function

Blood Reviews (2005) 19, 275–288

www.elsevierhealth.com/journals/blre

REVIEW

The molecular physiology and pathology of fibrin structure/function ¨ns, Peter J. Grant* Kristina F. Standeven, Robert A.S. Arie Academic Unit of Molecular Vascular Medicine, The LIGHT Laboratories, University of Leeds, Clarendon Way LS2 9JT, UK

KEYWORDS

Summary The formation of a fibrin clot is a pivotal event in atherothrombotic vascular disease and there is mounting evidence that the structure of clots is of importance in the development of disease. This review describes the crucial events in the formation and dissolution of a clot, with particular focus on genetic and environmental factors that have been identified as determinants of fibrin structure in vivo, and discusses the substantiation of the relationship between fibrin structure and disease in conjunction with a review of the current literature. c 2005 Elsevier Ltd. All rights reserved.

Fibrinogen; Fibrin; Fibrin structure; Factor XIII; Haemostasis; Genetic; Environment; Thrombosis; Vascular disease




Introduction The coagulation and fibrinolytic cascades form part of an important protective mechanism against blood loss and exsanguination, which in concert with the fluid and cellular phases of immunity also protect against invasion by microbes and systemic infection. In modern life, changes in the environments to which man is exposed has led to alterations in disease patterns, to the extent that both fibrin formation and inflammation have become in-

* Corresponding author. Tel.: +44 113 343 7721; fax: +44 113 343 7738. E-mail address: [email protected] (P.J. Grant).




volved in the pathogenesis of thrombotic disorders, the most important of which in the Western world is coronary artery disease. The development of coronary artery disease is dependent on complex interactions between inflammatory and coagulation pathways that ultimately lead to the formation of coronary artery plaques. Plaques which have a thin fibrous cap and an increased inflammatory infiltrate are more predisposed to rupture. When this occurs, platelet aggregation is seen on the plaque surface and the formation of a platelet rich fibrin mesh takes place which may lead to the symptoms of an acute coronary syndrome (ACS) or complete arterial occlusion with ST elevation myocardial infarction (STEMI). New evidence indicates important roles for the proteins that

0268-960X/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.blre.2005.01.003

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Figure 1 Potential role(s) of fibrinogen/fibrin in cardiovascular disease. Fibrinogen and fibrin are recruited to the atherosclerotic plaque throughout its development. The structural and functional characteristics of this fibrinogen and fibrin may in part determine cell-adhesion, infiltration, and stability of the plaque. Ultimately, when the plaque ruptures and its highly thrombogenic core containing tissue factor is exposed to the flowing blood, an acute occluding clot may occur consisting of fibrin and platelets. The structure of the clot will determine its stability and its susceptibility to the natural fibrinolytic system as well as therapeutic thrombolysis.

generate fibrin (fibrinogen, factor XIII) in the pathogenesis of coronary artery disease and myocardial infarction (Fig. 1). There is additionally emerging evidence that the structure of the fibrin clot may determine risk of thrombotic vascular disease, and that genetic variants of fibrinogen and factor XIII may impart alterations in fibrin structure. Formation of a stable clot that is capable of withstanding the pressure in a blood vessel is essential to prevent bleeding and to promote wound healing. If injury occurs in the vascular system, the coagulation cascade is activated, leading to the generation of thrombin, which in return results in the conversion of fibrinogen to polymerising fibrin. Emerging fibrin fibres are subsequently cross-linked to each other and an array of proteins are incorporated into the growing clot to further improve the strength of the clot and its resistance to dissolution. To achieve a dynamic equilibrium between clot formation and lysis, activation of the fibrinolytic system is initiated at the time of fibrin formation.

The fibrinogen molecule Fibrinogen is a glycoprotein with an unusual structure as it has the properties of both globular and fibrous proteins. It is made up of two identical subsets consisting of three chains, denominated Aa, Bb and c. The centre part of the molecule, the E-region, contains the N-termini of all six chains. From there, the chains intertwine and form a-helical coiled-coil structures, supported by disulfide bridges, which lead to the distal ‘D’ regions, where the Bb- and c-chains end. The Aa chains follow to the D-regions as part of the coiled

coil, but extend back to the E-region, with which they remain associated up to the point of fibrin formation.1 In the majority of fibrinogen molecules, the Aachain consists of 610, the Bb chain of 461 and the c chain of 411 residues. Consequently, the entire human fibrinogen molecule is built from 2964 amino acids, yielding a calculated molecular weight of 329 818.2 There are four carbohydrate clusters present – one on each Bb-and c chain – which contribute another 10 000, increasing the total molecular mass to approximately 340 000.2 A total of 29 disulfide bonds hold the six chains together, creating the dimeric structure of the molecule. Fibrinogen is primarily synthesised in the liver, where approximately 1.7–5 g per day are produced.3 In the hepatocyte, there is no difference in the expression of individual chains and the amounts of mRNA found for all three chains is equal in normal and acute phase conditions, regardless of the total amount of protein expressed. Chains are assembled rapidly in the ER, and assembly of the complete molecule takes place within five minutes.4 Fibrinogen is expressed at relatively high concentrations compared to other coagulation factors, the circulating level being normally maintained between 2.5 and 3 mg ml1 in plasma. Its expression is strongly up-regulated in response to pro-inflammatory agents.5 This occurs through IL-6 interaction with responsive elements located in all three fibrinogen genes leading to enhanced gene expression.6

Fibrin polymerisation and clot formation The conversion of fibrinogen to fibrin and the consequent formation of a fibrin clot are the ultimate

The molecular physiology and pathology of fibrin structure/function events in the coagulation cascade (Fig. 2). The trigger for this event is the thrombin-catalysed cleavage of the fibrinopeptides, (the short acidic N-terminal sequences on the fibrinogen Aa- and Bb-chain that shield specific polymerisation sites), which results in a dramatic change in solubility that causes the molecules to aggregate and form fibrin fibres. Thrombin cleaves the fibrinogen Aa chain between Arg16 and Gly17,7 leading to the formation of double-stranded twisting fibrils which are arranged in a half-staggered overlapping domain arrangement (Fig. 2).8 Fibrils branch out and create structures that result in a complex network of fibres. There are likely to be two different types of branching that define the structure of the clot:9 In the first type, double-stranded fibrils line up side-to-side and form a tetra-molecular or bilateral branch-point. This kind of branching supports strength and rigidity in the clot. The second kind of branching is trimolecular or equilateral. This is formed by the coalescence of three fibrin molecules that conjoin three double-stranded protofibrils of equal width and probably occurs more often when the rate of fibrinopeptide release is slow. It appears that during the initial phases of fibrinopeptide release, when des-A fibrin is formed, there is a higher degree of equilateral branching.10 Susequent cleavage of FpB from the Bb chain is associated with lateral aggregation of protofibrils.8 Removal of FpB occurs concurrently with liberation of the aC domains that interact with the central Ddomain in fibrinogen. Interactions between the aC domains change from intramolecular to intermolecular, which facilitates lateral aggregation of the fibres and also makes these parts of the achains more available to cross-linking by FXIII.11 It is not known how FpB release is involved in lateral aggregation of protofibrils, since this can actually also occur in the absence of FpB cleavage. FpB cleavage alone can also yield clot formation, but this is unlikely to happen under physiological conditions as thrombin will preferentially cleaves FpA first.8,12 It is likely that release of both fibrinopeptides strengthens the bonding between the fibrin molecules in protofibrils, which in return has an effect on lateral aggregation of fibres.8 Fibres are twisted structures and their radial growth is limited by the degree of twisting. Protofibrils are twisted and when several protofibrils aggregate, they maintain a periodicity of 22.5 nm. This means that protofibrils added to the growing fibres have to undergo a certain degree of stretching. The degree to which a protofibril can be stretched is likely to be one of the ultimate determinants of the thickness to which a fibre

