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MA, Haber E, Lee ME: Inhibition of growth and ~21’~ methylation in vascularendothelial cells by homocysteine but not cysteine. J Biol Chem 272:25380-25385, 1997. 23. Blundell G, Jones BG, Rose FA, Tudball N: Homocysteine mediated endothelial cell toxicity and its amelioration. Atherosclerosis 122: 163172.1996. 24. ljagi SC: Homocysteine redox receptor and regulation of extracellular matrix components in vascuhucells. Am J Physiol274:C3%-C405.1998. 25. Rohde LE. Lee RT, Rivero J, Jamawchian M, Arroyo LH, Briggs W, Rifai N, Libby R Creager MA, Ridker PM: Circulating cell adhesion molecules are correlated with ultrasound based assessment of carotid atherosclerosis. Arterioscler Tbromb Vast Biol l&1765-1770, 1998. 26. Ross R: Atherosclerosis - an inflammatory disease. N Engl J Med 340: 115- 126, 1999. 27. Ridker PM, Hennekens CH, Roitman-Johnson B, Stampfer MJ, Allen J: Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men. Lancer 351:88-92, 1998. 28. Tsakiris DA, Tschb;pl, Jiiger K, Haefeli WE, Wolf F, Marbet GA: Circulating cell adhesion

molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease. Atherosclerosis 142:193-200, 1999. 29. Ueland PM, Helland S, Broth OJ, Schanche JS: Homocysteine in tissues of the mouse and rat. J Biol Chem 259:2360-2364, 1984. 30. Ratter F, Germer M, Fischbach T, SchulzeOsthoff K, Peter ME, DrQe W, Kmmmer PH. Lehmann V: S-adenosylhomocysteine as a physiological modulator of Apo- 1 mediated apoptosis. Intl Immunol8:1139-1147, 1996. 3 1. Gao XM, Wordsworth P, McMichael AJ, Kyaw MM. Seifert M, Rees D, Dougan G: Homocysteine modification of HLA antigens and its immunological consequences. Eur J Immunol26:1443-1450, 1996. 32. Outinen PA, Sood SK, Liaw PC, Sarge KD, Maeda N, Hirsh 1, Ribau J. Podor TJ, Weitz JI, Austin RC: Characterization of the stress inducing effects of homocysteine. Biochem J 332:213-221.1998. 33. Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR. Maeda N: Mice deficient in cystathionine 8 synthase:Animal models for mild and severe homocyst(e)inemia. Proc

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Nat1Acad Sci USA 92:1585-1589, 1995. 34. Ambrosi P, Gwen D, Rib& A, Habib G, Barlatier A, Kreitmann B, Rolland PH, Bouvenot G, Luccioni R, M&as D: Association of mild hyperhomocysteinemia with cardiac graft vascular disease. Atherosclerosis 138:347-350, 1998. 35. Tang L, Mamotte CD, Bockxmeer FM, Taylor RR: The effect of homocysteine on DNA synthesis in cultured human vascular smooth muscle. Atherosclerosis 136:169-173, 1998. 36. Refsum H, Ueland PM. Nygard 0, Vollset SE: Homocysteine and cardiovascular disease.Ann Rev Medicine 49:3 l-62, 1998. 37. Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM, Malinow MR, Heistad DD: Vascular dysfunction in monkeys with dietinduced hyperhomocyst(e)inemia. I Clin Invest 98:24-29. 1996. 38. Zhang F, Slungaard A, Vercellotti GM, Iadecola C: Superoxide dependent cerebmvascular effects of homocysteine. Am J Physioi 274:R1704Rl711, 1998. 39. De Vriese AS, De Sutter JH, De Buyzere M, Duprez D: Mild to moderate hyperhomccysteinaemia in cardiovascular disease. Acta Cardiol 53:337-344, 1998.

Current

Understanding

Ashley P. DeAnglis and Gregory S. Retzinger Department of Pathology and Laboratory

R

Medicine,

ales for fibrin(ogen) in inflammatory processeshave beenpresumed since at least the first half of the nineteenth century. * Such presumption is well-founded: fibrinogen and fibrin accumulateat virtually all sitesof inflammation,*13including those elicited by physical or chemical disruption of tissue, tumors, atherosclerotic plaques,microbes, and implantedprostheticdevices.Although presumedfor many years, someof the roles attributed to fibrin(ogen) have only recently been substantiated.In this regard, much of the available evidence indicates fibrinfogen) operates at interfaces by design, a design we have proposedis intended to confer an adhesivequality to otherwise nonadhesivesurfaces.4,5 In this paper, we first review briefly generalfacts and principles of fibrin(ogen) physiology. We then presentknown roles of the protein in inflammatory phenomena, elaborating on two diseaseswith inflam-

Wniversity of Cincinnati, Cincinnati,

OH 45267-0529.

matory components,cancerandatherosclerosis. We end with a discussionof how fibrin(ogen), operating asan adhesive, might be contributing deleteriously to a host of diseasesthat have inflammatory components,and how such a contribution might be mitigated by specific therapy. Overview of Fibrin(ogen) Physiology Fibrinogen is a dimeric protein, each half of which is composedof disulfide-bonded polypeptide chainsdesignatedAa, B13, and y. “A” and “B” refer to the fibrinopeptidesA (FpA) and B (FpB) that constitute the amino terminal 16 and 14 residues,respectively, of the Aa- and BBchains.The three complete polypeptide chains are synthesizedin coordinated fashion by the parenchymal cells of the liver and, perhaps,by megakaryocytes.6 The chains are encodedby distinct but ancestrally related geneslocated within the long arm of human chromosome4 (L?IQQQ Elsevier Science Inc.

