction with the GLA-domain of FVIIa modulate substrate recognition.73 Taken together, these results suggest that the C-terminal portion of the TF ectodomain directly interacts with the GLA-domains (and possibly the adjacent EGF1 domains) of FIX and FX, and that the presence of the FVIIa GLA-domain may modulate these interactions, either directly or indirectly. Furthermore, the TF residues involved in substrate interactions with FIX and FX are similar or identical to those that interact with FVII during TF-mediated FVII autoactivation,53 indicating a similar mechanism of substrate binding.

A number of monoclonal anti-TF antibodies have been raised, with the vast majority blocking association between TF and FVII.74 However, two monoclonal antibodies, TF8-5G9 and TF8-11D12 (which came from the same fusion and which are probably identical) were shown not to inhibit TF-FVIIa binding, but to strongly inhibit activation of FIX and FX by TF-

FVIIa.75,76 Crystal structures of TF in complex with the antibody TF-5G977 indicates that the epitope on TF for this antibody overlaps significantly with the substrate interaction (exosites) region identified by Kirchhofer et al.70 More recently, two additional monoclonal anti-TF antibodies (D3 and 5G6) with similar properties have also been reported.78

Monoclonal antibody TF9-10H10 binds to TF but does not inhibit its procoagulant activity, which is unusual as almost all anti-TF antibodies are inhibitory.74 More recent studies using this antibody have shown that it does inhibit the ability of the TF-FVIIa complex to participate in signaling.79 These results suggest that TF-FVIIa signaling via integrins and PAR-2 is mediated by exosite-like interactions on TF distinct from those involved in TF-FVIIa procoagulant functions.

10

Protein-Membrane Interactions

Lipid bilayer composition plays an important role in activity of the TF-FVIIa complex.

GLA-domain containing coagulation proteins are well-known to preferentially bind anionic phospholipids in general, and PS in particular.12 PS is, however, actively sequestered to the inner leaflet of the plasma membrane, serving as an important point of regulation of blood clotting.12,18,19,80 Exposure of PS on the outer leaflet occurs either via physical disruption of the cell membrane as a consequence trauma, or via regulated cellular processes such as those that occur upon platelet activation. Interestingly, despite the high degree of sequence homology between GLA-domains of different clotting proteins, their membrane binding affinities vary by three orders of magnitude,81,82 with FVIIa and activated protein C (APC) displaying the weakest affinities for PS-containing bilayers. We recently reported that FVIIa and APC preferentially bind to bilayers containing phosphatidic acid (PA), a minor anionic lipid in cell membranes.83

PA has minimal effect on TF-FVIIa activity in vitro, however, likely due to the fact that protein- protein interactions between TF-FVIIa dominate the recruitment of FVIIa to the membrane.84

Further, when used pharmacologically to treat bleeding disorders, the mechanism of action of recombinant FVIIa has been shown to be independent of TF.85,86 Thus, the membrane binding characteristics of “free” (non-TF bound) FVIIa may be an important component of its in vivo efficacy, especially when high concentrations of recombinant FVIIa are employed to treat bleeding.

Direct interactions between the TF ectodomain and the membrane surface may also contribute to the activity of the TF-FVIIa complex. It is thought that there is considerable freedom of motion and autonomy of the TF ectodomain relative to the membrane due to the

11 structural flexibility of the short peptide linker between the ectodomain and the transmembrane domain.14 Furthermore, MD simulations have identified a number of TF residues in the C- terminal portion of the ectodomain that directly contact the phospholipid membrane surface.

These residues maintained association of sTF (i.e., the isolated ectodomain) with the membrane surface, suggesting that TF residues may associate directly with PS headgroups.16 Additionally, simulations suggest the orientation of TF with respect to the membrane is altered upon FVIIa binding, with TF leaning toward FVIIa. As a result, the TF residues in contact with the phospholipid membrane are proposed to change.16 This region of putative membrane-interacting

TF residues is immediately adjacent to the proposed substrate-binding exosite, and alanine scanning mutagenesis studies have identified mutations in this region that alter the ability of membrane-anchored TF-FVIIa to activate FX.20 Interestingly, increasing the PS content of TF- liposomes partially overcomes these deficiencies, suggesting that direct PS-TF interactions may either stabilize the complex or induce ideal conformational arrangements, promoting interaction of FIX and FX with the TF exosite.16,20

Biochemical studies of protein-membrane interactions in blood clotting often utilize liposomes with non-physiological membrane compositions. Thus, it typically requires 30% or more PS to achieve maximal TF-FVIIa enzymatic activity in vitro,87 while only ~10% of the total plasma membrane bilayer is composed PS in eukaryotic cells.88 Incorporation of phosphatidylethanolamine (PE, a plasma membrane phospholipid that is much more abundant than PS) into TF-liposomes markedly decreases the required PS content, although PE by itself does little to promote TF activity.87 Other lipids such as phosphatidic acid, phosphatidylglycerol, and phosphatidylinositol also reduce the PS requirement, indicating ‘synergy’ between PS and

PE is not a unique property of PE but a more broadly encompassing mechanism. This ‘ABC

12 hypothesis’ (Anything But Choline) postulates that any lipid not containing the bulky choline headgroup of PC or sphingomyelin can ‘synergize’ with PS and decrease the required PS content for maximal enzymatic activity.

Divalent Metal Ions

Calcium ions are required for assembly and function of TF-FVIIa. Although TF lacks any known divalent metal ion binding-sites, Ca2+ can occupy up to nine metal binding sites within FVIIa. Of these, seven reside in the GLA-domain of FVIIa and are critical for both structure and function of this domain. In particular, Gla7 and Gla9 coordination of Ca2+ induces formation of the ω-loop, which exposes hydrophobic residues that are believed to insert into the bilayer.87,89-91 Recent crystallographic, enzymatic, and equilibrium dialysis studies have indicated that the FVIIa GLA-domain actually binds a combination of 4-5 Ca2+ and 2-3 Mg2+ under physiologic divalent metal ion conditions.49,73 Ca2+ is absolutely required for GLA-domain structure and function, and can occupy all GLA-domain metal binding sites when it is the only divalent metal ion present (especially at supraphysiologic concentrations of Ca2+), but Mg2+ alone is unable to induce the correct GLA-domain structure. This suggests that a subset of metal binding sites in GLA domains are occupied with Mg2+ in vivo,40,73 and occupancy of these sites by Mg2+ even in the presence of vast excesses of Ca2+ suggest these sites preferentially bind Mg2+ in plasma.49,73 The reasons for differential metal ion specificity of GLA domains remain unclear, but are likely due to differences in coordination geometries of each binding site along with the

‘hardness’ properties of Ca2+ versus Mg2+.73,92 Mg2+ has been demonstrated to modulate both the membrane binding73 and enzymatic properties93 of FVIIa and FIX94.

13 One Ca2+ binds to the EGF1 domain of FVIIa, for which Mg2+ cannot substitute. 49 This

Ca2+ is implicated in optimizing TF-FVIIa binding interactions, likely through modulating the orientation of the FVIIa GLA-domain relative to the EGF1 domain.95 The protease domain of

FVIIa also contains one Ca2+ binding site; its occupancy results in allosteric activation through reregistration of the Ca2+ binding loop.52 Additionally, two Zn2+ binding sites have been identified in the protease domain, although Zn2+ occupancy of these sites inhibits both FVIIa enzymatic activity and TF binding.49,96

The Ca2+ binding sites in FVIIa are incompletely saturated at plasma concentrations of free Ca2+ (~1.25 mM).97,98 However, using the plasma concentrations of free Ca2+ and Mg2+

(~1.25 mM and ~0.6 mM, respectively) restores activity to maximal levels, indicating that Mg2+ likely plays a role in vivo.73,93,94,99 The GLA-domains of both FVIIa and FX mediate enzymatic rate enhancements due to Mg2+, suggesting that conformational changes of the FX GLA- domain upon Mg2+ occupancy of metal binding sites modifies its interactions with TF.99

Signaling

The role of TF in cellular signaling is incompletely understood, though it is known to play roles in tumor growth,79 metastasis,100,101 angiogenesis, and anti-apoptotic signaling;102 additionally, TF is known to be upregulated in many malignant tumor cell types.103-105 FVIIa binding to TF has been implicated in directly mediating PAR-2 cleavage via mechanisms both dependent106-109 and independent110,111 of the cytoplasmic domain of TF.112 Phosphorylation of the TF cytoplasmic domain subsequent to TF-FVIIa cleavage of PAR-2 is thought to release

TF-dependent negative regulation of PAR-2 mediated signaling.79,103,108 This results in mitogen- activated protein kinase (MAPK) pathway activation and downstream effects, including the

14 upregulation of cytokines and proangiogenic factors.103,113 Activation of the p44/42 MAPK pathway, as well as JAK2, p70/p85S6K, and p90RSK, can occur independent of the TF cytoplasmic domain.110,114,115 Though the cytoplasmic domain is not necessary for activation of these proteins, proteolytically active FVIIa is required. In addition, TF can also mediate signaling through the enzymatic activity of FXa in complex with TF-FVIIa, which plays an important role in the regulation of several pathways. TF-FVIIa-FXa-mediated cleavage of PARs, particularly PAR-2, has been shown to up-regulate IL-8 expression, resulting in increased cell migration.101 A number of detailed reviews can be found regarding TF-FVIIa-mediated signaling.101,103,116,117

Conclusion

The interaction between TF and FVIIa is a critical component of hemostasis. TF is expressed extensively on cells outside the vasculature, creating a ‘hemostatic envelope.’

Disruption of the blood vessel endothelium results in exposure of blood coagulation proteins, including FVIIa, to TF-bearing cells. Subsequent to TF-FVIIa complex formation, two blood clotting zymogens, FIX and FX, are proteolytically activated, propagating the coagulation cascade and resulting in fibrin deposition and clot formation. The physical separation of TF from the plasma clotting enzymes is undoubtedly a regulatory mechanism; without its protein cofactor,

FVIIa is a very weak enzyme and will not initiate coagulation at physiologic concentrations.

Expression of TF inside the vasculature results in aberrant coagulation cascade activity and is believed to play an important role in many thrombotic disorders.

TF modulates FVIIa activity through a number of mechanisms. FVIIa interaction with

TF has been demonstrated to position its active site ~75 Å above the membrane surface, ~6 Å closer than FVIIa in the absence of TF. Additionally, MD simulations have suggested that TF

15 restricts both FVIIa inter- and intra- domain flexibility, particularly within the protease domain.

TF has been shown to stabilize a number of loop regions in the protease domain of FVIIa as well as facilitate the insertion of the N-terminus of the FVIIa heavy chain into the activation pocket, which is critical for its enzymatic activity. In addition, an extended substrate binding exosite has been identified on TF, which has been shown to interact directly with both extrinsic tenase complex substrates, FIX and FX. The importance of the phospholipid membrane in mediating the activity of TF-FVIIa cannot be understated; recruitment of FX to the membrane surface is dependent upon the exposure of anionic phospholipids, particularly PS, which may interact with substrates (FIX and FX), enzyme (FVIIa) and protein cofactor (TF). All the interactions between TF, FVIIa, protein substrates and membrane surfaces are dependent upon divalent cations. Although several structures of TF-FVIIa have been solved, as of yet no tertiary TF-

FVIIa-FX or TF-FVIIa-FIX structures have been determined, nor has the role of the membrane surface in the formation of these complexes been determined structurally. Such results would provide significant information regarding the structure-function relationship of the extrinsic tenase in blood clotting.

PART 2: THE INTRINSIC PATHWAY

Introduction

The intrinsic, or contact, pathway of coagulation begins with the autoactivation of factor

XII (FXII) to FXIIa upon interaction with anionic surfaces.118,119 FXIIa cleavage of plasma prekallikrein (PPK) to plasma kallikrein (PK) creates a positive activation feedback loop,120 leading to further FXII activation by PK and high-molecular weight kininogen (HK). The cleavage of HK by PK releases bradykinin, a potent vasodilator and proinflammatory

16 peptide.121,122 After activation, FXIIa cleaves factor XI (FXI); subsequently, factor IX (FIX) is activated via limited proteolysis by FXIa. The newly generated FIXa then activates factor X (FX) in the presence of factor VIIIa (FVIIIa), initiating downstream proteolysis within the common pathway and clot formation. Figure 1.1 depicts both the extrinsic, intrinsic, and common pathways of coagulation.

For years the primary functions of the contact pathway in vivo appeared to be the release of bradykinin; the physiologic importance of the contact pathway to blood coagulation was unknown. Patients deficient in FXII demonstrate no bleeding phenotype, and mouse models support these observations.123 Furthermore, the contact pathway has been traditionally activated upon contact of blood plasma with non-physiologic, anionic surfaces such as glass, diatomaceous earth, or kaolin;124 thus, it was unclear what, if any, physiologic substances activated the pathway.

However, recent work has identified a number of possible physiologic activators of FXII.

Interestingly, the contact pathway appears to be dispensable for normal hemostasis but play important roles in thrombosis. Animal models suggest that FXII deficiency is thromboprotective,121,125 and numerous other investigations indicate a correlation between atherosclerosis or heart attack and elevated plasma protein levels of FXII, FXI, or PK.126-129 In addition to releasing bradykinin and initiating the contact pathway of coagulation, FXII has been demonstrated to modulate fibrinolysis and platelet function.123 Thus, the roles of the contact pathway are numerous and inextricably link coagulation and inflammation.

Structure and Function

FXII(a)

17 FXII, a 596 amino acid glycoprotein with a molecular weight of ~80 kDa, is synthesized in the liver and circulates in blood at a concentration of about 375 nM.123 The light and heavy chains contain 243 and 353 residues, respectively, and are held together by a single disulfide bond. The heavy chain contains 6 domains and is thought to mediate the interactions of FXII with negatively charged surfaces, while the light chain contains the active site. The activation of

FXII can occur via limited proteolysis by PK119,130 and by autoactivation, initiated by FXII interaction with anionic surfaces and subsequent conformational changes.131-134 It is believed that

FXII binding to anionic surfaces is mediated through its type II fibronectin domain, second

EGF domain, and kringle domain.135-139 The cognate substrates for FXIIa are PPK and FXI; importantly, FXII is known to exist in two forms, α-FXIIa and β-FXIIa. α-FXIIa is full length and fully active against PK and FXI, while β-FXIIa has been cleaved a second time, releasing a

28 kDa protein that is no longer able to activate FXI or bind anionic surfaces, but retains its full activity against PPK.140 Recent work suggests that FXIIa can mediate coagulation independent of

FXI in the presence of polyP, suggesting alternative substrates exist downstream of FXI in the coagulation cascade.141 FXII deficiency is not associated with bleeding in humans;142 FXII- deficient mice also demonstrate normal hemostasis,143,144 albeit with slightly impaired thrombus stabilization.145,146

PPK/PK

PPK, also made in the liver, is a 609 amino acid glycoprotein with varying degrees of glycosylation, leading to molecular weights of 85 kDa or 88 kDa.147 PPK circulates in plasma at a concentration of ~500 nM, 75% of which is bound to HK,147 and is activated to PK via cleavage by FXIIa and HK into a 248 amino acid light chain and 371 amino acid heavy chain; PPK can

18 also undergo autoactivation in the presence of anionic polymers.148 Creating a positive feedback loop, PK and its cofactor HK reciprocally activate FXII. The cognate substrates for PK are FXII and HK.121 PPK deficiency is not associated with bleeding in humans.

HK

HK, a 110 kDa plasma protein, circulates at a concentration of ~640 nM. HK serves as a cofactor for both FXII activation by PK and for PPK activation by FXIIa, and is required for PK formation via surface activation.123,149 PK cleavage of HK results in release of bradykinin from

HK, leading to propagation of the kallikrein-kinin pathway.121 In addition to circulating in complex with PPK, HK also circulates in complex with FXI,150 and acts to indirectly anchor both

FXI and PPK to the membrane surface through its own interactions with proteoglycans and heparan.151 Deficiencies in HK are not associated with bleeding.

FXI(a)

FXI, with a monomer molecular weight of 80 kDa, circulates as a homodimer in plasma at a concentration of ~30 nM. Activation of FXI occurs via limited proteolysis by FXIIa in the presence of Ca2+ and HK, or it can occur via autoactivation (i.e., FXI activation by FXIa);152-154 additionally, FXI can be activated by thrombin in the presence of negatively charged surfaces,155 particularly polyphosphate (polyP).156 Once activated, FXIa activates FIX in a Ca2+-dependent manner, leading to FX activation and subsequent clot formation. Deficiency in FXI results in a mild bleeding disorder, termed hemophilia C, which presents most often in tissues high in fibrinolytic activity after trauma or surgery.157-161

19 C1 Esterase Inhibitor

C1-Inhibitor is a 104 kDa member of the serpin superfamily, and is the main inhibitor responsible for regulating the contact and complement pathways. As an acute-phase protein its concentration can increase greatly during inflammation from its basal circulating concentration of ~1.8 µM.162 C1-Inhibitor is a key regulator of FXIIa, PK, and FXIa; deficiency in C1-

Inhibitor results in hereditary angioedema, a disorder resulting from the increased plasma PK concentration and subsequent bradykinin release.

Contact Pathway Activators

In vitro

A number of artificial anionic surfaces are known contact pathway activators; indeed, these substances have been used for years as the activators in the clinical aPTT assay, used to probe the contact and common pathways of coagulation, as well as monitor patients being treated with anticoagulants such as heparin. A shared property of most of these substances responsible for contact pathway activation is their polyanionic nature, which is thought to mediate FXII autoactivation, and subsequent reciprocal activation of FXII by PK and HK. One notable example is glass, as the clotting of blood collected into glass tubes in the absence of metal chelators can be attributed to glass-mediated contact pathway activation. In addition, kaolin, dextran sulfate, and diatomaceous earth are all activators of the contact pathway.163,164

In vivo

For decades the physiologic activators of the contact pathway in vivo were unknown.

Recently, renewed interest in the (patho)physiology of the contact pathway has led to the

20 identification of numerous candidate in vivo activators, including DNA,165-167 RNA,166,167 misfolded proteins,168 and polyP.169,170 These activators will be discussed in greater detail in the following sections.

Misfolded protein

A number of studies have identified misfolded proteins as potential activators of FXII.

Aggregated amyloid β peptide, believed to be play a role in Alzheimer’s disease, has been shown to activate FXII in vivo.171 Misfolded protein aggregates also activate FXII, leading to the production of PK;168 interestingly, these aggregates did not result in the generation of FXIa, suggesting differential regulation of the contact pathway of coagulation and the kallikrein-kinin pathway. Furthermore, misfolded protein interactions with FXII are mediated through the fibronectin type I domain of FXII,172 distinct from its anionic surface binding domains.

Nucleic Acids

Nucleic acids are released from cells into the extracellular space following a number of events, including apoptosis and extrusion; neutrophils have been shown to release their nuclear content –termed neutrophil extracellular traps, or NETs– under certain conditions, a process known as NET-osis.173 A number of studies have identified extracellular nucleic acids as potential in vivo activators of the contact pathway.166,174,175 Kannemeier et al166 provided both in vitro and in vivo evidence for the activation of contact proteins by extracellular nucleic acids, and demonstrated that FXII and FXI bound cell-derived RNA tightly while PPK and HK bound more weakly. Investigations into the possible role of nucleic acid structure towards its procoagulant ability suggest that secondary structure is an important consideration.176

21 Additionally, extracellular DNA purified from cellular sources has been shown to modulate clot structure and mediate fibrinolysis.177 Recent work from our lab has indicated that the procoagulant effects of cell-free nucleic acids may be due, in part, to the presence of contaminant polyP that co-purifies readily with DNA or RNA.

PolyP

PolyP is a linear anionic polymer of phosphate molecules, held together by high-energy phosphoanhydride bonds, and can be tens to thousands of phosphate residues long.178 PolyP is an ancient molecule and can be found across all branches of life.179,180 Microorganisms synthesize and store very long-chain polyP, up to thousands of units in length, in granules known as acidocalcisomes.181-183 In these subcellular compartments, polyP is often in complex with metal ions under acidic conditions, and is hypothesized to play important roles in metal and energy storage.184,185 Mammalian cells, such as platelets, store much shorter polyP –60 to 100 units long– in dense granules; these granules are evolutionarily related to acidocalcisomes in microorganisms.186,187 PolyP has also been found in mast cell granules,187 lysosomes,188 mitochondria, nuclei, and across numerous tissue types spanning heart, lung, brain, and kidneys.189

PolyP is a potent activator of the contact pathway of coagulation, promoting the activation of FXII and the kallikrein-kinin pathway. PolyP-mediated activation of FXII appears to occur via an allosteric activation mechanism, as proteolytically active single-chain FXII is generated in the presence of polyP. Upon disruption of the polyP-FXII interaction, the enzyme returns to its inactive, zymogen state.133 PolyP binds with high affinity to numerous proteins of the coagulation cascade, and contact pathway in particular.190-192

22 Interestingly, the effects of polyP are dependent upon the polymer length.190 Short-chain polyP, such as that found in and secreted from platelet dense granules, weakly activates the contact pathway but strongly supports Factor V activation and back-activation of FXI by thrombin.156,190,191 Conversely, long-chain polyP, up to thousands of phosphate monomers in length, potently activates the contact pathway, supports factor V activation, accelerates thrombin back-activation of FXI, and enhances fibrin polymerization.190,193 On a weight basis, long-chain polyP is more potent than kaolin, an artificial surface, in activating the contact pathway;190 polyP is also orders of magnitude more potent –on both a weight and molar basis– than cell-free DNA or RNA when compared in a modified aPTT clotting assay (unpublished data).