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can grow. Protofibrils are added up to the moment when the energy required for stretching exceeds the energy necessary for protofibril bonding. At this point, fibre growth ceases. To form a three-dimensional network, fibres also branch out.8

Cross-linking of fibrin by Factor XIIIa The covalent cross-linking of fibrin by activated coagulation Factor XIII (FXIIIa) leads to the formation of a rigid and elastic structure that is capable of stopping leakages in the circulatory system. Cross-linked fibrin is less susceptible to proteolytic or mechanical disruption. Zymogen FXIII is a transglutaminase with a heterodimeric structure, consisting of two A- and two B-subunits. Factor XIII circulates in plasma mainly bound to fibrinogen by its B-subunit as an inactive precursor and becomes activated at the same time as the fibrinopeptides are released from fibrinogen. To convert FXIII to activated enzyme FXIIIa, thrombin cleaves a 37 amino acid activation peptide from the N-termini of the A-subunit. After subunit dissociation, FXIIIa, the remaining, activated A-subunit dimer, cross-links the a- and c-, but not the

C

D

coiled coil

E

FPB

Monomers

Protofibrils

E D

D

Thrombin

FpA

E

FPA

D

Thrombin FpB

Fibres Lys406

Figure 2 Schematic diagram of fibrinogen and fibrin polymerisation: the fibrinogen molecule has the E region with the N-termini of all chains at its centre, from where the three intertwining chains in the form of a coiled coil lead to the distal D-regions. The Aa-chain, which is elongated further, leads back to the central E-region. Upon thrombin cleavage of fibrinopeptide A from the Nterminus of the a-chain, half-staggered, double-stranded protofibrils spontaneously form through D–E–D interactions. At the time of fibrinopeptide B cleavage, the Cterminal a-chain regions disengage from the central region and the protofibrils aggregate laterally to form the fibrin scaffolding of the clot.

278 b-chains of fibrin. c-Chain cross-linking occurs first, very soon after protofibril formation. Fibrin formation greatly accelerates the thrombin cleavage of the FXIII activation peptides, helping to achieve the co-ordination of events at this crucial moment in haemostasis.13,14 Correct alignment of the fibrin D–E–D regions, as it occurs during protofibril formation, is essential for the enhancement of FXIII activation. A schematic diagram of fibrin crosslinking is depicted in Fig. 3. Activated FXIII creates a bond between cLys406 on one and Glu398 and/or cGlu399 on the other chain, leading to the formation of mainly dimeric structures.15 There has been a long dispute in the fibrinogen research community on the orientation of these cross-links.16,17 Isopeptide bonds could be connecting the D-domains of two fibrin molecules in two possible ways: either FXIII introduces longitudinal cross-links that act as a junction between two adjacent D-domains,18,19 or it cross-links in a transverse fashion, in which case the bond would stretch from one protofibril strand to its opposite.20 c-Chains are also cross-linked into higher molecular weight formations as trimers and tetramers.21,22 Certain experimental conditions such as low ionic strength, increased CaCl2 concentrations and slow fibrin polymerisation favour c-polymer formation.10 Inter-chain cross-linking between the c- and Aa chain by FXIIIa has also been observed.22 In plasma, native fibrinogen molecules containing intramolec-

K.F. Standeven et al. ular cross-links between the Aa- and c-chains have been found, although these are most likely mediated by a tissue transglutaminase rather than FXIIIa.23 Studies of the incorporation of primary amines such as fluorescent dansylcadaverine have shown that glutamine residues involved in the cross-linking reaction of the a-chain include 221, 237, 328 and 366.24–26 Many lysine residues that potentially function as acceptor sites for the transglutaminase reaction have been reported, including lysine residues 208, 219, 224, 418, 427, 429, 446, 448, 508, 539, 556, 580, 583, 601 and 606.27,28 The multiplicity of available cross-linking sites in the Aa-chains may contribute to the mechanism of polymer formation.27 It it is possible that this variety of sites has evolved to circumvent the intrinsic structural heterogeneity of the aC domains, mainly caused by plasmic degradation. In vivo the structure of a-polymers is further complicated by the crosslinking of other plasma proteins, such as fibronectin,29,30 a2-antiplasmin,31 TAFI32 and von Willebrand factor.33 The rate and extent of a-chain cross-linking is significantly influenced by the concentration of FXIII. a-Polymers, when formed with higher than physiological concentrations of plasma and platelet FXIIIa, can become very large, possibly even reaching a molecular weight of several million.34 a-Chain cross-linking plays an important role in the regulation of fibrinolysis.35–37 For effective

Figure 3 Fibrin crosslinking: activated FXIIII introduces e-(glutamyl) lysine isopeptide bonds between the C-terminal c-chain crosslinking sites in the protofibrils to rapidly form c-dimers (a). Crosslinking of the a-chain to other a chains occurs more slowly and stabilises the three-dimensional fibre network (b). This process can be observed on SDS–PAGE, where with increasing time, more crosslinking products appear and the a- and c-chains disappear (c).

The molecular physiology and pathology of fibrin structure/function digestion, plasmin has to degrade fibrin in the coiled coil region between the D- and E-regions, a process that is interfered with by the presence of a-chain cross-links.38 It is also a determinant in the visco-elastic properties of the fibrin clot.39

Clot lysis and structure As fibrin is generated, it activates the fibrinolytic system and plays an active role in the tPA-mediated conversion of plasminogen to plasmin. At the same time, a2-antiplasmin40 and PAI-2,41,42 both inhibitors of plasmin, are cross-linked to fibrin in the clot. Fibrin therefore serves as co-activator and substrate for plasmin as well as participating in its inhibition. The presence of fibrin, but not fibrinogen, increases the slow activation of plasminogen by tPA by up to three orders of magnitudes. The inability of fibrinogen to participate in the activation of plasminogen is physiologically relevant as it causes the fibrinolytic activity consequently to become restricted to the location of clot formation. In vitro, cross-linked plasma fibrin fibres are dissected laterally rather than progressively. Clots with a loose fibrin conformation lyse at a faster rate than tight fibre networks consisting of thin fibres. Individually, however, thick fibres are cleaved at a slower rate.43,44 Although at first glance this appears as a contradiction, it is possibly due to the fact that not the diameter, but the three-dimensional configuration of the fibrin network is the main determinant for rates of fibrinolysis. When clots are made under conditions that favour the generation of either tight or loose networks, tight network configurations display significant higher fibrin fibre densities compared to clots with loose structure despite the equal amount of total protein used for clot formation.44 In other words, although thinner fibres lyse faster, there are more fibres to lyse. Lysis rates could also be prolonged in such structures because of slower permeation of fibrinolytic agents through the gel.

