Fibrin Network Formation Research Articles

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.

Dynamic Imaging of Fibrin Network Formation Correlated with Other Measures of Polymerization.

The clotting time or gel point is commonly used in a variety of clinical assays, but little has been known about what it represents structurally because of the limitations of imaging technologies. The necessity of fixation and dehydration for electron microscopy means that only final clot structures, often distorted by preparation artifacts, can be observed, and there are severe limitations of image quality for conventional light microscopy. For similar reasons, we know almost nothing about the branching and lateral aggregation of fibrin that are essential for clot or thrombus stability. Turbidity, light scattering, or clot stiffness are useful to follow the time course of polymerization but reflect only overall clot properties. We visualized in real time fibrin network formation in the hydrated state, using deconvolution microscopy, which allows optical sectioning without the bleaching that accompanies confocal microscopy. Thus, the events during polymerization could be followed quantitatively over long periods of time. Videos will demonstrate the major observations. Individual mobile fibers were observed before the gel point. After gelation, an initial fibrin network, or scaffold, was seen, which evolved over time by addition of new fibers and elongation and branching of others. Furthermore, some fibers in the network moved chaotically for some time. A detailed, quantitative morphological analysis of network formation was carried out by superposition of images from different time points colorized to distinguish changes. We quantified network formation by the number of branch points, and longitudinal and lateral growth of fibers as a function of time. The distributions of fibers that reached a maximum of longitudinal growth and branch point formation both had maxima at the gel point but, surprisingly, some longitudinal growth continued and new branch points appeared after the gel point, requiring modification of existing models of fibrin polymerization. The cumulative percentage of fibers reaching their final length and the number of branch points attained maximum values at the time corresponding to that at which the turbidity reached approximately 60% of its maximum. Lateral growth reached a plateau at the same time as turbidity. Measurements of clot mechanical properties revealed that the clots achieved maximum stiffness and minimum plasticity well after branch point, as well as length changes and lateral growth of fibers, were completed. These results provide new information on the time sequence of events during fibrin network formation, which is important to understand both clotting and thrombosis and to allow modulation of clot properties.

An In Vitro Study To Compare Argatroban with Bivalirudin, Lepirudin and Danaparoid Concerning the Effects on Fibrin Gel Permeability, APTT and INR.

Numerous thrombin/Xa-inhibitors have been used in prophylaxis/treatment of thromboembolic diseases. We were interested to compare argatroban (direct thrombin inhibitor, DTI) with bivalirudin (DTI), lepirudin (DTI) and danaparoid (indirect Xa inhibitor) concerning the effects on fibrin gel permeability (Ks), APTT and INR. We were also interested to assess whether the drug-induced impairment of haemostasis reacts differently to the addition of two coagulants-rVIII or rVIIa. Ks was measured in a normal plasma pool (NPP) mixed with one of the inhibitors in increasing concentrations. Coagulation was initiated by adding recombinant tissue factor (rTF, 5pM), purified phospholipids (PPL, 4mM) and CaCl2. Since the activity of rVIIa is dependent on the presence of activated platelets, rTF and PPL were replaced by washed-frozen platelets when the influence of rVIIa was evaluated. APTT and INR were tested in the same inhibitor-spiked NPP using reagents from Stago, France. We found that all inhibitors investigated made the fibrin network more porous in a concentration-dependent way. However, danaparoid was less potent to change Ks than argatroban or bivalirudin. When a comparison was performed between samples containing argatroban or bivaluridin, the former drug brought increases in Ks which appeared gentler than those by the latter. Regarding the effect by lepirudin, a dose-response curve in Ks assay was very steep. APTT and INR were prolonged by all the inhibitors examined, but only argatroban and bivaluridin led to prolongations which were significantly correlated to Ks. rFVIII overcame the anticoagulant effect on Ks to some degree. Similar influence by rVIIa was seen in samples containing argatroban or bivalirudin but not in those containing lepirudin. The effect of danaparoid on Ks was almost neutralised by addition of the two recombinant coagulants.Conclusions: In the in vitro study, argatroban, bivalirudin, lepirudin and danaparoid can inhibit thrombin generation and thus depress coagulation, shown as prolonged time-to-start of detectable fibrin formation and increased porosity of fibrin network. The latter effect may favour fibrinolysis partly by facilitating the transportation of fibrinolytic components. The steep dose-response curve for Ks shown in the samples containing lepirudin may implicate a narrow therapeutic window. Further work are needed to clarify 1) whether the dissimilarities between argatroban and other inhibitors concerning the effects on Ks, APTT or INR implicate different therapeutic effects or safety in patients; 2) whether the dose-dependent influence of argatroban/bivalirudin on APTT and INR can be shown in vivo, thereby determining if these two simple methods are sensitive enough for therapeutic monitoring; 3) whether rVIII or rVIIa can be used to treat patients with haemorrhagic complications to the therapies. Additionally, the fibrin gel assay which employs a tiny dose of TF (together with PPL) as the thrombin generation trigger to test the end stage of coagulation, i.e. fibrin network formation, may reflect the effects of multiple protease activation and inhibition in a physiologically relevant way. We consider that in contrast to the conventional methods such as APTT and INR, the fibrin gel assay can give additional and important information on drug effects, and thus contribute with important information in the development of new anticoagulant compounds.

