Marlien Pieters

Glycaemic control improves fibrin network characteristics in type 2 diabetes – A purified fibrinogen model

Diabetic subjects have been shown to have altered fibrin network structures. One proposed mechanism for this is non-enzymatic glycation of fibrinogen due to high blood glucose. We investigated whether glycaemic control would result in altered fibrin network structures due to decreased fibrinogen glycation. Twenty uncontrolled type 2 diabetic subjects were treated with insulin in order to achieve glycaemic control. Twenty age- and body mass index (BMI)-matched non-diabetic subjects were included as a reference group. Purified fibrinogen, isolated from plasma samples was used for analysis. There was a significant decrease in fibrinogen glycation (6.81 to 5.02 mol glucose/mol fibrinogen) with a corresponding decrease in rate of lateral aggregation (5.86 to 4.62) and increased permeability (2.45 to 2.85 x 10(-8) cm(2)) and lysis rate (3.08 to 3.27 microm/min) in the diabetic subjects after glycaemic control. These variables correlated with markers of glycaemic control. Fibrin clots of non-diabetic subjects had a significantly higher ratio of inelastic to elastic deformation than the diabetic subjects (0.10 vs. 0.09). Although there was no difference in median fiber diameter between diabetic and non-diabetic subjects, there was a small increase in the proportion of thicker fibers in the diabetic samples after glycaemic control. Results from SDS-PAGE indicated no detectable difference in factor XIIIa-crosslinking of fibrin clots between uncontrolled and controlled diabetic samples. Diabetic subjects may have altered fibrin network formation kinetics which contributes to decreased pore size and lysis rate of fibrin clots. Achievement of glycaemic control and decreased fibrinogen glycation level improves permeability and lysis rates in a purified fibrinogen model.

The effect of glycaemic control on fibrin network structure of type 2 diabetic subjects

Diabetic subjects have been shown to have altered fibrin network structures. One possible cause may be fibrinogen glycation resulting in altered structure/function properties. We investigated the effect of glucose control on fibrinogen glycation and fibrin network structure in type 2 diabetes. Blood samples were taken from twenty uncontrolled diabetic subjects at baseline to determine the levels of fibrinogen glycation and fibrin network structures. The subjects were then treated with insulin until blood glucose control was achieved before end blood samples were taken. Twenty age- and BMI-matched non-diabetic subjects were included as a reference group. The diabetic subjects had significantly higher mean fibrinogen glycation at baseline than the non-diabetic subjects (7.84 vs. 3.89 mol glucose / mol fibrinogen; p < 0.001). This was significantly reduced during the intervention (7.84 to 5.24 mol glucose / mol fibrinogen; p < 0.0002) in the diabetic group. Both groups had high mean fibrinogen concentrations (4.25 and 4.02 g/l, diabetic and non-diabetic subjects respectively). There was no difference in fibrinogen concentration, porosity, compaction and kinetics of clot formation between the diabetic subjects and non-diabetic subjects at baseline, nor were there any changes during the intervention despite the reduced fibrinogen glycation. Fibrin network characteristics correlated well with fibrinogen but not with any markers of glycaemic control. Improved glycaemic control resulted in decreased fibrinogen glycation but not fibrinogen concentration. It seems as though porosity, compaction and kinetics of clot formation are more related to fibrinogen concentration than fibrinogen glycation in this model.

Ultrastructural comparison of the morphology of three different platelet and fibrin fiber preparations

The aim of the current study was to investigate the ultrastructural morphology of three different sources of fibrin networks and platelets, namely, lypholized human platelet‐rich plasma (LPRP), freshly prepared human platelet‐rich plasma (FPRP), and human platelet concentrate (HPC). The ultrastructural morphology of the three different fibrin networks was studied using the scanning electron microscope (SEM). Turbidity curves were drawn at 405 nm at room temperature and fibrinogen concentrations were measured. Scanning electron micrographs showed that all clots produced thick major fibrin fibers as well as a well‐defined fine fibrin network, which appeared to be a superimposed process that occurred after the major fibrin network was established. These features were decidedly more pronounced in the HPC specimens. Turbidity curves of the three types of plasma showed differences in LPRP and FPRP. Fibrinogen concentrations of all three preparations were in the normal ranges. Because of the great similarity between LPRP, HPC, and FPRP, we suggest that LPRP could be used successfully to study morphological changes in fibrin fibers and platelets, which may occur after exposure to certain therapeutic agents. However, functionality studies such as turbidity curves should concurrently be included. We therefore conclude that from a basic science point of view, LPRP is a valuable research tool and that such results may add information that could be valuable for clinical application. Anat Rec 290:188–198, 2007.

An international study on the standardization of fibrin clot permeability measurement: methodological considerations and implications for healthy control values.