In vitro determinants of fibrin structure Much work has been done to investigate the effect of various modifications of the clotting process in vitro. Various proteins and ions have been added to in vitro systems to determine what effect on

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polymerisation they could have in vivo. Some examples identified as capable of altering clot properties are calcium,45,46 dextran,47,48 albumin,49,50 collagen,51 homocysteine,52 aspirin53 and metformin.54 Clots produced in plasma can be different from those made from purified fibrinogen and thrombin alone,55 whereby fibrin fibres made from plasma in general show a larger diameter compared to purified fibrin(ogen), but it is not known which plasma components are responsible for the modulation of the clot structure. Clot formation is strongly influenced by alterations of pH and ionic strength. Di Stasio et al.56 have identified a role for chloride ions as a basic physiological modulator of fibrin polymerisation, capable of controlling fibre size by inhibiting lateral aggregation and hence preventing the growth of thicker, stiffer and straighter fibres. In experimental work, chloride ions have been identified as the most important variable in solutions that control polymerisation,57 which is an important observation when considering the physiological presence of chloride ions in vivo. Variation in concentrations of fibrinogen and thrombin can also have a major influence on the polymerisation process. Alterations within the high range of thrombin concentrations have no effect on turbidity development (which is a measure of polymerisation rate of protofibrils, monitored over a period of time at a wavelength of 350 nm). Within a lower range of thrombin (0.001–1 U ml1), however, there is a marked effect on turbidity curves. Increasing the concentration of thrombin causes a decrease in the lag period, which reflects the time necessary for protofibrils to grow to a sufficient length before they aggregate.58 It also leads to an increase in the maximum rate of turbidity development and a decrease in the maximum final turbidity. Clot turbidity is proportional to the diameter of the average fibre59 and a decrease indicates the presence of finer fibres in clots produced with higher thrombin concentrations. Scanning electron micrographs of clots formed under conditions corresponding to these turbidity experiments have revealed that addition of thrombin at concentrations of 1 U ml1 results in fibre bundles that are thinner, whereas decreasing thrombin concentrations to a level of 0.001 U ml1 lead to thicker fibres, with intermediate fibre sizes at intermediate thrombin concentrations. This observation has been put into a physiological context by a recent study by Wolberg et al.60 who found that an increase of prothrombin levels in vivo could lead to alterations of fibrin structure that essentially corresponded to the in vitro structures generated from higher thrombin concentrations.

280 Variations in fibrinogen concentrations alter the polymerisation pattern and overall clot structure. Increasing fibrinogen concentration causes the lag period to become shorter, whilst the overall turbidity and maximum fibre size increases. There are more fibres present, and these fibres are longer compared to fibres formed at lower fibrinogen concentrations. The maximum rate of assembly is also increased.61 However, it is important to note that at very high fibrinogen concentrations the opposite effect may occur.62 The fact that fibrinogen concentrations can fundamentally change clot structure and properties is clearly of physiological importance and may help to explain why increased fibrinogen levels are a risk factor for cardiovascular disease.63

In vivo influences on fibrin structure It has been calculated that as a result of possible combinations of variations caused by post-translational modifications, splice variants or genetic polymorphic sites, fibrinogen in healthy individuals can occur in more than a million different forms.64 Further modifications of the fibrinogen molecule occur in association with disease, when it is often not clear whether the observed changes develop as a consequence or cause of the disease process. Some modifications have been shown to directly influence clot structure. Modifications of fibrinogen or FXIII can influence fibrin polymerisation, fibrin structure and FXIII cross-linking. A number of studies that examined the clinical consequences of altered clot structure have provided clear evidence that changes in clot properties are a risk factor for cardiovascular disease.65–68 In particular the formation of a tight network made of thin fibres and decreased porosity has been associated with cardiovascular events. This is probably in part because, as mentioned before, clot structure determines the rate of fibrinolysis in vitro.44 Under physiological conditions, clot formation is regulated by a precise balance of fibrin deposition and cross-linking on one hand, and fibrin degradation by plasmin on the other. The formation of clots relatively more resistant to fibrinolysis may shift this balance in favour of fibrin deposition and thrombosis. In addition, the presence of fibrinolysis-resistant clots has major implications for thrombolytic therapy in patients with occlusive atherothrombotic disease.

K.F. Standeven et al.

Genomic determinants of fibrin structure There is no single gene that independently determines the structure of the fibrin clot in vivo. Nonetheless, there is a relatively high degree of genetic regulation of the relatively complex molecular phenotype of fibrin clot structure/function, with a heritability of 39–46% as estimated in twins.91 Clot formation is the result of complex interactions between many proteins participating in the activated coagulation cascade. The concentration of plasma fibrinogen, a known determinant in fibrin structure, is to a certain extent determined by genetic heritability.69 Several polymorphisms in the Bb chain have been associated with circulating fibrinogen levels.70 In the genes encoding the mature fibrinogen molecule, two structural polymorphisms have been identified that have the potential to lead to alterations of clot properties:71 BbArg448Lys in the Bbchain and AaThr312Ala in the Aa-chain, both of which have been linked with vascular disease. Clinical studies of the latter have found an association of the variant encoding the alanine allele with increased post-stroke mortality in patients with atrial fibrillation72 and an increase in the occurrence of pulmonary embolism in patients suffering from deep vein thrombosis.73 In vitro experiments with purified fibrinogen homozygous for both forms showed there were significant differences in the ultrastructure of the variants. Ala312 fibrinogen produces stiffer clots, associated with increased alpha chain cross-linking. Ala312 clots were also characterised by thicker fibres and a lower number of fibres per square micrometer (Fig. 4). These changes in clot properties could potentially provide a molecular mechanism for the association of the genotype with disease in clinical studies, although the precise mechanism by which this occurs needs further investigation. The BbLys448 allele has been associated with coronary artery disease.74 Two investigations have been carried out recently to determine whether this association is due to alteration of fibrin properties. Lim et al.75 reported that fibrin clots formed from plasma and purified BbLys448 fibrinogen displayed different clot properties compared to BbArg448 fibrinogen. These findings were, however, challenged by Magzhal et al.76 who found no difference in fibrin structure between the genotypes. A polymorphism in the A-subunit of FXIII, which leads to a substitution of Val to Leu in codon 34, 3 amino acids away from the thrombin cleavage site between Arg37 and Gly38 has been the subject of extensive study. In Caucasians and American Indians, this polymorphism is relatively common,

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Figure 4 Morphology of clots from different variants of the AaThr312Ala polymorphism observed by scanning electron microscopy at a magnification of 5000·. Clot A, formed from homozygous Ala-fibrin shows subtle structural differences characterised by thicker fibrin fibres and larger pores compared to the Thr variant (b).

with an allele frequency ranging between 0.25 and 0.4. It is less common in South Asians and Africans (0.13 and 0.17) and barely present in Japanese (0.01).77 Results from several studies investigating the relationship between this polymorphism and myocardial infarction and stroke have found that possession of the Leu allele appears to be protective against both events.78–83 This effect appeared to be lost in the presence of increasing degrees of insulin resistance and high levels of PAI-1, a fibrinolytic inhibitor.84,85 On a molecular basis, it was found that Val34Leu had a significant effect on both FXIII function and fibrin clot structure.86–89 Activation of Leu34 FXIII by thrombin occurs more rapidly than Val34, with an increase in catalytic efficiency of 2.5-fold. In the presence of polymerising fibrin, the Leu34 FXIII activation peptide is cleaved at the same time as FpA, whereas Val34 FXIII is activated at the time of FpB release, which has an effect on subsequent fibre formation. Clots formed in the presence of Val34 form thicker fibres

with larger pores compared to clots stabilised by Leu34, which results in the formation of thinner fibres and a less porous fibrin gel. It appears that these alterations in structures are caused by the different kinetics of activation. Cleavage of the activation peptide from Leu34 FXIII concurrently with FpA could lead to an early stabilisation of the thinner des-A fibres with covalent isopeptide bonds, reducing molecular rearrangement and lateral aggregation of fibres. There is, however, a puzzling question about the protective effect of the Leu34 allele: from the in vitro work, it appears that clots cross-linked by Leu34 FXIII result in a structure that previously has been considered to be a fibrin phenotype that increases the risk of, rather than be protective against, vascular disease. This paradox was addressed by Lim et al.,90 who found that when fibrinogen concentrations were increased, the looser structure in clots formed with Val34 FXIII described earlier was exchanged for a tighter fibrin network compared with Leu34 FXIII