Conclusion

Traditionally, the contact pathway is initiated via the activation of FXIIa and propagated through the reciprocal activation of FXII and PK (in association with HK). Numerous potential physiologic activators of the contact pathway have come to light in recent years, increasing interest in the pathway and its potential role in vivo. PolyP, a highly negatively charged, linear polymer of orthophosphate residues held together by high-energy phosphoanhydride bonds is an exceptionally potent mediator of the contact pathway that is not only physiologically relevant, but exists across all branches of life. Fully understanding how this molecule mediates coagulation on a reaction level will be critical for a full appreciation of the role the contact pathway plays in both hemostasis and thrombosis.

Though the role of the contact pathway in normal hemostasis appears to be minimal, the opportunity for design of therapeutic approaches targeting components of the contact pathway to mitigate thrombosis are numerous. The contact pathway and its constituents are important

23 contributors to many thrombotic disorders and play important roles in linking coagulation and inflammation. Current antithrombotic therapies target common pathway coagulation factors; while this approach is often effective at lessening the risk of thrombotic events, it comes at the price of an increased risk of bleeding. In contrast, contact pathway coagulation factors and mediators are generally not required for normal hemostasis but have been shown to play important roles in the propagation of thrombosis. Thus, these components are enticing targets for new antithrombotic therapies.

24 CHAPTER 2: Mg2+ Effects on TF/FVIIa Activityb

Abstract

The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa

(FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their γ - carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound

Ca2+ ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca2+ concentrations to ensure saturation of these domains with bound Ca2+. Recently, it has become clear that, at plasma concentrations of metal ions, Mg2+ actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg2+ ions are required for full function of these clotting proteins.

In this study, we investigated how Mg2+ influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg2+ to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg2+. Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg2+ to enhance the activity of the TF/FVIIa complex. Our results suggest that these

TF residues interact with the GLA domain of FX in a Mg2+ -dependent manner (although

b This chapter has been adapted in its entirety from an article originally published online in the journal Biochemistry, and used in accordance with the publisher’s copyright privileges for publication in a dissertation thesis. Full Citation: Gajsiewicz, J. M., et al. (2015). "Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex." Biochemistry 54(30): 4665-4671.

25 effects of Mg2+ on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF.

Introduction

The importance of divalent metal ions in blood coagulation reactions is well documented, as they play key roles in both protein-membrane and protein-protein interactions.12,39 Seven blood coagulation proteins interact reversibly with anionic membranes via their GLA domains, which are rich in the post-translationally modified amino acid, γ-carboxyglutamate (Gla). GLA domains bind multiple divalent metal ions,90 causing them to fold and form a characteristic ω- loop structure, exposing hydrophobic residues that are believed to facilitate penetration of the membrane bilayer.194

The vast majority of biochemical studies of blood clotting proteins have employed supraphysiologic concentrations of Ca2+, which can occupy 7 to 9 metal ion-binding sites in the

GLA domains of factors VII (FVII), IX (FIX) and X (FX).41,90,195,196 Ca2+ is the most prevalent divalent metal ion in plasma, with a free concentration of ~1.25 mM.197 However, biochemical studies of blood clotting proteins using 1.25 mM Ca2+ result in submaximal enzymatic activity and membrane binding, apparently because of incomplete saturation of the metal ion binding sites in the GLA domains of these proteins. For this reason, supraphysiologic Ca2+ concentrations (typically, 2.5 to 5 mM Ca2+) are typically employed in studies of blood clotting reactions. Recent studies, however, have shown that using the plasma concentrations of free Ca2+

(1.25 mM) together with the plasma concentration of free Mg2+ (~0.6 mM) results in clotting factor activities that are at least as high as those observed in the presence of supraphysiologic

26 [Ca2+].49,94,99,198,199 These studies therefore indicate that Mg2+ plays important, albeit somewhat overlooked, roles in blood clotting reactions.

In the present study, we examined how Mg2+ modulates the initiation phase of the blood clotting cascade. Blood clotting is triggered when the integral membrane protein, tissue factor

(TF), binds FVIIa to form the TF/FVIIa complex. This complex activates FIX and FX by limited proteolysis. The mechanism(s) by which Mg2+ modulates the activity of clotting proteases is unclear, but recent crystal structures of FVIIa indicate that Mg2+ occupies 2 or 3 of the metal ion binding in its GLA domain under physiologic concentrations of Ca2+ and Mg2+,49 with structural implications.200 Persson et al.99 reported that removal of the FX GLA domain eliminated the ability of Mg2+ to increase the rate of FX activation by TF/FVIIa, suggesting that incorporation of Mg2+ into the GLA domain of FX may be the basis by which Mg2+ enhances

FX activation.

We utilized site-directed mutagenesis of TF to investigate how Mg2+ modulates the activity of the TF/FVIIa complex. We confirm that TF is a key mediator of the Mg2+ response, despite its lack of direct interaction with either Ca2+ or Mg2+. We now identify individual TF residues within or near its putative substrate-binding exosite that are necessary for Mg2+ to enhance of the rate of FX activation by TF/FVIIa. Furthermore, we demonstrate that physiologic concentrations of Ca2+ plus Mg2+ support higher TF/FVIIa enzymatic activities than in the presence of Ca2+ alone, an effect that was more pronounced when more physiologic lipid compositions were utilized. Our findings support the idea that TF is an integral component of macromolecular substrate recognition by the TF/FVIIa complex, and that Mg2+ contributes to

TF/FVIIa function.

27 Methods

Materials.

Materials were from the following sources: phospholipids 1-palmitoyl-2- oleoylphosphatidylcholine (PC) and 1-palmitoyl-2-oleoylphosphatidyl-L-serine (PS), Avanti

Polar Lipids (Alabaster, AL); Bio-Beads® SM-2 absorbent, Bio-Rad; human FVIIa, American

Diagnostica (now Sekisui Diagnostics, Lexington, MA); human FX, FXa, FIX, and FIXa,

Haematologic Technologies (Essex Junction, VT); Spectrozyme Xa, Bachem (Bubendorf,

Switzerland); Pefachrome FIXa, Enzyme Research Laboratories (South Bend, IN); and

Chromozym t-PA, Roche Diagnostics (Mannheim, Germany).

Production and relipidation of TF.

Recombinant human membrane-anchored TF (memTF, residues 3-244)201 and soluble

TF (sTF, residues 3-219)202 were expressed in Escherichia coli and purified as previously described. TF mutants were prepared using the Q5 site-directed mutagenesis kit from New

England Biolabs (Ipswich, MA). TF-liposomes were prepared by incorporating memTF into phospholipid vesicles of varying composition as described.203

FVIIa Amidolytic Activity and Quantifying TF/FVIIa Binding.

Initial rates of hydrolysis of Chromozym-tPA substrate (FVIIa amidolytic activity) were

61 measured essentially as previously described, in buffer containing either 1.25 mM CaCl2, 1.85 mM CaCl2, or 1.25 mM CaCl2 plus 0.6 mM MgCl2. The affinity of FVIIa for TF was measured as described,15 using 5 nM FVIIa, 0-20 nM memTF, 1 mM Chromozym-tPA, and 0.1% Triton

28 X-100 in HBSA (20 mM HEPES pH 7.4, 100 mM NaCl, 0.02% sodium azide, 0.1% bovine serum albumin), either with 1.85 mM CaCl2, or with 1.25 mM CaCl2 plus 0.6 mM MgCl2.

Rates of Activation of FX and FIX.

Initial rates of FX activation by TF/FVIIa using memTF, either in solution or relipidated into phospholipid vesicles, were quantified using a continuous FX activation assay as described,15,204 with slight modifications. Briefly, memTF and FVIIa were incubated essentially as described, but in buffers whose divalent metal ion concentrations were either 1.25 mM Ca2+,

1.85 mM Ca2+, or 1.25 mM Ca2+ plus 0.6 mM Mg2+. Reactions were initiated by addition of 100 nM FX and 1 mM Spectrozyme Xa. Reactions without membranes included 0.1% Triton X-100 and typically employed 10 nM FVIIa and 500 nM memTF. Reactions with membranes typically employed 30-100 pM FVIIa and excess relipidated memTF (>1 nM memTF, with 25 µM total phospholipid).

Initial rates of FIX activation were quantified in a two-stage assay as described,70 with some variations. Briefly, FVIIa and memTF were incubated at 37°C for 5 min in buffer containing 0.06% Triton X-100 together with either 1.25 mM Ca2+ or 1.25 mM Ca2+ plus 0.6 mM Mg2+. FIX was then added and incubated at 37°C, yielding typical final concentrations of

20-40 nM FVIIa, 500 nM TF, and 1 µM FIX. Timed 10 µL aliquots were removed and quenched on ice using 10 µL of 20 mM EDTA in 10×HBSA. To quantify the FIXa generated, final concentrations of 40% ethylene glycol and 1 mM Pefachrome FIXa were added to quenched samples (total volume, 100 µL), and the rate of change in A405 was measured at 35°C.

Solution NMR Spectroscopy.

29 Purified sTF was concentrated using Amicon Ultra-15 centrifugal filters (EMD

Millipore, Billerica, MA) with a Mr cutoff of 10 kDa, then dialyzed into 50 mM NaCl, 50 mM sodium phosphate (pH 6.5). Solution NMR samples were prepared with 100 µM sTF, 1 mM

2+ DSS (4,4,-dimethyl-4-silapentane-1-sulfonic acid) and 10% D2O along with 1.25 mM Ca , 0.5 mM Mg2+, or no divalent cations. 15N-1H 2D HSQC correlation spectra were acquired on a

Varian/Agilent VNMRS 17.6 T (750 MHz 1H frequency) spectrometer for 16.6 h each at

~30°C. The measured pH values were 6.74, 6.78, and 6.79 for the samples with 1.25 mM Ca2+,

0.5 mM Mg2+, and no divalent cations, respectively. Spectra were processed using NMRPipe205 and chemical shifts were analyzed in SPARKY.206

Results

Components of the TF/FVIIa Complex Required for Mg2+-Dependent Rate Enhancements.

Although Mg2+ has been shown to modulate the enzymatic activity of FVIIa towards its cognate substrates, FIX94 and FX,49,93 and removal of the FX GLA domain abrogates this effect,99 a thorough understanding of how each component of the TF/FVIIa/membrane complex responds to Mg2+ has not yet been achieved. We therefore varied the constituents of this complex and examined the ability of Mg2+ to modulate the rate of FX activation by FVIIa. Figure 2.1 shows the relative rates of FX activation by various components of the TF/FVIIa/membrane complex in the presence of physiologic concentrations of Ca2+ plus Mg2+ (1.25 mM and 0.6 mM, respectively), normalized to the rates observed with physiologic (1.25 mM) Ca2+ alone. For FVIIa in solution (no membranes or TF), the presence of Mg2+ plus Ca2+ actually decreased the rate of

FX activation by 35% relative to Ca2+ alone. Mg2+ did not significantly influence the rate of FX activation by FVIIa in the presence of 30% PS/70% PC liposomes (but in the absence of TF;

30 second bar in Figure 2.1). In the presence of sTF but without membranes, Mg2+ enhanced the rate of FX activation by FVIIa by approximately 4-fold, consistent with previous findings.49,99 In the presence of sTF plus 30% PS/70% PC liposomes, Mg2+ still enhanced the rate of FX activation by FVIIa, but to a lesser extent than in the absence of membranes. Mg2+ also enhanced

FX activation by FVIIa bound to relipidated TF, with the magnitude of the enhancement dependent on the lipid composition. Thus, Mg2+ enhanced the rate of FX activation in the presence of TF-liposomes containing either 100% PC or 5% PS/95% PC, but did not significantly enhance the rate of FX activation when TF-liposomes contained very high levels of

PS (i.e., 30% PS/70% PC).

TF Mutations That Diminish the Ability of Mg2+ to Enhance FX Activation by TF/FVIIa in

Solution.

Previous studies identified a putative substrate-binding region (exosite) in TF, consisting of a patch of surface-exposed residues in the C-terminal fibronectin type III domain of this protein that are critical for recognition of macromolecular substrates (FVII, FIX, and FX) by the

TF/FVIIa complex. In particular, mutating TF residues S163, K166, or Y185 to alanine substantially decreased the rates of FX activation by TF/FVIIa, while having little or no effect on the affinity of FVIIa for TF or on FVIIa amidolytic activity.70 We previously showed that if the

GLA domain of FX is removed, mutations in the TF exosite no longer influence the rate of FX activation, suggesting that the FX GLA domain interacts with the TF exosite.69 Furthermore,

Persson et al.99 showed that removing the FX GLA domain abrogated the ability of Mg2+ to enhance FX activation by TF/FVIIa. We therefore hypothesized that Mg2+ enhances the rate of

FX action by TF/FVIIa largely by enhancing the interaction between FX and the substrate-

31 binding exosite region of TF. To test this idea, we mutated 17 amino acids in memTF individually to alanine, chosen by their location within or adjacent to the putative substrate- binding exosite.70 We expressed and purified these memTF mutants, then assessed the ability of

Mg2+ to enhance the rate of FX activation by the resulting TF/FVIIa complexes. In particular, we compared three divalent metal ion conditions: 1.25 mM Ca2+ alone; 1.85 mM Ca2+ alone; and

1.25 mM Ca2+ + 0.6 mM Mg2+. When we measured the rates of FX activation by memTF/FVIIa complexes in solution (i.e., with Triton X-100 and no membranes) in the presence of 1.85 mM

Ca2+, we found that 13 of the 17 single amino acid mutations decreased the initial rate of FX activation by at least 50% relative to wild-type (WT) memTF, and that 6 of the mutations

(Y157A, K159A, S163A, G164A, K166A, and Y185A) decreased the rate by at least 90%

(Figure 2.2A). These results are in good agreement with those previously determined by

Kirchhofer et al,70 which were obtained in the presence of phospholipid membrane surfaces. The absolute rates of FX activation obtained under each divalent metal ion condition that we tested are given in Table 2.1.

To determine how these TF mutations affected the ability of Mg2+ to enhance the enzymatic activity of memTF/FVIIa, we quantified initial rates of FX activation in the presence of 1.25 mM Ca2+ plus 0.6 mM Mg2+ and normalized those data to the rates using 1.85 mM Ca2+ alone (Figure 2.2B) or 1.25 mM Ca2+ alone (Figure 2.2C). The purpose of the former condition is to compare the combination of Ca2+ plus Mg2+ to an identical concentration of Ca2+ alone, such that the total divalent metal ion concentration is held constant at 1.85 mM.

Similar to the effect of Mg2+ we observed with sTF in solution, the combination of 1.25 mM Ca2+ plus 0.6 mM Mg2+ enhanced the rate of FX activation by FVIIa bound to WT memTF in detergent solution by 4.4-fold compared to 1.85 mM Ca2+ alone (Figure 2.2B) and

32 6.0-fold compared to 1.25 mM Ca2+ alone (Figure 2.2C). The TF exosite mutants fell into two main categories with regard to their sensitivity to Mg2+. The first category included mutations that were highly deleterious for FX activation but which nevertheless showed essentially the same ability of Mg2+ to enhance the rate of FX activation observed with WT memTF/FVIIa complexes. Thus, TF mutants W158A, S160A, S162A, E174A, L176A and R200A exhibited absolute rates of FX activation less than 75% of WT memTF (Figure 2.2A), yet showed essentially the same response to the combination of Ca2+ plus Mg2+ versus Ca2+ alone that was observed for WT memTF (Figures 2.2B and 2.2C). The second category included mutants

Y157A, S163A, G164A, K159A, K165A, K166A and Y185A that exhibited both substantially decreased absolute rates of FX activation (Figure 2.2A) and substantially blunted ability of Mg2+ to enhance their rates of FX activation (Figures 2.2B and 2.2C). On the other hand, no TF mutants were identified that retained WT absolute rates of FX activation yet were deficient in their response to Mg2+. A similar pattern of results was obtained when we examined the ability of

Mg2+ to enhance the rate of FIX activation by this collection of memTF mutants (Figure 2.6).

To better understand our results, we also investigated the effect of simply increasing the

Ca2+ concentration from 1.25 to 1.85 mM on the rate of FX activation by memTF/FVIIa complexes in solution (Figure 2.7). With WT TF, increasing the Ca2+ concentration resulted in an approximately 1.3-fold increase in the rate of FX activation. Within experimental error, most of the TF mutants supported similar increases in the rate of FX activation upon increasing the

Ca2+ concentration. Two TF mutants (K159A and K165A) were significantly different, exhibiting approximately 30% slower rates of FX activation at 1.85 mM Ca2+ compared to

1.25 mM Ca2+ (Figure 2.7).

33 TF Mutations That Diminish the Ability of Mg2+ to Enhance FX Activation by TF/FVIIa on

Membranes.

Our studies thus far have examined the ability of Mg2+ to increase the rate of FX by

TF/FVIIa complexes in solution. We next examined the effects of eleven selected TF mutants in experiments in which memTF was incorporated into liposomes of varying phospholipid composition (Figure 2.3). As was also seen in Figure 2.1, the ability of the combination of 1.25 mM Ca2+ plus 0.6 mM Mg2+ to enhance the rate of FX activation relative to 1.85 mM Ca2+ alone using WT TF was a function of the PS content of the TF-liposomes. Thus, as the PS content increased, the ability of Mg2+ to enhance the rate of FX activation decreased, reaching an essentially negligible level of enhancement by Mg2+ at 15% PS. For clarity, we grouped the TF mutants in three panels of Figure 2.3 according to their ability to support Mg2+-dependent enhancement of the rate of FX activation in solution. Thus, those memTF mutants that supported essentially WT responses to Mg2+ in solution (W158A, S162A, E174A and D178A) also exhibited increased rates of FX activation in the presence of Mg2+ when employed in liposomes (Figure 2.3A). Although increasing the PS content of the TF-liposomes tended to blunt the Mg2+ response in these mutants, the effect of increasing the PS content on the Mg2+ response was generally less extensive than that observed with WT TF. This was most particularly true of the S162A mutant.

On the other hand, the memTF mutants that supported intermediate responses to Mg2+ in solution (Y157A, K159A, S163A, and K165A) exhibited essentially no response to Mg2+ in liposomes (Figure 2.3B). And finally, the memTF mutants that exhibited the most deficient responses to Mg2+ in solution (G164A, K166A and Y185A) actually exhibited, in liposomes, lower rates of FX activation in the presence of Mg2+ plus Ca2+ compared to Ca2+ alone (Figure

34 2.3C). Furthermore, the blunted responses to Mg2+ by the TF mutants in Figures 2.3B and 2.3C were observed irrespective of the PS content of the memTF-liposomes.

Solution NMR Spectroscopy of sTF in the Presence of Divalent Cations.

To investigate whether TF interacts directly with the divalent cations Ca2+ and Mg2+, we utilized solution NMR spectroscopy to compare 15N-1H 2D heteronuclear single quantum coherence (HSQC) correlation spectra in the presence and absence of these ions. As shown in

Figure 2.4, addition of physiologic divalent cation (either 1.25 mM Ca2+ or 0.5 mM Mg2+) to

100 µM sTF samples resulted in no significant shifts in peak positions in 15N-1H 2D correlation spectra. These results are consistent with the idea that neither Ca2+ nor Mg2+ interact directly with sTF in a specific manner, which in turn is consistent with available x-ray crystal structures in which neither Ca2+ nor Mg2+ have been observed bound to sTF.41,49

Discussion

Divalent cations such as Ca2+ and Mg2+ are critical components of blood coagulation, supporting both protein-protein and protein-membrane interactions. While the contributions of

Ca2+ to blood coagulation reactions have been extensively studied, the roles of Mg2+ are less well understood. In this study, we confirmed that TF is required for Mg2+ to enhance the rate of activation of FIX or FX by FVIIa,99 and we showed that specific residues in the putative substrate-binding region of TF contribute to this Mg2+-dependent rate enhancement. Although

TF is an allosteric regulator of the general catalytic activity of FVIIa, this function is independent of the presence of Mg2+ as long as Ca2+ is available, since adding Mg2+ has no significant effect on FVIIa amidolytic activity,99 nor does it enhance the affinity of FVIIa for TF

35 (Table 2.2). Thus, enhancement of TF/FVIIa catalytic activity by Mg2+ is restricted to macromolecular substrates (FIX and FX). Importantly, a previous study showed that removing the FX GLA domain eliminated the ability of Mg2+ to enhance the rate of FX activation by

TF/FVIIa,99 and our lab previously demonstrated that the GLA domain of FX must be intact for mutations in the TF exosite to have any effect on the rate of FX activation by TF/FVIIa.69 These results support the idea that the FX GLA domain interacts directly with the TF exosite and is of critical importance for Mg2+ recognition. This does not preclude the involvement of FVIIa in mediating these interactions; indeed, a recent study reported that the FVIIa GLA domain must also be intact for Mg2+ to enhance the rate of FX activation by TF/FVIIa.27

Our working hypothesis to explain all these findings is that Mg2+ promotes the binding of

FX (or FIX) to the exosite region of TF. To accomplish this, Mg2+ could, in principle, be contributing to increase rates of FX activation via its binding to the GLA domains of either

FVIIa or FX, or perhaps even binding to TF. (Since we observe the effect of Mg2+ in solution, it is not necessary to invoke association of Mg2+ with phospholipids, and therefore Mg2+ must be promoting protein-protein interactions.) Our NMR results, together with a wealth of x-ray crystal structures, argue strongly that Mg2+ does not associate measurably with TF. Under physiologic concentrations of divalent metal ions, Mg2+ is thought to occupy at least two, and possibly three or four metal ion-binding sites in the GLA domains of both FVIIa and FX.207

Although our results do not allow us to determine whether Mg2+ binding to FVIIa or FX is more important for enhancing the rate of FX activation, one can speculate that occupancy of the FX

GLA domain with Mg2+ may increase the affinity of this domain for the TF exosite. Mg2+ binding to the FVIIa GLA domain may also play a regulatory role, as comparison of crystal structures in the presence or absence of Mg2+ indicates that conformational changes occur in the

36 FVIIa GLA domain upon Mg2+ occupancy.200 Interestingly, the 159-165 loop of sTF was disordered when the sTF/FVIIa complex was crystallized with Ca2+ only, but was ordered when crystallized in the presence of both Ca2+ and Mg2+. How these structural changes might affect

TF/FVIIa activity towards its cognate substrates is currently unclear.

