Possible mechanisms through which dietary pectin influences fibrin network architecture in hypercholesterolaemic subjects.

It is suspected that not only fibrinogen concentration but also the quality of fibrin networks may contribute to cardiovascular risk. Evidence is accumulating that a "prudent" diet may protect against diseases associated with raised clotting factors. The effect of diet on fibrinogen is, however, still controversial. In a previous study performed in our laboratory, it was shown that dietary pectin influences fibrin network architecture in hypercholesterolaemic men without causing any changes in fibrinogen concentration. To elucidate the possible mechanisms, it was necessary to study the possibility that pectin may itself have indirect effects on fibrin network architecture. Pectin is fermented in the gastrointestinal tract to acetate, propionate, and butyrate. In humans, only acetate reaches the circulation beyond the liver. This investigation primarily examined the possibility that pectin may, through acetate, influence fibrin network architecture in vivo. The effects of pectin and acetate supplementation in hypercholesterolaemic subjects were compared. Furthermore, this study also aimed at describing the possible in vitro effects of acetate on fibrin network architecture. Two groups of 10 male hyperlipidaemic volunteers each received a pectin (15 g/day) or acetate (6.8 g/day) supplement for 4 weeks. Acetate supplementation did not cause a significant change in plasma fibrinogen levels. As in the pectin group, significant differences were found in the characteristics of fibrin networks developed in plasma after 4 weeks of acetate supplementation. Fibrin networks were more permeable (from 213+/-76 to 307+/-81 x 10(11) cm2), had lower tensile strength (from 23+/-3 to 32+/-9% compaction), and were more lyseable (from 252+/-11 to 130+/-15 minutes). These results strongly suggest that the effect of pectin on network architecture could partially be mediated by acetate. Progressive amounts of acetate were used in vitro to investigate the possibility that acetate may be directly responsible for changes that occurred in fibrin network architecture in the plasma medium. Results indicated that acetate influenced fibrin network architecture directly. From the results, it seems highly possible that acetate may be responsible in part for the beneficial effects of pectin supplementation in vivo. It is evident that pectin or acetate supplementation can be useful during the treatment or prevention of some clinical manifestations, especially those associated with raised total cholesterol and possibly also plasma fibrinogen.

Electrophoretic transfer of proteins across polyacrylamide membranes.

The electrophoretic transfer of purified proteins has been examined in a Gradiflow "Babyflow BF100" unit. A number of factors affect protein separation within this preparative electrophoresis system. We established that the rate of protein transfer was proportional to the applied voltage. The transfer is slowest at the isoelectric point (pI) and increased the further away the pH was from the pI of the protein. Protein transfer was found to be independent of the ionic strength of the buffer, for buffers that excluded the addition of strong acids or strong bases or sodium chloride. Transfer decreased as the pore size of the membrane decreased. Finally, transfer was inhibited at high salt concentrations in the protein solution, but remained unaffected when urea and non-ionic detergents were added to the solution. To increase the speed of protein separations, buffers with low conductivity should be used. A pH for the optimal separation should be selected on the basis of the relative pI and size of the target proteins and that of the major contaminants.

Dietary pectin influences fibrin network structure in hypercholesterolaemic subjects.

Fibrinogen is an important risk factor for atherosclerosis, stroke and cardiovascular heart disease (CHD). This risk is increased when associated with a high serum cholesterol. Furthermore, it is also believed that not only fibrinogen concentration, but also the quality of fibrin networks may be an important risk factor for the development of CHD. CHD and stroke as a result of atherosclerosis, plus the related problems of hyperinsulinaemia, hyperlipidaemia and hypertension are strongly related to diet. The "western" diet, defined by low fibre and high fat, sucrose and animal protein intakes, appears to be a major factor leading to death. It has been established that the water-soluble dietary fibre, pectin, significantly decrease the concentration of serum cholesterol levels. Evidence is also accumulating that a diet rich in fibre may protect against diseases associated with raised clotting factors. This investigation studied the possible effects of pectin on fibrinogen levels and fibrin network architecture. Two groups of 10 male hyperlipidaemic volunteers each, received a pectin supplement (15 g/day) or placebo (15 g/day) for 4 weeks. Lipid and fibrin network structure variables were measured at baseline and the end of supplementation. Pectin supplementation caused significant decreases in total cholesterol, low-density lipoprotein cholesterol, apolipoprotein A & B and lipoprotein (a). Significant changes in the characteristics of fibrin networks developed in the plasma of the pectin supplemented group indicated that networks were more permeable and had lower tensile strength. These network structures are believed to be less atherogenic. It is suspected that pectin modified network characteristics by a combination of its effects on metabolism and altered fibrin conversion. This confirms the therapeutic possibilities of dietary intervention. Furthermore, this study also showed that changes in plasma fibrinogen need not be present to induce alterations in fibrin network architecture.