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in clots formed from both plasma and purified fibrinogen. In other words, the effect of Val34Leu was reversed with increasing fibrinogen concentrations, leading to the hypothesis that the protective effect of Leu34 FXIII only occurs at high fibrinogen levels, which themselves are a factor for MI. It is important to consider all the functional effects of the polymorphisms in FXIII and fibrinogen on fibrin clot properties as they may be important genetic determinants of the molecular as well as clinical phenotypes associated with thrombotic vascular disease. However, one has to bear in mind that both in vivo clot formation (Fig. 5) and cardiovascular disease are complex processes, involving interactions between genetic and environmental factors. Indeed, the role that the environment plays in fibrin structure has been illustrated by our twin study, which estimated that contribution of the environment amounts to 54–61% of the total phenotypic variation in fibrin structure.91

Splice variations Approximately, 8–15% of plasma fibrinogen contains a variant c chain, known as c0 , that results from alternative processing at the boundary between exon 9 and exon 10. The more common variant, referred to as cA, results after translation of exon 9 followed by exon 10 up to the stop codon after the fourth residue on exon 10. In c0 fibrinogen, intron 9 is partly included in the translation process, leading to an extension of the c chain by 16 residues to a total of 427 as opposed to 411 residues. Both c0 and cA share the initial 407 resi-

IN VIVO INFLUCENCES ON FIBRIN STRUCTURE

Environmental influences

Genetic polymorphisms

Splice variants

Fibrin clot structure

Disease

Plasma fibrinogen levels

Genetic polymorphisms

Environmental influences

Disease

Figure 5 Several factors have been shown to influence the structure of fibrin clots in vivo. Genetic and environmental factors as well as disease can have a direct impact on structure, but also alter the plasma fibrinogen level, which itself is a major determinant of fibrin structure.

dues.92 Replacement of the C-terminal sequence in A fibrinogen from AGDV to VRPEHPAETEYDSLYPEDDL has functional implications for the fibrinogen molecule. c0 Fibrinogen chains have a high affinity for FXIII and it is believed that FXIII binds to it in circulation.93 The same region also contains a high affinity thrombin binding site situated between c0 414 and c0 427, with the sulphated tyrosine residues at c0 418 and c0 422 especially enhancing the thrombin binding potential.94,95 Fibrinogen c0 has a decreased platelet aggregation potential. Interaction of fibrinogen with platelet membrane glycoprotein IIb–IIIa, a member of the integrin family (integrin aIIbb3) is decreased in recombinant, homodimeric (AaBbc0 )2, indicating that the binding of platelets to the fibrinogen c chain is mediated by the four C-terminal residues of the cA chain.96 Approximately, 3–34% of the c0 chains occur as a shortened version of the full-length variant. This reduced form, also called c0 1-423P probably arises in plasma by post-secretory in vivo processing of c0 1-427L fibrinogen chains, possibly through cleavage by a prolyl carboxypeptidase, the identity of which is currently unknown. The c0 chain extension protrudes from the D region of the fibrinogen molecule. Additional to the two sulphated tyrosine residues, the overall negative charge of the chain is increased by the presence of several Asp and Glu residues. In vivo, c0 is nearly always found in the heterozygous form with cA, whereby one D-domain of the fibrinogen molecule contains the c0 and the other D-domain the cA variant (cA/c0 fibrinogen).97 The clinical relevance of fibrinogen c0 levels in plasma has yet to be fully elucidated. A recent study has indicated that fibrinogen c0 may play a role in vascular disease, as it demonstrated an increase in c0 levels in patients with cardiovascular disease independent of total plasma fibrinogen.98 In vitro studies have shown that cA/c0 fibrinogen is more extensively cross-linked by activated FXIII than cA/cA and that clots made from the c0 splice variant have a greater resistance against fibrinolysis.99 Analysis of clots made from both variants purified from plasma found that the structure of heterogeneous cA/c0 clots was characterised by the presence of thinner fibres with decreased pore sizes compared to cA/cA clots.100 Experiments using recombinant homogenous c0 /c0 fibrinogen, however, did not confirm these structural differences.101 The reasons for this apparent discrepancy are not entirely clear, but they could be caused by the presence of other (uncharacterised) heterogeneities in the plasma preparations, or by the differences caused by the heterodimer (cA/c0 ) nature of

The molecular physiology and pathology of fibrin structure/function the plasma-purified preparation compared with the homodimer (c/c) nature of the recombinant preparation, which may behave differently with regards to polymerisation and clot morphology due to differences in electrostatic interactions at the D–D interface. Whilst all three fibrinogen chains have strong N-terminal homologies, there are differences in their C-terminal structure: the Aa chain ends in coils, unlike the Bb and c chain, which have distinct globular features at their C-terminus. There is, however, a subclass or alternative form of human fibrinogen, fibrinogen-420, in which an extended Aa-chain (aE) has a differently shaped Cterminus, denoted as aEC that bears resemblance to the globular structure at the end of the Bb and c chain.102 In humans, the conventional achain and the aE chain are identical up to residue Val610, encoded by exon V. In the aE chain, this residue is followed by Arg611 and a further 236 residues, which are encoded by exon VI (Asp612–Gln847) and which form the aEC domains.103 This extension leads to a mass increase of 50% compared to the conventional a-chain, caused by the additional residues and by glycosylation of this part of the a-chain, which normally does not show any glycosylation.104 Unlike c0 , aE containing molecules are primarily homodimeric.105 The molecular mass of a fibrinogen molecule incorporating two aEC domains is 420 kDa, which has led to the term ‘fibrinogen-420’ for this particular isoform. It occurs in approximately 1–3% of all fibrinogen molecules. In the foetus, circulation of fibrinogen-420 is three times higher than in adults.106 Many aspects of the (patho-) physiological role of fibrinogen-420 and the aEC domains have yet to be elucidated, although there is evidence to suggest that the extended aC region plays a role in cellular adhesion.107 A recent study by Mosesson et al.108 states that whilst there is no difference in proteolytic cleavage and assembly of fibrin-420 and fibrin-340 molecules, the aEC domains in fibrin-420 occupy the fibre surface, which probably delays lateral aggregation of fibres, leading to a more highly branched network consisting of thinner fibres. This surface location could also facilitate interaction between the aEC domains and cellular integrins.

