1 Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds, U.K.
2 School of Biochemistry and Molecular Biology, University of Leeds, Leeds, U.K.
3 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subjects with MI are reported to exhibit differences in the structure of fibrin clots compared with control subjects (2). Fibrin clots are formed after thrombin-induced cleavage of fibrinopeptides A and B from fibrinogen, which results in the spontaneous polymerization of fibrin monomers and the formation of two-stranded protofibrils, which aggregate laterally to form thicker fibrils. These associate with each other and branch out, creating a gel (3), which is further stabilized by factor XIII (FXIII)-mediated cross-links between glutamine and lysine residues on the
and
chains of fibrin. Plasma FXIII is a transglutaminase consisting of two A- and two B-subunits. Thrombin activates FXIII by cleaving the peptide bond between Arg 37 and Gly38 on the A-subunit, which results in the release of an activation peptide. The A-subunit dimer dissociates from the B-subunits in the presence of calcium. Both steps are necessary to reveal the active site of the enzyme (4). FXIII cross-links fibrin and incorporates other proteins, such as
2-antiplasmin (5), von Willebrand factor (6), thrombospondin (7), and fibronectin (8,9), into the clot, increasing its mechanical strength and resistance to fibrinolysis. To determine whether the cardioprotective effect of DMB could be caused by interactions with the processes involved in clot formation and stability, we studied the FXIII activity, fibrin formation, and clot structure in vitro and the FXIII activity and antigen levels in vivo in response to DMB.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification of fibrinogen.
Fibrinogen was isolated from 100 ml platelet-poor plasma (obtained from volunteers after informed consent) by precipitating twice with 25% ammonium sulfate, pH 7.4, and a final precipitation step of 7% ethanol at 4°C. The resulting pellet was resuspended in 50 mmol/l Tris and 150 mmol/l NaCl, pH 7.4, dialyzed against the same buffer overnight at 4°C, and passed through a lysine-Sepharose and a gelatin-agarose column connected in tandem, as described by Matsuda et al. (11), in 30 mmol/l Tris, 100 mmol/l NaCl, 5 mmol/l benzamidine, and 2 mmol/l EDTA, pH 7.4. After this double-affinity chromatography step, fibrinogen free of plasminogen and fibronectin was concentrated by saturation with 25% ammonium sulfate, pH 7.4. The pellet was resuspended in 50 mmol/l Tris and 150 mmol/l NaCl and dialyzed overnight against the same buffer. Concentration of fibrinogen was measured by absorbency at 280 nm using an extinction coefficient of 1.61. The purity of the preparations was tested with SDS-PAGE (8%, bis-to-acrylamide ratio of 1:37.5). To regenerate the affinity columns, fibronectin was eluted from the gelatin-agarose column with 8 mol/l urea, 0.05 mol/l Tris, pH 7.4, and plasminogen from the lysine-Sepharose column with 0.02 mol/l -aminocaproic acid, 0.15 mol/l NaCl, and 0.05 mol/l Tris, pH 7.4.
Biotin-pentylamine incorporation assay.
FXIII cross-linking activity was determined using a sensitive functional assay, as previously described (12). The assay was based on the incorporation of 5-(biotinamido) pentylamine (Pierce, Rockford, IL) by FXIII into microtiter plates coated with fibrinogen. The amount of cross-linked 5-(biotinamido)pentylamine was detected by measuring phosphatase activity after incubation with a streptavidin-alkaline phosphatase conjugate (Sigma Chemical, St. Louis, MO). The intra- and interassay coefficients of variation were <10%.
Berichrom FXIII activity assay.
FXIII activity was also measured with a commercial FXIII activity assay kit (Berichrom FXIII; Behring Diagnostics, Marburg, Germany). The assay principles are different from the biotin-pentylamine incorporation assay. FXIII A-subunit links a specific peptide substrate with glycine ethyl ester, thereby releasing ammonia. The ammonia released is determined in a parallel enzymatic reaction. The variable measured is the decrease in NADH, which is detected by monitoring the absorbance at 340 nm.
Effect of DMB on FXIII activity.
