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Essentials The roles of β-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking β-barrel 2, both β-barrels, or full length FXIII, were made. Removing β-barrel 2 caused total loss of activity, removing both β-barrels returned 30% activity. β-barrel 2 is necessary for exposure of the active site cysteine during activation., Summary: Background Factor XIII is composed of an activation peptide segment, a β-sandwich domain, a catalytic core, and, finally, β-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of β-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to β-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to β-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of β-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of β-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the β-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the β-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual β-barrel domains in modulating access to the FXIII active site region., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Both type 2 diabetes (T2DM) and Bβ448Lys variant of fibrinogen are associated with dense fibrin clots, impaired fibrinolysis and increased cardiovascular risk. It was our objective to investigate whether BβArg448Lys adds to vascular risk by modulating fibrin network structure and/or fibrinolysis in diabetes. The primary aim was to study effects of BβArg448Lys on fibrin network characteristics in T2DM. Secondary aims investigated interactions between gender and BβArg448Lys substitution in relation to fibrin clot properties and vascular disease. Genotyping for BβArg448Lys and dynamic clot studies were carried out on 822 T2DM patients enrolled in the Edinburgh Type 2 Diabetes Study. Turbidimetric assays of individual plasma samples analysed fibrin clot characteristics with additional experiments conducted on clots made from purified fibrinogen, further examined by confocal and electron microscopy. Plasma clot lysis time in Bβ448Lys was longer than Bβ448Arg variant (mean ± SD; 763 ± 322 and 719 ± 351 seconds [s], respectively; p<0.05). Clots made from plasma-purified fibrinogen of individuals with Arg/Arg, Arg/Lys and Lys/Lys genotypes showed differences in fibre thickness (46.75 ± 8.07, 38.40 ± 6.04 and 25 ± 4.99 nm, respectively; p<0.001) and clot lysis time (419 ± 64, 442 ± 87 and 517 ± 65 s, respectively; p=0.02), directly implicating the polymorphism in the observed changes. Women with Bβ448Lys genotype had increased risk of cerebrovascular events and were younger compared with Bβ448Arg variant (67.2 ± 4.0 and 68.2 ± 4.4 years, respectively; p=0.035). In conclusion, fibrinogen Bβ448Lys variant is associated with thrombotic fibrin clots in diabetes independently of traditional risk factors. Prospective studies are warranted to fully understand the role of BβArg448Lys in predisposition to vascular ischaemia in T2DM with the potential to develop individualised antithrombotic management strategies.

Type 2 diabetes mellitus (T2DM) increases the risk of coronary thrombosis and both conditions are associated with altered fibrin clot properties. However, the influence of T2DM on fibrin clot properties in patients with coronary artery disease (CAD) remains unclear. We aimed to investigate the influence of T2DM on fibrin clot properties in patients with CAD. Fibrin clot structure and fibrinolysis were investigated in 581 CAD patients (148 with T2DM) using turbidimetric assays, confocal and scanning electron microscopy. Clots made from plasma and plasma-purified fibrinogen were studied, and plasma levels of inflammatory markers were analysed. T2DM patients had increased clot maximum absorbance compared with non-diabetic patients (0.36 ± 0.1 vs 0.33 ± 0.1 au; p=0.01), displayed longer lysis time (804 [618;1002] vs 750 [624;906] seconds; p=0.03) and showed more compact fibrin structure assessed by confocal and electron microscopy. Fibrinogen levels were elevated in T2DM (p< 0.001), but clots made from purified fibrinogen showed no differences in fibrin properties in the two populations. Adjusting for fibrinogen levels, T2DM was associated with C-reactive protein and complement C3 plasma levels, with the former correlating with clot maximum absorbance (r=0.24, p< 0.0001) and the latter with lysis time (r=0.30, p< 0.0001). Independent of fibrinogen levels, females had more compact clots with prolonged lysis time compared with males (all p-values< 0.001). In conclusion, T2DM is associated with prothrombotic changes in fibrin clot properties in patients with CAD. This is related to quantitative rather than qualitative changes in fibrinogen with a possible role for inflammatory proteins.