E-mail: deanglap@emailuc,edu

betweenq23 and q32.7 In the liver, the genesfor the Aor- and B&chains encode single products of 610 and 46 1 amino acid residues,respectively.* In contrast, alternative splicing of transcripts of the y-chain geneyield y-chain variants’ of slightly different lengths (411 and 427 residues),the shorter of which constitutes -90% of the final product.” In humans, intracellular poolsof free Aa- and y-chains aswell asAu-/y-chain complexesexist, and final assemblyof fibrinogen in the rough endoplasmicreticulum is rate-limited by the synthesis of the B&chain.” After post-translational glycosylation’* and phosphorylation,13the predominant form of fibrinogen is secretedinto the circulation with a molecular massof -340 l&a. Following its secretion from the liver, the protein exists not only in plasma,but also in lymph and interstitial fluid.‘4.*5In healthy individuals, the concentration of fibrinogen in plasmais between 4 and 0197- 1859/99 (see frontmatter)

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10 uM. Importantly, that concentration can increase by as much as 200 to 400% during times of physiological stress,16 a process mediated, in large part, by macrophage-derived interleukin-6 (IL-6)” The fibrinogen concentration of lymph and interstitial fluid is roughly 20 to 40% that of plasma. In plasma, fibrinogen has a half-life of -3 to 5 days.‘* Early electron micrographs” showed fibrinogen to be elongate, and suggested the protein has a trinodular structure composed of two larger terminal globular domains and a smaller central globular domain, all connected by intervening linear segments, The length of the molecule is -460 A along its major axis and 60 to 9OA along its minor axis. Although more recent electron microscopic and x-ray crystallographic studies indicate the structure of fibrinogen is somewhat more complex, 2othe trinodular depiction is still used generally as a model for the protein (Figure 1). In this model, the outermost globules are referred to as D-domains, and the central globule is referred to as the E-domain. Biochemical and physical chemical studies have revealed much about the fine structure of fibrinogen. The halves of the protein are linked in antiparallel fashion within the central E-domain.2’ That domain consists of the N-terminal portions of the six constituent chains and is stabilized by 11 inter-chain disullide bonds. The linear segments that join an E-domain to its flanking D-domains are suprahelical arrangements of the Aa-, BB- and y-chains, and are often referred to as “coiled-coil” regions. Whereas the carboxyl halves of the BL3- and y-chains fold extensively and terminate as the bulk of the globular Ddomains,20 the A&chains extend from the D-domains, fold back across the molecule, and appear to terminate juxtaposed to the E-domain, perhaps in close proximity to the FpA’s. 22That segment of an Aachain that projects beyond a D-domain is referred to as an Acx-chain extension or protuberance. In a solution-phase, physiologic milieu, fibrinogen to fibrin conversion manifests as a rather remarkable transformation of water-soluble monomers into a waterinsoluble polymeric gel. The initial steps of that transformation are catalyzed by thrombin, a trypsin-like serine proteinase. 0 197- 1859/99 (see frontmatter)

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a-chain protuberance

a-chain protuberance b 460 A Figure 1. Trinodular structure of fibrinogen showing D and E globular domains, a-chain protuber antes, and fibrinopeptides A (FpA) and B (FpB). ‘I’hrombin hydrolyzes at least two specific Arg-Gly bonds within fibrinogen. One of those bonds is located near the N-terminus of each of the Aa-chains and its cleavage liberates the FpA’s, the other is located near the N-terminus of each of the BBchains and its cleavage liberates the FpB’s. In solution, FpA’s are liberated at a rate that is at least an order of magnitude faster than that of F~B’s.‘~ The liberation of the two FpA’s results in the expression of two positively-charged nodes or “knobs” on the E-domain. Those knobs can then interact spontaneously with complementary “holes” pre-existing within the y-chain on D-domains of neighboring fibrin monomers.24 Such interactions yield, minimally, dimers in which the two monomeric subunits overlap. Linear propagation of dimers leads to the formation of fibrin protofibrils two molecules thick in which all of the monomers are aligned in halfstaggered array25 (Figure 2). Protofibrils can associate laterally, forming thicker fibers that, in turn, can associate to form even thicker and branched fibrin bundles.26 Although removal of FpB is not strictly required for fibrin formation, the liberation of that peptide appears to stabilize noncovalent lateral associations within higher-ordered fibrin polymers.27 Once formed, fibrin polymers are covalently stabilized by transglutamination, a process catalyzed by coagulation factor XIIIa. Specific Lys and Gln residues located near the C-termini of the y-chains of D-domains of adjacent fibrin molecules serve in reciprocal fashion as donors and acceptors, respectively, in that reaction.28 Q 1999

ElsevierScienceInc.