For better visual interpretation, we mapped the mutations in this study onto the x-ray crystal structure of the FVIIa/sTF complex, colored according to their impact either on the absolute rate of FX activation (Figure 2.5A) or on the ability of Mg2+ to enhance the rate of FX activation (Figure 2.5B) by memTF/FVIIa in solution. In Figure 2.5C, we also graphed the effects of these mutations, plotted as ability of Mg2+ to enhance the rate of FX activation (y-axis) versus absolute rate of FX activation (x-axis), with data points colored using the same scheme as

Figure 2.5B. The deleterious effects of these TF exosite mutations on the absolute rate of FX activation (Figure 2.5A) largely confirm the findings of Kirchhofer et al.70 Only a subset of these mutations, however, reduced the ability of Mg2+ to enhance the rate of FX activation, with mutations G164A, K166A and Y185A (colored blue in Figure 2.5B) essentially eliminating the

Mg2+ effect; notably, these three mutations also severely reduced the absolute rate of FX activation (colored blue in Figure 2.5A). TF mutations Y157A, S163A, K165A and D180A

(colored green in Figure 2.5B) exhibited a more moderate, yet still significant reduction in the

Mg2+ effect; of these, three (Y157A, S163A and K165A) greatly reduced the absolute rate of FX activation (colored blue in Figure 2.5A), while one (D180A) had a more moderate reduction in the absolute rate of FX activation (colored green in Figure 2.5A). TF mutant K159A severely reduced the absolute rate of FX activation, but did not significantly diminish the Mg2+ effect relative to WT TF. The remaining ten mutations also did not significantly reduce the ability of

37 Mg2+ to enhance the rate of FX activation relative to WT TF, irrespective of whether they decreased the absolute rate of FX activation.

The location of TF residues that mediate Mg2+-dependent rate enhancement suggests that a critical component of the TF/FVIIa interaction with Mg2+-bound FX substrate involves the flexible TF loop located at residues 160-165, with the adjacent residues Y185, K166, K159, and Y157 also involved. These residues, in particular 164-166, are highly conserved across mammalian species (Figure 2.8). The importance of G164 to Mg2+ recognition is particularly interesting, as glycine lacks a functional side chain with which to interact with macromolecular substrates. A possible explanation is that this highly conserved glycine permits flexibility or orientations of the adjacent loop(s) that are otherwise not possible with amino acids containing bulkier side chains.

When WT memTF was incorporated into liposomes, we found that increasing the PS content of the membranes blunted the response to Mg2+, confirming our previous findings.93 We also found that increasing the PS content of TF-liposomes blunted the Mg2+ response in these

TF mutants. With several of these mutants (especially S162A), this blunting effect of PS was less extensive than that observed with WT TF. We also found that the TF mutants that were most deficient in response to Mg2+ when tested in solution (G164A, K166A and Y185A) actually had lower rates of FX activation in TF-liposomes in the presence of Mg2+ plus Ca2+ compared to Ca2+ alone. Furthermore, the essentially negative effects of Mg2+ on this class of TF mutants were observed irrespective of the PS content of the memTF-liposomes.

Messer et al. have shown that occupancy of the FIXa GLA domain with Mg2+ enhances its affinity for PS-containing membranes, and have proposed that Mg2+ generally enhances the membrane binding of GLA domain-containing clotting proteins.208 If this is part of the

38 mechanism by which Mg2+ enhances the rate of FX activation by TF/FVIIa (i.e., by better recruiting FX to the membrane surface in the vicinity of TF/FVIIa and thereby raising the local substrate concentration), then it follows that increasing the PS content may blunt the Mg2+ effect, since greatly raising the PS content will likewise increase the affinity of FX for the membrane surface and thus overshadow the relative contribution from Mg2+. However, the fact that some TF mutations selectively abolish the ability of Mg2+ to enhance the rate of FX activation by the TF/FVIIa/membrane complex argues in favor of protein-protein interactions

(presumably, between the TF exosite and the GLA domain of FX) being also very important in mediating the ability of Mg2+ to enhance the proteolytic activity of TF/FVIIa toward FIX and

FX.

39 CHAPTER 3: Relative Contributions of Nucleic Acids and PolyP to Coagulation

Abstract

Polyphosphate and nucleic acids are linear, anionic polymers that contribute to thrombosis in animal models and may be important in thrombotic diseases in humans.

Polyphosphate plays several roles in coagulation and in particular may be one of the long-sought

(patho)physiologic initiators of the contact pathway; extracellular DNA and RNA have been implicated as initiators of coagulation and as biomarkers for disease states, including sepsis. In this study, we directly compared the procoagulant activities of polyphosphate, DNA and RNA, in order to understand their relative effectiveness in initiating and accelerating blood clotting reactions. Since polyphosphate, DNA, and RNA have similar physical properties, we hypothesized that polyphosphate and nucleic acids might co-purify using conventional techniques for isolating DNA or RNA from cells or tissues. We found that long-chain polyphosphate was a far more potent contact pathway activator than DNA or RNA.

Polyphosphate also potently abrogated the anticoagulant activity of tissue factor pathway inhibitor (TFPI), whereas DNA and RNA were relatively ineffective. Polyphosphate readily co- purified with DNA and RNA using several popular procedures for isolating nucleic acids.

Furthermore, treatment of human cell-derived DNA with either nucleases or phosphatases abrogated its procoagulant ability, and trace contamination of these samples with polyphosphate was confirmed and quantified. Our findings indicate that long-chain polyphosphate is two to three orders of magnitude more potent at triggering clotting than either DNA or RNA, and suggest that polyphosphate contamination of nucleic acid preparations may contribute to their observed procoagulant activities.

40 Introduction

Recent advances in understanding the contributions of the contact pathway of blood clotting to thrombosis, inflammation, and innate immunity209 have generated renewed interest in identifying the (patho)physiologic activators of this pathway. Proposed candidates include collagen,210 misfolded proteins,211 DNA, RNA,212 and polyphosphate (polyP).213 PolyP, consisting of linear polymers of inorganic phosphate held together by phosphoanhydride bonds, is widespread in biology.214 Work from our laboratory and others has identified important procoagulant and proinflammatory roles of polyP.215 We have also shown that the precise contributions of polyP to blood clotting are strongly dependent on its polymer length.215,216

Many microorganisms accumulate polyP that is heterogeneous in length.214,217 A number of mammalian tissues reportedly contain polyP, such as brain, heart, lung, bone and prostate.218-221

Long-chain polyP (up to thousands of phosphates long), such as that found in microorganisms,217 elaborated by prostate cancer cells,221 and possibly found in mammalian brain,218 is an especially potent activator of the contact pathway of coagulation.216 Shorter-chain polyP (roughly 60 to 120 phosphates), such as that secreted by activated platelets, mast cells and basophils,186,220,222 only very weakly activates the contact pathway.216 On the other hand, platelet-size polyP is highly active in modulating downstream clotting reactions, including promoting factor XI activation by thrombin,156 promoting factor V activation by multiple proteases,215 enhancing fibrin clot structure,193,223 and abrogating the anticoagulant function of tissue factor pathway inhibitor

(TFPI).213

PolyP and nucleic acids are both linear polymers with regularly spaced, anionic phosphate groups, so it is perhaps not surprising that they may have similar activities toward blood clotting reactions. The possible contributions of extracellular nucleic acids to coagulation and inflammation

41 are under intense investigation, with a number of studies reporting the procoagulant effects of cell- free RNA212 and DNA,176,224,225 particularly in the context of neutrophil extracellular traps.173,226,227

Although multiple groups have reported the ability of nucleic acids and polyP to activate the contact pathway, head-to-head comparisons of the relative procoagulant abilities of these anionic polymers are largely lacking.

The anionic nature of DNA and RNA is commonly exploited for their purification from cells and tissues, and many commercially available nucleic acid purification kits are based on silica or ion exchange resins. These same approaches are also employed for polyP purification, using either finely powdered silica216,228 or anion-exchange columns.221,229 We therefore hypothesized that polyP might co-purify with nucleic acids using standard DNA or RNA purification techniques. If so, then some or all of the previously reported procoagulant properties of nucleic acids might be due to polyP contamination.

In this study, we demonstrate that polyP is two to three orders of magnitude more active than DNA or RNA in triggering the contact pathway of blood clotting, and is also far more effective than nucleic acids at abrogating TFPI function. We also report that several commercially-available nucleic acid purification kits result in DNA or RNA preparations that are contaminated with sufficient amounts of polyP to substantially alter their apparent procoagulant properties.

Materials and Methods

Materials

PolyP was size-fractioned as described216 to obtain preparations indicated as: short-chain polyP (mode, 75 phosphates; range, 20 – 330 phosphates); medium-chain polyP (mode, 385

42 phosphates; range, 265 – 630 phosphates); and long-chain polyP (mode, 1320 phosphates; range, 430 to more than 2000 phosphates). All polyP concentrations are given in terms of phosphate monomers.

Citrated pooled normal plasma was from George King Biomedical. Liposomes were made by sonication using phospholipids from Avanti Polar Lipids (85% 1-palmitoyl-2- oleoylphosphatidylcholine/15% 1-palmitoyl-2-oleoylphosphatidyl-L-serine). Recombinant human TFPI was as described.230 Calf intestinal alkaline phosphatase (CIAP) was from Promega and lambda DNA, HindIII-digested lambda DNA, 1kb DNA ladder, Low MW DNA ladder, and 100 bp DNA ladder were from New England Biolabs. SYBR Green I (SYBR) was from

FMC BioProducts. Benzonase, 4',6-diamidino-2-phenylindole (DAPI), and RNA homopolymers (polyguanylate, polyG; polyinosinate, polyI; polyadenylate, polyA; or polycytidylate, polyC) were from Sigma-Aldrich. A size comparison of polyP and RNA homopolymers is given in Figure 3.7.

Isolation of cell-derived nucleic acids

HEK 293 cells were grown to confluence in Dulbecco's Modified Eagle Medium with

10% fetal bovine serum and penicillin-streptomycin, and harvested for isolation of DNA and

RNA. Cell-free DNA was purified using DNeasy Blood & Tissue kit (Qiagen), while total cellular RNA was purified using RNeasy Midi kit (Qiagen), according to the manufacturer’s instructions. DNA from multiple purifications was pooled and concentrated in 3,000 molecular weight-cutoff centrifugal filters (Millipore). Nucleic acid concentrations were determined by light absorption at 260 nm.

43 Comparison of polyP recovery rates using nucleic acid purification kits

DNeasy Blood and Tissue kit, RNeasy Mini kit, Qiaquick PCR Purification kit

(Qiagen), and TRIzol Reagent (Invitrogen) were evaluated for their recovery rates when used to

“purify” samples consisting of nucleic acid alone (either phage lambda DNA or polyG), or nucleic acid mixed with long-chain polyP. We employed 5 µg of each nucleic acid and polyP for such experiments with the DNeasy, RNeasy, and TRIzol kits, and 2.5 µg of each nucleic acid and polyP for the Qiaquick kit (because of its lower capacity). Instructions for the DNeasy kit vary according to source material, so the protocol for “cultured cells” was followed and the optional second elution of DNA was included. For the RNeasy kit, the protocol was followed for samples with less than 5 x 106 animal cells; the optional membrane-drying spin was included, but the optional on-column DNase digestion was omitted. Sample preparation for TRIzol purification used the protocol for suspension cells. The TRIzol reagent allows for isolation of both RNA and DNA; we performed sequential isolation of nucleic acids and/or polyP from both the aqueous (RNA-containing) and interphase/organic (DNA-containing) layers as described in the manufacturer’s instructions. Nucleic acid concentrations were determined by light absorption at 260 nm. PolyP concentrations were determined following complete digestion with recombinant yeast exopolyphosphatase (PPX1, purified as described231), after which the released monophosphate was quantified by PiBlue assay (BioAssay Systems).

Evaluation of polyP contamination in cell-derived DNA

DNA purified from HEK293 cells using the DNeasy Blood & Tissue kit was digested with Benzonase to hydrolyze the nucleic acids, after which any residual polyP was isolated as described221 with minor modifications. Briefly, 8M LiCl and 4.5M NaI were added to the

44 samples, which were then applied to EconoSpin DNA spin columns (Epoch Life Sciences).

Columns were washed 5 times with a solution of 50% ethanol, 100 mM NaCl, 10 mM Tris pH

7.5, 1 mM EDTA, after which polyP was eluted in purified water. The polyP was then digested with PPX1 and the released monophosphate quantified as described above. HEK293 cell DNA,

Benzonase-digested HEK293 cell DNA, and HindIII-digested lambda DNA were subjected to

PAGE on 4-20% gels and visualized with SYBR Green I for nucleic acid, or by DAPI staining for polyP (negative fluorescent staining after photobleaching) and nucleic acid (positive fluorescence) as described.232

Enzymatic digestions of DNA and polyP

DNA or polyP preparations were digested in 20 mM HEPES pH 7.4, 5 mM NaCl, 5 mM KCl for 30 minutes at 37°C with either 700 U/mL Benzonase, 300 U/mL CIAP, or no enzyme.

Contact pathway-initiated plasma clotting assays

Plasma clotting times were quantified in duplicate at 37°C by mixing 50 µL of activator

(nucleic acid or polyP in 20 mM HEPES pH 7.4, 5 mM NaCl, 5 mM KCl with 75 µM liposomes) and 50 µL of plasma in wells of medium-binding 96-well polystyrene microplates

(Corning). After 3 minutes, 50 µL of pre-warmed 25 mM CaCl2 in 20 mM HEPES was added and mixed in by trituration. Light scattering (absorbance at 405 nm) was monitored in a

SpectraMax 340PC microplate reader (Molecular Devices) at 37°C. Clot times were determined as previously described233 and normalized to the mean clot time for plasma without activator.

45 Figure 3.8 shows the distribution of clot times in the absence of activator. For evaluation of enzymatically digested preparations of polyP or nucleic acids, clot times in the presence of activator (nucleic acid or polyP) were normalized to the clot time in the absence of activator but with the corresponding enzymatic treatment.

Abrogation of the anticoagulant activity of TFPI

TFPI abrogation was evaluated using clotting assays essentially as described.216 Anionic polymers were diluted in nuclease-free water (Amresco). Final reactions contained 17 mM Tris-

HCl pH 7.4, 50 mM NaCl, 0.7% bovine serum albumin, 33 µM liposomes, 8.33 mM CaCl2, and 33% plasma, with or without 200 ng/mL TFPI. Clot times with added polyanion were normalized to clot times for plasma plus TFPI but without polyanion.

Results

PolyP is a much more potent and efficacious contact pathway activator than cellular DNA or

RNA

Previous work by our lab and others has shown that both polyP and nucleic acids trigger clotting via the contact pathway.166,191 Table 3.1 outlines the techniques used in a number of publications to isolate nucleic acids for investigations into their procoagulant properties. Though numerous tissue-, source-, and application-specific kits exist, in general, silica-based purification columns are most commonly utilized. Because of this, we isolated total cellular DNA and RNA from cultured HEK 293 cells using two of the most popular commercial kits from Qiagen:

DNeasy Blood & Tissue kit for DNA and RNeasy Midi kit for RNA.

46 To compare the relative procoagulant activities of nucleic acids and polyP, we utilized a modified activated partial thromboplastin time clotting assay in which varying concentrations of polyP, RNA or DNA were added to plasma to trigger clotting via the contact pathway. As previously reported,216 polyP shortened the time to clot formation in a manner that was dependent on both concentration and polymer length (Figure 3.1A). Short-chain polyP had limited ability to trigger clotting in this assay. Medium- and long- chain polyP were highly procoagulant, exhibiting maximal shortening of the clot time by 83% and 89%, respectively, at 2

µg/mL polyP

In contrast, HEK 293 cell-derived nucleic acids and lambda DNA were weaker and less potent than polyP in triggering plasma clotting, on both a mass (Figure 3.1A) and a molar

(Figure 3.9) basis. For HEK 293 cell-derived DNA, maximal shortening of clot time was 52% at

75 µg/mL, while for lambda DNA, maximal shortening was 25% at 50 µg/mL. HEK 293 cell- derived RNA was even less effective in triggering clotting, shortening the clot time by only 10% at a concentration of 100 µg/mL (Figure 3.1A).

To further investigate the proposed procoagulant activity of RNA,212,234 we evaluated the ability of synthetic RNA homopolymers to activate the contact pathway (Figure 3.1B). While polyA and polyC had virtually no detectable procoagulant effect, polyG and polyI were effective at triggering plasma clotting, exhibiting bell-shaped concentration dependences, similar to polyP.

In particular, polyG exhibited a maximal 85% decrease in clot time at 10 µg/mL, and was therefore almost as effective as long-chain polyP in triggering clotting.

47 Ability of polyP versus nucleic acids to abrogate TFPI anticoagulant activity

PolyP is highly effective at abrogating the anticoagulant activity of TFPI in plasma clotting assays, likely by accelerating factor V activation213,235 and blocking TFPI targeting of the prothrombinase complex.236 As we reported previously,216 short-chain polyP completely abrogated TFPI function at polyP concentrations above 5 µg/mL (Figure 3.2). In contrast, HEK

293 cell-derived DNA and RNA exhibited limited ability to reverse the anticoagulant activity of

TFPI over a range of concentrations tested. On the other hand, the synthetic RNA homopolymer, polyI, completely abrogated TFPI anticoagulant activity with a potency similar to that of polyP. PolyA and polyG showed more limited ability to reverse TFPI anticoagulant activity, requiring more than tenfold higher concentrations compared with polyP or polyI. PolyC was ineffective.

Digestion of cell-derived DNA with nuclease or polyphosphatase decreases its procoagulant activity

Because polyP and nucleic acids are both linear, anionic polymers, we hypothesized that methods for isolating nucleic acids might also co-purify polyP. If so, then some or all of the apparent procoagulant activity of cell-derived DNA might be attributable to contaminating polyP. DNA isolated from HEK 293 cells using the DNeasy kit had detectable procoagulant activity when tested at 50 µg/mL (Figure 3.3A). Digesting this DNA preparation with either a highly active nuclease (Benzonase), or CIAP, which is a highly active exopolyphosphatase,237 resulted in complete loss of clotting activity (Figure 3.3A). As a control, we found that

Benzonase digestion of 2 µg/mL long-chain polyP had an insignificant effect on its procoagulant activity, while CIAP digestion eliminated its procoagulant activity (Figure 3.3B). Interestingly,

48 the procoagulant effect of 20 µg/mL bacteriophage lambda DNA, which we hypothesized would be unlikely to contain polyP, was not diminished by CIAP but was abrogated by Benzonase

(Figure 3.3B). We confirmed that Benzonase had no detectable polyphosphatase activity and that CIAP had no detectable nuclease activity (Figure 3.10).

The procoagulant effects of polyP and DNA are additive

To examine if polyP and DNA might cooperate to initiate clotting, we evaluated the procoagulant activity of lambda DNA to which polyP had been added (Figure 3.4A). Mixing 20

µg/mL lambda DNA with varying concentrations of short-chain polyP consistently shortened the clot times across a range of polyP concentrations (0.1 to 10 µg/mL polyP). Mixing 20 µg/mL lambda DNA with medium-chain polyP did not alter the clotting times observed at polyP concentrations at or above 2 µg/mL, but did shorten the clotting times at all polyP concentrations from 0.1 to 1 µg/mL.

Small amounts of polyP enhance the procoagulant effects of DNA

Any possible contamination of nucleic acids with polyP would likely scale relative to the amount of DNA purified. Kumble and Kornberg218 found that the concentration of polyP in mammalian tissues and cell lines to be approximately 1% that of DNA. We consequently evaluated the procoagulant ability of medium-chain polyP added to lambda DNA at a fixed ratio of 1:100 on a mass basis (Figure 3.4B). Lambda DNA with 1% added medium-chain polyP was more procoagulant than either medium-chain polyP alone, HEK 293 cell DNA alone, or lambda

DNA alone.