Influence of environmental factors on fibrin structure and function Some drugs have the potential to alter fibrin clot structure and properties. Work from our group

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has found that in a purified system, the antihyperglycaemic drug metformin inhibited cross-linking of fibrin by FXIII, prolonged polymerisation and prolonged plasma clotting time induced by thrombin.54 Another antidiabetic drug, gliclazide, was shown to increase fibrin fibre thickness at the same time as creating a less permeable fibrin network, which was more rapidly lysed.109 Aspirin has been found to influence the clotting properties of purified fibrin. When purified fibrinogen was incubated with aspirin and converted to fibrin, the affinity of plasminogen to fibrin or thrombin inhibition by antithrombin increased if plasminogen or antithrombin, respectively, were present in the reaction system.53 In vivo, treatment with acetylsalicylic acid (aspirin) has been found to lead to an increase in permeability in fibrin clots.110 Demographic and environmental factors such as diet, age, drug use, smoking, alcohol consumption, body mass, gender, physical exercise, race and season have been shown to affect plasma fibrinogen levels.111 By raising fibrinogen levels, these factors may therefore also indirectly influence clot structure.

Fibrin structure and disease Perhaps the best documented evidence that alterations in fibrin structure directly contribute to disease derive from studies of hereditary dysfibrinogenaemias. Approximately, 55% of dysfibrinogenaemias are asymptomatic and 25% are associated with bleeding tendencies. In the remaining 20%, thrombotic events with or without haemorrhage can be found. The mechanism by which the risk for pathological events is increased is still unknown and is likely to be specific for each individual defect. However, in thrombophilic dysfibrinogenaemias, common features emerge: many mutations lie within the aC domains or within the thrombin cleavage sites of the Bb chain, leading to impaired polymerisation and structural alterations. In depth studies of the ultrastructure of some of the thrombophilic fibrinogens have consistently found the formation of networks characterised by thin fibres and small pores, heightened resistance to fibrinolysis and increased clot stiffness.112–115 These structural features were also found in clots formed of plasma taken from men who had suffered myocardial infarction at a young age.65,66 Rheological studies of purified116 and plasma117 fibrinogen have shown that the storage modulus (G0 ), which is inversely proportional to

284 the deformability of the clot, increases as the concentration of fibrinogen from which the gel is formed raises. As elevated fibrinogen levels are a risk factor for ischemic heart disease,63 this correlation may, in part, explain a relationship between increased plasma fibrinogen levels, fibrin clot structure and myocardial infarction. It has been suggested that less deformable clots formed from plasma containing high fibrinogen levels are more likely to lead to block of the coronary arterioles and cause subsequent myocardial infarction.118 Diabetic patients have a two to fourfold increased risk to of developing cardiovascular disease. The underlying mechanisms which determine the proatherogenic tendencies in diabetics have not been fully determined, but it is possible that alterations in fibrin clot properties may contribute. These could be caused by an observed increase in circulating fibrinogen levels119 and/or by direct molecular interaction between glucose and fibrinogen, leading to alterations in the structure and function of fibrinogen, possibly by glycation of lysine residues.64 Considering the involvement of lysine residues in both cross-linking and plasmin-induced lysis of fibrin, it is feasible that glycation could alter the polymerisation and lysis properties of clots as well as FXIII cross-linking and could therefore provide a direct mechanism by which fibrin structure could contribute to the increased cardiovascular risk in diabetics. In vivo studies of fibrinogen have shown that fibrinogen undergoes glycation, and that in diabetics, the degree of glycation correlated with glycaemic control.120,121 A study of polymerisation and crosslinking of fibrin in plasma of diabetic and control subjects by Lutjens et al.122 found that fibrinogen isolated from the diabetic patients was 35% more glycated compared to controls. The same study, however, found that a-chain cross-linking was impaired in the diabetic subjects, which resulted in increased susceptibility to clot lysis. The authors also observed no difference in the rate and extent of polymerisation between the groups. Structural analysis of fibrin clots formed from plasma of diabetic subjects revealed clots with decreased porosity compared to healthy controls.123,124 Recent work in our laboratory found that ambient glucose levels independently affect fibrin clot structure and that increasing glycosylated haemoglobin (HbA1c) is independently associated with the formation of clots characterised by a tight fibre network consisting of thin fibres.125 The mechanism by which fibrin structure in diabetics is altered is likely to be more complex and requires further investigation.

K.F. Standeven et al. Some conditions, such as advanced liver disease, including cirrhosis and hepatocellular carcinoma, are characterised by alterations in fibrin structure and function. It is believed that patients suffering from these conditions develop acquired dysfibrinogenaemia as a result of an abnormal increase of sialic acid residues, which causes impaired fibrin monomer formation126 that can be reversed by removal of the excess sialic acid. Multiple myeloma127 and Waldenstrom’s macroglobulinaemia128 are characterised by impaired polymerisation due to the presence of a paraprotein, and some autoimmune diseases129,130 produce abnormal antibodies that inhibit fibrin polymerisation or delay fibrinopeptide release.

Summary and conclusions The ability to regulate the generation and destruction of a fibrin clot in both the arterial and venous vascular beds is an essential protective mechanism against the threat of exsanguination and infection. The formation of a stable fibrin clot is a complex process involving multiple genes and their coded proteins, splice variants and post-translational modifications including glycation and oxidation. In addition, various cellular elements including platelets and leukocytes are involved and it remains to be established whether other cells such as endothelial cells or macrophages secrete proteins that modulate these processes when activated or damaged. In modern life thrombosis has become the greatest threat to the integrity of the organism, where once haemorrhage held sway. Recent advances in the fields of vascular biology and molecular medicine have enhanced our understanding of the mechanisms underpinning the development of vascular disease and the specific role that fibrin formation has. It is likely that post-translational modifications to the proteins involved in fibrin formation are going to have a major part to play in translating environmental influences into disease. This is particularly true in relation to diabetes mellitus, a disorder predicted to affect 300m worldwide by 2025.131 Diabetes is characterised by hyperglycaemia and the long term development of glycation, which has marked effects on fibrin structure and function and reduces the capability to lyse and model fibrin. Further understanding of these specific processes as well as the more general mechanisms affecting fibrin structure/function will provide opportunities in the medium term for the development of novel therapeutics

The molecular physiology and pathology of fibrin structure/function to ameliorate the adverse effects of fibrin formation in relation to arterial and venous thrombotic disease.