Thrombus formation without platelets under inflammatory condition: An in vitro study

Platelet derived microvesicles, which are shed from platelets upon platelet activation, interact with monocytes in the blood. In this study the nature of this interaction was characterized in a model system with the monocytic cell line MM6 and isolated platelet derived microvesicles (PMV). The interaction of PMV with MM6 is separated in two consecutive steps, which are partially overlapped in time. In a first step there is an immediate conjugate formation with single MM6 and PMV, which was proved microscopically and by cytometry measurements. This process is dependent on CD62P, determined by an inhibition after pre-incubation with anti-CD62P. After a lag time of 4min this process is supplemented by an aggregate formation of single conjugates, which leads finally to one macroscopic visible aggregate. The Nature of this aggregate was characterized by immunohistochemistry and laser aggregatometry. An addition of GPRP blocks the formation of a fibrin network and also the aggregate formation, proving the necessity of fibrin network formation. This was also shown by diminishing the aggregate formation by addition of hirudin. Finally fluorescent microscopic images proved the necessity of a fibrin network holding MM6 cell/PMV aggregates together. Even pure PMV can form such an aggregate only visible as thin film and less stable as the cell PMV aggregate. The described process might be important in vivo causing thrombotic events without direct involvement of platelets. Especially in situations with extreme PMV levels, such as acute coronary heart disease, trauma and sepsis, these events could lead to the appearance of haemostatic complications.

Synergistic platelet integrin signaling and factor XII activation in poly- N-acetyl glucosamine fiber-mediated hemostasis

The polymer poly- N-acetylglucosamine (pGlcNAc) containing fiber material is becoming increasingly important as a topical agent for hemostasis at wound sites. The pGlcNAc polymeric fiber provides hemostasis through redundant mechanisms that include platelet activation for fibrin network formation. The research presented here better defines the mechanism for the effect of pGlcNAc containing fibers on platelet-mediated processes. Adsorption experiments demonstrated that pGlcNAc fibers tightly bind most major plasma proteins and a specific sub-set of platelet surface proteins, including the integrin β 3 subunit (CD61) and the von Willebrand receptor GP1b (CD42b). The result of this interaction is a platelet-dependent acceleration of fibrin gel formation. Accelerated fibrin polymerization is sensitive to factor XII inhibition by corn trypsin inhibitor and integrin inactivation with integrilin. Confocal microscopy studies show that when platelet integrins contact plasma protein-saturated pGlcNAc fibers, an increase in intracellular free calcium for platelet activation occurs to drive surface expression of phosphatidyl serine (PS). Thus, a catalytic surface for thrombin generation and accelerated fibrin clot formation results from the interaction of platelets with pGlcNAc. These findings, when considered with the observation that pGlcNAc fibers also induce red blood cell agglutination and vasoconstriction, provides an explanation for the ability of the pGlcNAc material to provide hemostasis in a wide variety of clinical applications.

Improving mechanical stability and density distribution of hepatocyte microcapsules by fibrin clot and gold nano-particles

Bio-artificial livers (BAL) with microencapsulated hepatocytes have the typical limitations in maintaining hepatocyte functions, mechanical stability and uniform perfusion in packed or fluidized-bed bioreactors. We have previously developed microcapsules with enhanced hepatocyte functions. Here we have introduced a fibrin network inside microcapsules by (1) mixing collagen and fibrinogen with the encapsulated hepatocytes to support the cells; (2) submerging the microcapsules into a thrombin solution to induce the formation of an insoluble fibrin network inside the microcapsules. Fracture analysis on the microcapsules revealed significant improvement in mechanical stability. We have also introduced different amounts of gold nano-particles into microcapsules to achieve different densities for uniform bioreactor perfusion. These gold nano-particles also improved the mechanical stability of the microcapsules. Both the fibrin network and gold nano-particles exhibited the additional benefits of enhancing certain bio-functions of the encapsulated hepatocytes. The applications of these improved microcapsules in the development of bio-artificial livers are discussed.