The effect of DMB on both purified and pooled normal plasma FXIII activity was determined with the biotin-pentylamine incorporation assay and the Berichrom assay. For both assays, a range of concentrations between 0 and 0.25 mol/l of DMB (hydrochloride salt; Sigma) was prepared. In the incorporation assay, DMB was added to the plasma dilution buffer. In the Berichrom assay, DMB was added to the reaction mixture. Included were controls that contained the Tris-buffered saline (TBS)/reaction mixture without plasma, the TBS/reaction mixture without DMB, and glucose or sorbitol instead of DMB.
Mass spectrometry.
Mass spectrometry was performed on mixtures of DMB with FXIII or activated FXIII. Purified FXIII was activated with human -thrombin (Sigma) at a concentration of 10 units/ml for 1 h at 37°C and dialyzed against 5 mmol/l ammonium acetate pH 7.4. DMB was mixed with 0.0041 mol/l of activated or zymogen FXIII at various concentrations (0, 0.05, 0.5, 5, and 50 µmol/l), and the mixtures were analyzed on a single-quadrupole bench-top mass spectrometer (Platform II, Micromass UK, Cheshire, U.K.), as previously described (13).
Investigation of binding of DMB to FXIII by magnetic separation.
Amine-terminated BioMag particles (Warrington, PA) were washed three times with 0.01 mol/l pyridine buffer, pH 6, and incubated while rotating at room temperature in pyridine buffer containing 5% glutaraldehyde. Next, 200 µg purified FXIII in pyridine buffer was coupled to the magnetic particles in an overnight step. Binding efficiency was determined by comparing the precoupling FXIII solution to the postcoupling supernatant by bicinchoninic acid assay (Sigma). After five washes in pyridine buffer, DMB (at a concentration of 0.05 mmol/l in phosphate-buffered saline (PBS), pH 7.4) was added and incubated for 60 min at room temperature. After magnetic separation of the particles, DMB in the supernatant was measured at 234 nm, and the reading was compared with the absorbency of the original solution of 0.05 mmol/l DMB in PBS, pH 7.4.
FXIII activation peptide release.
Various concentrations of DMB (125, 62.5, 31.25, 15.6, 1.56, and 0 mmol/l DMB) in 9.47 mmol/l sodium phosphate, 137 mmol/l NaCl, 2.5 mmol/l KCl, and 0.1% (wt/vol) polyethlene glycol, pH 7.4, were added to 1.6 µmol/l purified FXIII, which had been dialyzed against the same buffer. Human -thrombin (final concentration 0.1 units/ml) was added, and the samples were incubated at 37°C for 60 min. The reaction was stopped by the addition of 1:10 (vol:vol) of 3 mol/l HClO4 and centrifuged for 10 min at maximum speed in an Eppendorf centrifuge. Activation peptide release was analyzed by reverse-phase high-performance liquid chromatography (HPLC), using a 0.46 x 25cm silica C18 (5 µm, 300 Å) column (Pepmap C18; Perseptive Biosystems, Framingham, MA) on a Biocad Sprint automated chromatography system (Perseptive Biosystems), as described earlier (10). For each sample, the molar quantity of activation peptide was determined by integrating the area under the curve of the peak on the chromatogram and comparing it with those obtained with the peptide loaded at a known concentration.
Thrombin-induced fibrinopeptides A and B release.
Fibrinogen at 1 mg/l was incubated with various concentrations of DMB (125, 62.5, 31.25, 15.6, and 1.56 and 0 mmol/l DMB) and human -thrombin (final concentration 0.1 unit mol/l) at 37°C. The reaction was stopped after 1, 5, and 20 min by the addition of 1:10 (vol:vol) of 3 mol/l HClO4. HPLC was performed using the same method as that used for FXIII activation peptide release (described above).
Ancrod-induced fibrinopeptide A release.
Ancrod is the venom of the Malayan pit viper (Agkistrodon rhodostoma) and specifically cleaves fibrinopeptide A, but not B. For the analysis, 0.1 unit/ml ancrod (Sigma) was incubated at 37°C with 1 mg/ml fibrinogen and 125, 62.5, 31.25, 15.6, 1.56, and 0 mmol/l DMB. The reaction was stopped after 5 min by the addition of 1:10 (vol:vol) of 3 mol/l HClO4. After centrifugation, the release of fibrinopeptide A was analyzed by reverse-phase HPLC, as described above.
Thrombin-induced clotting time of normal plasma.