M. P IETERS ,* 1 A . UN D A S , 1 R . MARCHI , M. P . M. DE MAAT ,§ J . W. WE ISEL– and R . A . S . AR I ËNS** ON BEHALF OF THE FA CTOR X I I I AND F I BR I NOGEN SUBCOMMITT EE OF THE SC IENT I F IC A ND STA ND ARD ISA T I ON CO MMI TTEE O F TH E I NT ERNA T IO N AL SO CI ETY FOR T HRO MBO S IS AN D HAEMOSTAS I S *Centre of Excellence for Nutrition, North-West University, Potchefstroom, South Africa; Department of Medicine, Jagiellonian University School of Medicine, Krakow, Poland; Department of Hematology, Erasmus University Medical Centre, Rotterdam, the Netherlands; §Centro de Medicina Experimental, Laboratorio de Hemostasia, Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela; –Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine, Philadelphia, PA, USA; and **Division of Cardiovascular and Diabetes Research, Section on Mechanisms of Thrombosis, University of Leeds, Leeds, UK

Fibrinogen concentration and its role in CVD risk in black South Africans – effect of urbanisation

The aim of this study was to investigate correlates of fibrinogen concentration in black South Africans, as well as its association with cardiovascular disease (CVD) risk and whether urbanisation influences this association. A total of 1,006 rural and 1,004 urban black South Africans from the PURE study were cross-sectionally analysed. The association of fibrinogen with CVD risk was determined by investigating the association of fibrinogen with other CVD risk markers as well as with predicted CVD risk using the Reynolds Risk score. The rural group had a significantly higher fibrinogen concentration than the urban group, despite higher levels of risk factors and increased predicted CVD risk in the urban group. Increased levels of CVD risk factors were, however, still associated with increased fibrinogen concentration. Fibrinogen correlated significantly, but weakly, with overall predicted CVD risk. This correlation was stronger in the urban than in the rural group. Multiple regression analysis showed that a smaller percentage of the variance in fibrinogen is explained by the traditional CVD risk factors in the rural than in the urban group. In conclusion, fibrinogen is weakly associated with CVD risk (predicted overall risk as well with individual risk factors) in black South Africans, and is related to the degree of urbanisation. Increased fibrinogen concentration, in black South Africans, especially in rural areas, is largely unexplained, and likely not strongly correlated with traditional CVD-related lifestyle and pathophysiological processes. This does, however, not exclude the possibility that once increased, the fibrinogen concentration contributes to future development of CVD.

Is HIV-1 infection associated with endothelial dysfunction in a population of African ancestry in South Africa?

Abstract The chronic infection status suffered by HIV-infected individuals promotes chronic arterial inflammation and injury, which leads to dysfunction of the endothelium, atherosclerosis and thrombosis. Although HIV-1 subtype C is prevalent in South Africa and accounts for almost a third of the infections worldwide, this subtype differs genetically from HIV-1 subtype B on which the majority of studies have been done. The objective of this study was to assess whether newly identified, never-treated, HIV-1-infected South African participants showed signs of endothelial dysfunction, accelerated atherosclerosis and increased blood coagulation. We compared 300 newly diagnosed (never antiretroviral-treated) HIV-infected participants to 300 age-, gender-, body mass index- and locality-matched uninfected controls. Levels of high-density lipoprotein cholesterol (HDL-C), triglycerides, interleukin-6 (IL-6), C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), fibrinogen and plasminogen activator inhibitor-1 (PAI-1), and carotid radialis pulse wave velocity (cr-PWV) were determined. The HIV-infected participants showed lower HDL-C and higher IL-6, CRP, ICAM-1 and VCAM-1 levels compared to the uninfected controls. No differences in fibrinogen and PAI-1 levels were detected. A continuous positive trend of increasing age with cr-PWV was detected in the HIV-infected group. Our findings suggest inflammatory injury of the endothelium, pointing to endothelial dysfunction of never-treated HIV-1-infected South Africans of African ancestry. Although no indication of a prothrombotic state could be detected, there was an indication of accelerated vascular aging and probable early atherosclerosis in the older HIV-infected participants.

Effect of freeze-drying, freezing and frozen storage of blood plasma on fibrin network characteristics

INTRODUCTION We investigated the effect of freezing, freeze-drying and the duration of frozen storage of blood plasma on fibrin network characteristics of clots subsequently produced. MATERIALS AND METHODS Fibrin network characteristics of clots made from freeze-dried and frozen plasma were compared to those made from fresh plasma. Freeze-dried pooled plasma was reconstituted and frozen each month for 4 months to describe the differences in fibrin networks that occur as a result of storage of the plasma over this period. RESULTS Compared to freezing, freeze-drying of plasma had fewer undesirable effects on the fibrin network characteristics measured. Only the permeability of the clots from freeze-dried plasma was significantly less compared to the values of clots from the fresh plasma (p=0.005). Fibrinogen activity and mass-length ratio, compaction and fibrin content of the clots made from frozen plasma were, however, all significantly affected by freezing. Mass-length ratio and compaction showed a linear decrease and fibrin content a linear increase over a 4-month frozen storage period, thereby indicating that these variables were probably not stable. Large variation found in the data from each month indicates that there may be other factors, apart from storage time, that have a larger influence on these fibrin network characteristics, than frozen storage of plasma for 4 months. Storage of plasma in the freeze-dried form for 4 months resulted in a significant increase in fibrinogen (p=0.0004) but significant decrease in fibrin content (p=0.0002). CONCLUSIONS Although the process of freeze-drying had fewer undesirable effects on the measured fibrin network characteristics compared to freezing, storage in both forms resulted in altered activity upon rehydration and thawing.