Additional polymer stability is conferred, albeit more slowly, by cross-links formed between Lys and Gln residues located within the a-chains of neighboring fibrin molecules.28 Cross-linking effectively increases the resistance of the polymers to denaturation, mechanical stress, and chemical and enzymatic lysis, and yields a three-dimensional meshwork of waterinsoluble fibers capable of trapping particulates and, consequently, of preventing/ limiting blood flow. Even as fibrin polymers are formed in the physiologic milieu, mechanisms are initiated there that are intended to disrupt those polymers. One of the mechanisms involves limited enzymatic digestion, and is mediated by the serine proteinase, plasmin. Specific Lys-X and Arg-X bonds common to fibrin polymers, fibrin monomers and fibrinogen are susceptible to the enzyme. All but one of those bonds are located within coiled-coil regions, the sole exception being a Lys-Met bond that constitutes the amino termini of the (A)achain protuberances.29 Although the final mix of plasmin-resistant fragments that derives from a particular fibrin(ogen) species depends on the extent to which that species had been cross-linked, several well-defined fibrin(ogen) degradation, or “split,” products (FDP’s or FSP’s) have been identified. They include monomeric D- and E-domains, dimeric D-domains (“D-dimers”), the (A)a-chain protuberance, BBl-42, B 15-42, and lower molecular weight peptides from within the coiled-coil regions.29

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and Inflammation

Inflammation can be defined broadly as a protective response of the body to tissue injury or destruction. Phenomenologically, several coordinated processes characterize it. Immediately following tissue damage, blood vessels serving the affected area constrict, limiting - by purely physical means - blood loss from the site. In the short term, vessel constriction restricts the propagation/dissemination of the damaging event/agent and renders local to the site of tissue damage those molecular and cellular events that constitute early i&ammation. During this early time, various effector molecules liberated by active and passive mechanisms from cells and macromolecules resident at the site accumulate there. Some of those molecules will represent the initial wave of signals to which nonresident cells and macromolecules that mediate later inflammatory processes will respond. Toward that end, vasodilatation follows vasoconstriction. Vasodilatation increases blood flow to the now-localized inflammatory site, facilitating ingress and egress of molecular and cellular reactants and products. The movement of molecules and cells into and out of the site is further facilitated by the retraction locally of endothelial cells, a process that increases vessel permeability. Leukocytes within the vascular volume adhere to, and exit via, the walls of the leaky vessels and, following the established chemical gradients, migrate along neighboring cells and extracellular matrix to the damaged tissues. Once there, those cells effect a number of important processes, including recruitment of additional responding elements, intracellular and extracellular degradation of debris, tissue repair/restoration, and presentation and/or translocation of debris to lymphoid tissue for immunologic processing. Indirect evidence for the participation of fibrin(ogen) in inflammation comes from numerous histological studies showing that fibrin accumulates within inflamed tissues. More direct evidence for fibrin(ogen) participation comes from studies of other sorts. For example, animals depleted of fibrin(ogen) by proteolytic means have markedly attenuated inflammatory responses, and those responses are restored by fibrinogen.30 Another example shows anticoagulants

Fibrinogen

Fibrin Monomer

Fibrin Polymer E.

Cross-Linked Fibrin Polymer

Factor XIJIa 1

Plasmin

Plasmin

:

Fragment

D

Fragment

E

1 D Dimer

!:;&+ 4

4

Fragment

E

Figure 2. Fibrin formation, cross-linking and degradation.

reduce inflammation.3’ Other studies address specifically the role of adsorbed fibrin(ogen), and show measures that prevent the binding of fibrin(ogen) to otherwise proinflammatory surfaces reduce inflammation elicited by those surfaces.32 Recent studies have provided mechanistic insight on the contributions of fibrin(ogen) to the inflammatory processes described earlier. Thrombin and factor XIIIa, generated immediately at the site of tissue damage by various mechanisms, convert local intra- and extravascular librinogen to cross-linked fibrin. Acting in large part by physical means, the developing fibrin meshwork entraps blood cells, limiting blood loss from the site and confining it to inflammatory cells that would otherwise circulate, i.e., platelets, granulocytes, monocytes, and lymphocytes. If the 0 1999 Elsevier Science Inc.

inflammation is of infectious or malignant etiology, the fibrin matrix may also impede dissemination of the offending agent.33,34 To facilitate further the retention and migration of platelets and leukocytes at/to an inflammatory site, those cells, as well as endothelial cells, express on their outer surface, cellular adhesion molecules (CAM’s) that, when “activated,” have significant affinity for fibrin(ogen). The platelet CAM, an integrin (a,,,13,), recognizes the final 12 residues of the C-terminus of the y-chains3’ of the dominant form of fibrin(ogen). Some believe it also recognizes Arg-Gly-Asp sequences within fibrin(ogen) (A)a-chains.36 Neutrophils, monocytes and lymphocytes express at least two relevant CAM’s, also integrins. One, CQ~~ (CD 11b/CD1 8, Mac-l), recognizes ~190-202

and y377-395

within

the

0197-1859/99 (see frontmatter)