For this analysis, 100 µl of different concentrations of DMB in barbiturate-buffered saline (0.028 mol/l sodium diethyl barbiturate, 0.05 mol/l HCl, and 0.125 mol/l NaCl) were added to 100 µl pooled normal plasma. To induce clotting of the plasma, 100 µl bovine thrombin (Sigma) at a concentration of 10 units/ml were added, and the time required for the formation of a clot was measured with an Amelung coagulometer (Amelung, Lemgo, Germany). DMB was used at final concentrations of 0, 0.65, 1.3, 2.6, 5.2, 10.4, 20.8, 41.6, and 83.2 mmol/l.
Analysis of the effect of DMB on the active site of thrombin.
The thrombin-specific chromogenic substrate H-D-Phe-Pip-Arg-pNA·2HCl (Chromogenix Instrumentation Laboratory, Milan, Italy) was used to determine the effect of DMB on thrombin. For the analysis, 20 µl chromogenic substrate (final concentration 1 mmol/l) was added to 100 µl of 250 mmol/l DMB in 50 mmol/l Tris, 100 mmol/l NaCl, and 0.1% (wt/vol) bovine serum albumin. After the addition of 100 µl human -thrombin at a concentration of 0.5 units/ml, the reaction was stopped at 0, 1, 2, 4, 8, and 16 min with 50 µl 50% (vol/vol) acetic acid (whereby at 0 min, acetic acid was added before thrombin). The thrombin-induced color change on hydrolysis of the substrate was measured at a wavelength of 405 nm. The experiment was repeated without DMB and, to check the specificity of the substrate, with hirudin because hirudin acts as a direct inhibitor of the active site of thrombin. For this, 100 µl of hirudin, at a concentration of 1 unit/ml in 50 mmol/l Tris, 100 mmol/l NaCl, and 0.1% (wt/vol) bovine serum albumin was used.
Cross-linking of - and
-chains by FXIII.
SDS-PAGE was performed using a Miniprotean 3 (Biorad, Hercules, CA) electrophoresis unit. Gels were cast at a polyacrylamide concentration of 7% (bis-to-acrylamide ratio of 1:37.5) in 1.5 mol/l Tris-HCl, pH 8.8, with a stacking gel of 4% in 0.5 mol/l Tris-Cl, pH 6.8, and run at 100 V for 80 min. Gels were stained with GelCode Blue Stain Reagent (Pierce) for 60 min at room temperature and washed overnight with deionized water. Prior to electrophoresis, 1 mg/ml fibrinogen (final concentration) was incubated with 22 µg/ml purified FXIII and 0.3 units/ml human -thrombin (Sigma) in 0.05 mol/l Tris-HCl and 0.1 mol/l NaCl, pH 7.4, for 5 and 20 min at 37°C. The reactions were stopped by the addition of a one-half volume of reducing loading buffer (100 mmol/l Tris-HCl, 0.1 mol/l dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol, pH 6.8) with immediate boiling for 5 min, and 10 µl was loaded on the gel. Spot densitometry was used to quantitate the bands representing
and
cross-links. Bands were analyzed using an AlphaImager 950 Documentation System (Alpha Innotech, San Leandro, CA). Band intensity was established by measuring the integrated density value (the sum of all pixel values after background correction) of each band. The density of the bands (expressed in percent intensity) was plotted against the concentration of DMB used, with maximum cross-linking representing 100% band intensity.
Permeation properties of plasma clots.
Permeation properties of plasma clots formed in the presence of DMB were investigated as previously described (10). Human -thrombin (final concentration 1.18 units/ml) and calcium chloride (final concentration 10 mmol/l) in 100 mmol/l NaCl, 50 mmol/l Tris, pH 7.4, and DMB (final concentrations of 0, 0.5, 1, 5, 10, 20, 40, and 80 mmol/l) were added to pooled normal plasma. The mixture was filled in specially prepared preetched plastic tubes, which were sealed on one side with parafilm. The clots were incubated in a moist chamber for 2 h. After removing the parafilm, each tube was connected to plastic tubing, which itself was attached to a container holding 100 mmol/l NaCl, 50 mmol/l Tris, pH 7.4. The pressure drop in the buffer reservoir was 4 cm. After washing the clots, the time required for six drops to permeate through the gel was measured, and each of the six drops was weighed on an analytical balance. Plotting the accumulative weight of the drops against the accumulative time of permeation allowed calculating the flow rate (Q/t), which was used to establish the Darcy constant (Ks):
![]() |
where Q is the volume of liquid (milliliters), with the viscosity (10-2 poise) flowing through a clot with length L (1.3 cm) and a cross-sectional area A (0.049 cm2) in time t (s) under pressure
P (dyne/cm2). The unit of the resulting Darcy constant is in centimeters squared.