49 Nucleic acids and polyP co-purify using several popular kits for nucleic acid isolation

To examine whether polyP will co-purify with nucleic acids, we quantified the recovery of nucleic acids and polyP using a variety of commercially available kits for isolating DNA or

RNA (Figure 3.5). In these experiments, known quantities of lambda DNA or polyG, either alone or mixed with long-chain polyP, were re-purified using Qiaquick, DNeasy, or TRIzol kits, following the manufacturer’s instructions. Purification of DNA with either the Qiaquick or

DNeasy kit resulted in 40-60% recovery of the applied DNA, and adding polyP to the sample did not diminish the DNA recovery. Purification of RNA with the RNeasy Miniprep kit resulted in ~50% RNA recovery, regardless of whether polyP was present. Similarly, the presence of polyP did not diminish RNA recovery from either the aqueous or organic phase when using

TRIzol Reagent (Figure 3.5).

When polyP was present in the starting nucleic acid sample, it was readily recovered with the DNA or RNA using all the kits tested (Figure 3.5). The Qiaquick kit yielded ~55% of the starting polyP, when purified alone or in the presence of DNA. With the DNeasy kit, polyP recovery was higher (91%) when DNA was present in the sample than when DNA was absent

(79%). PolyP (58%) was also recovered with the RNeasy kit. Little or no polyP was recovered in the aqueous phase of TRIzol (which is typically used to isolate RNA), while 50-60% of polyP was recovered in the organic phase (which typically contains DNA).

HEK 293 cell-free DNA purified using the DNeasy kit and lambda DNA were resolved by PAGE, after which the gel was stained sequentially with DAPI (which stains DNA fluorescently and which shows strong photobleaching for polyP232) followed by SYBR Green I

(which fluorescently stains DNA but not polyP) (Figure 3.6). Lambda DNA fluoresced with both stains, which was abolished by Benzonase pretreatment. HEK 293 cell DNA was also

50 positively fluorescent with both stains, but we also observed photobleaching during DAPI staining in the faster-migrating portions of this lane beginning immediately below the DNA and continuing as a streak towards the bottom of the gel, suggesting the presence of polyP (Figure

3.6A, lane 2) Pretreatment of HEK 293 cell DNA with Benzonase eliminated the positive fluorescence and left only the faster-migrating, photobleached material near the bottom of the gel, indicative of the presence of polyP (Figure 3.6A, lane 4). HEK 293 gave no fluorescence with SYBR Green I after Benzonase treatment, confirming its complete degradation by this nuclease (Figure 3.6B, lane 4).

We next quantified the polyP present in DNA that had been isolated from HEK 293 cells with the DNeasy Blood and Tissue Kit. After treatment with Benzonase to digest DNA, polyP was purified and quantified as described in Materials and Methods. We recovered 1.9 nmoles of polyP-derived monophosphate from the DNA sample that contained 1.23 µmoles of nucleotides (assuming an average nucleotide molecular weight of 327 Da). Thus, the polyP content in this sample, on a molar basis, was 0.16% that of DNA (i.e., moles of phosphate in polyP relative to moles of nucleotide in DNA).

Discussion

Here we report a head-to-head comparison of the procoagulant activities of DNA, RNA and polyP, all of which have been proposed to be significant pathophysiologic activators of the contact pathway and modulators of downstream clotting reactions. Medium- and long-chain polyP were two to three orders of magnitude more potent than human cell-derived DNA or

RNA, and resulted in considerably shorter clotting times. We also tested DNA purified from bacteriophage lambda, reasoning that it was likely not to be contaminated with polyP, and found

51 that lambda DNA was indeed appreciably less procoagulant than an equivalent concentration of human cell-derived DNA. One possible explanation for this difference is that the human cell- derived DNA is contaminated with polyP. Given the much higher procoagulant activity of polyP, even trace contamination of DNA with polyP might significantly alter its apparent procoagulant activity. Digestion of HEK 293 cell-derived DNA with the nuclease, Benzonase, rendered its clotting activity undetectable; however, digestion with CIAP, which is a very active exopolyphosphatase, also rendered its procoagulant activity undetectable. Notably, treatment of lambda DNA with CIAP had no effect on its procoagulant activity, while Benzonase treatment completely abolished its activity. These results support the notion that contaminating polyP contributes to the contact pathway activation observed with mammalian cell-derived DNA. Our results can be explained if the observed procoagulant activity is due to a combination of a high concentration of DNA (which has weak potency as a procoagulant) plus trace concentrations of contaminating polyP (which has high potency as a procoagulant). If so, then elimination of either component leaves undetectable procoagulant activity.

Previous work has focused on the possible procoagulant activity of RNA, in part because

RNase A decreased the procoagulant activity of cell lysates and was protective in in vivo models of thrombosis.212 In contrast, we found cell-derived RNA to be a weak contact pathway activator, reducing the clot time by only 10% even at concentrations that were orders of magnitude higher than optimal concentrations of long- or medium-chain polyP. On the other hand, we found that

RNA homopolymers composed of polyG or polyI were highly procoagulant, both activating the contact pathway and abrogating TFPI anticoagulant activity. These results indicate that the procoagulant nature of these molecules is not an intrinsic property of RNA in general, but rather highly dependent on base composition. The finding that the homopolymers polyG and polyI

52 have procoagulant properties similar to polyP, but that polyA and polyC have little to no procoagulant activity, suggests that polynucleotide structure plays an important role in determining the procoagulant activity of nucleic acids. Indeed, Gansler et al.176 recently reported that hairpin-containing RNA and DNA oligonucleotides, as well as oligonucleotides that adopt quadruplex structures and small nucleolar RNAs, exhibited the greatest ability to shorten plasma clotting times and promote PPK autoactivation. Both polyG and polyI can form G-quadruplexes in the presence of Na+ or K+,238,239 and similar structures have previously been shown to bind thrombin.240,241 Furthermore, telomeric DNA and its RNA transcription products are rich in G- quadruplex repeats,242 and DNA G-quadruplexes have been visualized in human cells.243 Our findings confirm and extend those of Gansler et al.176 and suggest that non-canonically base- pairing sequences of DNA or RNA are particularly procoagulant when released from cells.

We determined that polyP contamination of HEK 293 cell-derived DNA constituted about 0.16% of the total phosphate. This is slightly lower than that previously suggested by

Kumble and Kornberg;218 however, this level of contamination is consistent with the procoagulant activity we measured when adding polyP to purified bacteriophage lambda DNA at a 1/100 mass ratio, which had a procoagulant activity that exceeded that of DNA isolated from

HEK 293 cells.

In summary, we have demonstrated that polyP co-purifies with nucleic acids when utilizing traditional purification methods that have been used by others for coagulation experiments,212,225,244 and that the observed procoagulant effects of mammalian cell-derived DNA are due in part to contaminant polyP. Medium- and long-chain polyP are 2 to 3 orders of magnitude more potent contact pathway activators than total cell DNA or RNA, and yield far shorter clotting times. PolyP is present in mammalian cells at much lower concentrations than

53 DNA;218 however, the greater potency and efficacy of polyP suggests that even trace contamination of cell-derived nucleic acid preparations with polyP could contribute substantially to the observed procoagulant properties. Indeed, it seems likely that a combination of extracellular nucleic acids and polyP contribute to clotting reactions in vivo, but this needs to be tested explicitly. Future studies exploring the procoagulant effects of nucleic acids derived from cellular sources should include removal or neutralization of possible contaminating polyP.

54 CHAPTER 4: Characterization of Polyphosphate- and Ribonucleic Acid-Mediated Contact

Pathway Activation

Abstract

The contact pathway of the blood clotting cascade is dispensable for normal hemostasis but contributes to thrombosis and serves as a bridge between inflammation and coagulation. This pathway is triggered upon exposure of plasma to certain anionic polymers and artificial surfaces.

Recently, several potential (patho)physiologically relevant activators of this pathway have been identified, including extracellular nucleic acids and inorganic polyphosphate (polyP). However, mechanistic details regarding how nucleic acids or polyP modulate the individual reactions of the contact pathway have been lacking. In this study, we investigate the ability of RNA homopolymers and polyP to bind the primary constituents of the contact pathway: factor XI, factor XII, and plasma prekallikrein, in the presence and absence of high molecular weight kininogen (HK), an important cofactor in this pathway. We examined seven proteolytic activation reactions within the contact pathway and report that polyP greatly enhances the rate of all seven, while RNA is effective in supporting only a subset of these reactions. HK both enhances and suppresses these proteolytic activation reactions, depending on the specific reaction evaluated. Overall, we find that polyP is a potent mediator of contact pathway activation reactions in general, that RNA secondary structure may be important to its procoagulant activity, and that nucleic acids versus polyP may differentially modulate specific enzyme activation events within the contact pathway.

55 Introduction

The contact pathway of blood clotting is initiated when plasma is exposed to anionic surfaces and substances such as glass and clay.245,246 This mechanism for triggering clotting is irrelevant to normal hemostasis, since severe deficiencies in either of the two key enzymes that trigger this pathway, factor XII (FXII)247 or plasma prekallikrein (PPK),248 are not associated with bleeding tendencies in humans. However, recent findings have implicated the contact pathway in thrombosis, inflammation and innate immunity. Thus, in models of venous thrombosis, mice deficient in FXII249-251 are protected, and pharmacologic targeting of either

FXII or PPK is protective.252 Animal models of arterial thrombosis have also indicated a protective effect of FXII deficiency;251,253,254 FXII contributes to thrombus stability on atherosclerotic plaques;255 and several studies have reported a correlation between myocardial infarction and elevated plasma levels of contact pathway proteins.126,129,256 FXII also plays a central role in hereditary angioedema.209

This renaissance in the contact pathway has led to renewed interest in understanding its true pathophysiologic triggers. Recently identified candidates include collagen210 and misfolded proteins,211 as well as the linear, anionic polymers, polyphosphate (polyP),213,222,257 DNA,173,225-227 and RNA.212 Long-chain polyP and extracellular RNA appear to be particularly active at triggering the contact pathway and are implicated in thrombosis and inflammation.209,257 The goal of the present study is to achieve better mechanistic insights into how polyP and RNA trigger the contact pathway.

Figure 4.1 is a schematic overview of how this pathway is initiated, with the present study examining how polyP and RNA influence the rates of each of the numbered proteolytic events in this figure. The majority of PPK and factor XI (FXI) circulate in plasma bound to high

56 molecular weight kininogen (HK),258-260 so PPK and FXI are depicted in complex with HK.

Whether the active enzymes – plasma kallikrein (PK) or factor XIa (FXIa) – remain associated with HK is not so clear, but we have chosen to depict them as complexes in this figure. This pathway is initiated when zymogen factor FXII undergoes autoactivation and/or allosteric activation upon interaction with certain anionic surfaces or polymers.118,246 The resulting active enzyme, FXIIa, converts PPK to PK, creating a positive feedback loop through reciprocal activation of PPK by FXIIa, and FXII by PK.261 Once sufficient FXIIa is generated, it activates factor XI (FXI), leading to propagation of the coagulation cascade and, ultimately, fibrin formation.

HK is not only an important cofactor for triggering the contact pathway, but it is also acted upon proteolytically by PK, resulting in the release of the vasodilator peptide, bradykinin.250,262 Because bradykinin is a potent vasoactive mediator, it represents an important link between the coagulation cascade and inflammation. Binding of PPK and FXI to certain anionic surfaces is greatly enhanced by HK263,264 and HK is required in stoichiometric amounts for maximal enzymatic activity in vitro.264 Although single-chain HK can bind to, and serve as, a cofactor for FXI and PPK, the proteolytically cleaved two-chain form of HK is a much more efficient cofactor, likely due to the fact that the two-chain protein binds with higher affinity to anionic polymers and surfaces.263

To date, limited studies have investigated the ability of polyP or RNA to accelerate the rates of the individual reactions of the contact pathway. In the present study, we show that polyP and RNA exert differential effects on these individual enzyme activation reactions, both in the presence and absence of HK.

57 Materials and Methods

Materials.

Synthetic RNA homopolymers (polyguanylic acid, polyG; polyinosinic acid, polyI; polyadenylic acid, polyA; and polycytidylic acid, polyC), hirudin, and soybean trypsin inhibitor

(STI) were from Sigma (St. Louis, MO). Human FXII, α-thrombin, FXI, FXIa, and corn trypsin inhibitor (CTI) were from Haematologic Technologies (Essex Junction, VT); PPK, PK,

α-FXIIa and single-chain HK were from Enzyme Research Laboratories (South Bend, IN).

Chromogenic substrates for FXIa (Pyr-Pro-Arg-pNA) and FXIIa or PK (H-D-Pro-Phe-Arg- pNA) were from Bachem (Bubendorf, Switzerland).

Polyanion concentrations are given in terms of the concentration of phosphate monomers, i.e. phosphate concentration for polyP and nucleotide concentration for nucleic acids.

Narrowly size-fractionated long-chain polyP (size range: 1110 - 1540) and biotinylated polyP

(size range: 110 – 385 phosphates) were prepared as previously described.192,216

Competition Binding Assays.

The ability of RNA homopolymers and polyP to bind to proteins of the contact pathway was investigated using a plate-based competition assay essentially as described.192,265 Briefly, 10

μM biotin-polyP was captured on streptavidin-coated microplate wells. FXIa, FXIIa, or PK (at

50 nM) were incubated in the wells together with various competitors at ambient temperature for 1 h in 20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM NaCl, 10 μM ZnCl2, 1% polyethylene glycol (8000 MW), 0.05% Tween-20. In some experiments, 50 nM HK was also included. Wells were washed and the bound enzyme was quantified via amidolytic activity (rate

58 of cleavage of the appropriate chromogenic substrate), compared to a standard curve with known enzyme concentrations.

General Conditions for Enzyme-Substrate Reactions.

Enzyme activation reactions (including autoactivation reactions) were generally carried out at 37°C in 20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM NaCl, 1% polyethylene glycol

(8000 MW). 10 μM ZnCl2.

In discontinuous assays, the ice-cold quench buffer (HEP) was 20 mM HEPES pH 7.4,

5 mM EDTA, and 15 μg/mL polybrene (as an inhibitor of the polyanions). Titration experiments were performed to determine the polyanion concentration (tested from 1.25 to

100 μM) that resulted in the maximal enzyme activation rate, and that polyanions concentration was used for further experiments. Amidolytic activities of the various enzymes were quantified in a Spectramax M2 96-well spectophotometer (Molecular Devices) by measuring the change in

A405 for 1 h using 0.4 mM of the appropriate chromogenic substrate, typically at ambient temperature (except for PK, which was quantified at 37 °C in order to prevent temperature- dependent autoinhibition of this enzyme266).

Activation of FXI by Thrombin or FXIIa.

Rates of FXI activation were quantified using a discontinuous assay as previously described,156,267 with minor modifications. Briefly, 40 nM of FXI dimer was incubated with 15 nM thrombin or 5 nM FXIIa in the presence or absence of polyanion. In some experiments, 40 nM HK was also included. Timed aliquots of 20 μL were removed and quenched on ice in

59 80 μL HEP plus an inhibitor of either thrombin (18 U/mL hirudin) or FXIIa (500 nM CTI) as appropriate. The FXIa concentration at each quenched time point was determined by measuring its amidolytic activity, with reference to a standard curve.

Autoactivation of FXI.

Second-order rate constants for FXI autoactivation (FXI activation by FXIa) were determined using a discontinuous chromogenic assay. 100 pM FXIa dimer was incubated with

40 nM FXI dimer in the presence or absence of polyanion; 40 nM HK was included in some reaction mixtures. Timed aliquots were removed and quenched on ice in HEP, after which FXIa amidolytic activity was quantified. Data were analyzed according to a second-order reaction mechanism (FXIa + FXI à 2FXIa) and as described previously for autoactivation reactions.6,268

FXI Autolysis.

FXI autolysis was measured essentially as previously described.156 Briefly, FXIa (40 nM) and HK (40 nM) were incubated at 37°C in the presence of 1.25 μM polyI or 5 μM polyP, and timed aliquots were removed and quenched with in HEP buffer on ice. Residual FXIa activity was quantified using 0.4 mM FXIa chromogenic substrate with reference to a standard curve.

Autoactivation of FXII or PPK.

A discontinuous assay was used to quantify FXII autoactivation similarly to that previously described.269 200 nM FXII and 200 nM HK were incubated with polyanion. Timed aliquots of 20 μL were quenched on ice in 80 μL HEP, after which FXIIa amidolytic activity

60 was quantified. The FXIIa concentration at each quenched time point was determined by measuring its amidolytic activity, with reference to a standard curve. In separate experiments, continuous assays for FXII or PPK autoactivation employed zymogen (either 200 nM FXII or

100 nM PPK) pre-mixed with chromogenic substrate. Solutions were warmed at 37°C for 3 min, and polyanion was added to initiate the reaction. Rates of chromogenic substrate hydrolysis were measured at 37°C. Enzyme concentrations were then calculated from the first derivative of A405 vs. time; second-order rate constants were derived as described.6,268

Activation of FXII by PK, and PPK by FXIIa.

In discontinuous assays, zymogen and enzyme were incubated with polyanions, with or without 200 nM HK. For PK activation of FXII, the concentrations were 1 nM and 200 nM respectively. For FXIIa activation of PPK, the concentrations were 250 pM and 50 nM respectively. Timed aliquots were quenched on ice in HEP plus inhibitor for the activating enzyme (50 μg/mL STI for PK, 500 nM CTI for FXIIa). Enzyme concentrations were quantified via amidolytic activity, with reference to a standard curve.

Results

FXI Binding and Rates of FXI Activation.

Polyanions such as polyP192,270 and dextran sulfate271 have been reported to facilitate FXI activation. To investigate the ability of FXI to bind to RNA, we utilized a microplate-based binding assay in which various RNA homopolymer (or polyP) in solution competed with immobilized polyP for binding to FXI (Figure 4.2). Since Zn2+ has been reported to enhance the

61 binding affinities and activities of several of the proteins of the contact pathway,272,273 including

FXI(a) (unpublished data), we employed 10 μM ZnCl2 in essentially all the assays in this study.

PolyG, polyI, and polyP in solution were effective competitors for the binding of FXIa to immobilized polyP, while neither polyA nor polyC appreciably reduced FXIa binding. Addition of HK did not appreciably alter the outcome of this competition binding experiment with regard to any of the polymers tested.

FXI can be activated in three reactions, all of which are enhanced by polyanions; the relevant proteases, numbered as in Figure 4.1, are: (5) FXIa (autoactivation), (6) FXIIa, and (7) thrombin. PolyP strongly enhanced the rate of FXI autoactivation (confirming our previous reports156,267), while polyI was much less effective and polyG did not support this reaction (Figure

4.2B). Inclusion of HK reduced the rate of polyP-mediated FXI autoactivation by as much as

60% (Figure 4.2C). HK also shifted the polyP concentration dependence such that maximal autoactivation required twice as much polyP (5 μM polyP in the presence to HK). On the other hand, HK did not appreciably alter the rate of polyI-mediated FXI autoactivation (Figure 4.2C).

PolyG, polyI and polyP greatly enhanced the rate of FXI activation by thrombin (Figure

4.2D), by at least 75-fold compared to vehicle control. Of these, polyG was the least effective, supporting rates about one-quarter of those with polyP or polyI. Adding HK reduced the rate at which thrombin activated FXI by 30-40% for all three polyanions (Figure 4.2D). This is consistent with a previous report in which HK reduced the rate of FXI activation by thrombin by about 50% in the presence of sulfatides or heparin.271

At a suboptimal concentration of polyP, 1.25 μM, polyP enhanced the rate of FXI activation by FXIIa relative to the vehicle control, but the rate gradually slowed over 5 to 10 min

62 (Figure 4.2E). The same trend was also seen with both polyG and polyI, but occurred at all concentrations of polyG and polyI tested (Figure 4.2E). In contrast, in the presence of polyP at the optimal concentration of 2.5 μM, linear rates of FXIa generation were observed over the 30 min time course (Figure 4.2F); polyP supported initial rates of 0.41 ± 0.06 and 0.44 ± 0.06 nM/min in the presence and absence of HK, respectively (compared to 0.01 ± 0.001 nM/min in the absence of polyP or HK; Figure 4.2F). Adding HK did not appreciably alter the rates of FXI activation with any of the polyanions tested (Figure 4.2E,F).

PolyP-mediated FXI autolysis had been previously demonstrated in the absence of HK.156

We found that both polyP and polyG were able to support FXI autolysis in the presence of HK, with rates of 1.22 ± 0.16 and 0.55 ± 0.18 nM/min, respectively. The polyP-mediated rate of autolysis in the presence of HK was comparable to that in its absence.

FXII Binding and Rates of FXII Activation.

PolyG, polyI and polyP in solution were effective competitors of FXIIa binding to immobilized polyP, while polyA and polyC were ineffective (Figure 4.3A). HK did not appreciably alter the competition binding results.

Autoactivation of single-chain FXII on anionic surfaces to the two-chain active enzyme,

α-FXIIa, was thought to be the initial enzymatic step in the contact pathway. However, a recent study reported significant enzymatic activity of single-chain FXII in complex with polyP, which appears to function as an allosteric activator of this zymogen.269 In discontinuous assays of FXIIa generation in which time aliquots were quenched with polybrene (to disrupt FXII(a) binding to polyP), we found that polyP supported very little FXIIa generation via autoactivation in the

63 presence or absence of HK (Figure 4.3B), consistent with the findings of Engel et al.269 On the other hand, both polyG and polyI supported generation of readily detectable FXIIa activity, although this was observed only in the presence of HK (Figure 4.3B).