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Newdefter

fibrin(ogen) D-domains;37 the other, o$32 (CDllcKJD18, p150/95), recognizes a Gly-Pro-At-g sequence at the N-terminus of fibrin a-chains.38 Finally, endothelial cells express two receptors for fibrin(ogen). CX.,~~,another integrin, is claimed to recognize Arg-Gly-Asp sequences within the fibrin(ogen) (A)cr-cham3’ and intercellular adhesion molecule 1 (ICAM-l), a member of the immunoglobulin gene superfamily, recognizes yl17- 133.‘@ Thus, there exists within/near sites of inflammation an abundance of cells that have receptors for fibrin(ogen). And because fibrin(ogen) is bivalent/multivalent, those cells can be tethered to each other and/or to solution phase fibrin matrices, thereby maintaining/increasing for an extended period their concentration locally. Movements of blood elements to/from an inflammatory site are also facilitated by “fenestrations” created within the walls of regional vessels and by gradients of chemoattractants established at the siteS4’ The fenestrations are, in part, consequences of endothelial cell retraction. Fibrin(ogen) likely influences endothelial cell retraction by both chemical and physical means. Evidence from studies in vitro indicates at least three fibrin(ogen)-derived peptides, i.e., FpB, BBl-42, and 615-42, are operative.42 Importantly, those same peptides signal leukocyte chemotaxis.43*44 That the peptides derive from both “early” (thrombin) and “late” (plasmin) proteolysis of fibrin(ogen) ensures circulating blood elements will percolate through the affected tissues during the course of the protein’s processing. Furthermore, as the developing complex of fibrin and platelets itself retracts,45 tension is necessarily placed on the structures to which that complex is anchored. That tension undoubtedly facilitates the disjunction of neighboring endothelial cells and, consequently, the influx/ efflux of relevant molecules and cells. Many other processes affecting initiation, propagation, maintenance, and abatement of inflammation are served by fibrin(ogen)-derived peptides. Those processes include signaling endothelial cells to spread and release von Willebrand factor and growth factors,46 prompting smooth muscle cells to contract,47 suppressing lymphocyte functions,48 inhibiting thrombin activity and platelet aggregation,47 and regulating fibrinogen synthesis by

hepatocytes. l7 Taken together, these lindings argue unequivocally that fibrin(ogen) does indeed play key roles in inflammation. Fibrin(ogen), Cancer, and Atherosclerosis That malignant tumors initiate inflammation has been appreciated by pathologists for well over a century, and in the late 1950’s fibrin(ogen) was shown to be a component of tumor-induced inflammation.49 More recent 1y , fibrin(ogen)-derived materials that accumulate within tumor stroma and envelop tumor cells have been identified.3 The data indicate that much of the tumor-localized fibrinogen is converted in situ to cross-linked fibrin. That conversion likely involves coagulant/procoagulant activities generated both by the malignant cells5’ and by tumor-infiltrating leukocytes, probably macrophages.5”52 Although envelopment of tumor cells by fibrin matrix need not involve any specific interactions between the protein and the cells, many malignant cells express integrin-like receptors that promote such interactions.53 Fibrin matrix also facilitates angiogenesis within tumors, a process essential for tumor growth and viability. From these and other findings, we4,5*54and many others34,50have concluded that fibrin(ogen) and its processing are important to tumor biology, and we4v5*54and others34 have speculated that promotion of tumor growth and propagation by fibrin(ogen) may be an inadvertent consequence of the protein’s role in inflammation. Atherosclerosis is another disease in which fibrin(ogen) and inflammation go hand-in-hand.55 Indeed, the prevailing hypothesis on atherogenesis has as its underpinning the notion of vascular “response-to-injury.““6 The signature lesion of atherosclerosis, the plaque, consists of a deposit of extracellular hydrophobic lipids, lipid-laden macrophages, smooth muscle cells, and proteins embedded just beneath the endothelial lining of larger arteries. Of the proteins present in plaques, fibrin(ogen) and its degradation products are some of the most conspicuous. 57While the amounts of the different components of the plaque vary to some extent as the lesion matures, hydrophobic lipids and fibrin(ogen) are invariably present at all stages of plaque development.58,5g

Moreover, the fibrin(ogen) content of a plaque correlates positively with its lipid content,57J9’60 and plasma fibrinogen level is an independent risk factor for atherosclerotic cardiovascular disease.61 Thus, and perhaps analogous to its involvement in the pathophysiology of malignant tumors, fibrin(ogen) may promote plaque initiation, development, and growth as an inadvertent consequence of its role in inflammation. “Connections” Fibrinogen adsorbs rapidly, near-irreversibly, and, of all the plasma proteins, somewhat preferentially to the blood-contacting surfaces of implanted synthetic polymeric materials.62 Indeed, fibrinogen binds to the surface of most materials - even liquid hydrophobic phases - when they are brought into contact with blood.63Y64Such binding is attended by all of the inflammatory processes noted earlier and may, in fact, be required for their occurrence.32 Thus, with regard to inflammation, there appears to exist by design a synergism between fibrinogen and surfaces in general. The binding of fibrinogen to most materials is not mediated by any specific receptor. Instead, it is mediated by physicochemical properties of the protein itself, most notably amphiphilicity,65 a property shared by all proteins as a consequence of their content of both hydrophilic and hydrophobic amino acid residues. However, whereas most proteins that operate in an aqueous environment denature and lose function following adsorption to a surface, 66fibrinogen undergoesonly limited denaturation at an interface6’ and remainsoperational there in the classic senseof fibrin gelation.67Thus, adsorbed fibrinogen confers an adhesivepotential to otherwise non-adhesivesurfaces.In our opinion, it is this adhesive or “tethering” potential that rendersfibrinogen special in the context of inflammation. We speculatethat this potential is realized in a host of diseasesand, once realized, contributes to the progressionof those diseases.In this context, we now re-address cancer and atherosclerosis,presenting new paradigmsfor the involvement of fibrin(ogen) in those disorders. Metastasis: Of Motor Boats, Towlines, and Water Skiers Metastasisis the unequivocal hallmark of