Effect of DMB on turbidity measurements.
Pooled normal plasma was diluted two-thirds with 50 mmol/l Tris and 100 mmol/l NaCl, pH 7.5, and incubated with either 0.7 units/ml human thrombin or 0.12 units/ml ancrod and 16 mmol/l calcium. DMB or NaCl, dissolved in 50 mmol/l Tris and 100 mmol/l NaCl, pH 7.5, was added to the dilution buffer at the following concentrations: 0, 1, 10, and 40 mmol/l. Immediately on addition of the thrombin/calcium and ancrod/calcium, absorbency was read every 10 s at 350 nm for 15 min on a MRX Microplate Reader (Dynex Technologies, Ashford, Middlesex, U.K.). From the turbidity curves, parameters can be calculated to characterize polymerization, including the lag phase before the increase in turbidity, the maximum rate of increase of turbidity, and the maximum absorbency. Two replicate measurements were performed for each sample.
Effect of DMB on clot structure determined by scanning electron microscopy.
Activation mixtures consisting of human -thrombin (final concentration 1 unit/ml), calcium chloride (final concentration 10 mmol/l), and DMB (final concentrations 50, 10, 1, and 0 mmol/l) in 50 mmol/l Tris, 100 mmol/l NaCl, pH 7.4, were added to pooled normal plasma and purified fibrinogen (final concentration 1 mg/ml). After a 2-h incubation in a moist environment, the resulting clots were prepared for scanning electron microscopy by fixation, dehydration, critical pointdrying, and sputter-coating with gold palladium, as described previously (14). Clots for each DMB concentration were prepared in duplicate. All clots were observed and photographed digitally in at least two different areas per clot, using a scanning electron microscope (XL 20; Philips Electron Optics, Eindhoven, the Netherlands).
FXIII A- and B-subunit antigen assays.
FXIII A- and B-subunit antigen levels were determined by in-house sandwichenzyme-linked immunosorbent assays, as described previously (12). The assays were specific for either A- or B-subunits, and they did not cross-react with the other subunit nor with any other plasma protein. For the FXIII A-subunit assay, the intra- and interassay coefficients of variation were 5.4 and 9.3%, respectively; for the B-subunit assay, the intra- and interassay coefficients of variation were 6.2 and 9.8%, respectively.
Patients and in vivo study design.
Samples from 39 patients with type 2 diabetes, previously enrolled for a study of DMB and cardiovascular risk, were available (15). The study had been approved by the United Leeds Teaching Hospitals Research Ethics Committee. Exclusion criteria included insulin therapy, BMI 25, and fasting blood glucose on treatment <6 mmol/l. After a baseline visit, patients were randomly allocated in a double-blind manner to receive 1.5 g DMB daily for the first 3 weeks (n = 26) or placebo (n = 13) for the study period. Subsequently, patients on DMB were either increased to 3.0 g daily or remained on 1.5 g for the remainder of the study period. At the baseline visit, patients were examined, and a full clinical history was taken. Venous blood samples were taken between 8:00 A.M and 9:00 A.M after an overnight fast at baseline and again after 3, 6, and 12 weeks. Blood samples were processed at room temperature by centrifugation at 2,540g for 20 min within 2 h from collection. Platelet-poor plasma was separated and stored at -40°C until analysis.
Statistical analysis.
Data were tested for conformity to the normal distribution using the Kolmogorov-Smirnov test. All data sets showed a sufficient fit to the normal distribution (P > 0.2) to allow for parametric analysis. Correlation between baseline and final values were assessed for every end point, and a correlation coefficient of r > 0.5 permitted the use of analysis of covariance (ANCOVA) to compare final values adjusted for baseline between treatment groups (16). A test of multiple comparisons between means (Student-Newman-Keuls tests) was used to detect a dosage effect. Finally, as the three FXIII measures vary together, a multivariate ANCOVA on these three values in the same time priod was used by adjusting for baseline. Statistical analyses were performed using SAS (Version 8.0) running on Windows NT.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Investigation of binding of DMB to FXIII by magnetic separation.