In a continuous assay in which a chromogenic substrate was included with FXII and polyP, we found that polyP led to robust FXII(a) amidolytic activity (Figure 4.3C), though this could not be fit to a second order rate equation. PolyG and polyI also supported this reaction, though to a lesser extent (Figure 4.3C), and the data was fit to a second order rate equation and quantified (Figure 4.3D). In the presence of HK, polyG enhanced the rate of FXII autoactivation 8-fold when compared to the vehicle control, while polyI mediated a 6.8-fold increase. Though we were unable to quantify the second order rate constant for polyP-mediated autoactivation, a qualitative assessment of substrate generation (Figure 4.3C) indicates that the fold enhancement would be greater than that for either polyG or polyI. Inclusion of HK enhanced the FXII(a) activity 68% and 76% in the presence of polyG and polyI, respectively

(Figure 4.3D).

We next evaluated the abilities of polyG, polyI and polyP to support FXII activation by

PK. In the absence of HK, all three anions enhanced the rate of FXIIa generation, with polyG and polyI, yielding 5- and 27-fold greater activity, respectively, than with polyP (Figure 4.3E).

HK further enhanced the rates of FXII activation by PK in the presence of all three polyanions, but most profoundly for polyP (17-fold increase by HK) (Figure 4.3E).

(P)PK Binding and Rates of PPK Activation.

PK at 50 nM did not bind detectably to immobilized polyP in the absence of HK (not shown), but did bind in the presence of 50 nM HK (Figure 4.4A). In the presence of HK,

64 polyG, polyI and polyP in solution were equally effective competitors of PK binding to immobilized polyP, while polyA and polyC were ineffective.

In a continuous assay of PPK autoactivation, none of the polyanions supported appreciable substrate hydrolysis in the absence of HK (Figure 4.4B). In the presence of HK, polyG, polyI and polyP all supported robust PK enzyme activity. The second-order rate constants for PPK autoactivation in the presence of HK supported by polyG, polyI or polyP are presented in Figure 4.4C. The rate supported by polyP was approximately 40% higher than that with polyG or polyI.

We next examined the effect of polyanions and HK on the rate of activation of 50 nM

PPK by 250 pM FXIIa. In the absence of HK, polyP, polyG, and polyI all substantially enhanced the rate of PPK activation, to a roughly equal extent. Adding 50 nM HK enhanced the rate of PPK activation even in the absence of anions. Adding polyG or polyI plus HK resulted in little further enhancement of the rate compared with HK alone, while adding polyP plus HK roughly doubled the rate compared with HK alone (Figure 4.4D).

Discussion

The contact pathway acts as a bridge between coagulation and inflammation and plays important roles in thrombosis. Recent work has identified a number of physiologic activators of the contact pathway; however, a systematic interrogation of the effects of procoagulant polyanions on each step in the contact pathway was lacking. In this study, we compared the interactions between various procoagulant polyanions and the enzymes of the contact pathway, and assessed the contributions of HK to these reactions. We found that procoagulant polyanions differ greatly in how they impact the enzymatic steps constituting contact pathway activation,

65 and that HK both potentiates and suppresses polyanion-mediated activation depending on the reaction being investigated.

An intriguing observation in this work is the related to the multivariate nature of the effects of the polyanions on the contact pathway. PolyP supported every reaction tested, and was the most potent polyanion for all autoactivation reactions. PolyI also supported every reaction, and was most effective at supporting FXI back-activation by thrombin and FXII activation by

PK. PolyG, on the other hand, was a relatively poor supporter of FXI back-activation by thrombin and did not support FXI autoactivation. A visual representation of the contact pathway is shown in Figure 4.1, with each reaction labeled. In Figure 4.5, a qualitative table summarizes the relative effects of the various polyanions and HK on the contact pathway enzyme/substrate reactions. Our findings highlight the mechanistic heterogeneity with which the anionic surfaces function and reinforce the need to study the contact pathway at both the pathway and protein level. They also open the door to the possibility that in vivo activation of the contact pathway should not be considered a single event, but rather as a collection of reciprocal and feedback reactions, the balance of which may result in small –but potentially important –phenotypic differences.

Our investigations into the rate of FXI activation by FXIIa produced interesting results, with FXIa activity over time plateauing in the presence of polyG or polyI. The endpoint measurement of this experiment, FXIa activity, is likely affected by the combination of three reactions: FXI activation by FXIIa, FXI autoactivation, and FXI autolysis. We previously found that FXI undergoes autolysis in the presence of polyP,156 and have found that polyI also supports

FXIa autolysis. Furthermore, the activation of FXI by FXIIa in the presence of suboptimal

66 quantities of polyP (1.25 μM) resulted in non-linear FXIa activity curves similar to those seen with polyG and polyI (Figure 4.2E). At this lower concentration (1.25 μM), polyP-mediated

FXI autoactivation is less than 10% of the maximal observed rate when using 2.5 μM polyP

(unpublished results). Taken together, these results suggest that the non-linear FXI activation curves seen for polyG and polyI in Figure 4.2E are a function of poorly supported FXI autoactivation and shifting equilibriums between the three concurrent reactions.

When comparing the qualitative results shown in Figure 4.5, a potentially important observation can be seen in the differing abilities of polyP and nucleic acids to support the activation of FXI by FXIIa (Figure 4.5, reaction 6). FXI activation by FXIIa is believed to be an important activation mechanism, especially with regard to thrombosis,254 and serves as the single, critical bridge between the contact and common pathways of coagulation. Our findings show that polyP mediates a strong and sustained FXI activation, whereas polyG and polyI only support an initial, small burst of FXIa generation. These differences may contribute greatly to the increased procoagulant effects of polyP compared to nucleic acids, and also suggest that downregulating FXIIa-mediated activation of FXI through the inhibition of polyP might be a potentially useful antithrombotic approach.

Our findings here confirm those of Engel et al.,269 who reported that polyP “activates”

FXII by inducing reversible conformational changes in the single-chain protein. This mechanism of action appears to be unique to polyP; polyG and polyI both induced FXIIa activity in the discontinuous FXII activation assay, indicating proteolytic cleavage and formation of two-chain

FXIIa. Interestingly, FXII autoactivation by either mechanism appears to be greatly enhanced in

67 the presence of HK despite the fact that HK is not generally thought to be a relevant cofactor for

FXII(a).

Our findings indicate that PK only binds to polyanions in the presence of HK, and that

HK is required for PPK autoactivation. HK also enhances polyP-mediated activation of PPK by

FXIIa 2-fold. These results indicate that the activation of PPK is enhanced by HK regardless of the activating enzyme, and that future in vitro studies of polyanion-mediated contact pathway activation using purified proteins should be carried out in the presence of HK.

A comparison of second-order rate constants for FXII and PPK autoactivation in the presence of HK under ideal activating conditions highlights a fascinating finding; rates of PPK autoactivation with polyG, and polyI are 7.3-, and 11.2-fold higher than the autoactivation rates for FXII. Furthermore, PPK is present at slightly higher plasma concentrations than FXII (~580 vs. ~375 nM), and the majority of PPK in plasma is bound to HK, conceivably able to undergo autoactivation. These findings open the door to the possibility that the contact pathway is initiated, at least in part, through the autoactivation of PPK in the presence of physiologic polyanions.

We previously reported that cell-derived nucleic acids have only limited ability to activate that contact pathway (Unpublished experiments). Among the RNA homopolymers, only polyG and polyI had detectable contact activation in plasma-based assays. The purified protein studies presented here confirm and extend those observations. PolyA and polyC did not bind to any of the contact pathway proteins, thus only polyG and polyI were able to support the various enzymatic reactions of the contact pathway. This is further evidence that the procoagulant nature of RNA is sequence-specific and not an intrinsic property of RNA.

68 Although polyP and RNA homopolymers are both polyanions, the size of the repeating unit in polyP is markedly smaller than that in RNA. The differences between the polyP and

RNAs with regards to their spectra of effects may be in part related to the charge spacing within the polymers. Alternatively, it may be more a function of flexibility and the capacity to form secondary structure. Previous work by Gansler et al.176 demonstrated the importance of secondary structure on the procoagulant ability of nucleic acids; hairpin forming RNA oligomers are not only more resistant to degradation but are also more procoagulant, as structure-forming RNA aptamers were shown to be able to mediate the autoactivation of PPK. PolyG and polyI are known to form stable, non-canonical base-pairing structures termed G-quadruplexes.238,239

Though our studies did not specifically probe structural considerations, our findings suggest that secondary structure is likely a factor contributing to the procoagulant properties of nucleic acids.

Our findings also suggest the intriguing notion that possible secondary structures of polyP in solution might contribute to its interactions with contact factors.

69 FIGURES AND TABLES

Figure 1.1. Crystal structures of FVIIa and sTF arranged on a membrane surface. A) FVIIa (blue) has an extended conformation and binds to anionic phospholipids in membrane bilayers through its N-terminal GLA-domain (depicted here in contact with the membrane). Coordination of divalent cations such as Ca2+ (red spheres) and Mg2+ (gray spheres) by the GLA domain is critical for proper domain folding and function. In addition, a Ca2+ ion is bound to the first EGF-like domain of FVIIa and also to the protease domain of this protein (the domain farthest from the membrane). The isolated ectodomain of TF (sTF, orange) is depicted here as anchored to the membrane surface via a single transmembrane helix, which has been modeled in. Full-length TF also contains a 21 amino acid-long cytoplasmic tail (not shown) which is implicated in interactions with the cytoskeleton. B) Crystal structure of the sTF-FVIIa complex, with the transmembrane helix of TF modeled in. FVIIa interacts extensively with sTF, with a binding interface that spans all domains of FVIIa and sTF. C) Close-up of TF residues putatively involved in substrate recognition (i.e., the substrate-binding exosite region of TF). In addition to allosterically activating FVIIa, TF is thought to directly interact with the protein substrates, FIX and FX, through membrane-proximal residues. Thus, TF residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185 (shown as van Der Waals radii and colored according to identity as follows; Teal: Lysine, White: Glycine, Yellow: Serine, Green: Tyrosine) contribute significantly to interactions with substrate as demonstrated by mutagenesis studies. (Panel C is rotated ~45° from panel B.) The structure of the sTF-FVIIa complex in panels B and C is rendered from pdb file 3TH2274 using VMD Molecular Graphics Viewer.275 The isolated structures of FVIIa and sTF shown in panel A are a separation of the two from the TF-FVIIa complex. The transmembrane helix attached to sTF is adopted from pdb file 1A11.276

70

Figure 2.1. Effect of Mg2+ on FX activation by FVIIa in the presence or absence of TF or membranes. FX activation by FVIIa was measured in the presence of the components listed, with a dash (—) indicating the component was not present. Data are mean initial rates of FX activation in the presence of 1.25 mM Ca2+ + 0.6 mM Mg2+ normalized to rates using 1.25 mM Ca2+ alone. Error bars are one standard error (n ≥ 3). Normalized rates that are statistically significantly different from 1.0 are indicated with an asterisk (one-sample t-tests; p < 0.05).

71

Figure 2.2. Effect of TF mutations on rates of FX activation by memTF/FVIIa in solution (with 0.1% Triton X-100), measured using different divalent metal ion concentrations. (A) FX activation by memTF/FVIIa using 1.85 mM Ca2+ alone, graphed as initial rates of FX activation divided by the memTF/FVIIa concentration. (B) Relative rates of FX activation by memTF/FVIIa in solution using 1.25 mM Ca2+ + 0.6 mM Mg2+, normalized to rates using 1.85 mM Ca2+ alone. (C) Relative rates of FX activation by memTF/FVIIa in solution using 1.25 mM Ca2+ + 0.6 mM Mg2+, normalized to rates using 1.25 mM Ca2+ alone. Data are mean ± standard error (n ≥ 3).

72

Figure 2.3. Effect of eleven selected TF mutations on the ability of Mg2+ to enhance the rate of FX activation by TF/FVIIa/membrane complexes. WT or mutant memTF was incorporated into liposomes containing 0-15% PS, with the balance being PC. Initial rates of FX activation by the resulting memTF/FVIIa complexes were measured with 1.25 mM Ca2+ plus 0.6 mM Mg2+, and normalized to the rates observed with 1.85 mM Ca2+ alone. The memTF mutants are grouped in the three panels as described in the text, with the same data for WT memTF (˜) plotted in each panel for comparison. (A) Normalized rates of FX activation observed with memTF mutants S162A (Î), W158A (q), E174A (s), and D178A (ó). (B) Normalized rates of FX activation observed with memTF mutants S163A (£), K165A (¿), K159A (™), and Y157A (p). (C) Normalized rates of FX activation observed with memTF mutants Y185A (r), K166A (¢), and G164A (¯). Data are mean ± standard error (n ≥ 3).

73

Figure 2.4. Lack of detectable effect of divalent metal ions on NMR spectra of sTF. (A) 15N-1H 2D HSQC correlation spectrum of 100 µM sTF in 50 mM sodium phosphate, 50 mM NaCl, 1 mM DSS, 10% D2O and no divalent metal ions (blue) overlaid with the sTF spectrum in the same buffer plus 1.25 mM Ca2+ (red). (B) 15N-1H 2D HSQC correlation spectrum of 100 µM sTF in the same buffer as in panel A, without divalent metal ions (blue) overlaid with the spectrum in the presence of 0.5 mM Mg2+ (green). (C) Chemical shift perturbations upon 2+ 2 2 1/2 titration with 1.25 mM Ca (from panel A) calculated as δ = ([0.1δN] + δH ) ), with an average chemical shift perturbation of 0.0014 ppm. (D) Chemical shift perturbations upon titration with 0.5 mM Mg2+ (from panel B), with an average chemical shift perturbation of 0.0015 ppm. Of the 208 expected non-proline resonances in sTF, 196 resonances were assigned.

74 Figure 2.5. Localization and properties of TF residues investigated. The structure is from PDB file 3TH2, in which sTF/FVIIa was crystallized in the presence of 5 mM Ca2+ and 2.5 mM Mg2+.200 FVIIa is depicted as orange ribbons, with bound Ca2+ ions in teal and Mg2+ ions in beige. (A) Localization of the TF residues tested in this study for their effect on the absolute rate of FX activation by memTF/FVIIa in solution. TF residues are color-coded according to their rate of FX activation in the presence of 1.85 mM Ca2+ alone (from Figure 2.2A). Unmutated TF residues are white. TF residues are colored red which, when mutated, retained essentially WT activity (i.e., ≥ 75% of WT rate of FX activation). Residues with moderate effect on FX activation rate are green (30-75% of WT); residues with severe defects are blue (≤ 30% of WT). Residues W158 and S160, obstructed in the visualization above, are colored blue and green, respectively. (B) Localization of TF residues tested in this study as being important for the ability of Mg2+ to enhance the rate of FX activation by memTF/FVIIa in solution. TF residues are color-coded according to their rate of FX activation in the presence of Ca2+ plus Mg2+ normalized to the rate at 1.85 mM Ca2+ alone (from Figure 2.2B). Unmutated TF residues are white. TF residues are colored red which, when mutated, retained essentially WT responses to Mg2+ (i.e., 4- to 6-fold enhancement in FX activation rate). Residues with blunted responses to Mg2+ are colored green (2- to 4-fold enhancement by Mg2+) or blue (1- to 2-fold enhancement by Mg2+). Residues W158 and S160, obstructed in the visualization above, are colored red. (C) Relative rate of FX activation (rate with 1.25 mM Ca2+ + 0.6 mM Mg2+, divided by rate with 1.85 mM Ca2+) versus the absolute rate of FX activation, in both cases by memTF/FVIIa in solution (i.e., data re-plotted from Figures 2.2A and 2.2B, respectively). Residues are color-coded as in panel B of this figure. Vertical black dotted lines correspond to the color-coding cutoff values from panel A; horizontal black dotted lines correspond to the color-coding cutoff values from panel B. Vertical dotted red line marks the location of WT TF. Experiments were performed by Kristin Nuzzio.

75 Table 2.1. Effect of Mg2+ on Rates of FX Activation by memTF/FVIIa in Solution Initial rates of FX activation (nM/min/nM ×103)a TF 1.25 mM Ca2+ + 0.6 mM 1.85 mM Ca2+ 1.25 mM Ca2+ (WT or mutant) Mg2+ WT 122.1 ± 4.0 27.8 ± 3.9 20.4 ± 1.3 Y157A 5.3 ± 0.4 2.2 ± 0.1 1.7 ± 0.1 W158A 30.8 ± 2.8 7.2 ± 0.3 5.4 ± 0.6 K159A 10.3 ± 2.0 2.8 ± 0.2 4.0 ± 1.9 S160A 58.2 ± 4.0 12.1 ± 2.2 7.1 ± 0.2 S161A 189.4 ± 17.2 31.4 ± 2.8 24.3 ± 1.1 S162A 47.3 ± 1.8 9.4 ± 0.6 6.5 ± 0.4 S163A 2.8 ± 0.5 1.0 ± 0.1 0.9 ± 0.1 G164A 1.1 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 K165A 4.8 ± 0.2 1.8 ± 0.1 2.4 ± 1.0 K166A 2.5 ± 0.4 1.5 ± 0.2 1.4 ± 0.3 K169A 133.9 ± 12.9 25.0 ± 1.4 17.4 ± 1.1 E174A 98.3 ± 3.3 18.9 ± 1.0 13.0 ± 1.1 L176A 60.8 ± 4.2 12.5 ± 0.5 7.4 ± 0.4 D178A 129.5 ± 15.0 24.2 ± 1.5 19.1 ± 2.5 D180A 34.4 ± 4.9 13.1 ± 2.3 8.8 ± 0.8 Y185A 2.3 ± 0.2 1.8 ± 0.1 1.4 ± 0.1 R200A 29.0 ± 1.8 5.9± 0.5 3.8 ± 0.3

aData are initial rates of FX activation in (nM/min) divided by the memTF/FVIIa concentration (in nM), expressed as mean ± standard error (n ≥ 3). Reaction conditions were 10 nM FVIIa, 500 nM memTF, 0.1% Triton X-100, 100 nM FX, and 1 mM Spectrozyme Xa.

76

Figure 2.6. Effect of TF mutations on Mg2+-dependent rate enhancements of FIX activation. (A) Initial rates of FIX activation by WT or mutant memTF/FVIIa in solution (with 0.06% Triton X-100) using 1.25 mM Ca2+. (B) Relative rates of FIX activation by WT or mutant memTF/FVIIa in solution (with 0.06% Triton X-100). In this panel, the rates using 1.25 mM Ca2+ + 0.6 mM Mg2+ were normalized to those using 1.25 mM Ca2+ alone. Data are mean ± standard error (n = 3).

77

Figure 2.7. Effect of increased Ca2+ concentration on FX activation by memTF/FVIIa in solution. In each case, the initial rates of FX activation by memTF/FVIIa complexes in detergent solution (0.1% Triton X-100) using 1.85 mM Ca2+ were normalized to the rates observed with the same version of memTF (mutant or WT) at 1.25 mM Ca2+. The rates of FX activation supported by WT and most memTF mutants were increased approximately 1.3-fold upon increasing the Ca2+ concentration from 1.25 to 1.85 mM. However, the rates of FX activation supported by two TF mutants (K159A, p = 0.018; and K165A, p = 0.029) were significantly decreased upon increasing the Ca2+ concentration (asterisks). Data are mean ± standard error (n ≥ 3), with statistical significance determined using Student’s t-test.

78

Figure 2.8. TF sequence alignment of the region of interest across selected mammals and birds. BLAST search was performed using human TF as the standard (with the amino acid numbering also representative of human TF). Highlighted in blue (164-166, 185) were residues most important for Mg2+ recognition. Residues in green (157, 159, 163, 180) were intermediate in their importance towards recognizing Mg2+. Mutations of residues in red resulted in no identifiable defect in Mg2+ response. We note that the TF residues most important for Mg2+ recognition are all highly conserved.

79 Table 2.2: Kd Values for TF/FVIIa under Varying Divalent Metal Ion Conditions

a Kd (nM) 1.25 mM Ca2+ + TF (WT or mutant) 1.85 mM Ca2+ Ratiob 0.6 mM Mg2+ WT 0.39 ± 0.1 0.58 ± 0.1 1.48 ± 0.4 Y157A 0.13 ± 0.05 0.23 ± 0.02 1.87 ± 0.7 S163A 0.70 ± 0.3 0.57 ± 0.1 0.81 ± 0.4 G164A 1.03 ± 0.3 1.26 ± 0.06 1.22 ± 0.3 K165A 0.66 ± 0.3 0.94 ± 0.5 1.42 ± 1 K166A 0.44 ± 0.08 0.47 ± 0.04 1.07 ± 0.2 D180A 1.07 ± 0.3 0.86 ± 0.2 0.80 ± 0.3 Y185A 0.43 ± 0.1 0.49 ± 0.1 1.15 ± 0.4 a b 2+ Values for Kd are mean ± standard error (n ≥ 3). Ratios are mean Kd at 1.25 mM Ca + 0.6 mM 2+ 2+ Mg divided by mean Kd at 1.85 mM Ca (± standard error).