Interaction of endothelial cells and fibroblasts with modified fibrin networks: role in atherosclerosis.

Fibrinogen has been recognised in recent years as an independent risk factor in athero/thrombogenesis. However, the mechanism by which elevated fibrinogen translates into higher incidence of atherosclerosis is not known. One possible mechanism may be through the modification of fibrin. While it is already known that fibrin network is altered in disease states like peripheral vascular disease, diabetes, hypercholesterolaemia and myocardial infarction, the influence of altered fibrin network structure on growth and function of endothelial cells (EC) and fibroblasts (FB) requires investigation. Fibrin network structure in plasma clots was modified by changing pH and characterised using established biophysical methods. PGI(2), von Willebrand Factor (vWF), t-PA and PAI-1 were measured to evaluate changes in cell function induced by modified fibrin structure. In general, networks composed of thin fibres induced growth over their entire layer. Networks composed of thick fibres and open matrix promoted infiltration of cells into gel matrix and growth of macrovascular structures. Furthermore, thin fibres promoted a more prothrombotic environment as observed from changes in cell biochemical function. Fibrin, whilst initially acting as a scaffolding for cellular and biochemical processes, may also alter cell function and determine the progress of atherosclerosis.

Compaction as a method to characterise fibrin network structure: kinetic studies and relationship to crosslinking.

While it has been shown that compaction is inversely correlated to the Young's modulus of elasticity of the network and to the final strength at break, the relationship between collapsibility of the network subjected to a standardised centrifugal force and the degree of crosslinking (i.e., compaction) has not been properly addressed. Three sets of networks developed from plasma and pure fibrinogen solution, with varying degree of crosslinking induced by the addition of different amounts of calcium, were subjected to centrifugation at 8000g. In networks developed from plasma, compaction correlated with the degree of crosslinking. Whilst totally crosslinked clots were most resistant to collapse, partially crosslinked clots were far less resistant to collapse. In purified fibrinogen, however, the effect crosslinking was all or nothing. Both totally crosslinked and partially crosslinked clots were equally resistant to collapse. Calcium induced crosslinking provides fibrin with the required strengthening of the fibrin network. However, there are also fibre to fibre interactions as can be observed in networks developed in the presence of drugs like gliclazide. Compaction is a simple technique which can be used in any clinical laboratory to characterise the degree of crosslinking and also the tensile properties of the network.

Colloid determination of fibrin network permeability.

The effects of polymers, dextran and polyvinylpyrolidone (PVP) and of albumin on the permeability of thrombin-induced fibrin networks developed in plasma were examined. Both PVP and dextran increased the network permeability and turbidity and increased the fibrin fibre thickness. The effect was molecular weight dependent. Derivation of the dimensionless permeability (permeability/fibre radius2) indicated that the increase in network permeability was mainly from altered arrangement of fibres and not from increased fibre thickness. The effects of albumin on network structure were similar to those of the polymers. Scanning electron microscopy of networks developed in plasma under the influence of dextran and poloxamer 188 showed fibres with increased thickness and a coarse nodular appearance. There was an increased tendency for fibres to be aggregated into clumps. It is suggested that during polymerization fibrin fibres and fibrin polymerization intermediaries behave as colloidal particles. Attractive forces between the particles are generated by soluble macromolecules such as plasma proteins or polymers. Attractive forces increase the thickness of fibrin fibres and induce a more permeable arrangement of the fibres in the network. The most likely colloidal mechanism is depletion flocculation. This would account for (1) the molecular weight dependence and concentration dependence of the effects of macromolecules, (2) the effects of macromolecules which do not bind to fibrin, (3) the effects of the surfactant poloxamer 188. Depletion flocculation may be a significant mechanism for biological regulation of fibrin network permeability by non-specific macromolecules such as soluble proteins or fibrin intermediaries.

