Pro-thrombotic state induced by post-translational modification of fibrinogen by reactive nitrogen species

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G' value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.     

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.     

Proteolytic degradation of tyrosine nitrated proteins

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.     

S-nitrosoglutathione acts as a small molecule modulator of human fibrin clot architecture

Background

Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S-nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure.

Methodology/Principal Findings

Accordingly, we treated human platelet-poor plasma with GSNO (0.01–3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates.

Conclusions/Significance

Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range.     

Functional consequences of alpha-synuclein tyrosine nitration: diminished binding to lipid vesicles and increased fibril formation

Previous studies have shown the presence of nitrated alpha-synuclein (alpha-syn) in human Lewy bodies and other alpha-syn inclusions. Herein, the effects of tyrosine nitration on alpha-syn fibril formation, lipid binding, chaperone-like function, and proteolytic degradation were systematically examined by employing chromatographically isolated nitrated monomeric, dimeric, and oligomeric alpha-syn. Nitrated alpha-syn monomers and dimers but not oligomers accelerated the rate of fibril formation of unmodified alpha-syn when present at low concentrations. Immunoelectron microscopy revealed that nitrated monomers and dimers are incorporated into the fibrils. However, the purified nitrated alpha-syn monomer by itself was unable to form fibrils. Nitration of the tyrosine residue at position 39 was largely responsible for decreased binding of nitrated monomeric alpha-syn to synthetic vesicles, which correlated with an impairment of the nitrated protein to adopt alpha-helical conformation in the presence of liposomes. The chaperone-like activity of alpha-syn was not inhibited by nitration or oxidation. Furthermore, the 20 S proteasome and calpain I degraded nitrated monomeric alpha-syn, although at a slower rate compared with control alpha-syn. Collectively, these data suggest that post-translational modification of alpha-syn by nitration can promote the formation of intracytoplasmic inclusions that constitute the hallmark of Parkinson disease and other synucleinopathies.     

Protein tyrosine nitration--functional alteration or just a biomarker?

Protein 3-nitrotyrosine is a posttranslational modification found in many pathological conditions from acute to chronic diseases. Could 3-nitrotyrosine formation participate on the basis of these diseases or is it just a marker connected with the associated nitroxidative stress? In vitro and in vivo data, including proteomic research, show that protein tyrosine nitration is a selective process where only a small amount of proteins is found nitrated and one or a few tyrosine residues are modified in each. Accumulating data suggest a strong link between protein 3-nitrotyrosine and the mechanism involved in disease development. In this review, we analyze the factors determining protein 3-nitrotyrosine formation, the functional and biological outcome associated with protein tyrosine nitration, and the fate of the nitrated proteins.     

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.     

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.     

Incorporation of thrombospondin into fibrin clots

Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.     

Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation

We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha(IIb)beta(3), based on absence of retraction of alpha(IIb)beta(3)-deficient thrombasthenic platelets. However, only a subset of alpha(IIb)beta(3)-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha(IIb)beta(3) in fibrin clot retraction versus aggregation. Immunoblotting demonstrated a phosphorylated protein pattern comparable with aggregation at early time points. However, as opposed to aggregation, tyrosine phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was alpha(IIb)beta(3)-dependent, since it was blocked by alpha(IIb)beta(3)-specific inhibitors and was absent in thrombasthenic platelets. Inhibition of platelet clot retraction by phenyl-arsine oxide and peroxovanadate, suggested a role for tyrosine phosphatases. Cytochalasin D and E (5 microm) blocked fibrin clot retraction and tyrosine dephosphorylation, suggesting regulation by actin cytoskeleton assembly. Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were alpha(IIb)beta(3)-dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that alpha(IIb)beta(3) engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.     

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.     

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.     

Protein and lipid nitration: role in redox signaling and injury

Protein and lipid nitration represent novel footprints of oxidative and nitrative stress processes. In this review, we first discuss the mechanisms of formation of protein 3-nitrotyrosine and nitrated fatty acids as well as their key biological and signaling actions. Elevation of protein 3-nitrotyrosine levels is associated to tissue injury, and some specific nitrated proteins play a causative role in disease progression; on the other hand, the substantiation on the role of tyrosine nitration on redox signaling is rather scarce. Herein, we also provide evidence to support that the nitration of lipids (i.e. to nitrofatty acids) results in the formation of novel endogenous modulators of redox processes, partially counteracting pro-inflammatory effects of oxidant exposure.     

Proteomic detection of nitroproteins as potential biomarkers for cardiovascular disease

Increased levels of reactive oxygen and nitrogen species are linked to many human diseases and can be formed as an indirect result of the disease process. The accumulation of specific nitroproteins which correlate with pathological processes suggests that nitration of protein tyrosine represents a dynamic and selective process, rather than a random event. Indeed, in numerous clinical disorders associated with an upregulation in oxidative stress, tyrosine nitration has been limited to certain cell types and to selective sites of injury. Additionally, proteomic studies show that only certain proteins are nitrated in selective tissue extracts. A growing list of nitrated proteins link the negative effects of protein nitration with their accumulation in a wide variety of diseases related to oxidation. Nitration of tyrosine has been demonstrated in diverse proteins such as cytochrome c, actin, histone, superoxide dismutase, α-synuclein, albumin, and angiotensin II. In vitro and in vivo aspects of redox-proteomics of specific nitroproteins that could be relevant to biomarker analysis and understanding of cardiovascular disease mechanism will be discussed within this review.     