References 1. Weisel JW, Medved L. The structure and function of the alpha C domains of fibrinogen. Ann N Y Acad Sci 2001;936:312–27. 2. Doolittle RF. Fibrinogen and fibrin. In: Bloom AL, Forbes DP, Thomas DP, Tuddenham EGD, editors. Thrombosis and haemostasis. London: Churchill Livingstone; 1994. p. 491–521. 3. Takeda Y. Studies of the metabolism and distribution of fibrinogen in healthy men with autologous 125-I-labeled fibrinogen. J Clin Invest 1966;45:103–11. 4. Redman CM, Xia H. Fibrinogen biosynthesis: assembly, intracellular degradation, and association with lipid synthesis and secretion. Ann NY Acad Sci 2001;936:480–95. 5. Chung DW, Harris JE, Davie EW. Nucleotide sequence of the three genes coding for human fibrinogen. In: Liu CY, Chien S, editors. Fibrinogen, thrombosis, coagulation, and fibrinolysis. New York: Plenum Press; 1991. p. 39–48. 6. Fuller GM, Zhang ZHIX. Transcriptional control mechanism of fibrinogen gene expression. Ann NY Acad Sci 2001;936:469–79. 7. Blomback B, Hessel B, Hogg D, Therkildsen L. A two-step fibrinogen–fibrin transition in blood coagulation. Nature 1978;275:501–5. 8. Weisel JW. Fibrin assembly: lateral aggregation and the role of the two pairs of fibrinopeptides. Biophys J 1986;50:1079–93. 9. Mosesson MW, DiOrio JP, Siebenlist KR, Wall JS, Hainfeld JF. Evidence for a second type of fibril branch point in fibrin polymer networks, the trimolecular junction. Blood 1993;82:1517–21. 10. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci 2001;936:11–30. 11. Gorkun OV, Veklich YI, Medved LV, Henschen AH, Weisel JW. Role of the alpha C domains of fibrin in clot formation. Biochemistry 1994;33:6986–97. 12. Shainoff JR, Dardik BN. Fibrinopeptide B in fibrin assembly and metabolism: physiologic significance in delayed release of the peptide. Ann N Y Acad Sci 1983;408:254–68. 13. Lewis SD, Janus TJ, Lorand L, Shafer JA. Regulation of formation of factor XIIIa by its fibrin substrates. Biochemistry 1985;24:6772–7. 14. Curtis CG, Janus TJ, Credo RB, Lorand L. Regulation of factor XIIIa generation by fibrinogen. Ann N Y Acad Sci 1983;408:567–76. 15. Chen R, Doolittle RF. Cross-linking sites in human and bovine fibrin. Biochemistry 1971;10:4487–91. 16. Mosesson MW. Cross-linked c-chains in fibrin fibrils bridge ‘transversely’ between strands: yes. J Thromb Haemost 2004;2:388–93. 17. Weisel JW. Cross-linked c-chains in fibrin fibrils bridge transversely between strands: no. J Thromb Haemost 2004;2:394–9. 18. Weisel JW, Francis CW, Nagaswami C, Marder VJ. Determination of the topology of factor XIIIa-induced fibrin c-chain cross-links by electron microscopy of ligated fragments. J Biol Chem 1993;268:26618–24.

285

19. Veklich Y, Ang EK, Lorand L, Weisel JW. The complementary aggregation sites of fibrin investigated through examination of polymers of fibrinogen with fragment E. Proc Natl Acad Sci USA 1998;95:1438–42. 20. Mosesson MW, Siebenlist KR, Meh DA, Wall JS, Hainfeld JF. The location of the carboxy-terminal region of c chains in fibrinogen and fibrin D domains. Proc Natl Acad Sci USA 1998;95:10511–6. 21. Shainoff JR, Urbanic DA, DiBello PM. Immunoelectrophoretic characterizations of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid a-/rgamma-chain cross-linking that is promoted by the c-chain cross-linking. J Biol Chem 1991;266:6429–37. 22. Mosesson MW, Siebenlist KR, Amrani DL, DiOrio JP. Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin. Proc Natl Acad Sci USA 1989;86:1113–7. 23. Siebenlist KR, Mosesson MW. Evidence of intramolecular cross-linked A alpha.gamma chain heterodimers in plasma fibrinogen. Biochemistry 1996;35:5817–21. 24. Cottrell BA, Strong DD, Watt KW, Doolittle RF. Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites. Biochemistry 1979;18:5405–10. 25. Matsuka YV, Medved LV, Migliorini MM, Ingham KC. Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen. Biochemistry 1996;35:5810–6. 26. Fretto LJ, Ferguson EW, Steinman HM, McKee PA. Localization of the alpha-chain cross-link acceptor sites of human fibrin. J Biol Chem 1978;253:2184–95. 27. Sobel JH, Gawinowicz MA. Identification of the alpha chain lysine donor sites involved in factor XIIIa fibrin crosslinking. J Biol Chem 1996;271:19288–97. 28. Lorand L. Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Ann N Y Acad Sci 2001;936:291–311. 29. Procyk R, Blomback B. Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin. Biochim Biophys Acta 1988;967:304–13. 30. Okada M, Blomback B, Chang MD, Horowitz B. Fibronectin and fibrin gel structure. J Biol Chem 1985;260:1811–20. 31. Tamaki T, Aoki N. Cross-linking of alpha 2-plasmin inhibitor to fibrin catalyzed by activated fibrin-stabilizing factor. J Biol Chem 1982;257:14767–72. 32. Valnickova Z, Enghild JJ. Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases. Evidence for transglutaminase-catalyzed cross-linking to fibrin. J Biol Chem 1998;273:27220–4. 33. Hada M, Kaminski M, Bockenstedt P, McDonagh J. Covalent crosslinking of von Willebrand factor to fibrin. Blood 1986;68:95–101. 34. Francis CW, Marder VJ. Rapid formation of large molecular weight alpha-polymers in cross-linked fibrin induced by high factor XIII concentrations. Role of platelet factor XIII. J Clin Invest 1987;80:1459–65. 35. Gormsen J, Laursen B. Fibrinogen break-down products and clotting parameters. Thromb Diath Haemorrh 1967;17:467–81. 36. Gaffney PJ, Whitaker AN. Fibrin crosslinks and lysis rates. Thromb Res 1979;14:85–94. 37. McDonagh Jr RP, McDonagh J, Duckert F. The influence of fibrin crosslinking on the kinetics of urokinase-induced clot lysis. Br J Haematol 1971;21:323–32. 38. Francis CW, Marder VJ, Barlow GH. Plasmic degradation of crosslinked fibrin. Characterization of new macromolecular

286

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

K.F. Standeven et al. soluble complexes and a model of their structure. J Clin Invest 1980;66:1033–43. Shen LL, Hermans J, McDonagh J, McDonagh RP, Carr M. Effects of calcium ion and covalent crosslinking on formation and elasticity of fibrin cells. Thromb Res 1975; 6:255–65. Sakata Y, Aoki N. Cross-linking of alpha 2-plasmin inhibitor to fibrin by fibrin-stabilizing factor. J Clin Invest 1980; 65:290–7. Ritchie H, Lawrie LC, Mosesson MW, Booth NA. Characterization of crosslinking sites in fibrinogen for plasminogen activator inhibitor 2 (PAI-2). Ann N Y Acad Sci 2001; 936:215–8. Ritchie H, Lawrie LC, Crombie PW, Mosesson MW, Booth NA. Cross-linking of plasminogen activator inhibitor2 and alpha2-antiplasmin to fibrin(ogen). J Biol Chem 2000; 275:24915–20. Collet JP, Lesty C, Montalescot G, Weisel JW. Dynamic changes of fibrin architecture during fibrin formation and intrinsic fibrinolysis of fibrin-rich clots. J Biol Chem 2003; 278:21331–5. Collet JP, Park D, Lesty C, et al. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol 2000;20: 1354–1361. Hardy JJ, Carrell NA, McDonagh J. Calcium ion functions in fibrinogen conversion to fibrin. Ann N Y Acad Sci 1983; 408:279–87. Carr Jr ME, Gabriel DA, McDonagh J. Influence of Ca2+ on the structure of reptilase-derived and thrombin-derived fibrin gels. Biochem J 1986;239:513–6. Dhall TZ, Bryce WA, Dhall DP. Effects of dextran on the molecular structure and tensile behaviour of human fibrin. Thromb Haemost 1976;35:737–45. Carr Jr ME, Alving BM. Effect of fibrin structure on plasminmediated dissolution of plasma clots. Blood Coagul Fibrin 1995;6:567–73. Wilf J, Gladner JA, Minton AP. Acceleration of fibrin gel formation by unrelated proteins. Thromb Res 1985;37: 681–688. Galanakis DK, Lane BP, Simon SR. Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen. Biochemistry 1987;26:2389–400. Jones M, Gabriel DA. Influence of the subendothelial basement membrane components on fibrin assembly. Evidence for a fibrin binding site on type IV collagen. J Biol Chem 1988;263:7043–8. Lauricella AM, Quintana IL, Kordich LC. Effects of homocysteine thiol group on fibrin networks: another possible mechanism of harm. Thromb Res 2002;107: 75–79. He S, Blomback M, Yoo G, Sinha R, Henschen-Edman AH. Modified clotting properties of fibrinogen in the presence of acetylsalicylic acid in a purified system. Ann N Y Acad Sci 2001;936:531–5. Standeven KF, Ariens RA, Whitaker P, et al. The effect of dimethylbiguanide on thrombin activity, FXIII activation, fibrin polymerization, and fibrin clot formation. Diabetes 2002;51:189–97. Carr ME. Fibrin formed in plasma is composed of fibers more massive than those formed from purified fibrinogen. Thromb Haemost 1988;59:535–9. Di Stasio E, Nagaswami C, Weisel JW, Di Cera E. Cl regulates the structure of the fibrin clot. Biophys J 1998; 75:1973–9.