Background: Factor XIII is a 320 kDa tetramer, comprising two enzymatic A-subunits and two carrier B-subunits (FXIII A₂ B₂). Activated FXIII (FXIIIa) catalyses the formation of ε-(γ-glutamyl)lysyl covalent bonds between γ-γ, γ-α and α-α chains of adjacent fibrin molecules and also cross-links the major plasmin inhibitor, α2-antiplasmin, to fibrin., Objectives: We investigated the role of FXIII cross-linking of fibrin directly in clot morphology and its functional effect on clot formation and lysis, in the absence of α2-antiplasmin., Results and Conclusions: Our data show that the presence of FXIII during clot formation results in fibrin clots that have a significant 2.1-fold reduction in pore size, as determined by the Darcy constant, Ks, and formed thinner fibers (74.7 ± 1.5 nm) and higher density of fibers compared with those without FXIII (86.0 ± 1.7 nm, P < 0.001), as determined by scanning electron microscopy. Additionally, fibrinolysis showed a significant increase in the time to lysis for clots formed in the presence of FXIII in both static and flow systems. These data demonstrate that independent of α2-antiplasmin, FXIII activity plays a role in increasing the stability of the fibrin clot by altering its structure and increasing the resistance to fibrinolysis., (© 2013 International Society on Thrombosis and Haemostasis.)

Context: Diabetes is associated with increased incidence of atherothrombotic disease. The fibrin network forms the backbone of the arterial thrombus, and fibrin clot structure determines predisposition to cardiovascular events., Objectives: The aim of the study was to investigate fibrin clot structure/fibrinolysis in the largest type 2 diabetes cohort and analyze associations with cardiometabolic risk factors and vascular pathology., Design: Clot structure/fibrinolysis was assessed in 875 participants of the Edinburgh Type 2 Diabetes Study [age, 68 (range, 60-75) yr; 450 males] by turbidimetric assays, and clots were visualized by confocal microscopy. Four parameters of clot structure/fibrinolysis were analyzed, and plasma levels of fibrinogen and plasminogen activator inhibitor-1 were studied by Clauss assay and ELISA, respectively., Results: Clot maximum absorbance was increased in females compared with males (0.37 ± 0.005 and 0.34 ± 0.005 arbitrary unit, respectively; P < 0.001), and took longer to lyse (803 ± 20 and 665 ± 12 sec, respectively; P < 0.001). These gender differences in clot structure and fibrinolysis were still evident after correcting for fibrinogen and plasminogen activator inhibitor-1 plasma levels. A prothrombotic fibrin structure profile was associated with increased body mass index and low levels of high-density lipoprotein in women and with inadequate diabetes control in men. Clot formation time was related to previous cardiac ischemic events in both men and women after adjusting for traditional risk factors [odds ratio, 1.22 (95% confidence interval, 1.07, 1.38); and 1.33 (1.15, 1.50), respectively], and prothrombotic clots were associated with low ankle brachial index, renal impairment, and smoking, regardless of gender., Conclusions: Women with type 2 diabetes have compact clots with compromised fibrinolysis compared with men. There are gender-specific associations between clotting parameters and cardiometabolic risk factors in this population, whereas vascular abnormalities, impaired renal function, and smoking are associated with prothrombotic clot structure profile regardless of gender.

Aims/hypothesis: Impaired fibrin clot lysis is a key abnormality in diabetes and complement C3 is one protein identified in blood clots. This work investigates the mechanistic pathways linking C3 and hypofibrinolysis in diabetes using ex vivo/in vitro studies., Methods: Fibrinolysis and C3 plasma levels were determined in type 1 diabetic patients and healthy controls, and the effects of glycaemia investigated. C3 incorporation into fibrin clots and modulation of fibrinolysis were analysed by ELISA, immunoblotting, turbidimetric assays and electron and confocal microscopy., Results: Clot lysis time was longer in diabetic children than in controls (599 ± 18 and 516 ± 12 s respectively; p < 0.01), C3 levels were higher in diabetic children (0.55 ± 0.02 and 0.43 ± 0.02 g/l respectively; p < 0.01) and both were affected by improving glycaemia. An interaction between C3 and fibrin was confirmed by the presence of lower protein levels in sera compared with corresponding plasma and C3 detection in plasma clots by immunoblot. In a purified system, C3 was associated with thinner fibrin fibres and more prolongation of lysis time of clots made from fibrinogen from diabetic participants compared with controls (244 ± 64 and 92 ± 23 s respectively; p < 0.05). Confocal microscopy showed higher C3 incorporation into diabetic clots compared with controls, and fully formed clot lysis was prolonged by 764 ± 76 and 428 ± 105 s respectively (p < 0.05). Differences in lysis, comparing diabetes and controls, were not related to altered plasmin generation or C3-fibrinogen binding assessed by plasmon resonance., Conclusions/interpretation: C3 incorporation into clots from diabetic fibrinogen is enhanced and adversely affects fibrinolysis. This may be one novel mechanism for compromised clot lysis in diabetes, potentially offering a new therapeutic target.