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the phenotypically malignant cell. By as yet incompletely understood mechanisms, a primary tumor sheds cells that then disseminate, i.e., metastasize, to distant sites. Once at those sites and provided the local microenvironment is accommodating, the metastatic cells may take up residence and propagate, forming secondary tumors that may themselves spread. Prevailing notions have cancer cells being disjoined from a primary tumor by various hydrolytic enzymes,68 and those liberated cells then either passively “spilling” or actively migrating into vascular and/or lymphatic channels where they flow to their final place of residence. As for the involvement of fibrin(ogen) in malignancy, it has been proposed that the protein: (i) envelops tumor cells thereby protecting them from immune surveillance,‘j9 and (ii) provides an intravascular matrix for trapping and anchoring malignant cells following their embolization from a parent tumor.” We propose another mechanism by which fibrinogen may contribute to the malignant process. This proposal, which addresses metastasis, stems from our study of the dissemination of fibrin(ogen)-coated latex particles in mice following intraperitoneal injection of those particles.” Despite their rather large size, -6 urn, and the absence of similarly sized pores in the peritoneal lining, many of the particles escape the peritoneum within days of injection and can be found in virtually all organs of the body. Particles are most prominent in the sinuses of thoracic lymph nodes that drain the peritoneum (Figure 3). Outside of the peritoneum and especially obvious in lymph nodes, particles are always associated with macrophages, some within those cells, others adjacent to those cells. Measures that inhibit the binding of fibrinogen to the particles inhibit particIe dissemination, as does anticoagulant therapy.54 Our working hypothesis is that the particles are tramported from the peritoneum by macrophages, and that at least some of the particles are transported by those cells in extracellular fashion. We theorize that fibrin tethers existing between and fixed to a particle and a macrophage allows the particle to be pulled/pushed by the macrophage when the cell migrates. Then, if the macrophage leaves the peritoneum, it drags/pushes the particle along with it.

Figure 3. Fibrln(ogen)-coated latex beads (diame sinuses of a thoracic lymph node. See text for det ails. The presence of the particles in lymph nodes is a consequence of the targeted trafficking there of the macrophages. If such a mechanism operates to move nonliving fibrin-coated particles, so might it operate to move fibrin-coated cancer cells (Figure 4). It is our belief that macrophages, acting synergistically and as intended with fibrin(ogen) to scavenge particulate debris and cells during inflammation, unwittingly facilitate metastasis. Atherosclerotic Plaques: Of Burrs and Knit Sweaters

That lipid deposits form starting at birth within the intima of arteries of otherwise healthy individuals who eat high fat diets is universally accepted. The mechanisms accounting for the nucleation and propagation of those deposits, however, are far from certain. As noted above, current opinion favors the idea that circulating cholesterol- and cholesterol ester-rich lipoproteins are somehow retained at sites of vascular injury and accumulate there during life. The long-term deposition of lipid ultimately leads in many older individuals to the formation of gruel-like arterial plaques with their attending morbid, often mortal, consequences. As for the involvement of fibrin(ogen) in plaque ini-

in subcapsular (A) and rnedullary (B

tiation and growth, there is growing consensus that some relationship between plaques and fibrin(ogen) does exist, but the nature of that relationship is elusive. Indeed, many seem willing to relegate the role of the protein to one of only a nonspecific indicator of inflammation. We think differently. Fibrinogen adsorbs from any aqueous medium containing it - most importantly, plasma63@ and lymph (G.S. Retzinger, unpublished) - to droplets of liquid hydrophobic phases, i.e., oils, dispersed in that same medium. Because the bound protein remains coagulable, the oil droplets can be incorporated into any developing clot. We exploited these phenomena to develop fibrinogen-coated oil droplets and fibrinogen-coated liposomes as drug delivery systems that target sites of fibrin deposition.2 That development prompted us to consider the possibility that there exist in vivo natural analogues of the lipid-based particles we were creating. It occurred to us that if such analogues do exist, then they might be contributing to the deposition of lipids within arteries, i.e., they might be contributing to plaque initiation and growth. With this as our working hypothesis, we are following two investigative paths.

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f

/

M0

)

Figure 4. Pictorial representation of the authors’ proposal that fibrinogen tethers facilitate the extracellular translocation of tumor cells (TC) by macrophages (Me)). According to this proposal, fibrinogen, which is known to bind to both tumor cells and macrophages, forms intercellular fibrin strands. Fibrin strand formation is catalyzed by thrombin and factor XIIIa present in the tumor microenvironment, perhaps released from the macrophage. When the macrophage leaves the viciky of the tumor, it 1)ulls/pushes the tethered tumor cell along side it, the:reby promoting metastasis. See text for details. One path has us assessing in vitro the interactions of fibrinogen with lipoproteins, the lipid particles believed most involved in atherogenesis. In this regard, the results of preliminary studies have been gratifying because they suggest fibrinogen binds stoichiometrically with an equilibrium dissociation constant, I&. of -8 to 10 pm to low density lipoproteins (LDL) (G.S. Retzinger, unpublished). This & is particularly meaningful because it provides a rationale for the observation that individuals with a “high normal” plasma level of fibrinogen (normal reference interval: 4.0 to 10.0 pm) are predisposed to cardiovascular disease.7’ We have still to determine the functionality of fibrinogen when it is bound to LDL. The other investigative path has us exploring relationships between dietary lipids and fibrinogen in gastrointestinal lymph. While reasons for such exploration may not be obvious, there are at least three compelling ones. First, there is a substantial body of evidence that plaque propagation occurs as a post-prandial event involving chylomicrons and/or chylomicron remnants.72q73Second, newly ingested lipids are ideally positioned to interact with fibrinogen in the gastrointestinal lymph because it is there the emulsified lipids initially encounter the protein. Third, the natural history of atherosclerosis - a disease of the high pressure arterial system - has lipid depots forming centrally first, i.e., in the coronary