Interactions between surface proteins of Streptococcus pyogenes and coagulation factors modulate clotting of human plasma

Invasive and toxic infections caused by Streptococcus pyogenes are connected with high morbidity and mortality. Typical symptoms of these infections are hypotension, edema formation, tissue necrosis, and bleeding disorders. Here we report that components of the coagulation system including fibrinogen, factors V, XI, and XII, and H-kininogen, are assembled at the surface of S. pyogenes through specific interactions with bacterial surface proteins. In plasma environment, absorption of fibrinogen by S. pyogenes causes a hypocoagulatory state resulting in prolonged clotting times and impaired fibrin network formation. Moreover, the binding of coagulation factors and the subsequent activation of the coagulation system at the bacterial surface lead to the formation of a fibrin network covering S. pyogenes bacteria adhering to epithelial cells. The results suggest that interactions between S. pyogenes and components of the coagulation system contribute to some of the symptoms seen in severe infections caused by this important human pathogen.

Effects of homocysteine thiol group on fibrin networks: another possible mechanism of harm

On the basis of growing clinical evidence, it is well known that elevated plasma homocysteine (Hcy) levels are associated with higher risk of venous and arterial thrombosis. Several experimental studies have been carried out in order to elucidate the mechanisms involved that still remain unclear. The aim of our study was to evaluate the homocysteine effects on formation and structure of plasmatic fibrin network. We also assayed homocystine and cysteine to determinate possible participation of thiol group in the tested activity. Aliquots of a pool of plasma incubated separately with sulfur compounds were clotting with thrombin. Fibringeneration and fibrin networks were evaluated by kinetic studies and scanning electronic microscopy, respectively. No significant differences were observed on fibrin generation of the substances assayed in comparison to control. The scanning electronic microscopy showed that Hcy-associated networks were different from control, with shorter, thicker and more branched fibers, resulting in a more compact structure and probably more resistant to fibrinolysis. The thiol group would be involved in this effect. Our findings would be a new contribution to elucidate the mechanisms involved in harmful effects associated to hyperhomocysteinemia.

Abnormal Formation of Fibrin Network from X-ray Irradiated Fibrinogen: Two-color Fluorescence Analysis of the Dynamic Process and the Incorporation of Fibrinolytic Components into Fibrin by Confocal Laser-Scanning Microscopy

Abnormal Formation of Fibrin Network from X-ray Irradiated Fibrinogen: Two-color Fluorescence Analysis of the Dynamic Process and the Incorporation of Fibrinolytic Components into Fibrin by Confocal Laser-Scanning Microscopy

The mechanism of anticoagulant activity of a novel heparinoid sulfated glucoside-bearing polymer

In our previous study on glycoside polymer, it was discovered that sulfated poly(glucosyl-oxyethyl methacrylate) [poly(GEMA)-sulfate], which bears sulfated D-(+)-glucose, exhibited anticoagulant activity. The anticoagulant activity of poly(GEMA)-sulfate in solution was prolonged by increasing the dose or degree of sulfation of the polymer. In this study, to recognize the mechanism of anticoagulant activity of poly(GEMA)-sulfate, we estimated the binding capacity of antithrombin III to thrombin and some in vitro clotting tests in the presence of poly(GEMA)-sulfate. These results revealed that poly(GEMA)-sulfate had an anticoagulant mechanism which differed to that of heparin. We concluded that the anticoagulant activity of poly(GEMA)-sulfate is responsible for inhibiting fibrin network formation by the insoluble ion complex between fibrinogen and poly(GEMA)-sulfate.

The effects of pH on the generation of turbidity and elasticity associated with fibrinogen-fibrin conversion by thrombin are remarkably influenced by sialic acid in fibrinogen.

In fibrinogen-fibrin conversion by thrombin, the polymerization of a fibrin monomer is accompanied by gelation and an increase turbidity. Since sialic acids at the terminal of the carbohydrate chains bound to fibrinogen are part of the low affinity calcium binding site necessary for polymerization, they are closely involved in the network structure of fibrin clots. Fibrin clots derived from asialofibrinogen exhibited definite differences in turbidity and elasticity compared with those derived from intact fibrinogen, and were markedly dependent on the pH during the reaction. The turbidity during polymerization of fibrin, evaluated according to the absorbance at 350 nm, was maximum at pH 6.5-7.0, but it decreased in the other pH ranges, with the changes being unremarkable at higher pH levels but remarkable at lower pH ranges. The turbidity of fibrin derived from asialofibrinogen was far higher than that from intact fibrinogen near neutrality, but decreased rapidly and was lower than in intact fibrinogen at higher and lower pH ranges. Concerning the elasticity evaluated by thromboelastography, the coagulation time (k) and the maximum amplitude (ma) were lower in asialofibrinogen, indicating a deterioration of the clotting function of fibrinogen with the loss of sialic acid. These results suggest that sialic acid bound to fibrinogen is closely related to the fibrin network formation in blood coagulation, which is the most important function of fibrinogen, and plays a functional role in the stabilization of fibrin clot formation against environmental changes, including pH.