Objective: Studies report clustering of cardiovascular risk factors and increased cardiovascular events in healthy first-degree relatives (FDR) of subjects with intermittent claudication (IC). Family history is an independent risk factor in coronary artery disease but the role of genetic factors is undefined in peripheral arterial disease. The fibrin clot is the final product of the atherothrombotic process and is subject to genetic influence. We proposed that healthy male FDR of subjects with IC possess abnormalities in their fibrin clots., Methods: This was a case-control family study. The FDR were recruited from claudicants attending vascular surgery out-patient clinics with the control subjects being recruited from the local primary care register. A total of 106 white European male FDR of male subjects with IC were age matched with 107 white European male control subjects from an identical geographic area. The control subjects had no FDR with a history of symptomatic cardiovascular disease, and subjects from both groups were free from a personal history of symptomatic cardiovascular disease or diabetes mellitus. Ex vivo assays for fibrin clot permeation, fiber thickness, factor XIII cross-linking activity, and fibrinolysis were performed on the plasma of the above subjects. In addition, linear regression analysis was undertaken to determine factors associated with clot parameters., Results: For controls and FDR, respectively, fiber thickness by turbidity was 0.75 (0.67-0.93) vs 0.86 (0.75-0.98) (P < .001), and FXIII cross-linking activity was 105% (87-141) vs 133% (103-155) (P < .001). On confocal microscopy, fibers measured 315.8 (307.0-324.6) vs 405.1 (397.6-412.6) nm (P < .001), and lysis front velocity was 12.66 (6.38-18.94) vs 4.83 (2.50-7.17), mum/min (P = .018). Linear regression analysis revealed cholesterol was associated with changes in certain clot parameters., Conclusion: The healthy FDR of subjects with IC produce clots which have thicker fibers, increased cross-linking, and resistance to fibrinolysis when compared to controls. This supports the potential genetic basis of peripheral arterial disease and highlights that cholesterol may contribute to this abnormal structure. This suggests that the FDR of subjects with IC, an apparently healthy sub-group of the population, have an elevated cardiovascular risk associated with abnormalities in their clot structure.

Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.

Activated coagulation factor XIII (FXIIIa) cross-links the gamma-chains of fibrin early in clot formation. Cross-linking of the alpha-chains occurs more slowly, leading to high molecular weight multimer formations that can also contain gamma-chains. To study the contribution of FXIIIa-induced gamma-chain cross-linking on fibrin structure and function, we created 2 recombinant fibrinogens (gammaQ398N/Q399N/K406R and gammaK406R) that modify the gamma-chain cross-linking process. In gammaK406R, gamma-dimer cross-links were absent, but FXIIIa produced a cross-linking pattern similar to that observed in tissue transglutaminase cross-linked fibrin(ogen) with mainly alpha-gamma cross-links. In Q398N/Q399N/K406R, cross-links with any gamma-chain involvement were completely absent, and only alpha-chain cross-linking occurred. Upon cross-linking, recombinant normal fibrin yielded a 3.5-fold increase in stiffness, compared with a 2.5-fold increase by alpha-chain cross-linking alone (gammaQ398N/Q399N/K406R). gammaK406R fibrin showed a 1.5-fold increase in stiffness after cross-linking. No major differences in clot morphology, polymerization, and lysis rates were observed, although fiber diameter was slightly lower in cross-linked normal fibrin relative to the variants. Our results show that gamma-chain cross-linking contributes significantly to clot stiffness, in particular through gamma-dimer formation; alpha-gamma hybrid cross-links had the smallest impact on clot stiffness.