arteries and aorta, and then spreading peripherally. In the context of our understanding of fibrin(ogen) and hydrophobic surfaces, we propose the following scenario, a schematic of which is given in Figure 5. Immediately following a fat rich meal, emulsified fat particles in the gastrointestinal lymph absorb fibrinogen pre-existing in that medium. Those coated particles which, following a meal, would be most concentrated - then enter the great veins, the right heart, and the pulmonary vessels where the particles, because of their size and the low ambient pressures, are unable to penetrate the endothelial lining. Under the influence of the high pressures in the left heart and the arteries, however, some of the coated particles are able to penetrate the endothelial barrier on that side of the vasculature. Properties of the arterial subendothelial matrix and cells impede further translocation of the fibrinogencoated particles across the vessel wall so the particles accumulate there over time. If, for any reason, thrombin is generated in the vicinity of a growing lipid depot, the buildup is made worse by fibrin-mediated “capture” of additional particles. According to this scenario, proximal arteries, i.e., the coronary arteries and the aortic arch, develop plaques first because of their early position in the high pressure arterial tree, when the particles are still relatively concentrated. It follows that veins are spared from plaques not only because of

the low pressure that exists in those vessels, but also because the operative particles are significantly diluted or removed before they reach the venous system. Anti-adhesive

Therapy

Clearly, we are willing to attribute much of the pathogenesis of atherosclerosis and cancer to adhesive inflammatory events involving fibrin(ogen). Presuming our ideas are correct, then interfering with those events should mitigate disease. Indeed, there already exists evidence supporting such a notion. Heparin and other polyanionic molecules inhibit the mutual adhesion of fibrin-coated surfaces.4 Those same molecules inhibit inflammation in general,75 and both metastasis76 and atherosclerosis77 in particular. It appears that heparin and heparin-like molecules recognize and bind to adsorbed fibrin(ogen) rather specifically and by intent. By so doing, they prevent fibrin polymerization at interfaces, i.e., they prevent fibrin tether formation. It is our hope and belief that as fibrin-mediated adhesive events are better defined, interested parties will work both to delineate and optimize existing antiadhesive therapies and to develop new anti-adhesive agents and strategies. In closing, we have reviewed roles for fibrin(ogen) in inflammation, and we have presented new perspectives on those roles with a focus on cancer and atherosclerosis. We believe understanding the interfacial operation of fibrin(ogen) in

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inflammation is important and will lead to the development of effective modalities for the treatment of a host of diseases that have inflammatory components.

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Acknowledgments

This work was supported by a grant to GSR from the Ohio Affiliate of the American Heart Association, and by a Focused Giving Award to GSR from Johnson & Johnson, Inc. GSR thanks Ruth Mary Retzinger for inspiration.

FAT

t Low Pressure System

References 1. Addison W: On the colourless corpuscules, and on the molecules and cytoblasts in the blood. London Med Gaz 30:144-14X, 1842. 2. DeAnglis AP, Fox MD, Retzinger GS: Accumulation of fibrinogen-coated microparticles at a tibrin(ogen)-rich inflammatory site. Biotechnol Appl Biochem 29:251-161.1999. 3. Brown LF, VanDeWater L, Harvey VS, Dvorak HF: Fibrinogen influx and accumulation of cross-linked fibrin in healing wounds and tumor stroma. Am J Path01 130:455-465, 1988. 4. Retzinger GS, Chandler LJ, Cook BC: Complexation with heparin prevents adhesion between fibrin-coated surfaces. J Bioi Chem 26724356-24362, 1992. 5. Cook BC. Retzinger GS: Lipid microenvironment influences the processivity of adsorbed fibrin(ogen): enzymatic processing and adhesivity of the bound protein. J Colloid Interface Sci 162:171-1X1, 1994. 6. Leven RM, Schick PK. Budzynski AZ: Fibrinogen biosynthesis in the isolated guinea pig megakaryocytes. Blood 65:501-504, 1985. 7. Kant JA, Fomace AJ, Saxe D, Simon MI. McBride OW, Crabtree GR: Evolution and organization of the fibtinogen locus on chromosome 4: Gene duplication accompanied by transposition and inversion. Proc Nat1 Acad Sci USA X2:2344-2348, 1985. 8. Doolittle RF: The structure and evolution of vertebrate fibrinogen. Ann NY Acad Sci 408: 1326, 1983. 9. Francis CW, Miiller E, Hens&en A. Simpson PJ, Marder VJ: Carhoxyl-terminal aminoacid

sequences of twovariantformsof theychain of human plasma fibrinogen. Proc Nat1Acad Sci USA 85:335X-3362, 1988. 10. Wolfenstein-Todel C, Mosesson MW: Human plasma fibrinogen heterogeneity: Evidence for an extended carboxyl-terminal sequence in a normal ychain variant (y’). Proc Nat1Acad Sci USA 77~5069-5073, 1980. II. Yu S, Sher B, Kudryk B, Redman CM: Fibrinogen precursors. Order of assembly of fibiinogen chains. J Biol Chem 259:10574-105X1, 1984. 12. Nickerson JM, Fuller GM: Modification of fibrinogen chains during synthesis:Glycosylation of BB andychains. Biochemistry 20:2X18-2821, 1981.