Coupling of purified FXIII to the BioMag particles was 88% efficient. After incubation of 0.05 mmol/l DMB with the FXIII-coupled particles, no difference in absorbency at 234 nm between the pre- and postincubation solution could be detected. These results showed that DMB did not bind to FXIII in physiological conditions.
FXIII activation peptide release.
Reverse-phase HPLC was used to measure the effect of DMB on FXIII activation peptide release. After incubation of purified FXIII with human -thrombin for 60 min at 37°C, a large peak for the FXIII activation peptide was detected. The activation peptide release reaction was measured as the ratio of the molar quantity of released peptide over the molar quantity of peptide released at maximum activation. The addition of DMB resulted in a significant decrease of peak size. Inhibition of the release of the activation peptide by DMB started at a concentration of 7.5 mmol/l, and the effect was dose-dependent (Fig. 2).
|
|
Clotting time.
Clotting times for pooled normal plasma with added thrombin in the presence and absence of DMB were recorded. Increasing concentrations of DMB prolonged the plasma clotting time induced by thrombin (Fig. 4). The effect was dose-dependent and appeared with concentrations of 1020 mmol/l or higher.
|
|
|
Effect of DMB on turbidity curves.
DMB at a concentration of 0.04 mol/l in 0.05 mol/l Tris and 0.1 mol/l NaCl, pH 7.5, caused an increase in the lag period, a small decrease in the maximum rate, and a decrease in the maximum turbidity (Fig. 7). The addition of NaCl to test whether the effect was specific for DMB or caused by an increase of ionic strength resulted in a change of turbidity, but to a lesser degree than DMB at the same concentration. Effects on turbidity of samples containing 0.04 mol/l that had been clotted with 0.1 unit/ml ancrod were even stronger (Fig. 7).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results from the current study demonstrate that DMB has marked effects on the processes that regulate the formation of cross-linked fibrin, to provide a further potential mechanism for the cardioprotection described in the UKPDS. The in vitro addition of DMB to either pooled normal plasma or to a purified system of fibrinogen, FXIII, and human -thrombin resulted in a decrease of FXIII cross-linking activity. Activation of FXIII was at least partly reduced by the dose-dependent decrease in activation peptide release in the presence of DMB. No molecular interaction between DMB and FXIII could be detected by either mass spectrometry or analysis of the binding of DMB to FXIII-coupled magnetic particles.
These observations suggest that DMB inhibits FXIII cross-linking activity by interfering with thrombin-induced activation of FXIII. We analyzed the thrombin-induced cleavage of the fibrinopeptides from fibrinogen because this is a reaction similar to the release of the FXIII activation peptide from the A-subunit, and we found that DMB also inhibited cleavage of fibrinopeptides A and B from fibrinogen in a dose-dependent manner. Ancrod cleaved fibrinopeptide A from fibrinogen, regardless of the amount of DMB added, which implies that DMB has a direct inhibitory effect on thrombin. However, experiments with a chromogenic substrate showed that the active site of thrombin was unaffected by the presence of DMB. Because the chromogenic substrate is smaller than the natural substrates of thrombin, it is possible that inhibition occurred via a different mechanism, such as binding to an exosite.
Measuring thrombin-induced clotting time of pooled normal plasma showed that DMB influenced fibrin polymerization in vitro. DMB also caused changes in turbidity measurements of clotting fibrin, which gave further information about the changes in the polymerization process. Interestingly, the effects on turbidity were more dramatic when ancrod was used, even though ancrods ability to cleave fibrinopeptide A (and therefore its enzymatic activity) was not affected by DMB. This indicated that a decline in thrombin activity might not be the only reason for the changes in polymerization. However, to generate turbidity curves, ancrod had to be used at a lower concentration than thrombin, and therefore effects on fibrin formation may have been accentuated.
Chloride ions are known to influence polymerization of fibrin and clot structure (23). To exclude the possibility of the observed effects of DMB being caused by ionic strength, we performed turbidity experiments with NaCl as a control instead of DMB. The addition of NaCl changed turbidity curves, but to a minor degree compared with DMB, indicating that factors other than ionic strength were responsible for the changes in polymerization.