Aims/hypothesis: The aim of this study was to determine the influence of type 2 diabetes on fibrinolysis by assessing interactions between the regulatory components of fibrinolysis and the fibrin clot, using fibrinogen purified from 150 patients with type 2 diabetes and 50 matched controls., Methods: Clot lysis rates were determined by confocal microscopy. Plasmin generation was measured using a plasmin-specific chromogenic substrate. Surface plasmon resonance was used to determine the binding interactions between fibrin, tissue-type plasminogen activator (t-PA) and Glu-plasminogen; cross-linkage of plasmin inhibitor to fibrin by factor XIII was determined using a microtitre plate assay., Results: Lysis of diabetic clots was significantly slower than that of controls (1.35 vs 2.92 microm/min, p<0.0001) and plasmin generation was significantly reduced. The equilibrium binding affinity between both t-PA and Glu-plasminogen and fibrin was reduced in diabetic subjects: t-PA, K (D)=0.91+/-0.3 micromol/l (control subjects), 1.21+/-0.5 micromol/l (diabetic subjects), p=0.001; Glu-plasminogen, K (D)=97+/-19 nmol/l (control subjects), 156+/-66 nmol/l (diabetic subjects), p=0.001. Cross-linkage of plasmin inhibitor to fibrin by factor XIII was enhanced in diabetic subjects, with the extent of in vitro cross-linkage correlating with in vivo glycaemic control (HbA(1c)) (r=0.59, p=0.001)., Conclusions/interpretation: These results indicate that impairment of the fibrinolytic process in diabetic patients is mediated via a number of different mechanisms; these may be a consequence of post-translational modifications to fibrinogen molecules, resulting from their exposure to the abnormal metabolic milieu associated with diabetes.

Atherothrombotic disease is a multifactorial disorder that develops secondary to a complex gene-environment interaction. The formation of an obstructive thrombus represents the final stage of the atherothrombotic process, and understanding the mechanisms involved in clot formation is essential in order to develop new preventive and therapeutic strategies aimed at decreasing mortality and morbidity from the disease. Studies have demonstrated an important correlation between final clot structure and predisposition to atherothrombotic disease. Both genetic and environmental factors contribute to the final ultrastructure of the clot, which, in turn, influences an individual's risk of the disease. This paper reviews the factors involved in determining clot structure. The role of commonly used therapeutic agents in modulating clot structure will also be discussed.

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.

Aims/hypothesis: The precise mechanisms underlying the increased risk of cardiovascular disease (CVD) in type 2 diabetes are unclear. Fibrin clot structure has been related to CVD risk in the general population. We therefore assessed this in type 2 diabetic patients as a potential mechanism whereby diabetes influences CVD risk., Methods: Fibrin clots were formed from fibrinogen purified from 150 subjects with type 2 diabetes and varying degrees of glycaemic control (assessed by HbA1c), and from 50 matched control subjects. Clot structure was assessed by turbidity, permeation and confocal microscopy. The specific effect of glucose itself was assessed by analysing the structure of clots formed from purified fibrinogen in the presence of increasing concentrations of the sugar., Results: Clots formed by fibrinogen purified from type 2 diabetic subjects had a denser, less porous structure than those from control subjects. The structural changes found were related to the individual's glycaemic control; HbA1c correlated negatively with permeation coefficient (Ks) values (indicates clot pore size) (r = -0.57, p < 0.0001) and positively with maximum absorbance (indicator of fibre size) (r = 0.33, p < 0.0001), branch point number (r = 0.78, p < 0.0001) and fibre density (r = 0.63, p < 0.0001). The ambient glucose level influenced clot structure; hypo- (< 5 mmol) and hyperglycaemia (> or = 10 mmol/l) were both associated with a reduction in Ks values and maximum absorbance, and with increased fibre density and branch point number within clots., Conclusions/interpretation: The structural differences found to occur in type 2 diabetes and in association with hypo- and hyperglycaemia may confer increased resistance to fibrinolysis, and in consequence contribute to the increase in CVD risk in diabetic patients.

The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. The structure of the fibrin clot and the genetic and environmental factors that modify it have effects on its biological function. Alterations in fibrin structure and function have implications for the clinical presentation of vascular disease. This review briefly describes the key features involved in the formation of a fibrin clot, its typical structure, and function. This is followed by a review of the current literature on genetic and environmental influences on fibrin structure/function and the relationship to clinical disease. The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. This review discusses how genetic and environmental factors alter fibrin structure and function and the implications this has for the clinical presentation of vascular disease.