Lipid Deposition (Plaque)

+ No Lipid Deposition

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pignre5. Pictorialrepresentation of the authors’proposalthat fibrinogenin thegastrointestinal ymphcontributesto the nucleationandgrowthof atherosclerotic plaquesin arteries.According D the proposal, fibrinogenin the lymphadsorbsto newly ingestedandemulsifiedlipids.The fibinogen-coated lipid particlesthenleavethe lymphaticsand“rain” on the vasculature.The high bressure existingwithin the arterialsystemfacilitatesthe percolationof the particlesacrossthe :ndotheliumof thearteriesin whichthe lipidsarefinally deposited. Any thrombingeneratedin hevicinity of a growinglipid depositwouldcontributeto the furtheraccumulationof fibrinogenC:oated particlesat thes&See text for details. 13. Seydewitz HH, Kaiser C, Rothweiler H, Witt I: The location of a second in vivo phosphorylation site in the Aa-chain of human fibrinogen. Thrombo Res 33:48749X, 1984. 14. Chrobak L. Bartos V, Brzek V, Hnizdova D: Coagulation properties of human thoracic duct lymph. Am J Med Sci 250:99-105, 1967. I 5. Le DT, Borgs P, Toneff TW, Witte MH. Rapaport SI: Hemostatic factors in rabbit limb lymph: Relationship to mechanisms regulating extravascular coagulation. Am J Physiol 274:H769-H776, 1998. 16. Schultz DR, Arnold PI: Properties of four acute phase proteins: C-reactive protein, serum amyloid A protein, at-acid glycoprotein, and tibrinogen. Semin Arthritis Rheum 20:129-147, 1990. 17. Ritchie DG, Levy BA, Adams MA, Fuller GM: Regulation of fibrinogen synthesisby plasminderived fragments of fibrinogen and fibrin: An indirect feedback pathway. Proc Nat1Acad Sci USA 79:1530-1534, 1982. 18. Collen D, Tytgat GN, Claeys H, Piessens R: Metabolism and distribution of fibrinogen. I. Fibrinogen turnover in physiological conditions in humans. Br J Haematol22:681-700, 1972. I 9. Hall CE, Slayter HS: The fibrinogen molecule: Its size, shape and mode of polymerization. J Biophys Biochem Cytol5: 11-15, 1959. 20. Weisei JW, Stauffacher CV. Bullitt E, Cohen C: A model for fibrinogen: domains and sequence. Science 230:138X-1391, 1985. 2 1. Hoeprich PD, Doohttle RF: Dimeric half-molecules of human fibrinogen am joined through disulfide bonds in an antiparallel orientation.

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Biochemistry 22:2049-2055, 1983. 22. Lorand L: New approaches to old problems in the clotting of fibrinogen. AnnNYAcadSci 40X:226-232, 1983. 23. Hanna LS, Scheraga HA, Francis CW, Marder VJ: Comparison of structures of various human fibrinogens and a derivative thereof by a study of the kinetics of release of fibrinopeptides. Biochemistry 2346X1-4687, 1984. 24. Yamazumi K, Doolittle RF: Photoafftnity labeling of the primary fibrin polymerization site: Localization of the label to y-chain Tyr-363. Proc Nat1 Acad Sci USA X9:2893-2896, 1992. 25. Doolittle RF: Fibrinogen and fibrin, Sci Am 245:126-135, 1981. 26. Fowler WE, Hantgan RR, Hermans J, Erickson HP: Structure of the fibrin protofibtil. Proc Nat1 Acad Sci USA 78:4X72-4876, 1981. 27. Mtiller MF, Ris H, Ferry JD: Electton microscopy of fine tibrin clots and fine and course fibrin films. J MolBiol 174:369-384, 1984.

28.Francis CW,MarderVJ: Increased resistance to plamic degradation of fibrin with highly crosslinked a-polymer chains formed at high factor XIII concentrations. Blood 71:1361-1365, 1988. 29. Hantgan RH, Francis CW. Marder VJ: Fibrinogen structure and physiology. In; Colman RW, Hiih I, Marder VJ, Saizman EW, eds.. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia, JB Lippincott Company. p. 277-300, 1994. 30. McRitchie DI, Girotti MJ, Glynn MFX, Goldberg JM, Rotstein OD: Effect of systemic