Erythrocyte aggregation and erythrocyte deformability modify the permeability of erythrocyte enriched fibrin network.

Intravascular thrombus formed under low shear conditions consists of red cells enmeshed within a fibrin network. Since red cells reduce the permeability of fibrin network by surface drag and by volume occupancy the significance of red cell aggregability and deformability in network permeability needs examination. In this study networks were developed by the addition of thrombin to washed red cells suspended in platelet free plasma. The effects of the polymers polyvinylpyrrolidone (PVP) and poloxamer 188 on network permeability were compared to gauge the influence of red cell aggregation. Both polymers increase network permeability by an action on fibrin polymerisation but PVP alone enhances red cell aggregation. PVP was found to increase network permeability significantly both by increasing the permeability of the fibrin component of the network and by increasing red cell aggregation and thus reducing red cell surface drag. In separate experiments red cells were pre-treated with heat, glutaraldehyde, or diamide to reduce cell deformability. Decreased cell deformability caused significant reductions in network permeability. This was ascribed to the reduced aggregability of hardened red cells. Red cell aggregation during coagulation enhances molecular transport through modifying the network. This may have implications for the penetration of fibrinolytic agents.

The permeability of fibrin network developed in human plasma.

Fibrin network permeability has an important role in thrombosis and inflammation since it influences the rate of transport of macromolecules through the network by convection. The conditions of polymerization of fibrin determine the network permeability and this has been attributed to variability in fibrin fibre thickness. Inconsistencies between values for fibrin fibre thickness derived from turbidity and permeability were examined. Networks were developed from human plasma by the addition of thrombin and network polymerization was modified pharmacologically. Dextran (MW 70,000) and poloxamer 188 both increased, and lauryl sulphate decreased, network permeability and network turbidity. Network fibre thickness was consistently higher when derived from permeability than from turbidity. Network permeability was significantly more susceptible to pharmacological manipulation by these agents than network turbidity. These inconsistencies were attributed to variation in the arrangement of the network fibres such as inhomogeneity of network fibre distribution and to fibre aggregation or alignment. Collectively these factors prohibit the derivation of fibrin fibre thickness from permeability. The dimensionless permeability (network permeability/(fibre radius)2) was used as an index of network fibre arrangement and found to be readily modified pharmacologically. Physiological and pharmacological regulation of fibrin network permeability may be predominantly mediated through modification of fibre arrangement and not through fibre thickness.

Effects of gliclazide on fibrin network.

Fibrin network structure is altered by diabetes, peripheral vascular disease, and by some drugs. The antidiabetic drug, gliclazide, increases fibrin fiber thickness but reduces whole network permeability. The networks are, however, more lysable. These effects are further examined in this study using electron microscopy. Changes were observed in protein concentrations in fibrin fibers, in fibrin fiber alignment and in fiber porosity. These results show that gliclazide modifies fibrin monomer polymerization so that the fibrin network is rendered more susceptible to fibrinolysis. This pharmacological action of gliclazide may be useful in the treatment of thromboembolism.

Complement activation: a new participant in the modulation of fibrin gel characteristics and the progression of atherosclerosis?

The activation of the complement system has been implicated as an important mechanism in the progression of atherosclerosis. The relationship between fibrin gel characteristics and complement activation has, however, not been investigated. Zymosan-treated plasma with 87% complement activation as measured by C3a production using radioimmunoassay was found to induce changes in biophysical characteristics of fibrin network developed in both plasma and purified fibrinogen solution. Using already established methods to measure fibre thickness (mu T), permeability (T) and compaction, it was found that these networks are made of thinner fibres with increased tensile strength arranged into a matrix which renders the networks less permeable. Such networks are resistant to streptokinase-induced lysis. Fibrin networks with thin fibres in turn induced 3.7 times higher production of C3a than unmodified networks. These observations suggest the existence of positive feedback; complement activation induces major alterations in fibrin structure which in turn can induce further activation of the complement system. The detailed mechanism underlying this interrelationship is not clear at present, but this positive feedback system may play an important role in establishing a fibrin infrastructure ultimately responsible for the progression of atherosclerosis.