The Kd values for FVIIa binding to TF in Table 2.2 were determined using memTF in solution (with 0.1% Triton X-100), under conditions of equal total divalent metal ion concentration with either no Mg2+ (1.85 mM Ca2+) or with physiologic concentrations of Mg2+ and Ca2+ (1.25 mM Ca2+ + 0.6 mM Mg2+).

80

Figure 3.1. Ability of DNA, RNA and polyP to initiate clotting via the contact pathway. Varying concentrations of DNA, RNA, or polyP were incubated with pooled normal plasma in microplate wells for 3 minutes at 37°C, after which CaCl2 was added and clotting was detected. Shortening of the clot time is expressed as percent of the clot time with buffer alone. (See supplemental Figure S2 for the distribution of clot times in the absence of activator.) (A) Clotting observed with polyP, cell-derived nucleic acids, or lambda DNA: long-chain polyP (˜), medium-chain polyP (n), short-chain polyP (u), HEK 293 cell DNA isolated with DNeasy Blood & Tissue kit (q), HEK 293 cell RNA isolated with RNeasy Midi kit (r), or lambda DNA ( ¢). (B) Clotting observed with synthetic RNA homopolymers: polyG (s), polyI (¯), polyA (£), or polyC (™). Data are mean ± SEM (n ≥ 3).

81

Figure 3.2. Ability of DNA, RNA and polyP to abrogate the anticoagulant activity of TFPI. The indicated polymers and 200 ng/mL TFPI were added to pooled normal plasma at 37°C, after which clotting was initiated by addition of factor Xa and CaCl2 and detected using a coagulometer. The clot time of plasma with TFPI but no added polymer was defined as 100% (q+TFPI); for comparison, clotting of plasma without added TFPI or polymer is also plotted (q-TFPI). Polymers added were: short-chain polyP (u), HEK 293 cell DNA isolated with DNeasy Blood & Tissue kit (q), HEK 293 cell RNA isolated with RNeasy Midi kit (r), lambda DNA (¢), polyG (s), polyI (¯), polyA (£), or polyC (™). Data are mean ± SEM (n ≥ 3).

82

Figure 3.3. Effect of enzyme digestion on the procoagulant activity of polyP, cell-derived DNA and lambda DNA. HEK 293 cell DNA isolated with DNeasy Blood & Tissue kit (50 µg/mL), lambda DNA (20 µg/mL), or long-chain polyP (2 µg/mL) was incubated with pooled normal plasma in microplate wells for 3 minutes at 37°C, after which CaCl2 was added and clotting was detected. Shortening of the clot time is expressed as percent of the clot time with buffer alone. Effects of enzymatic predigestion of DNA or polyP for 30 minutes at 37°C on clotting, using: no enzyme (black bars), 700 U/mL Benzonase (grey), or 300 U/mL calf intestinal alkaline phosphatase (CIAP, white). Statistical significance was assessed using a two-tailed t-test against the no enzyme sample. *P < 0.05; **P < 0.01; ***P < 0.001. Data are mean ± SEM (n ≥ 3).

83

Figure 3.4. Ability of mixtures of DNA and polyP to initiate clotting via the contact pathway. Plasma clotting was evaluated in a microplate-based assay in which the contact pathway was triggered by DNA plus polyP. Activators were incubated with pooled normal plasma in microplate wells for 3 minutes at 37°C, after which CaCl2 was added and clotting was detected. Shortening of the clot time is expressed as percent of the clot time with buffer alone. (A) Clotting observed with varying concentrations of polyP alone or with a constant concentration of lambda DNA: short-chain polyP alone (u) or with 20 µg/mL lambda DNA (¯); medium- chain polyP alone (n) or with 20 µg/mL lambda DNA (£). (B) Clotting observed with varying concentrations of a combination of medium-chain polyP and lambda DNA at a fixed mass ratio of 1:100 (£). PolyP concentrations are given on the upper x-axis and DNA concentrations on the lower x-axis. For comparison, clotting observed with medium-chain polyP alone (n), HEK 293 cell DNA alone (isolated with DNeasy Blood & Tissue kit) (s), and lambda DNA alone (p) are also plotted using the corresponding x-axes. Data are mean ± SEM (n ≥ 3).

84

Figure 3.5. Recovery of nucleic acids and polyP with commercial nucleic acid purification kits. Known quantities of lambda DNA (DNA, gray bars), polyG (RNA, white bars) and/or long- chain polyP (black bars) were processed for nucleic acid purification using Qiaquick, DNeasy, or TRIzol kits, following the manufacturer’s instructions. Recovered nucleic acids or polyP were quantified as described in Materials and Methods and plotted as percent of the input amount in each experiment. The 3 samples were: (A) nucleic acid alone, consisting of 5 µg of either lambda DNA or polyG (RNA); (B) 5 µg polyP alone; or (C) a mixture of 5 µg polyP plus 5 µg of either lambda DNA or polyG (RNA). Experiments with the Qiaquick kit employed 2.5 µg of polyP and/or nucleic acid. Data are mean ± SEM (n ≥ 3).

85

Figure 3.6. Traces of polyP in cell-derived DNA. DNA isolated from HEK 293 cells using the DNeasy Blood & Tissue kit was concentrated to 3 mg/mL and resolved by electrophoresis on a 4-20% polyacrylamide gel. The same gel was stained sequentially, using: (A) DAPI and (B) SYBR Green I (after removing DAPI by repeated rinsing). Samples were: 1 kb DNA ladder (lane 1); HEK 293 DNA (lane 2); HindIII-digested lambda DNA (lane 3); Benzonase-digested HEK 293 DNA (lane 4); and Benzonase-digested, HindIII-digested lambda DNA (lane 5).

86

Figure 3.7. Urea-PAGE of RNA homopolymers and polyP. PolyP preparations and RNA homopolymers used in this study were resolved by PAGE using TBE-urea gels (10% polyacrylamide). 2 µg of each sample was loaded into the following lanes, with 2M urea in the loading buffer: 1, Low MW DNA Ladder; 2, long-chain polyP; 3, medium-chain polyP; 4, short-chain polyP; 5, polyI; 6, polyG; 7, polyC; 8, polyA. PolyP and nucleic acids were stained for 5 minutes with 0.05% toluidine blue in 5% glycerol/25% methanol, and de-stained overnight in 5% glycerol/25% methanol.

87

Plasma clot times in the absence of any added activator

Number of replicates 54

Minimum clot time (sec) 218.5

Maximum clot time (sec) 719.2

Median clot time (sec) 331.0

Mean clot time (sec) 366.8

Standard Deviation (sec) 106.4

Standard Error (sec) 14.48

Figure 3.8. Distribution of clot times in the absence of activator. The clotting times in Figures 1, 3, and 4 (and in Supplemental Figure S3) were normalized to the clot times in the absence of any added activator (polyanion) within each experimental run. Data on observed plasma clotting times in the absence of activator are plotted on the left as a box-and-whisker plot (depicting the minimum, 25th percentile, median, 75th percentile, and maximum), and shown in detail on the right in tabular form.

88

Figure 3.9. Ability of DNA, RNA and polyP to initiate clotting via the contact pathway, plotted on a molar basis. Alternative representation of the data presented in Figure 1, with clot times plotted vs. molar concentration of polyP (in terms of phosphate monomer) or molar concentration of DNA (also in terms of phosphate, which is identical to the molar concentration of nucleotides). Varying concentrations of DNA, RNA, or polyP were incubated with pooled normal plasma in microplate wells for 3 minutes at 37°C, after which CaCl2 was added and clotting was detected. Shortening of the clot time is expressed as percent of the clot time with buffer alone. (A) Clotting observed with polyP, cell-derived nucleic acids, or lambda DNA: long-chain polyP (˜), medium-chain polyP (n), short-chain polyP (u), HEK 293 cell DNA isolated with DNeasy Blood & Tissue kit (q), HEK 293 cell RNA isolated with RNeasy Midi kit (r), or lambda DNA ( ¢). (B) Clotting observed with synthetic RNA homopolymers: polyG (s), polyI (¯), polyA (£), or polyC (™). Data are mean ± SEM (n ≥ 3).

89

A

B

Figure 3.10. CIAP and Benzonase digestion of DNA and polyP. Lambda DNA-HindIII digest and long-chain polyP were incubated with CIAP, Benzonase, or no enzyme, then resolved on 4- 20% polyacrylamide gels. PolyP and DNA were visualized via DAPI staining, which fluoresces in the presence of DNA, but which rapidly photobleaches in the presence of polyP (negative staining). (A) Lambda DNA: Lanes 1 and 10, 100 bp DNA ladder; lanes 2 and 3, Lambda DNA-Hind III digest, further digested with CIAP; lanes 4 and 5, Lambda DNA-Hind III digest, further digested with Benzonase; lanes 6 and 7, Lambda DNA-Hind III digest, further digested with PPX1; lanes 8 and 9, no enzyme. (B) Long-chain polyP (mode = 1125). Lanes 1 and 10, 100 bp DNA ladder; lanes 2 and 3, polyP incubated in buffer without enzyme; lanes 4 and 5, polyP after PPX1 digestion; lanes 6 and 7, polyP after Benzonase digestion; lanes 8 and 9, polyP after CIAP digestion.

90 Table 3.1. Nucleic acid purification kits used in studies of the procoagulant activity of DNA and RNA

Kit Name Manufacturer Type/Principle Used By Qiaquick PCR Qiagen Silica Modified protocol used by 221 Purification Nickel et al. (2015) *

DNeasy Blood Qiagen Silica Vu et al. (2015)277 and Tissue Bhagirath et al. (2015)225

QIAamp Blood Qiagen Silica Gould et al. (2014)175 Gould et al. (2015)177 Swystun et al. (2011)278

RNeasy Mini Qiagen Silica Kannemeier et al. (2007)166

RNeasy Plus Qiagen Silica Vu et al. (2015)277 Mini

TRIzol Invitrogen Guanidinium isothiocyanate Kannemeier et al. (2007)166 and acidic phenol/chloroform extraction

*Utilized for the purification of polyP

91

Figure 4.1. Schematic overview of the contact pathway. Specific proteolytic activation events that were examined in this study are numbered. PPK can be activated by PK (1) or FXIIa (2). FXII can be activated by FXIIa (3) or PK (4). FXI can be activated by FXIa (5), FXIIa (6), or thrombin (7). The common pathway represents the bulk of the clotting cascade, from factor IX activation through thrombin generation.

92

Figure 4.2. FXI(a) interactions with RNA and polyP. (A) Ability of 50 μM solution-phase polyP or RNA homopolymers to compete with immobilized biotin-polyP for binding to 50 nM FXIa in the presence (black bars) or absence (white bars) of 50 nM HK. Bound FXIa in microplate wells was normalized to the amount bound in the absence of any competing polyanion (“None”). (B) Second-order rate constants for FXI autoactivation in the absence of HK. 40 nM FXI was incubated with 100 pM FXIa and either 2.5 μM polyP, 1.25 μM polyG, 1.25 μM polyI or no polymer (“Vehicle”). (C) Second-order rate constants for polyP- and polyI-mediated FXI autoactivation. 40 nM FXI was incubated with 100 pM FXIa and either 2.5 to 10 μM polyP or 1.25 μM polyI, in the presence (black bars) or absence (white bars) of 40 nM HK. (D) FXI

93 Figure 4.2 (cont.) activation by thrombin, plotted as initial rates of FXI activation divided by the thrombin concentration. 40 nM FXI with (black bars) or without (white bars) 40 nM HK was incubated with 15 nM thrombin in the presence of either 15 μM polyP, 6 μM polyG, 10 μM polyI, or no polymer. (E) FXI activation by FXIIa in the presence (dotted lines), or absence (solid lines) of 40 nM HK. 40 nM FXI was incubated with 5 nM FXII plus either 1.25 μM polyP (green ¢), 1.25 μM polyI (purple ˜), 1.25 μM polyG (blue ¿), or no polymer (red ▲). (F) Data in panel F is the same progress curves of FXIa generation with the exception of polyP (green ¢), which is 2.5 μM in panel F. Panel E represents the first 10 min, and panel F represents the full 30-min time course. Data in all panels are mean ± standard error (n ≥ 3).

94

Figure 4.3. FXII(a) interactions with RNA and polyP. (A) Ability of 50 μM solution-phase polyP or RNA homopolymers to compete with immobilized biotin-polyP for binding to 50 nM FXIIa in the presence (black bars) or absence (white bars) of 50 nM HK. Bound FXIIa in microplate wells was normalized to the amount bound in the absence of any competing polyanion (“None”). (B) Discontinuous assay of FXII autoactivation (using 200nM FXII) in the presence (solid lines) or absence (dotted lines) of 200 nM HK and 10 μM of either polyP (green q), polyG (purple ¢), or polyI (blue ˜). FXIIa activity (rate of chromogenic substrate hydrolysis) is plotted as mOD/min versus time. (C) Continuous assay of FXII autoactivation (using 200 nM FXII) in the presence (solid lines) or absence (dotted lines) of 200 nM HK and either 100 μM polyP (green), 5 μM polyG (purple), or 5 μM polyI (blue). FXIIa activity is

95 Figure 4.3 (cont.) plotted as the A405 generation versus time. (D) Second-order rate constants in the continuous activation assay for 5 μM polyG-, 5 μM polyI-, or no polymer (“Vehicle”) mediated autoactivation of FXII, in the presence or absence of HK. Initial concentrations of FXII and HK were 200 nM. Rates were derived using the continuous autoactivation assay as shown in Figure 3C. (E) Initial rates of activation of 200 nM FXII by 1 nM PK in the discontinuous assay in the presence (black) or absence (white) of 200 nM HK and either 20 μM polyP, 10 μM polyG, 10 μM polyI, or no polymer (“Vehicle”). Data in all panels are mean ± standard error (n ≥ 3), except for the vehicle control in panel D (n = 2).

96

Figure 4.4. (P)PK interactions with RNA and polyP. (A) Ability of 50 μM solution-phase polyP or RNA homopolymers to compete with immobilized biotin-polyP for binding to 50 nM PK in the presence of 50 nM HK. Bound PK in microplate wells was normalized to the amount bound in the absence of any competing polyanion (“None”). (PK did not bind appreciably to polyP in the absence of HK.) (B) Raw data from continuous PPK autoactivation assays, in triplicate. Autoactivation of 100 nM PPK plus 100 nM HK was examined in the presence of either 10 μM polyP (green), 5 μM polyG (purple), or 5 μM polyI (blue). (No appreciable PK activity developed in the absence of HK, even in the presence of polyP, polyG or polyI.) (C) Second- order rate constants from the continuous assay for PPK autoactivation by 10 μM polyP, 2.5 μM polyG, 2.5 μM polyI or no polymer (“Vehicle”) in the presence of equimolar HK. (D) Initial rates of activation of 50 nM PPK by 250 pM FXIIa in a discontinuous assay in the presence (black) or absence (white) of 50 nM HK and either 10 μM polyP, 2.5 μM polyG, 2.5 μM polyI, or no polymer. Data in all panels are mean ± standard error (n ≥ 3).

97

Figure 4.5. Qualitative summary of our findings on the effects of polyP or RNA (in the presence or absence of HK) on the various activation reactions of the contact pathway. The number of plus signs (+) indicates the rate of the reaction relative to the maximum rate supported in the presence of HK, while a minus sign (−) indicates that the reaction was not detected under that condition. Approximately, reaction rates above the 80th percentile received (+++), above the 33rd percentile received (++), and below the 33rd percentile received (+). The intensities of the green or red colors correspond qualitatively to the change in rate upon addition of HK (red = rate decreased; green = rate increased; white = no effect). Reaction numbers in the first column correspond to the reaction numbers depicted in Figure 1.

98 REFERENCES

1. Mackman N. The role of tissue factor and factor VIIa in hemostasis. Anesth Analg. 2009;108(5):1447-1452. 2. Morrissey JH. Tissue factor: a key molecule in hemostatic and nonhemostatic systems. International Journal of Hematology. 2004;79(2):103-108. 3. Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis. Am J Pathol. 1989;134(5):1087-1097. 4. Fleck RA, Rao LVM, Rapaport SI, Varki N. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody. Thromb Res. 1990;59(2):421-437. 5. Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proceedings of the National Academy of Sciences of the United States of America. 1989;86(8):2839-2843. 6. Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood. 1993;81(3):734-744. 7. Kondo S, Kisiel W. Regulation of factor VIIa activity in plasma: Evidence that antithrombin III is the sole plasma protease inhibitor of human factor VIIa. Thromb Res. 1987;46(2):325-335. 8. Seligsohn U, Kasper CK, Osterud B, Rapaport SI. Activated factor VII: presence in factor IX concentrates and persistence in the circulation after infusion. Blood. 1979;53(5):828- 837. 9. Lawson JH, Butenas S, Mann KG. The evaluation of complex-dependent alterations in human factor VIIa. Journal of Biological Chemistry. 1992;267(7):4834-4843. 10. Neuenschwander PF, Branam DE, Morrissey JH. Importance of substrate composition, pH and other variables on tissue factor enhancement of factor VIIa activity. Thrombosis and haemostasis. 1993;70(6):970-977. 11. Ruf W, Rehemtulla A, Morrissey JH, Edgington TS. Phospholipid-independent and - dependent interactions required for tissue factor receptor and cofactor function. Journal of Biological Chemistry. 1991;266(4):2158-2166. 12. Zwaal RFA, Comfurius P, Bevers EM. Lipid-protein interactions in blood coagulation. Biochim Biophys Acta. 1998;1376(3):433-453. 13. Morrissey JH, Neuenschwander PF, Huang Q, McCallum CD, Su B, Johnson AE. Factor VIIa-tissue factor: functional importance of protein-membrane interactions. Thrombosis and Haemostasis. 1997;78(1):112-116. 14. Paborsky LR, Caras IW, Fisher KL, Gorman CM. Lipid association, but not the transmembrane domain, is required for tissue factor activity. Substitution of the transmembrane domain with a phosphatidylinositol anchor. J Biol Chem. 1991;266(32):21911-21916. 15. Waters EK, Morrissey JH. Restoring full biological activity to the isolated ectodomain of an integral membrane protein. Biochemistry. 2006;45(11):3769-3774.

99 16. Ohkubo YZ, Morrissey JH, Tajkhorshid E. Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor. J Thromb Haemost. 2010;8(5):1044-1053. 17. Bazan JF. Structural design and molecular evolution of a cytokine receptor superfamily. Proceedings of the National Academy of Sciences of the United States of America. 1990;87(18):6934-6938. 18. Lhermusier T, Chap H, Payrastre B. Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome. Journal of Thrombosis and Haemostasis. 2011;9(10):1883- 1891. 19. Fadeel B, Xue D. The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease. Critical Reviews in Biochemistry and Molecular Biology. 2009;44(5):264-277. 20. Ke K, Yuan J, Morrissey JH. Tissue factor residues that putatively interact with membrane phospholipids. PLoS ONE. 2014;9(2):e88675. 21. Al-Hashimi AA, Caldwell J, Gonzalez-Gronow M, et al. Binding of anti-GRP78 autoantibodies to cell surface GRP78 increases tissue factor procoagulant activity via the release of calcium from endoplasmic reticulum stores. J Biol Chem. 2010;285(37):28912- 28923. 22. Pozza LM, Austin RC. Getting a GRP on tissue factor activation. Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25(8):1529-1531. 23. Butenas S. Tissue factor structure and function. Scientifica (Cairo). 2012;2012:964862. 24. Chen VM, Ahamed J, Versteeg HH, Berndt MC, Ruf W, Hogg PJ. Evidence for activation of tissue factor by an allosteric disulfide bond. Biochemistry. 2006;45(39):12020-12028. 25. Versteeg HH, Ruf W. Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity. J Biol Chem. 2007;282(35):25416- 25424. 26. Bach RR, Monroe D. What is wrong with the allosteric disulfide bond hypothesis? Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29(12):1997-1998. 27. Persson E. Protein disulfide isomerase has no stimulatory chaperone effect on factor X activation by factor VIIa-soluble tissue factor. Thromb Res. 2008;123(1):171-176. 28. Pendurthi UR, Ghosh S, Mandal SK, Rao LV. Tissue factor activation: is disulfide bond switching a regulatory mechanism? Blood. 2007;110(12):3900-3908. 29. Rao LV, Kothari H, Pendurthi UR. Tissue factor: mechanisms of decryption. Front Biosci. 2012;4:1513-1527. 30. Pendurthi UR, Rao LV. Role of tissue factor disulfides and lipid rafts in signaling. Thromb Res. 2008;122 Suppl 1:S14-18. 31. Butenas S, Krudysz-Amblo J. Decryption of tissue factor. Thromb Res. 2012;129 Suppl 2:S18-20. 32. Rao LV, Kothari H, Pendurthi UR. Tissue factor encryption and decryption: facts and controversies. Thromb Res. 2012;129 Suppl 2:S13-17. 33. Radcliffe R, Nemerson Y. Activation and control of factor VII by activated factor X and thrombin: Isolation and characterization of a single chain form of factor VII. J Biol Chem. 1975;250(2):388-395.