Coronary artery thrombosis following plaque rupture is an important feature of myocardial infarction, and studies have highlighted the role of coagulation in this condition. Although genetic and environmental influences on the variance in coagulation protein concentrations have been reported, there are no data on the heritability of structure/function of the final phenotype of the coagulation cascade, the fibrin clot. To assess genetic and environmental contributions to fibrin structure, permeation and turbidity studies were performed in 137 twin pairs (66 monozygotic, 71 dizygotic). The environmental influence (e2) on pore size (Ks) (e2 = 0.61 [95% confidence interval (CI), 0.45-0.80]) and fiber size (e2 = 0.54 [95% CI, 0.39-0.73]) was greater than the heritability (h2 = 0.39 [95% CI, 0.20-0.55] and 0.46 [95% CI, 0.27-0.62], respectively). After correction for fibrinogen levels, the environmental effect persisted for Ks (e2 = 0.61), but genetic influence assumed a greater importance in determining fiber size (h2 = 0.73). Multivariate analysis revealed an overlap in the influence of genetic and environmental factors on fibrinogen levels, Ks, and fiber size. Factor XIII B subunit showed environmental and genetic correlation with fibrinogen and fiber size and a genetic correlation with Ks. The results indicate that genetic and environmental influences are important in determining fibrin clot structure/function.

Background: The fibrinogen Aalpha Thr312Ala polymorphism occurs within the alphaC domain of fibrinogen, which is important for lateral aggregation and factor XIII-induced cross-linking of fibrin fibers. We have previously shown an association of Ala312 fibrinogen with poststroke mortality in subjects with atrial fibrillation and with pulmonary embolism in subjects with venous thrombosis., Methods and Results: We studied the properties of clots formed from purified Ala312 and Thr312 fibrinogen and found that Ala312 fibrinogen produces stiffer clots, associated with increased alpha chain cross-linking, as demonstrated by SDS-Page. On electron microscopy analysis, we found larger fiber diameters in the Ala312 clots and observed a lower number of fibers per square micrometer. The number of branch points per square micrometer was similar between genotypes., Conclusions: Our study shows that Ala312 influences clot structure and properties by increased factor XIII cross-linking and formation of thicker fibrin fibers. These findings may provide a mechanism by which Ala312 fibrinogen could predispose to clot embolization.

Background: A family history of premature coronary artery disease (CAD) is an independent cardiovascular risk factor. Fibrin clots composed of dense fiber networks are found in young CAD patients and may occur in the relatives of such individuals., Methods and Results: The ex vivo fibrin structure of 100 healthy male relatives of patients with premature CAD and 100 age-matched control subjects was assessed by measurement of permeability (K(s)), fiber mass-length ratio ( micro ), and turbidity (lag phase and maximum absorbency [max DeltaAbs]). Scanning electron microscopy was performed on selected samples. Relatives and controls shared similar levels of conventional cardiovascular risk factors. K(s) was lower in relatives than in controls, 12.2 (11.1 to 13.3) versus 15.2 (14.0 to 16.5) x10(-9) cm(2) (P<0.001), associated with a smaller decrease in micro, 8.5 (7.7 to 9.2) versus 9.7 (8.9 to 10.5) x 10(13) Da/cm (P<0.05), respectively. Lag phase was shorter in relatives than in controls, 39 (37 to 41) versus 47 (44 to 50) seconds (P<0.001), and max DeltaAbs was higher in relatives, 0.78 (0.74 to 0.82) versus 0.71 (0.67 to 0.74) in controls (P=0.02), which indicates the presence of thicker fibers in relatives. After adjustment for fibrinogen levels, lag phase and K(s) remained significantly different between relatives and control subjects. Scanning electron microscopy images confirmed increased fiber diameter in relatives, possibly of reduced density. Factor XIII Val34Leu and fibrinogen Aalpha Thr312Ala and Bbeta -455 G/A showed no association with clot structure., Conclusions: The male relatives of patients with premature CAD form fibrin clots that begin polymerization more quickly, have thicker fibers, and are less permeable than those of control subjects.