Fibrin Fiber Stiffness Is Strongly Affected by Fiber Diameter, but Not by Fibrinogen Glycation

The major structural component of a blood clot is a mesh of fibrin fibers. Our goal was to determine whether fibrinogen glycation and fibrin fiber diameter have an effect on the mechanical properties of single fibrin fibers. We used a combined atomic force microscopy/fluorescence microscopy technique to determine the mechanical properties of individual fibrin fibers formed from blood plasma. Blood samples were taken from uncontrolled diabetic patients as well as age-, gender-, and body-mass-index-matched healthy individuals. The patients then underwent treatment to control blood glucose levels before end blood samples were taken. The fibrinogen glycation of the diabetic patients was reduced from 8.8 to 5.0 mol glucose/mol fibrinogen, and the healthy individuals had a mean fibrinogen glycation of 4.0 mol glucose/mol fibrinogen. We found that fibrinogen glycation had no significant systematic effect on single-fiber modulus, extensibility, or stress relaxation times. However, we did find that the fiber modulus, Y, strongly decreases with increasing fiber diameter, D, as Y∝D−1.6. Thin fibers can be 100 times stiffer than thick fibers. This is unusual because the modulus is a material constant and should not depend on the sample dimensions (diameter) for homogeneous materials. Our finding, therefore, implies that fibrin fibers do not have a homogeneous cross section of uniformly connected protofibrils, as is commonly thought. Instead, the density of protofibril connections, ρPb, strongly decreases with increasing diameter, as ρPb∝D−1.6. Thin fibers are denser and/or have more strongly connected protofibrils than thick fibers. This implies that it is easier to dissolve clots that consist of fewer thick fibers than those that consist of many thin fibers, which is consistent with experimental and clinical observations.

Overfatness, stunting and physical inactivity are determinants of plasminogen activator inhibitor-1activity, fibrinogen and thrombin-antithrombin complex in African adolescents.

We examined fibrinogen, thrombin–antithrombin complex, factor VIIIc and plasminogen activator inhibitor-1 activity in African children in order to determine haemostatic profile patterning and to identify possible subdivisions at high risk for cardiovascular disease. In a cross-sectional analysis, a convenience sample of 117 girls and 78 boys (15.6 ± 1.35 years) in a South African township was investigated within the Physical Activity in Youth study. Haemostatic variables were investigated in the total group and subdivisions for physical activity levels, maturity (Tanner staging), sex, fat percentage and height for age. Overfatness (53.6%) coexisted with stunting (17.5%). Plasminogen activator inhibitor-1 activity differed significantly between the sexes after adjustments for fat percentage and physical activity levels. Sex explained 10% and muscle mass 1% of the variance in plasminogen activator inhibitor-1 activity. Fibrinogen was significantly higher in girls than in boys (before adjustment for fat percentage), in overfat than in lean children and in stunted than in the nonstunted children (even after adjustment for fat percentage). C-reactive protein, sex and height for age were predictors of fibrinogen. Thrombin–antithrombin complex was significantly higher in girls than in boys, but after separate adjustment for physical activity and fat percentage there were no significant differences. Fitness and muscle mass explained the variance in thrombin–antithrombin complex the best. No significant differences were seen between the groups for C-reactive protein and factor VIIIc. Overfatness, stunting and inactivity negatively influenced plasminogen activator inhibitor-1 activity, fibrinogen and thrombin–antithrombin complex possibly increased the risk for cardiovascular disease. These factors are modifiable through behavioural changes and optimal nutritional status throughout the early life.

Clustering of haemostatic variables and the effect of high cashew and walnut diets on these variables in metabolic syndrome patients.

We investigated the effect of a high walnut and cashew diet on haemostatic variables in people with the metabolic syndrome. Factor analysis was used to determine how the haemostatic variables cluster with other components of the metabolic syndrome and multiple regression to determine possible predictors. This randomized, control, parallel, controlled-feeding trial included 68 subjects who complied with the Third National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol criteria. After a 3-week run-in following the control diet, subjects were divided into three groups receiving either walnuts or cashews (20 energy%) or a control diet for 8 weeks. The nut intervention had no significant effect on von Willebrand factor antigen, fibrinogen, factor VII coagulant activity, plasminogen activator inhibitor 1 activity, tissue plasminogen activator activity or thrombin activatable fibrinolysis inhibitor. Statistically, fibrinogen clustered with the body-mass-correlates and acute phase response factors, and factor VII coagulant activity clustered with high-density lipoprotein cholesterol (HDL-C). Tissue plasminogen activator activity, plasminogen activator inhibitor 1 activity and von Willebrand factor antigen clustered into a separate endothelial function factor. HDL-C and markers of obesity were the strongest predictors of the haemostatic variables. We conclude that high walnut and cashew diets did not influence haemostatic factors in this group of metabolic syndrome subjects. The HDL-C increase and weight loss may be the main focus of dietary intervention for the metabolic syndrome. Furthermore, diet composition may have only limited effects if weight loss is not achieved.