100 34. Broze GJ, Jr., Majerus PW. Purification and properties of human coagulation factor VII. J Biol Chem. 1980;255(4):1242-1247. 35. Kisiel W, Davie EW. Isolation and characterization of bovine factor VII. Biochemistry. 1975;14(22):4928-4934. 36. Gladhaug A, Prydz H. Purification of the coagulation factors VII and X from human serum. Some properties of factor VII. Biochim Biophys Acta. 1970;215(1):105-111. 37. Hagen FS, Gray CL, O'Hara P, et al. Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci U S A. 1986;83:2412-2416. 38. O'Hara PJ, Grant FJ, Haldeman BA, et al. Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation. Proc Natl Acad Sci U S A. 1987;84(15):5158-5162. 39. Bloom JW, Mann KG. Metal ion induced conformational transitions of prothrombin and prothrombin fragment 1. Biochemistry. 1978;17(21):4430-4438. 40. Freedman SJ, Furie BC, Furie B, Baleja JD. Structure of the metal-free gamma- carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy. J Biol Chem. 1995;270(14):7980-7987. 41. Banner DW, D'Arcy A, Chène C, et al. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature. 1996;380(6569):41-46. 42. Schiodt J, Harrit N, Christensen U, Petersen LC. Two different Ca2+ ion binding sites in factor VIIa and in des(1-38) factor VIIa. FEBS Letters. 1992;306(2-3):265-268. 43. Persson E, Nielsen LS. Ca2+ in the first epidermal growth factor-like domain of activated factor VII. Blood Coagul Fibrinolysis. 1998;9 Suppl 1:S79-S81. 44. Sabharwal AK, Birktoft JJ, Gorka J, Wildgoose P, Petersen LC, Bajaj SP. High affinity Ca2+- binding site in the serine protease domain of human factor VIIa and its role in tissue factor binding and development of catalytic activity. J Biol Chem. 1995;270(26):15523- 15530. 45. Higashi S, Matsumoto N, Iwanaga S. Molecular mechanism of tissue factor-mediated acceleration of factor VIIa activity. J Biol Chem. 1996;271(43):26569-26574. 46. Persson E. Macromolecular substrate affinity for free factor VIIa is independent of a buried protease domain N-terminus. Biochemical and Biophysical Research Communications. 2006;341(1):28-32. 47. Toso R, Bernardi F, Tidd T, et al. Factor VII mutant V154G models a zymogen-like form of factor VIIa. Biochemical Journal. 2003;369(3):563-571. 48. Song H, Olsen OH, Persson E, Rand KD. Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry. J Biol Chem. 2014;289(51):35388-35396. 49. Bajaj SP, Schmidt AE, Agah S, Bajaj MS, Padmanabhan K. High Resolution Structures of p- Aminobenzamidine- and Benzamidine-VIIa/Soluble Tissue Factor: UNPREDICTED CONFORMATION OF THE 192-193 PEPTIDE BOND AND MAPPING OF Ca2+, Mg2+, Na+, AND Zn2+ SITES IN FACTOR VIIa. Journal of Biological Chemistry. 2006;281(34):24873- 24888. 50. Mather T, Oganessyan V, Hof P, et al. The 2.8 Å crystal structure of Gla-domainless activated protein C. EMBO Journal. 1996;15(24):6822-6831.

101 51. Walker CP, Royston D. Thrombin generation and its inhibition: a review of the scientific basis and mechanism of action of anticoagulant therapies. British Journal of Anaesthesia. 2002;88(6):848-863. 52. Rand KD, Jorgensen TJ, Olsen OH, et al. Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange. J Biol Chem. 2006;281(32):23018-23024. 53. Kirchhofer D, Eigenbrot C, Lipari MT, Moran P, Peek M, Kelley RF. The tissue factor region that interacts with factor Xa in the activation of factor VII. Biochemistry. 2001;40(3):675-682. 54. Persson E, Kjalke M, Olsen OH. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc Natl Acad Sci U S A. 2001;98(24):13583- 13588. 55. Neuenschwander PF, Vernon JT, Morrissey JH. Tissue factor alters the pKa values of catalytically important factor VIIa residues. Biochemistry. 2002;41(10):3364-3371. 56. McCallum CD, Hapak RC, Neuenschwander PF, Morrissey JH, Johnson AE. The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study. J Biol Chem. 1996;271(45):28168-28175. 57. Mutucumarana VP, Duffy EJ, Lollar P, Johnson AE. The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study. J Biol Chem. 1992;267(24):17012-17021. 58. Husten EJ, Esmon CT, Johnson AE. The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex. Journal of Biological Chemistry. 1987;262(27):12953-12961. 59. McCallum CD, Su B, Neuenschwander PF, Morrissey JH, Johnson AE. Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain. A fluorescence resonance energy transfer study. J Biol Chem. 1997;272(48):30160-30166. 60. Huang X, Ding WQ, Vaught JL, et al. A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties. Blood. 2006;107(3):980-986. 61. Waters EK, Yegneswaran S, Morrissey JH. Raising the active site of factor VIIa above the membrane surface reduces its procoagulant activity but not factor VII autoactivation. J Biol Chem. 2006;281(36):26062-26068. 62. Waxman E, Laws WR, Laue TM, Nemerson Y, Ross JBA. Human factor VIIa and its complex with soluble tissue factor: Evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods. Biochemistry. 1993;32(12):3005-3012. 63. Olsen OH, Rand KD, Østergaard H, Persson E. A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation. Protein Science. 2007;16(4):671-682. 64. Colina CM, Venkateswarlu D, Duke R, Perera L, Pedersen LG. What causes the enhancement of activity of factor VIIa by tissue factor? Journal of Thrombosis and Haemostasis. 2006;4(12):2726-2729.

102 65. Soejima K, Yuguchi M, Mizuguchi J, et al. The 99 and 170 loop-modified factor VIIa mutants show enhanced catalytic activity without tissue factor. J Biol Chem. 2002;277(50):49027-49035. 66. Dickinson CD, Kelly CR, Ruf W. Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(25):14379- 14384. 67. Pike ACW, Brzozowski AM, Roberts SM, Olsen OH, Persson E. Structure of human factor VIIa and its implications for the triggering of blood coagulation. Proc Natl Acad Sci U S A. 1999;96(16):8925-8930. 68. Ruf W, Miles DJ, Rehemtulla A, Edgington TS. Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII. Journal of Biological Chemistry. 1992;267(31):22206-22210. 69. Huang Q, Neuenschwander PF, Rezaie AR, Morrissey JH. Substrate recognition by tissue factor-factor VIIa. Evidence for interaction of residues Lys165 and Lys166 of tissue factor with the 4-carboxyglutamate-rich domain of factor X. J Biol Chem. 1996;271(36):21752- 21757. 70. Kirchhofer D, Lipari MT, Moran P, Eigenbrot C, Kelley RF. The tissue factor region that interacts with substrates factor IX and factor X. Biochemistry. 2000;39(25):7380-7387. 71. Roy S, Hass PE, Bourell JH, Henzel WJ, Vehar GA. Lysine residues 165 and 166 are essential for the cofactor function of tissue factor. J Biol Chem. 1991;266(32):22063- 22066. 72. Ruf W, Miles DJ, Rehemtulla A, Edgington TS. Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex. J Biol Chem. 1992;267(9):6375-6381. 73. Vadivel K, Agah S, Messer AS, et al. Structural and functional studies of γ- carboxyglutamic acid domains of factor VIIa and activated protein C: Role of magnesium at physiological calcium. Journal of Molecular Biology. 2013;425(11):1961-1981. 74. Morrissey JH, Fair DS, Edgington TS. Monoclonal antibody analysis of purified and cell- associated tissue factor. Thromb Res. 1988;52(3):247-261. 75. Fiore MM, Neuenschwander PF, Morrissey JH. An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates. Blood. 1992;80(12):3127-3134. 76. Ruf W, Edgington TS. An anti-tissue factor monoclonal antibody which inhibits TF.VIIa complex is a potent anticoagulant in plasma. Thromb Haemost. 1991;66:529-533. 77. Huang M, Syed R, Stura EA, et al. The mechanism of an inhibitory antibody on TF- initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF 5G9 complex. Journal of Molecular Biology. 1998;275(5):873-894. 78. Kirchhofer D, Moran P, Chiang N, et al. Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thromb Haemost. 2000;84(6):1072-1081. 79. Versteeg HH, Schaffner F, Kerver M, et al. Inhibition of tissue factor signaling suppresses tumor growth. Blood. 2008;111(1):190-199.

103 80. Contreras FX, Sánchez-Magraner L, Alonso A, Goñi FM. Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes. FEBS Letters. 2010;584(9):1779-1786. 81. McDonald JF, Shah AM, Schwalbe RA, Kisiel W, Dahlback B, Nelsestuen GL. Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site. Biochemistry. 1997;36(17):5120-5127. 82. Nelsestuen GL, Kisiel W, Di Scipio RG. Interaction of vitamin K dependent proteins with membranes. Biochemistry. 1978;17(11):2134-2138. 83. Tavoosi N, Smith SA, Davis-Harrison RL, Morrissey JH. Factor VII and protein C are phosphatidic acid-binding proteins. Biochemistry. 2013;52(33):5545-5552. 84. Neuenschwander PF, Morrissey JH. Roles of the membrane-interactive regions of factor VIIa and tissue factor. The factor VIIa Gla domain is dispensable for binding to tissue factor but important for activation of factor X. Journal of Biological Chemistry. 1994;269(11):8007-8013. 85. Hedner U. Mechanism of action, development and clinical experience of recombinant FVIIa. Journal of Biotechnology. 2006;124(4):747-757. 86. Feng D, Whinna H, Monroe D, Stafford DW. FVIIa as used pharmacologically is not TF dependent in hemophilia B mice. Blood. 2014;123(11):1764-1766. 87. Tavoosi N, Davis-Harrison RL, Pogorelov TV, et al. Molecular determinants of phospholipid synergy in blood clotting. Journal of Biological Chemistry. 2011;286(26):23247-23253. 88. Kay JG, Koivusalo M, Ma X, Wohland T, Grinstein S. Phosphatidylserine dynamics in cellular membranes. Mol Biol Cell. 2012;23(11):2198-2212. 89. Huang M, Rigby AC, Morelli X, et al. Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins. Nature Structural Biology. 2003;10(9):751- 756. 90. Sunnerhagen M, Forsén S, Hoffrén AM, Drakenberg T, Teleman O, Stenflo J. Structure of the Ca2+-free GLA domain sheds light on membrane binding of blood coagulation proteins. Nature Structural Biology. 1995;2(6):504-509. 91. Persson E, Petersen LC. Structurally and functionally distinct Ca2+ binding sites in the gamma-carboxyglutamic acid-containing domain of factor VIIa. European Journal of Biochemistry. 1995;234(1):293-300. 92. de Courcy B, Pedersen LG, Parisel O, et al. Understanding selectivity of hard and soft metal cations within biological systems using the subvalence concept. I. Application to blood coagulation: direct cation-protein electronic effects vs. indirect interactions through water networks. Journal of chemical theory and computation. 2010;6(4):1048- 1063. 93. Tavoosi N, Morrissey JH. Influence of membrane composition on the enhancement of factor VIIa/tissue factor activity by magnesium ions. Thromb Haemost. 2014;111(4):770- 772. 94. Sekiya F, Yoshida M, Yamashita T, Morita T. Magnesium(II) is a crucial constituent of the blood coagulation cascade - Potentiation of coagulant activities of factor IX by Mg2+ ions. Journal of Biological Chemistry. 1996;271(15):8541-8544.

104 95. Persson E, Olsen OH, Østergaard A, Nielsen LS. Ca2+ binding to the first epidermal growth factor-like domain of factor VIIa increases amidolytic activity and tissue factor affinity. J Biol Chem. 1997;272(32):19919-19924. 96. Petersen LC, Olsen OH, Nielsen LS, Freskgård PO, Persson E. Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor. Protein Science. 2000;9(5):859-866. 97. Prendergast FG, Mann KG. Differentiation of metal ion-induced transitions of prothrombin fragment 1. J Biol Chem. 1977;252(3):840-850. 98. Persson E, Bjork I, Stenflo J. Protein structural requirements for Ca2+ binding to the light chain of factor X. Studies using isolated intact fragments containing the gamma- carboxyglutamic acid region and/or the epidermal growth factor-like domains. J Biol Chem. 1991;266(4):2444-2452. 99. Persson E, Østergaard A. Mg2+ binding to the Gla domain of factor X influences the interaction with tissue factor. Journal of Thrombosis and Haemostasis. 2007;5(9):1977- 1978. 100. Bromberg ME, Konigsberg WH, Madison JF, Pawashe A, Garen A. Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation. Proc Natl Acad Sci U S A. 1995;92(18):8205-8209. 101. Hjortoe GM, Petersen LC, Albrektsen T, et al. Tissue factor-factor VIIa-specific up- regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration. Blood. 2004;103(8):3029-3037. 102. Hembrough TA, Swartz GM, Papathanassiu A, et al. Tissue factor/factor VIIa inhibitors block angiogenesis and tumor growth through a nonhemostatic mechanism. Cancer Research. 2003;63(11):2997-3000. 103. Bluff JE, Brown NJ, Reed MW, Staton CA. Tissue factor, angiogenesis and tumour progression. Breast Cancer Res. 2008;10(2):204. 104. Ueno T, Toi M, Koike M, Nakamura S, Tominaga T. Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration. British Journal of Cancer. 2000;83(2):164-170. 105. Shoji M, Hancock WW, Abe K, et al. Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer. Am J Pathol. 1998;152(2):399-411. 106. Dorfleutner A, Ruf W. Regulation of tissue factor cytoplasmic domain phosphorylation by palmitoylation. Blood. 2003;102(12):3998-4005. 107. Ahamed J, Ruf W. Protease-activated receptor 2-dependent phosphorylation of the tissue factor cytoplasmic domain. J Biol Chem. 2004;279(22):23038-23044. 108. Belting M, Dorrell MI, Sandgren S, et al. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling. Nature Medicine. 2004;10(5):502-509. 109. Ryden L, Grabau D, Schaffner F, Jonsson PE, Ruf W, Belting M. Evidence for tissue factor phosphorylation and its correlation with protease-activated receptor expression and the prognosis of primary breast cancer. International Journal of Cancer. 2010;126(10):2330- 2340. 110. Sorensen BB, Freskgård PO, Nielsen LS, Rao LVM, Ezban M, Petersen LC. Factor VIIa- induced p44/42 mitogen-activated protein kinase activation requires the proteolytic

105 activity of factor VIIa and is independent of the tissue factor cytoplasmic domain. J Biol Chem. 1999;274(30):21349-21354. 111. Camerer E, Rottingen JA, Iversen JG, Prydz H. Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells only when proteolytically active. J Biol Chem. 1996;271(46):29034-29042. 112. Åberg M, Siegbahn A. Tissue factor non-coagulant signaling – molecular mechanisms and biological consequences with a focus on cell migration and apoptosis. Journal of Thrombosis and Haemostasis. 2013;11(5):817-825. 113. de Jonge E, Friederich PW, Vlasuk GP, et al. Activation of coagulation by administration of recombinant factor VIIa elicits interleukin 6 (IL-6) and IL-8 release in healthy human subjects. Clinical and Diagnostic Laboratory Immunology. 2003;10(3):495-497. 114. Versteeg HH, Spek CA, Slofstra SH, Diks SH, Richel DJ, Peppelenbosch MP. FVIIa:TF induces cell survival via G12/G13-dependent Jak/STAT activation and BclXL production. Circulation Research. 2004;94(8):1032-1040. 115. Versteeg HH, Sorensen BB, Slofstra SH, et al. VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis, p70, and p90 S6 kinase phosphorylation. J Biol Chem. 2002;277(30):27065-27072. 116. Kasthuri RS, Taubman MB, Mackman N. Role of tissue factor in cancer. J Clin Oncol. 2009;27(29):4834-4838. 117. Rao LV, Pendurthi UR. Tissue factor-factor VIIa signaling. Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25(1):47-56. 118. Miller G, Silverberg M, Kaplan AP. Autoactivatability of human Hageman factor (factor XII). Biochem Biophys Res Commun. 1980;92(3):803-810. 119. Tankersley DL, Finlayson JS. Kinetics of activation and autoactivation of human factor XII. Biochemistry. 1984;23(2):273-279. 120. Muller F, Gailani D, Renne T. Factor XI and XII as antithrombotic targets. Curr Opin Hematol. 2011;18(5):349-355. 121. Muller F, Renne T. Novel roles for factor XII-driven plasma contact activation system. Curr Opin Hematol. 2008;15(5):516-521. 122. Leeb-Lundberg LM, Marceau F, Muller-Esterl W, Pettibone DJ, Zuraw BL. International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol Rev. 2005;57(1):27-77. 123. Renne T, Schmaier AH, Nickel KF, Blomback M, Maas C. In vivo roles of factor XII. Blood. 2012;120(22):4296-4303. 124. Nossel HL. Differential consumption of coagulation factors resulting from activation of the extrinsic (tissue thromboplastin) or the intrinsic (foreign surface contact) pathways. Blood. 1967;29(3):331-340. 125. Renne T, Gailani D. Role of Factor XII in hemostasis and thrombosis: clinical implications. Expert Rev Cardiovasc Ther. 2007;5(4):733-741. 126. Doggen CJ, Rosendaal FR, Meijers JC. Levels of intrinsic coagulation factors and the risk of myocardial infarction among men: Opposite and synergistic effects of factors XI and XII. Blood. 2006;108(13):4045-4051. 127. Colhoun HM, Zito F, Norman Chan N, Rubens MB, Fuller JH, Humphries SE. Activated factor XII levels and factor XII 46C>T genotype in relation to coronary artery calcification

106 in patients with type 1 diabetes and healthy subjects. Atherosclerosis. 2002;163(2):363- 369. 128. Grundt H, Nilsen DW, Hetland O, Valente E, Fagertun HE. Activated factor 12 (FXIIa) predicts recurrent coronary events after an acute myocardial infarction. Am Heart J. 2004;147(2):260-266. 129. Merlo C, Wuillemin WA, Redondo M, et al. Elevated levels of plasma prekallikrein, high molecular weight kininogen and factor XI in coronary heart disease. Atherosclerosis. 2002;161(2):261-267. 130. Fujikawa K, Heimark RL, Kurachi K, Davie EW. Activation of bovine factor XII (Hageman factor) by plasma kallikrein. Biochemistry. 1980;19(7):1322-1330. 131. Samuel M, Pixley RA, Villanueva MA, Colman RW, Villanueva GB. Human factor XII (Hageman factor) autoactivation by dextran sulfate. Circular dichroism, fluorescence, and ultraviolet difference spectroscopic studies. J Biol Chem. 1992;267(27):19691- 19697. 132. Citarella F, Wuillemin WA, Lubbers YT, Hack CE. Initiation of contact system activation in plasma is dependent on factor XII autoactivation and not on enhanced susceptibility of factor XII for kallikrein cleavage. Br J Haematol. 1997;99(1):197-205. 133. Engel R, Brain CM, Paget J, Lionikiene AS, Mutch NJ. Single-chain factor XII exhibits activity when complexed to polyphosphate. J Thromb Haemost. 2014;12(9):1513-1522. 134. Hojima Y, Cochrane CG, Wiggins RC, Austen KF, Stevens RL. In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E. Blood. 1984;63(6):1453-1459. 135. Citarella F, Ravon DM, Pascucci B, Felici A, Fantoni A, Hack CE. Structure/function analysis of human factor XII using recombinant deletion mutants. Evidence for an additional region involved in the binding to negatively charged surfaces. Eur J Biochem. 1996;238(1):240-249. 136. Citarella F, te Velthuis H, Helmer-Citterich M, Hack CE. Identification of a putative binding site for negatively charged surfaces in the fibronectin type II domain of human factor XII--an immunochemical and homology modeling approach. Thromb Haemost. 2000;84(6):1057-1065. 137. Clarke BJ, Cote HC, Cool DE, et al. Mapping of a putative surface-binding site of human coagulation factor XII. J Biol Chem. 1989;264(19):11497-11502. 138. Samuel M, Samuel E, Villanueva GB. Histidine residues are essential for the surface binding and autoactivation of human coagulation factor XII. Biochem Biophys Res Commun. 1993;191(1):110-117. 139. Pixley RA, Stumpo LG, Birkmeyer K, Silver L, Colman RW. A monoclonal antibody recognizing an icosapeptide sequence in the heavy chain of human factor XII inhibits surface-catalyzed activation. J Biol Chem. 1987;262(21):10140-10145. 140. Revak SD, Cochrane CG, Bouma BN, Griffin JH. Surface and fluid phase activities of two forms of activated Hageman factor produced during contact activation of plasma. J Exp Med. 1978;147(3):719-729. 141. Puy C, Tucker EI, Wong ZC, et al. Factor XII promotes blood coagulation independent of factor XI in the presence of long-chain polyphosphates. J Thromb Haemost. 2013;11(7):1341-1352.