In vitro non-thrombogenicity of a thrombin-substrate-immobilized polymer surface by the inhibition of thrombin activity

Derivatives of thrombin substrate were synthesized and immobilized on a poly(acrylic acid)-grafted polyurethane film. The carboxyl terminal of the thrombin substrate peptide should be blocked for a higher inhibitory effect of the thrombin activity. Immobilization of the thrombin substrate peptide enhanced adsorption and inactivation of thrombin on the polymer film to prolong the time for fibrin network formation, and suppressed adhesion and deformation of platelets on the film. Consequently, in vitro thrombus formation on the polymer film was strongly suppressed.

Experimental Study of Effects of the Ultrasonic Surgical Aspirator on the Vascular System

The effects of an ultrasonic surgical aspirator on the vascular system was evaluated in 12 mongrel dogs. The pathologic effects of the device were investi gated in femoral vessels by light and electron scanning microscopies and the physiologic effects by measuring the internal mammary arterial and femoral venous blood flow. Changes in the intimal surface of arteries (platelet aggregation and fibrin network formation) were more frequent at higher vibration amplitudes, while such changes were less common in the vein. Disintegration and loss of the vascu lar wall did not extend beyond the adventitia. There was no obvious change in the blood flow pattern or flow rate after administration of ultrasonic vibrations. The results showed that by keeping the output below 110 microns and using the aspirator together with other conventional surgical devices for dissection, damages to the vascular wall may be held to a minimum. This device can be a very useful supplementary tool in cardiovascular surgery.

Platelet Microtubules in Clot Structure Formation and Contractile Force Generation: Investigation of a Controversy

Whether platelet microtubules are involved in clot retraction/contraction has been controversial. To address this question we have simultaneously measured two clotting parameters, clot structural rigidity and isometric contractile force, using a rheological technique. For recalcified PRP clots these two parameters began rising together at about 15 min after CaCl2 addition. In the concentration range affecting microtubule organization in platelets, colchicine, vinca alkaloids and taxol demonstrated insignificant effects on both clotting parameters of a recalcified PRP clot. For PRP clots induced by adding small amounts of exogenous thrombin, the kinetic curves of clot rigidity were biphasic and without a lag time. The first phase corresponded to a platelet-independent network forming process, while the second phase corresponded to a platelet-dependent process. These PRP clots began generating contractile force at the onset of the second phase. For both rigidity and force parameters, only the second phase of clotting kinetics was retarded by microtubule affecting reagents. When PRP samples were clotted by adding a mixture of CaCl2 and thrombin, the second phase clotting was accelerated and became superimposed on the first phase. The inhibitory effects of microtubule affecting reagents became less pronounced. Thrombin clotting of a two-component system (washed platelets/purified fibrinogen) was also biphasic, with the second phase being microtubule-dependent. In conclusion, platelet microtubules are important in PRP clotted with low concentrations of thrombin, during which fibrin network formation precedes platelet-fibrin interactions. On the other hand they are unimportant if a PRP clot is induced by recalcification, during which the fibrin network is constructed in the presence of platelet-fibrin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

The Dynamic Viscoelastic Study of Clotting Reaction in Fibrinogen-Thrombin Systems

The reaction order in the formation of fibrin network in the fibrinogen-thrombin solution is determined by the analysis of clotting curves obtained by dynamic viscoelasticity measurements at various concentrations of fibrinogen. It is concluded from the rheological point of view that the clotting curves for elastic modulus and loss modulus can be represented by superposition of two first-order reaction processes with different rate constants respectively. The rate constants obtained from the loss modulus are larger than those from the elastic modulus. The saturated values of the elastic moduli in the two processes increase with the increase of the concentration of fibrinogen. The amplitude dependence of the elastic modulus of the gel supports the presence of the two processes.The elastic modulus in the first process is proportional to the number of crosslinks between the fibrin fibers, and that in the second process is related to the non-linear elongation of fibrin fibers between the crosslinks. The loss modulus in the first process is associated with the polymerization of fibrin, and that in the second process is related to the increasing interaction between the crosslinked fibrin fibers.