57. Vindigni A, Di Cera E. Release of fibrinopeptides by the slow and fast forms of thrombin. Biochemistry 1996;35: 4417–26. 58. Hantgan RR, Hermans J. Assembly of fibrin: a light scattering study. J Biol Chem 1979;254:11272–81. 59. Carr Jr ME, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules 1978;11:46–50. 60. Wolberg AS, Monroe DM, Roberts HR, Hoffman M. Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk. Blood 2003;101:3008–13. 61. Weisel JW, Nagaswami C. Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophys J 1992;63: 111–28. 62. Ferry JD, Morrison PR. Preparation and properties of serum and plasma proteins. VIII. The conversion of human fibrinogen to fibrin under various conditions [abstract]. J Am Chem Soc:388–400. 63. Meade TW, Mellows S, Brozovic M, et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986;2:533–7. 64. Henschen-Edman AH. Fibrinogen non-inherited heterogeneity and its relationship to function in health and disease. Ann N Y Acad Sci 2001;936:580–93. 65. Fatah K, Silveira A, Tornvall P, et al. Proneness to formation of tight and rigid fibrin gel structures in men with myocardial infarction at a young age. Thromb Haemost 1996;76:535–40. 66. Fatah K, Hamsten A, Blomback B, Blomback M. Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis. Thromb Haemost 1992;68:130–5. 67. Collet JP, Soria J, Mirshahi M, et al. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood 1993;82: 2462–9. 68. Greilich PE, Carr ME, Zekert SL, Dent RM. Quantitative assessment of platelet function and clot structure in patients with severe coronary artery disease. Am J Med Sci 1994;307:15–20. 69. de Lange M, Snieder H, Ariens RA, Spector TD, Grant PJ. The genetics of haemostasis: a twin study. Lancet 2001; 357:101–5. 70. Green FR. Fibrinogen polymorphisms and atherothrombotic disease. Ann N Y Acad Sci 2001;936:549–59. 71. Baumann RE, Henschen AH. Human fibrinogen polymorphic site analysis by restriction endonuclease digestion and allele-specific polymerase chain reaction amplification: identification of polymorphisms at positions A alpha 312 and B beta 448. Blood 1993;82:2117–24. 72. Carter AM, Catto AJ, Grant PJ. Association of the alphafibrinogen Thr312Ala polymorphism with poststroke mortality in subjects with atrial fibrillation. Circulation 1999; 99:2423–6. 73. Carter AM, Catto AJ, Kohler HP, et al. Alpha-fibrinogen Thr312Ala polymorphism and venous thromboembolism. Blood 2000;96:1177–9. 74. Behague I, Poirier O, Nicaud V, et al. Beta fibrinogen gene polymorphisms are associated with plasma fibrinogen and coronary artery disease in patients with myocardial infarction. The ECTIM Study. Etude CasTemoins sur l’Infarctus du Myocarde. Circulation 1996; 93:440–9.

The molecular physiology and pathology of fibrin structure/function 75. Lim BC, Ariens RA, Weisel JW, Grant PJ. Bb448 Lys/Lys variant alters coronary risk by an effect on fibrin structure/ function due to conformational changes in fibrinogen [abstract]. J Thromb Haemost 2003;Suppl. 1. Abstract No. OCO29. 76. Maghzal GJ, Brennan SO, George PM. Fibrinogen B beta polymorphisms do not directly contribute to an altered in vitro clot structure in humans. Thromb Haemost 2003; 90:1021–8. 77. Ariens RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood 2002;100:743–54. 78. Kohler HP, Stickland MH, Ossei-Gerning N, et al. Association of a common polymorphism in the factor XIII gene with myocardial infarction. Thromb Haemost 1998;79:8–13. 79. Kohler HP, Schroder V. Role of coagulation factor XIII in cardio- and cerebrovascular diseases. Hamostaseologie 2002;22:53–8. 80. Franco RF, Pazin-Filho A, Tavella MH, et al. Factor XIII val34leu and the risk of myocardial infarction. Haematologica 2000;85:67–71. 81. Elbaz A, Poirier O, Canaple S, et al. The association between the Val34Leu polymorphism in the factor XIII gene and brain infarction. Blood 2000;95:586–91. 82. Wartiovaara U, Perola M, Mikkola H, et al. Association of FXIII Val34Leu with decreased risk of myocardial infarction in Finnish males. Atherosclerosis 1999;142:295–300. 83. Catto AJ, Kohler HP, Coore J, et al. Association of a common polymorphism in the factor XIII gene with venous thrombosis. Blood 1999;93:906–8. 84. Kohler HP, Mansfield MW, Clark PS, Grant PJ. Interaction between insulin resistance and factor XIII Val34Leu in patients with coronary artery disease. Thromb Haemost 1999;82:1202–3. 85. Kohler HP, Grant PJ. Plasminogen-activator inhibitor type 1 and coronary artery disease. N Engl J Med 2000;342: 1792–1801. 86. Ariens RA, Philippou H, Nagaswami C, et al. The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure. Blood 2000;96:988–95. 87. Trumbo TA, Maurer MC. Examining thrombin hydrolysis of the factor XIII activation peptide segment leads to a proposal for explaining the cardioprotective effects observed with the factor XIII V34L mutation. J Biol Chem 2000;275:20627–31. 88. Wartiovaara U, Mikkola H, Szoke G, et al. Effect of Val34Leu polymorphism on the activation of the coagulation factor XIII-A. Thromb Haemost 2000;84: 595–600. 89. Balogh I, Szoke G, Karpati L, et al. Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia. Blood 2000;96:2479–86. 90. Lim BC, Ariens RA, Carter AM, Weisel JW, Grant PJ. Genetic regulation of fibrin structure and function: complex gene– environment interactions may modulate vascular risk. Lancet 2003;361:1424–31. 91. Dunn EJ, Ariens RA, de Lange M, et al. Genetics of fibrin clot structure: a twin study. Blood 2004;103:1735–40. 92. Wolfenstein-Todel C, Mosesson MW. Carboxy-terminal amino acid sequence of a human fibrinogen c-chain variant (c0 ). Biochemistry 1981;20:6146–9. 93. Siebenlist KR, Meh DA, Mosesson MW. Plasma factor XIII binds specifically to fibrinogen molecules containing c chains. Biochemistry 1996;35:10448–53. 94. Meh DA, Siebenlist KR, Brennan SO, Holyst T, Mosesson MW. The amino acid sequence in fibrin responsible for

95. 96. 97.