107 142. Ratnoff OD, Colopy JE. A familial hemorrhagic trait associated with a deficiency of a clot- promoting fraction of plasma. J Clin Invest. 1955;34(4):602-613. 143. Gailani D, Lasky NM, Broze GJ, Jr. A murine model of factor XI deficiency. Blood Coagul Fibrinolysis. 1997;8(2):134-144. 144. Pauer HU, Renne T, Hemmerlein B, et al. Targeted deletion of murine coagulation factor XII gene-a model for contact phase activation in vivo. Thromb Haemost. 2004;92(3):503- 508. 145. Renne T, Pozgajova M, Gruner S, et al. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med. 2005;202(2):271-281. 146. Kleinschnitz C, Stoll G, Bendszus M, et al. Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J Exp Med. 2006;203(3):513-518. 147. Mandle R, Jr., Kaplan AP. Hageman factor substrates. Human plasma prekallikrein: mechanism of activation by Hageman factor and participation in hageman factor- dependent fibrinolysis. J Biol Chem. 1977;252(17):6097-6104. 148. Tans G, Rosing J, Berrettini M, Lammle B, Griffin JH. Autoactivation of human plasma prekallikrein. J Biol Chem. 1987;262(23):11308-11314. 149. Griffin JH, Cochrane CG. Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor. Proc Natl Acad Sci U S A. 1976;73(8):2554-2558. 150. Renne T, Gailani D, Meijers JC, Muller-Esterl W. Characterization of the H-kininogen- binding site on factor XI: a comparison of factor XI and plasma prekallikrein. J Biol Chem. 2002;277(7):4892-4899. 151. Henderson LM, Figueroa CD, Muller-Esterl W, Bhoola KD. Assembly of contact-phase factors on the surface of the human neutrophil membrane. Blood. 1994;84(2):474-482. 152. Bouma BN, Griffin JH. Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII. J Biol Chem. 1977;252(18):6432-6437. 153. Kurachi K, Davie EW. Activation of human factor XI (plasma thromboplastin antecedent) by factor XIIa (activated Hageman factor). Biochemistry. 1977;16(26):5831-5839. 154. McMullen BA, Fujikawa K, Davie EW. Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains. Biochemistry. 1991;30(8):2056-2060. 155. Gailani D, Broze GJ, Jr. Factor XI activation in a revised model of blood coagulation. Science. 1991;253(5022):909-912. 156. Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011;118(26):6963-6970. 157. Ragni MV, Sinha D, Seaman F, Lewis JH, Spero JA, Walsh PN. Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds. Blood. 1985;65(3):719-724. 158. Salomon O, Steinberg DM, Seligshon U. Variable bleeding manifestations characterize different types of surgery in patients with severe factor XI deficiency enabling parsimonious use of replacement therapy. Haemophilia. 2006;12(5):490-493.

108 159. Rosenthal RL, Dreskin OH, Rosenthal N. Plasma thromboplastin antecedent (PTA) deficiency; clinical, coagulation, therapeutic and hereditary aspects of a new hemophilia-like disease. Blood. 1955;10(2):120-131. 160. Asakai R, Chung DW, Davie EW, Seligsohn U. Factor XI deficiency in Ashkenazi Jews in Israel. N Engl J Med. 1991;325(3):153-158. 161. Bolton-Maggs PH. Factor XI deficiency--resolving the enigma? Hematology Am Soc Hematol Educ Program. 2009:97-105. 162. Zeerleder S. C1-inhibitor: more than a serine protease inhibitor. Semin Thromb Hemost. 2011;37(4):362-374. 163. Maas C, Renne T. Regulatory mechanisms of the plasma contact system. Thromb Res. 2012;129 Suppl 2:S73-76. 164. Long AT, Kenne E, Jung R, Fuchs TA, Renne T. Contact system revisited: an interface between inflammation, coagulation, and innate immunity. J Thromb Haemost. 2016;14(3):427-437. 165. Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107(36):15880-15885. 166. Kannemeier C, Shibamiya A, Nakazawa F, et al. Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation. Proc Natl Acad Sci U S A. 2007;104(15):6388-6393. 167. Pavlov V, Zorn M, Kramer R. Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade. Biochem Biophys Res Commun. 2006;349(3):1011-1015. 168. Maas C, Govers-Riemslag JW, Bouma B, et al. Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation. J Clin Invest. 2008;118(9):3208-3218. 169. Müller F, Mutch NJ, Schenk WA, et al. Platelet Polyphosphates Are Proinflammatory and Procoagulant Mediators In Vivo. Cell. 2009;139(6):1143-1156. 170. Nickel KF, Ronquist G, Langer F, et al. The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis. Blood. 2015;126(11):1379-1389. 171. Shibayama Y, Joseph K, Nakazawa Y, Ghebreihiwet B, Peerschke EI, Kaplan AP. Zinc- dependent activation of the plasma kinin-forming cascade by aggregated beta amyloid protein. Clin Immunol. 1999;90(1):89-99. 172. Maas C, Schiks B, Strangi RD, et al. Identification of fibronectin type I domains as amyloid-binding modules on tissue-type plasminogen activator and three homologs. Amyloid. 2008;15(3):166-180. 173. Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood. 2014;123(18):2768- 2776. 174. Oehmcke S, Morgelin M, Herwald H. Activation of the human contact system on neutrophil extracellular traps. J Innate Immun. 2009;1(3):225-230. 175. Gould TJ, Vu TT, Swystun LL, et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arterioscler Thromb Vasc Biol. 2014;34(9):1977-1984. 176. Gansler J, Jaax M, Leiting S, et al. Structural requirements for the procoagulant activity of nucleic acids. PLoS One. 2012;7(11):e50399.

109 177. Gould TJ, Vu TT, Stafford AR, et al. Cell-Free DNA Modulates Clot Structure and Impairs Fibrinolysis in Sepsis. Arterioscler Thromb Vasc Biol. 2015;35(12):2544-2553. 178. Kornberg A. Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J Bacteriol. 1995;177(3):491-496. 179. Ault-Riche D, Fraley CD, Tzeng CM, Kornberg A. Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli. J Bacteriol. 1998;180(7):1841-1847. 180. Brown MR, Kornberg A. Inorganic polyphosphate in the origin and survival of species. Proc Natl Acad Sci U S A. 2004;101(46):16085-16087. 181. Docampo R, Moreno SN. Acidocalcisomes. Cell Calcium. 2011;50(2):113-119. 182. Kornberg A. Inorganic polyphosphate: a molecule of many functions. Prog Mol Subcell Biol. 1999;23:1-18. 183. Docampo R, Moreno SN. The acidocalcisome. Mol Biochem Parasitol. 2001;114(2):151- 159. 184. Docampo R, Ulrich P, Moreno SN. Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes. Philos Trans R Soc Lond B Biol Sci. 2010;365(1541):775-784. 185. Achbergerova L, Nahalka J. Polyphosphate--an ancient energy source and active metabolic regulator. Microb Cell Fact. 2011;10:63. 186. Ruiz FA, Lea CR, Oldfield E, Docampo R. Human Platelet Dense Granules Contain Polyphosphate and Are Similar to Acidocalcisomes of Bacteria and Unicellular Eukaryotes. Journal of Biological Chemistry. 2004;279(43):44250-44257. 187. Moreno-Sanchez D, Hernandez-Ruiz L, Ruiz FA, Docampo R. Polyphosphate is a novel pro-inflammatory regulator of mast cells and is located in acidocalcisomes. J Biol Chem. 2012;287(34):28435-28444. 188. Pisoni RL, Lindley ER. Incorporation of [32P]orthophosphate into long chains of inorganic polyphosphate within lysosomes of human fibroblasts. J Biol Chem. 1992;267(6):3626- 3631. 189. Kumble KD, Kornberg A. Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem. 1995;270(11):5818-5822. 190. Smith SA, Choi SH, Davis-Harrison R, et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood. 2010;116(20):4353-4359. 191. Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(4):903-908. 192. Choi SH, Collins JN, Smith SA, Davis-Harrison RL, Rienstra CM, Morrissey JH. Phosphoramidate end labeling of inorganic polyphosphates: facile manipulation of polyphosphate for investigating and modulating its biological activities. Biochemistry. 2010;49(45):9935-9941. 193. Smith SA, Morrissey JH. Polyphosphate enhances fibrin clot structure. Blood. 2008;112(7):2810-2816. 194. Falls LA, Furie BC, Jacobs M, Furie B, Rigby AC. The ω-loop region of the human prothrombin γ-carboxyglutamic acid domain penetrates anionic phospholipid membranes. J Biol Chem. 2001;276(26):23895-23902.

110 195. Furie B, Furie BC. The molecular basis of blood coagulation. Cell. 1988;53(4):505-518. 196. Stenflo J, Suttie JW. Vitamin K-dependent formation of gamma-carboxyglutamic acid. Annual Review of Biochemistry. 1977;46:157-172. 197. Wang S, McDonnell EH, Sedor FA, Toffaletti JG. pH effects on measurements of ionized calcium and ionized magnesium in blood. Arch Pathol Lab Med. 2002;126(8):947-950. 198. van den Besselaar AMHP. Magnesium and manganese ions accelerate tissue factor- induced coagulation independently of factor IX. Blood Coagul Fibrinolysis. 2002;13(1):19-23. 199. Sekiya F, Yamashita T, Atoda H, Komiyama Y, Morita T. Regulation of the tertiary structure and function of coagulation factor IX by magnesium(II) ions. J Biol Chem. 1995;270:14325-14331. 200. Vadivel K, Agah S, Messer AS, et al. Structural and functional studies of γ- carboxyglutamic acid domains of factor VIIa and activated protein C: Role of magnesium at physiological calcium. Journal of Molecular Biology. 2013;425(11):1961-1981. 201. Neuenschwander PF, Bianco-Fisher E, Rezaie AR, Morrissey JH. Phosphatidylethanolamine augments factor VIIa-tissue factor activity: enhancement of sensitivity to phosphatidylserine. Biochemistry. 1995;34(43):13988-13993. 202. Rezaie AR, Fiore MM, Neuenschwander PF, Esmon CT, Morrissey JH. Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody. Protein Expr Purif. 1992;3(6):453-460. 203. Smith SA, Morrissey JH. Rapid and efficient incorporation of tissue factor into liposomes. J Thromb Haemost. 2004;2(7):1155-1162. 204. Shaw AW, Pureza VS, Sligar SG, Morrissey JH. The local phospholipid environment modulates the activation of blood clotting. J Biol Chem. 2007;282(9):6556-6563. 205. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6(3):277-293. 206. Goddard TD, Kneller DG. SPARKY 3. University of California, San Francisco. 2006. 207. Vadivel K, Bajaj SP. Structural biology of factor VIIa/tissue factor initiated coagulation. Front Biosci. 2012;17:2476-2494. 208. Messer AS, Velander WH, Bajaj SP. Contribution of magnesium in binding of factor IXa to the phospholipid surface: implications for vitamin K-dependent coagulation proteins. J Thromb Haemost. 2009;7(12):2151-2153. 209. Long AT, Kenne E, Jung R, Fuchs TA, Renné T. Contact system revisited: an interface between inflammation, coagulation, and innate immunity. J Thromb Haemost. 2016;14(3):427-437. 210. van der Meijden PEJ, Munnix ICA, Auger JM, et al. Dual role of collagen in factor XII- dependent thrombus formation. Blood. 2009;114(4):881-890. 211. Maas C, Govers-Riemslag JW, Bouma B, et al. Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation. J Clin Invest. 2008;118(9):3208-3218. 212. Kannemeier C, Shibamiya A, Nakazawa F, et al. Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation. Proc Natl Acad Sci U S A. 2007;104(15):6388-6393.

111 213. Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci U S A. 2006;103(4):903- 908. 214. Rao NN, Gomez-Garcia MR, Kornberg A. Inorganic polyphosphate: essential for growth and survival. Annu Rev Biochem. 2009;78:605-647. 215. Morrissey JH, Smith SA. Polyphosphate as modulator of hemostasis, thrombosis, and inflammation. J Thromb Haemost. 2015;13 Suppl 1:S92-97. 216. Smith SA, Choi SH, Davis-Harrison R, et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood. 2010;116(20):4353-4359. 217. Kornberg A, Rao NN, Ault-Riché D. Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem. 1999;68:89-125. 218. Kumble KD, Kornberg A. Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem. 1995;270(11):5818-5822. 219. Leyhausen G, Lorenz B, Zhu H, et al. Inorganic polyphosphate in human osteoblast-like cells. Journal of Bone and Mineral Research. 1998;13(5):803-812. 220. Moreno-Sanchez D, Hernandez-Ruiz L, Ruiz FA, Docampo R. Polyphosphate is a novel pro-inflammatory regulator of mast cells and is located in acidocalcisomes. J Biol Chem. 2012;287(34):28435-28444. 221. Nickel KF, Ronquist G, Langer F, et al. The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis. Blood. 2015;126(11):1379-1389. 222. Müller F, Mutch NJ, Schenk WA, et al. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell. 2009;139(6):1143-1156. 223. Mutch NJ, Engel R, Uitte de Willige S, Philippou H, Ariens RA. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood. 2010;115(19):3980-3988. 224. Gould TJ, Vu TT, Stafford AR, et al. Cell-free DNA modulates clot structure and impairs fibrinolysis in sepsis. Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35(12):2544-2553. 225. Bhagirath VC, Dwivedi DJ, Liaw PC. Comparison of the proinflammatory and procoagulant properties of nuclear, mitochondrial, and bacterial DNA. Shock. 2015;44(3):265-271. 226. Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107(36):15880-15885. 227. Gould TJ, Vu TT, Swystun LL, et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34(9):1977-1984. 228. Rao NN, Kornberg A. Inorganic polyphosphate regulates responses of Escherichia coli to nutritional stringencies, environmental stresses and survival in the stationary phase. Prog Mol Subcell Biol. 1999;23:183-195. 229. Werner TP, Amrhein N, Freimoser FM. Specific localization of inorganic polyphosphate (poly P) in fungal cell walls by selective extraction and immunohistochemistry. Fungal Genetics and Biology. 2007;44(9):845-852. 230. Lockett JM, Mast AE. Contribution of regions distal to glycine-160 to the anticoagulant activity of tissue factor pathway inhibitor. Biochemistry. 2002;41(15):4989-4997.

112 231. Wurst H, Shiba T, Kornberg A. The gene for a major exopolyphosphatase of Saccharomyces cerevisiae. Journal of Bacteriology. 1995;177(4):898-906. 232. Smith SA, Morrissey JH. Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4',6-diamidino-2-phenylindol. Electrophoresis. 2007;28(19):3461-3465. 233. Smith SA, Morrissey JH. Polyphosphate as a general procoagulant agent. Journal of Thrombosis and Haemostasis. 2008;6(10):1750-1756. 234. Simsekyilmaz S, Cabrera-Fuentes HA, Meiler S, et al. Role of extracellular RNA in atherosclerotic plaque formation in mice. Circulation. 2014;129(5):598-606. 235. Choi SH, Smith SA, Morrissey JH. Polyphosphate accelerates factor V activation by factor XIa. Thromb Haemost. 2015;113(3):599-604. 236. Wood JP, Bunce MW, Maroney SA, Tracy PB, Camire RM, Mast AE. Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation. Proc Natl Acad Sci U S A. 2013;110(44):17838-17843. 237. Lorenz B, Schröder HC. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta. 2001;1547(2):254-261. 238. Burge S, Parkinson GN, Hazel P, Todd AK, Neidle S. Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res. 2006;34(19):5402-5415. 239. Petrovic AG, Polavarapu PL. Quadruplex structure of polyriboinosinic acid: dependence on alkali metal ion concentration, pH and temperature. Journal of Physical Chemistry B. 2008;112(7):2255-2260. 240. Nagatoishi S, Isono N, Tsumoto K, Sugimoto N. Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding. Biochimie. 2011;93(8):1231-1238. 241. Tasset DM, Kubik MF, Steiner W. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. Journal of Molecular Biology. 1997;272(5):688-698. 242. Collie GW, Haider SM, Neidle S, Parkinson GN. A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex. Nucleic Acids Res. 2010;38(16):5569-5580. 243. Biffi G, Tannahill D, McCafferty J, Balasubramanian S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry. 2013;5(3):182-186. 244. Vu TT, Leslie BA, Stafford AR, Zhou J, Fredenburgh JC, Weitz JI. Histidine-rich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway. Thromb Haemost. 2015;115(1):89-98. 245. Miller G, Silverberg M, Kaplan AP. Autoactivatability of human Hageman factor (factor XII). Biochemical and Biophysical Research Communications. 1980;92(3):803-810. 246. Tankersley DL, Finlayson JS. Kinetics of activation and autoactivation of human factor XII. Biochemistry. 1984;23(2):273-279. 247. Ratnoff OD, Colopy JE. A familial hemorrhagic trait associated with a deficiency of a clot- promoting fraction of plasma. J Clin Invest. 1955;34(4):602-613. 248. Kokoye Y, Ivanov I, Cheng Q, et al. A comparison of the effects of factor XII deficiency and prekallikrein deficiency on thrombus formation. Thromb Res. 2016;140:118-124. 249. Renné T, Gailani D. Role of factor XII in hemostasis and thrombosis: clinical implications. Expert Review of Cardiovascular Therapy. 2007;5(4):733-741.

113 250. Müller F, Renné T. Novel roles for factor XII-driven plasma contact activation system. Curr Opin Hematol. 2008;15(5):516-521. 251. Renné T, Pozgajová M, Grüner S, et al. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med. 2005;202(2):271-281. 252. Revenko AS, Gao D, Crosby JR, et al. Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding. Blood. 2011;118(19):5302-5311. 253. Matafonov A, Leung PY, Gailani AE, et al. Factor XII inhibition reduces thrombus formation in a primate thrombosis model. Blood. 2014;123(11):1739-1746. 254. Cheng Q, Tucker EI, Pine MS, et al. A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo. Blood. 2010;116(19):3981-3989. 255. Kuijpers MJ, van der Meijden PE, Feijge MA, et al. Factor XII regulates the pathological process of thrombus formation on ruptured plaques. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34(8):1674-1680. 256. Grundt H, Nilsen DW, Hetland O, Valente E, Fagertun HE. Activated factor 12 (FXIIa) predicts recurrent coronary events after an acute myocardial infarction. Am Heart J. 2004;147(2):260-266. 257. Morrissey JH, Choi SH, Smith SA. Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation. Blood. 2012;119(25):5972-5979. 258. Thompson RE, Mandle R, Jr., Kaplan AP. Studies of binding of prekallikrein and factor XI to high molecular weight kininogen and its light chain. Proc Natl Acad Sci U S A. 1979;76(10):4862-4866. 259. Schapira M, Scott CF, Colman RW. Protection of human plasma kallikrein from inactivation by C1 inhibitor and other protease inhibitors. The role of high molecular weight kininogen. Biochemistry. 1981;20(10):2738-2743. 260. Scott CF, Schapira M, James HL, Cohen AB, Colman RW. Inactivation of factor XIa by plasma protease inhibitors: predominant role of alpha 1-protease inhibitor and protective effect of high molecular weight kininogen. J Clin Invest. 1982;69(4):844-852. 261. Müller F, Gailani D, Renné T. Factor XI and XII as antithrombotic targets. Curr Opin Hematol. 2011;18(5):349-355. 262. Leeb-Lundberg LM, Marceau F, Muller-Esterl W, Pettibone DJ, Zuraw BL. International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacological Reviews. 2005;57(1):27-77. 263. Scott CF, Silver LD, Schapira M, Colman RW. Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor. J Clin Invest. 1984;73(4):954-962. 264. Griffin JH, Cochrane CG. Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor. Proc Natl Acad Sci U S A. 1976;73(8):2554-2558. 265. Smith SA, Choi SH, Collins JN, Travers RJ, Cooley BC, Morrissey JH. Inhibition of polyphosphate as a novel strategy for preventing thrombosis and inflammation. Blood. 2012;120(26):5103-5110.

114 266. Levison PR, Tomalin G. Studies on the temperature-dependent autoinhibition of human plasma kallikrein I. Biochemical Journal. 1982;205(3):529-534. 267. Geng Y, Verhamme IM, Smith SB, et al. The dimeric structure of factor XI and zymogen activation. Blood. 2013;121(19):3962-3969. 268. Tans G, Rosing J, Berrettini M, Lämmle B, Griffin JH. Autoactivation of human plasma prekallikrein. J Biol Chem. 1987;262(23):11308-11314. 269. Engel R, Brain CM, Paget J, Lionikiene AS, Mutch NJ. Single-chain factor XII exhibits activity when complexed to polyphosphate. J Thromb Haemost. 2014;12(9):1513-1522. 270. Geng Y, Verhamme IM, Smith SA, et al. Factor XI anion-binding sites are required for productive interactions with polyphosphate. J Thromb Haemost. 2013;11(11):2020- 2028. 271. Naito K, Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces. J Biol Chem. 1991;266(12):7353-7358. 272. Bernardo MM, Day DE, Halvorson HR, Olson ST, Shore JD. Surface-independent acceleration of factor XII activation by zinc ions. II. Direct binding and fluorescence studies. J Biol Chem. 1993;268(17):12477-12483. 273. Vu TT, Fredenburgh JC, Weitz JI. Zinc: an important cofactor in haemostasis and thrombosis. Thromb Haemost. 2013;109(3):421-430. 274. Vadivel K, Agah S, Messer AS, et al. Structural and functional studies of gamma- carboxyglutamic acid domains of factor VIIa and activated Protein C: role of magnesium at physiological calcium. J Mol Biol. 2013;425(11):1961-1981. 275. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14(1):33-38, 27-38. 276. Gu Z, Opella SJ. Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy. J Magn Reson. 1999;138(2):193-198. 277. Vu TT, Leslie BA, Stafford AR, Zhou J, Fredenburgh JC, Weitz JI. Histidine-rich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway. Thromb Haemost. 2015;115(1):89-98. 278. Swystun LL, Mukherjee S, Liaw PC. Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus. J Thromb Haemost. 2011;9(11):2313-2321.

115