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fibrinogen depletion on intmabdominal abscess formation. J Lab Clin Med 118:48-55, 1991. 3 1. Dunn CJ, Wtlioughby DA: Leukocyte and macrophage migration inhibitory activities in inflammatory exudates - Involvement of the coagulation system Lymphokiues 4231267.1981. 32. Tang L, Eaton Iw: Fibrin(ogen) mediates acute inflammatory responses to biomaterials. J Exp Med 178:2147-2156, 1993. 33. Dunn DL, Simmons RL: Fibrin in peritonitis. III. The mechanism of bacterial trapping by polymerized fibrin. Surgery 92:513-519.1982. 34. Dvorak HF. Nagy JA, Berse B, Brown LF, Yeo K, Yeo T, Dvorak AM, VanDeWater L, Sioussat TM, Senger DR: Vascular permeability factor, fibrin, and the pathogenesis of tumor stroma formation.AnnNYAcadSci667:101-111.1992. 35. Peers&e EIB: The. platelet fibrinogen receptor. Semin Hematol22:241-259. 1985. 36. Calvete JJ: On the structure and function of platelet integrin a,,&, the fibrinogen receptor. Pm Sot Exp Biol Med 208:346-360, 1995. 37. Ugarova TP, Solovjov DA, Zhang L, Loukinov DI, Yee VC, Medved LV, Plow EF: Identiftcation of a novel recognition sequence for the integrin a& with the y-chain of fibrinogen. J Biol Chem 273:22519-22527, 1998. 38. Loike JD, Sodeik B, Cao L. Leucona S, Weitz JI, Detmers PA, Wright SD, Silverstein SC: CD1 lc/CD18 on neutrophils recognizes a domain at the N terminus of the Aa chain of fibrinogen. Proc Natl Acad Sci USA 88:10441048, 1991. 39. Hawiger J: Adhesive interactions of blood cells and the vascular wall. in: Colman RW, Hirsh J, Marder VJ, Saixman EW, eds., Hemostasis and thrombosis: basic principles and clinical practice. Philadelphia, J. B. Lippincott Company, p. 7627%. 1994. 40. Languino LR, Duperray A, Joganic KJ, Fomam M, Thornton GB, Altieri DC: Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition. Proc Nat1Acad Sci USA 92: 1505-I 509, 1995. 41. Springer TA: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301-314, 1994. 42. Francis CW, Bunce LA, Spom LA: Endothelial cell response to fibrin mediated by FPB cleavage and the amino terminus of the 5 chain. Blood Cells 19:291-307, 1993. 43. Senior RM. Skogen WF, Griflin GL. Wilner GD: Effects of fibrinogen derivatives upon the inflammatory response. Studies with human tibrinopeptide B. J Clin Invest 77: 1014-1019, 1986. 44. Skogen WF, Senior RM, Griffin GL, Wilner GD: Fibrinogen-derived peptide Bl31-42 is a multidomained neutrophil chemoattractant. Blood 71:1475-1479, 1988. 45. Cohen I, Gerrard JM, White JG: Ultrastruct~ of clots during isometric contraction. J Cell BioI 93:775-787, 1982. 46. Loremet R, Sobet JH, Bini A, Wine LD: Low 0 197- 1859/99 (see frontmatter)

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molecular weight fibrinogen degradation products stimulate the release of growth factors horn endothelial ceils. Thromb Haemostas 68:357363,1992. 47. Kowalski E: Fibrinogen derivatives and their biologic activities. Semin Hematol5:45-59, 1968. 48. Plow EF, Edgington TS: Lymphocyte suppressive peptides from fibrinogen are derived pmdominantly from the Aa chain. J Immunol 137:19101915,1986. 49. Day ED, Planinsek JA, Pressman D: Localization in vivo of radioiodinated anti-rat-fibrin antibodies and radioiodinated rat fibrinogen in the Murphy rat lymphosarcoma and in other transplantable rat tumors. J Nat1 Cancer but 22:413-426, 1959. 50. Rickles FR, Hancock WW, Edwards RL. Zacharski LR: Antimetastatic agents. I. Role of cellular procoagulants in the pathogenesis of fibrin deposition in cancer and the use of anticoagulants and/or antiplatelet drugs in cancer treatment. Semin Thromb Hemost 14:88-94, 1988. 5 1. Hogg N: Human monocytes have protbrombin cleaving activity. Clin Exp Immunol53:725730, 1983. 52. Giuntoli DL, Retzinger GS: Evidence for prothrombin production and tbrombin expression by phorbol ester-treated THP-1 cells. Exper Mol Pathol64:53-62, 1997. 53. Grossi IM, Hatfield JS, Fitzgerald LA, Newcombe M, Taylor JD, HOM KV: Role of tumor cell glycoproteins immunologically related to glycoproteins Ib and IIb/IIIa in tumor cellplatelet and tumor cell-matrix interactions, FASEB J 2:2385-2395, 1988. 54. Retzinger GS: Dissemination of beads coated with trehalose 6,6’-dimycolate: a possible role for coagulation in the dissemination process. Exp Molecular Path01 46: 190-198, 1987. 55. Schwartz CJ, Valente AJ, Sprague EA. Kelley JL, Suenram CA, Graves DT, Rozek MM, Edwards EH, Delgado R: Monocytemacrophage participation in atherogenesis: Inflammatory components of pathogenesis. Semin Thrombo Hemost 12:79-85, 1986. 56. Ross R: Atherosclerosis: A problem of the biology of arterial wall cells and their interactions with blood components. Arteriosclerosis 1:293-311. 1981. 57. Smith EB, Keen GA, Grant A, Stirk C: Fate of fibrinogen in human atterial intima. Arteriosclerosis 10:263-275. 1990. 58. Small DM: Progression and regression of atherosclerotic lesions. Arteriosclerosis 8:103-129, 1988. 59. Bini A, Fenoglio JJ, Mesa-Tejada R, Kudryk B, Kaplan KL: Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis: use of monoclonal antibodies. Arteriosclerosis 9:109-121, 1989. 60. Lassila R, Peltonen S, Lep&ntalo M, Saarinen 0, Kauhanen P, Manninen V: Severity of peripheral atherosclerosis is associated with fibrinogen and degradation of cross-linked fibrin. Arterios&r Q 1999 Elsevier Science Inc.

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