98.

99. 100.

101.

102.

103.

104.

105.

106.

107.

108.

109. 110.

111. 112.

113.

114.

115.

287

high affinity thrombin binding. Thromb Haemost 2001;85: 470–4. Lovely RS, Moaddel M, Farrell DH. Fibrinogen c0 chain binds thrombin exosite II. J Thromb Haemost 2003;1:124–31. Farrell DH, Thiagarajan P. Binding of recombinant fibrinogen mutants to platelets. J Biol Chem 1994;269:226–31. Mosesson MW, Finlayson JS, Umfleet RA. Human fibrinogen heterogeneities. 3. Identification of chain variants. J Biol Chem 1972;247:5223–7. Lovely RS, Falls LA, Al Mondhiry HA, et al. Association of cA/c0 fibrinogen levels and coronary artery disease. Thromb Haemost 2002;88:26–31. Falls LA, Farrell DH. Resistance of cA/c0 fibrin clots to fibrinolysis. J Biol Chem 1997;272:14251–6. Cooper AV, Standeven KF, Ariens RA. Fibrinogen c-chain splice variant c0 alters fibrin formation and structure. Blood 2003;102:535–40. Collet JP, Nagaswami C, Farrell DH, Montalescot G, Weisel JW. Influence of c0 fibrinogen splice variant on fibrin physical properties and fibrinolysis rate. Arterioscler Thromb Vasc Biol 2004;24:382–6. Grieninger G. Contribution of the alpha EC domain to the structure and function of fibrinogen-420. Ann N Y Acad Sci 2001;936:44–64. Fu Y, Weissbach L, Plant PW, et al. Carboxy-terminalextended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and c subunits. Biochemistry 1992;31:11968–72. Fu Y, Zhang JZ, Redman CM, Grieninger G. Formation of the human fibrinogen subclass fib420: disulfide bonds and glycosylation in its unique (alphaE chain) domains. Blood 1998;92:3302–8. Fu Y, Grieninger G. Fib420: a normal human variant of fibrinogen with two extended alpha chains. Proc Natl Acad Sci USA 1994;91:2625–8. Grieninger G, Lu X, Cao Y, et al. Fib420, the novel fibrinogen subclass: newborn levels are higher than adult. Blood 1997;90:2609–14. Lishko VK, Yakubenko VP, Hertzberg KM, Grieninger G, Ugarova TP. The alternatively spliced alpha (E)C domain of human fibrinogen-420 is a novel ligand for leukocyte integrins alpha (M)beta (2) and alpha (X)beta (2). Blood 2001;98:2448–55. Mosesson MW, DiOrio JP, Hernandez I, et al. The ultrastructure of fibrinogen-420 and the fibrin-420 clot. Biophys Chem 2004;112:209–14. Dhall DP, Nair CH. Effects of gliclazide on fibrin network. J Diabetes Complicat 1994;8:231–4. Williams S, Fatah K, Hjemdahl P, Blomback M. Better increase in fibrin gel porosity by low dose than intermediate dose acetylsalicylic acid. Eur Heart J 1998;19: 1666–1672. de Maat MP. Effects of diet, drugs, and genes on plasma fibrinogen levels. Ann N Y Acad Sci 2001;936:509–21. Collet JP, Woodhead JL, Soria J, et al. Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots. Biophys J 1996;70:500–10. Marchi R, Lundberg U, Grimbergen J, et al. Fibrinogen Caracas V, an abnormal fibrinogen with an Aalpha 532 Ser fi Cys substitution associated with thrombosis. Thromb Haemost 2000;84:263–70. Lefebvre P, Velasco PT, Dear A, et al. Severe hypodysfibrinogenemia in compound heterozygotes of the fibrinogen AalphaIVS4 + 1G > T mutation and an AalphaGln328 truncation (fibrinogen Keokuk). Blood 2004;103:2571–6. Sugo T, Nakamikawa C, Takebe M, et al. Factor XIIIa crosslinking of the Marburg fibrin: formation of alpham.gam-

288

116.

117.

118. 119. 120. 121.

122.

123.

K.F. Standeven et al. man-heteromultimers and the alpha-chain-linked albumin. Gamma complex, and disturbed protofibril assembly resulting in acquisition of plasmin resistance relevant to thrombophila. Blood 1998;91:3282–8. Ferry JD, Miller M, Shulman S. The conversion of fibrinogen to fibrin. VII. Rigidity and stress relaxation of fibrin clots, effects of calcium. Arch Biochem 1951;34:424–36. Scrutton MC, Ross-Murphy SB, Bennett GM, Stirling Y, Meade TW. Changes in clot deformability – a possible explanation for the epidemiological association between plasma fibrinogen concentration and myocardial infarction. Blood Coagul Fibrin 1994;5:719–23. Scrutton MC, Ross-Murphy SB. Why is plasma fibrinogen a cardiovascular risk factor?. Thromb Haemost 1994;72:650–1. Dunn EJ, Ariens RA. Fibrinogen and fibrin clot structure in diabetes. Herz 2004;29:470–9. Lutjens A, te Velde AA, vd Veen EA, vd MJ. Glycosylation of human fibrinogen in vivo. Diabetologia 1985;28:87–9. Hammer MR, John PN, Flynn MD, Bellingham AJ, Leslie RD. Glycated fibrinogen: a new index of short-term diabetic control. Ann Clin Biochem 1989;26(Pt 1):58–62. Lutjens A, Jonkhoff-Slok TW, Sandkuijl C, vd Veen EA, vd MJ. Polymerisation and crosslinking of fibrin monomers in diabetes mellitus. Diabetologia 1988;31:825–30. Jorneskog G, Egberg N, Fagrell B, et al. Altered properties of the fibrin gel structure in patients with IDDM. Diabetologia 1996;39:1519–23.

124. Nair CH, Azhar A, Wilson JD, Dhall DP. Studies on fibrin network structure in human plasma. Part II – Clinical application: diabetes and antidiabetic drugs. Thromb Res 1991;64:477–85. 125. Dunn EJ, Ariens RA, Grant PJ. The influence of ambient glucose concentration and glycation on fibrin structure – a mechanism for altered cardiovascular risk in diabetes [abstract]. Am Heart Assoc. 126. Martinez J, Keane PM, Gilman PB, Palascak JE. The abnormal carbohydrate composition of the dysfibrinogenemia associated with liver disease. Ann N Y Acad Sci 1983;408:388–96. 127. Coleman M, Vigliano EM, Weksler ME, Nachman RL. Inhibition of fibrin monomer polymerization by lambda myeloma globulins. Blood 1972;39:210–23. 128. Gabriel DA, Smith LA, Folds JD, Davis L, Cancelosi SE. The influence of immunoglobulin (IgG) on the assembly of fibrin gels. J Lab Clin Med 1983;101: 545–52. 129. Galanakis DK, Ginzler EM, Fikrig SM. Monoclonal IgG anticoagulants delaying fibrin aggregation in two patients with systemic lupus erythematosus (SLE). Blood 1978;52: 1037–46. 130. Marciniak E, Greenwood MF. Acquired coagulation inhibitor delaying fibrinopeptide release. Blood 1979; 53:81–92. 131. World Health Organisation. World Health Report; 2002.