Permeation experiments and scanning electron microscopy showed that DMB altered clot structure and function. The effects on clot structure could be directly caused by the influence of DMB on polymerization, primarily the lateral aggregation of the protofibrils. Reduced thrombin activity may also play a role, although it has previously been shown that lower thrombin concentrations cause an increase in fiber thickness (14). The effect of changes in fibrin structure on the risk for thrombotic disease is not yet fully understood. Fibrin structures with thinner fibers and reduced permeation characteristics have been related to increased thrombotic risk (2,24). However, there is also evidence for similar changes in fibrin structure to have a possible antithrombotic effect. A common polymorphism of the FXIII A-subunit, Val34Leu, has been associated with decreased prevalence of thrombotic disease (25,26,27,28,29,30), and carriers of the protective Leu allele produce a finer fibrin meshwork, with thinner fibers and reduced pore size between the fibrin strands (10), findings similar to those observed with DMB in the current study. This may indicate that DMB influences the development of a fibrin structure that is easier to lyse, an effect that would be accentuated in the presence of the described effects of DMB on PAI-1. This is an area that requires further study.
The outstanding question arising from these findings is their relevance to the clinical management of diabetic subjects. Orally administered DMB is rapidly distributed throughout the body and has a bioavailability of 5060% (31). Therapeutic plasma concentrations of DMB fall within the range of 0.0060.01 mmol/l (31), which is significantly less than the concentrations required in vitro to affect the assay systems used in this study. There are several possible explanations for these discrepancies that might support a clinical relevance to these findings. For all of the systems studied, we have demonstrated clear evidence of dose-related effects, albeit at higher molarity than used clinically. It is possible that our assay systems are not sensitive enough to pick up very small differences, which may only become clinically important in an environment in which DMB is also affecting other components of this pathway (PAI-1 and factor VII). In vivo, the lowering of a procoagulant tendency by DMB may enhance the effect of even minor changes in clot structure/function, leading to less activation of coagulation and enhanced fibrinolysis with, ultimately, a less stable clot. To support these arguments, our study demonstrates that in vivo administration of DMB at pharmacological doses was associated with a decrease in the concentration of both FXIII A- and B-subunits as well as a sustained reduction in FXIII cross-linking activity. It has to be taken into consideration that the present study size is small for a clinical trial, indicating the value of a larger sample to confirm these findings. The decrease in the A- and B-subunits may be related to hepatic effects of DMB, leading to a decrease in insulin resistance and an associated reduction in levels of the B-subunit, which acts as the carrier protein for the A-subunit. In support of this, we have recently described the B-subunit levels as being associated with features of insulin resistance in type 2 subjects and first-degree relatives (32). However, activity levels of FXIII, when measured by the pentylamine-incorporation assay, have previously been found to correlate poorly with antigen levels (12), and an alternative explanation, such as the in vitro effects of DMB on FXIII activation, needs to be invoked to explain this observation.
Several studies have reported that DMB reduces risk factors involved in the thrombotic response to atheroma. Increased fibrinolysis caused by reduced PAI-1 has been found in patients with type 2 diabetes after DMB (19,20,33). In addition, circulating factor VII levels are reduced by DMB (21). Other studies have shown decreased platelet density, platelet aggregability, blood pressure, and peripheral arterial resistance after DMB treatment (34,35). These effects contribute to reduction of thrombotic risk associated with type 2 diabetes. In the present study, we found that DMB influenced thrombin activity and fibrin polymerization, which consequently led to decreased FXIII activity and altered fibrin clot structure. These findings, in combination with previously reported increased fibrinolysis, may constitute a major mechanism by which DMB decreases risk of cardiovascular disease in patients with type 2 diabetes, as observed in the UKPDS.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank Dr. Ph. Lehert (Statistics and Computer Department, FUCAM, Mons, Belgium) for statistical advice. We also thank Dr. S. MacLennan and E. Lee from the Regional Blood Transfusion Center of Yorkshire for the provision of outdated transfusion plasma, Dr. Sekar Nagaswami for assistance with the scanning electron microscope, and Dr. N. Goode and M. Shires for their help with the BioMag particles.
![]() |
FOOTNOTES |
---|
Received for publication 19 December 2000 and accepted in revised form 22 October 2001.
ANCOVA, analysis of covariance; DMB, dimethylbiguanide; FXIII, factor XIII; HPLC, high-performance liquid chromatography; MI, myocardial infarction; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; UKPDS, U.K. Prospective Diabetes Study.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|