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background

Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis.

Methodology/Principal Findings

We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis.

Conclusions/Significance

We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.     

Gel formation by fibrin oligomers without addition of monomers

Soluble fibrin oligomers were formed by reacting fibrinogen with thrombin under fine clotting conditions where the action of thrombin is the rate-determining step for polymerization, and by inhibiting the reaction shortly before gelation. Oligomeric fibrin was separated from unreacted fibrinogen and small oligomers by gel permeation chromatography. Electron microscopy revealed that the largest soluble fibrin oligomers resemble the protofibrils present in fine clots, but are somewhat shorter and entirely lack the twisted, trifunctional junctions that contribute to the elastic properties of fine clots. When thrombin was added to the soluble fibrin oligomers, polymerization resumed and clots were formed at a more rapid rate than from fibrinogen at the same concentration and resulted in a less-opaque clot under coarse clotting conditions. The results confirm a prediction of a theory for the polymerization of fibrin and provide additional evidence that the final state of a coarse fibrin clot depends on the mobility of protofibrils during its formation.     

Functional impact of oxidative posttranslational modifications on fibrinogen and fibrin clots

Fibrinogen is a circulating multifunctional plasma protein vital for hemostasis. Activation of the coagulation cascade converts soluble fibrinogen to insoluble polymerized fibrin, which, along with platelets, forms the hemostatic clot. However, inappropriate formation of fibrin clots may result in arterial and venous thrombotic disorders that may progress to life-threatening adverse events. Often thrombotic disorders are associated with inflammation and the production of oxidants. Fibrinogen represents a potential target for oxidants, and several oxidative posttranslational modifications that influence fibrinogen structure and function have been associated with disease pathogenesis. Here, we review various oxidative modifications of fibrinogen and the consequences of these modifications on protein structure and the ability to form fibrin and how the resulting alterations affect fibrin architecture and viscoelastic and biochemical properties that may contribute to disease.     

High glucose induced rat aorta vascular smooth muscle cell oxidative injury: involvement of protein tyrosine nitration

The alteration and further damage of vascular smooth muscle function have been implicated in the development of vascular complications and diabetes. Little is known about protein tyrosine nitration in vascular smooth muscle cell injury induced by high glucose. In this article, vascular smooth muscle cell was exposed to 30 and 40 mM high glucose for 72 h, and then the cell injury in vascular smooth muscle cell induced by high glucose was studied. It was found that high glucose stimulated vascular smooth muscle cell injury in a dose-dependent manner, including decreasing intracellular and extracellular glutathione contents, increasing malondialdehyde and intracellular reactive oxygen species content, increasing the production of nitric oxide (increased nitrite content in cell and medium), as well as increasing protein tyrosine nitration. By comparing protein tyrosine nitration induced by high glucose conditions and extrinsic factors (hemin–nitrite–glucose oxidase system and 3-morpholinosydnonimine), it may be speculated that protein is nitrated selectively, and specific protein tyrosine nitration is involved in diabetic vascular complications.     

Human fibrinogen and asialo-fibrinogen: a comparison of coagulation parameters.

The effect of desialylation of fibrinogen on its conversion to fibrin has been investigated with particular reference to the kinetics of clot formation and structure. Also examined was the role of sialic acid in fibrinogen (factor I) poor in factor XIII (fibrinstabilizing factor) and factor I containing F XIII. The removal of more than 90% of the sialic acid of fibrinogen does not alter the thrombin clotting time, the clot solubility in monochloroacetic acid, the extent of cross-linking in the fibrin polymer, or the firmness and elasticity of the evolved clot. The data indicate that the sialic acid residues of fibrinogen do not contribute significantly to its conversion to fibrin by thrombin.     

FXIII polymorphisms, fibrin clot structure and thrombotic risk

Fibrin clot structure is highly dependent on factor XIII activity. Activated FXIII catalyzes the formation of the peptide bonds between the gamma and alpha chains in noncovalently bound fibrin polymers and incorporates various adhesive and antifibrinolytic proteins into the final fibrin clot. In the absence of activated FXIII, clots are unstable and susceptible to fibrinolysis. Several studies have examined the effects of FXIII polymorphisms on final fibrin clot structure and clinical thrombotic risk. The Val34Leu FXIII polymorphism is associated with increased activation by thrombin. In the presence of saturating thrombin concentrations, however, FXIIIa specific enzyme activity is not affected by genetic polymorphisms. Fibrin clots formed in the presence of the FXIII 34Leu polymorphisms do tend to be thinner and less porous, however. The effects of prothrombin concentrations on clot structure have suggested that thinner clots are more resistant to fibrinolysis and associated with increased thrombotic risk. Most clinical studies of 34Leu FXIII carriers, however, have demonstrated a lower incidence of both venous and arterial thrombosis in carriers of the mutant allele compared to Val/Val carriers. One recent study has suggested that the interactions between FXIII phenotype and plasma fibrinogen concentrations significantly influence clinical thrombotic risk.