The significance of red cell surface area to the permeability of fibrin network.

Thrombus formed under low shear conditions in the intravascular compartment consists of red cells enmeshed within a fibrin network framework. The permeability of the network determines the rate of molecular transport by convection. The effect of red cells on the permeability of the fibrin network was examined in networks developed from red cell suspensions in platelet-rich (PRP) and platelet-poor plasma (PPP). Red cell-rich networks developed in PRP were significantly more permeable than those developed in PPP. Network permeability decreased linearly with increase in hematocrit due to volume exclusion and cell surface hydraulic drag. The hydraulic resistance of the red cells was similar in order of magnitude to that of the fibrin fibers. The hydraulic resistance was calculated to be dependent on the surface area of the red cells, as well as on red cell concentration. Calculation of red cell surface area from permeability (45-90 microns2) was found to be lower than the known surface area (145 microns2). From these studies, it is suggested that red cells entrapped within the network are aggregated. Aggregation is promoted by low shear plasma conditions during fibrin polymerization and by fibrin polymerization intermediaries. The degree of red cell aggregation regulates the hydraulic resistivity of the red cells, and the fibrin fiber structure regulates the hydraulic resistivity of the fibrin network. Both are significant determinants of the network permeability and, therefore, of molecular transport in thrombi.

Effects of Poloxamer 188 on fibrin network structure, whole blood clot premeability and fibrinolysis.

The effects of Poloxamer 188 (0-5 mg/ml) on the permeability, turbidity, compaction, and fibrinolysis of fibrin network developed in human plasma, and on the permeability and fibrinolysis of network developed in whole blood were examined. Poloxamer 188 was found to increase network permeability and compaction in plasma. In networks in plasma, effects on the fibre mass-length ratio from turbidity and fibrinolysis with recombinant tissue plasminogen activator were small. Poloxamer did not alter the fibrinolysis with streptokinase. The increase in fibrin network permeability at low poloxamer concentrations was not attributable to an increase in fibre thickness, but results from alterations in the arrangement of fibrin fibres. Poloxamer also significantly increased the permeability of networks developed in whole blood. Studies with the platelet inhibitor cytochalasin B demonstrated that this effect in whole blood networks was partly from facilitation of platelet induced clot retraction. Poloxamer was not found to affect streptokinase induced fibrinolysis of whole blood networks. The effects of poloxamer support the hypothesis that depletion flocculation of fibrin intermediaries by soluble macromolecules is a significant determinant of network permeability. The therapeutic use of poloxamer will result in altered fibrin function in particular its permeability and mechanical stability. These alterations may contribute to its described antithrombotic and rheological effects.

Studies on fibrin network structure in human plasma. Part One: Methods for clinical application.

Methods based on turbidity and permeability, for measurement of mass-length ratio of fibrin fibres developed in pure fibrinogen solution, have been evaluated in respect of their applicability to human plasma. Theoretical assumptions made in the calculation of mass-length ratios in plasma have been critically examined. Methods of handling plasma, reproducibility of technique and the influence of age and sex have been investigated. The anticoagulant used, as well as other factors such as time, venepuncture and effects of calcium and fibrinolytic inhibitors are fully explored. With suitable standardization the methods are acceptable for application to clinical studies and are reproducible.

Studies on fibrin network structure in human plasma. Part II--Clinical application: diabetes and antidiabetic drugs.

Using measurements of fibrin fibre thickness (microT) derived from turbidity and permeability (tau) of clotted plasma, it has been found that glucose in vitro added to plasma decreases permeability of the network despite unaltered fibrinogen conversion. Fibrin fibre thickness (microT) in uncontrolled diabetes is found significantly reduced. In diabetic plasma the degree of conversion to fibrin is similar to that in age and sex matched plasma from non-diabetics: the effect on fibrin network and fibre thickness probably arises from glycosylation of fibrinogen. Studies with Gliclazide, Metformin, Glibenclamide and insulin have shown that while all other drugs tested have no effect, Gliclazide increases fibrin fibre thickness (microT) significantly, diminishes tensile strength and reduces permeability. In separate experiments lysability of 125I-labelled fibrin networks developed in the presence of all four hypoglycaemic agents by tissue activator was tested. Networks developed in the presence of Metformin were found to lyse more quickly, followed by insulin and Gliclazide. Alterations induced in fibrin networks in diabetes may be nullified by some oral hypoglycaemic agents such as Gliclazide and not by others. Whether nullification of such changes has long-term effects in reducing the incidence of vascular disease in diabetics remains to be established.