Factor XIII and fibrinogen are unusual among clotting factors in that neither is a serine protease. Fibrin is the main protein constituent of the blood clot, which is stabilized by factor XIIIa through an amide or isopeptide bond that ligates adjacent fibrin monomers. Many of the structural and functional features of factor XIII and fibrin(ogen) have been elucidated by protein and gene analysis, site-directed mutagenesis, and x-ray crystallography. However, some of the molecular aspects involved in the complex processes of insoluble fibrin formation in vivo and in vitro remain unresolved. The findings of a relationship between fibrinogen, factor XIII, and cardiovascular or other thrombotic disorders have focused much attention on these 2 proteins. Of particular interest are associations between common variations in the genes of factor XIII and altered risk profiles for thrombosis. Although there is much debate regarding these observations, the implications for our understanding of clot formation and therapeutic intervention may be of major importance. In this review, we have summarized recent findings on the structure and function of factor XIII. This is followed by a review of the effects of genetic polymorphisms on protein structure/function and their relationship to disease.

The antihyperglycemic drug dimethylbiguanide (DMB, also known as metformin) reduces the risk of cardiovascular complications in type 2 diabetes, although the mechanism(s) involved are unclear. DMB reduces glycosylation-related protein cross-linking, a process similar to fibrin cross-linking catalyzed by activated factor XIII (FXIII). To investigate whether the cardioprotective effect of DMB could be related to effects on clot stabilization, we studied the effects of DMB on FXIII, thrombin activity, and cleavage of fibrin(ogen). Activity of purified and plasma FXIII was inhibited by DMB. Analysis by mass spectrometry and FXIII-coupled magnetic particles excluded binding of DMB to FXIII. Thrombin-induced cleavage of the activation peptide from FXIII was inhibited in a dose-dependent manner, as was fibrinopeptide cleavage from fibrinogen. Ancrod-induced cleavage of fibrinopeptide A was not affected. DMB prolonged clotting time of normal plasma. Fiber thickness and pore size of fibrin clots, measured by permeation experiments and visualized by scanning electron microscopy, decreased significantly with DMB. No interactions between DMB and the active site of thrombin were found. Turbidity experiments demonstrated that DMB changed polymerization and lateral aggregation of protofibrils. These results suggest that DMB interferes with FXIII activation and fibrin polymerization, but not only by binding to thrombin on a different location than the active site. In patients on DMB therapy, FXIII antigen and activity levels in vivo were reduced over a 12-week period. These findings indicate that part of the cardioprotective effect of DMB in patients with type 2 diabetes may be attributed to alterations in fibrin structure/function.

Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (k(cat)/K(m)) of thrombin was 0.5 for FXIII 34Leu and 0.2 (micromol/L)(-1) x sec(-1) for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (micromol/L)(-1) x sec(-1), respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of gamma- and alpha-chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.

To investigate the effect of blood glucose concentration on thrombin generation and fibrinolytic activity, six Type 1 patients had the blood glucose concentration maintained for 1 h at 5, 15, and 25 mmol l-1, and 8 patients underwent hypoglycaemia of 20 min duration after the blood glucose had been kept at 8 mmol l-1 for 1 h. During hyperglycaemia plasminogen activator activity rose from 214 (11-625) (median, range) to 478 (18-772) units (p less than 0.05) at a blood glucose of 5 mmol l-1 and to 511 (89-816) (p less than 0.05) and 535 (33-976) (p less than 0.05) units at a blood glucose of 15 and 25 mmol l-1, respectively. Cross-linked fibrin degradation products (FDP) were 45 and 53 micrograms l-1 at a blood glucose of 5 mmol l-1 and remained unchanged at higher glucose levels. Fibrinopeptide A was 1.3 (0.6-2.8) nmol l-1 at a blood glucose of 5 mmol l-1, and remained unchanged with hyperglycaemia, being 1.3 (0.9-1.3) nmol l-1 after 1h at 25 mmol l-1. During hypoglycaemia, plasminogen activator activity rose from 155 to 745 units (p less than 0.05) while both fibrinopeptide A and cross-linked FDP remained unchanged. The results indicate that acute fluctuations in blood glucose concentration do not lead to thrombin generation. Additionally, increased fibrinolytic activity measured in vitro is not associated with an increase in cross-linked FDP. This suggests that short-term hyper- and hypoglycaemia do not affect the end-products of the coagulation and fibrinolytic pathways.