Studies on fibrin network structure: the effect of some plasma proteins.

Significant differences were found between characteristics of networks developed in plasma and those developed in pure fibrinogen solution. Networks in plasma have thicker fibres, are more permeable and have lower tensile strength. In this investigation determinants of network structure under physiological conditions of clotting have been examined in an attempt to account for the differences in network structure in plasma and fibrinogen solution. Effect of antithrombin III and of physiological concentrations of fibronectin, albumin and gamma-globulin on fibrin network structure was examined using mass-length ratio (microT) from turbidity, bulk permeability of networks (tau) and kinetics of network development. At physiological concentrations effect of ATIII was too pronounced to allow this method to quantify changes in network characteristics. Whilst fibronectin and gamma-globulins increased fibrin fibre thickness and network permeability, albumin caused opposite effects. It is concluded that the final network structure is determined by kinetics of fibrin fibre growth and is highly responsive to the presence of plasma proteins.

The effects of some plasma proteins on fibrin network structure.

Pronounced differences are found between characteristics of networks developed in plasma and those developed in pure fibrinogen solution. Networks in plasma have thicker fibres, are more permeable and have lower tensile strength. In this investigation the role of some plasma proteins as determinants of network structure under physiological conditions of clotting has been examined in an attempt to account for the differences in network structure in plasma and fibrinogen solution. The effect of physiological concentrations of antithrombin III, fibronectin, albumin, alpha globulin and gamma globulin on fibrin network structure was examined using mass-length ratio (muT) from turbidity, bulk network permeability (tau) and kinetics of network development. It was found that differences in fibrin network structure developed in plasma and pure fibrinogen solution could not be accounted for by alterations induced in network properties by albumin, gamma globulin, alpha globulin, fibronectin and antithrombin III. It is concluded that the final network structure is determined by the kinetics of fibrin fibre growth and is highly responsive to the presence of plasma proteins.

Comparison of fibrin networks in plasma and fibrinogen solution.

Mass-Length ratio of fibrin fibres (microT and microP) derived independently from turbidity and permeability respectively, in networks made in fibrinogen solution and plasma, have been compared under similar conditions of clotting. Amount of fibrinogen conversion to fibrin was similar in both systems when high thrombin concentrations were used. But networks in plasma had significantly thicker fibres (higher microT and microP) than those in fibrinogen solution. This difference arises from differing kinetics of fibrin assembly in the two systems. When lower thrombin concentration is used, fibrin fibre thickness (as indicated by microP and microT) is increased in fibrinogen solution but the fibrin content of the network remains unaltered. In plasma, on the other hand, the fibrin content of the network is decreased, microT remains relatively unchanged while microP increases. The thrombin concentration dependence of the fibrin content of the network in plasma and the bimodal distribution of fibrin fibre thickness explain the breakdown in correlation between microP and microT.

Effect of temperature, pH and ionic strength and composition on fibrin network structure and its development.

Networks were developed in fibrinogen solution under pathophysiological conditions of clotting. Mass-length ratio (a measure of fibre thickness) was derived independently from the turbidity (microT) and from the permeability (microP) of the network. Kinetics of network growth were investigated turbidimetrically. Physiological concentrations of Ca++and Mg++increased microT while those of K+, Na+, Cl-, HCO-3, H2PO-4 and SO--4 had no effect. As pH and ionic strength were increased stepwise within the pathophysiological range, network development was delayed. Under these conditions the turbidity curves did not cross and both microT and microP were progressively decreased. When temperature was lowered, although network growth was delayed, the turbidy curves crossed: the equilibrium turbidity was higher at lower temperatures. It appears that while pH and ionic strength affect network structure by influencing fibrin assembly, lowering the temperature influences both the rate of fibrin monomer generation and fibrin assembly.