Thick Fibrin Fibers Research Articles

Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin

BackgroundThe aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (μm2), the area of the fibrin fibers (μm2), and the width of the fibrin fibers (μm) were determined for the dog clots. The platelet area (μm2), the area of fibrin fibers (μm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots.ResultsCat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB.ConclusionsThe findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.

Recombinant Human Fibrinogen That Produces Thick Fibrin Fibers with Increased Wound Adhesion and Clot Density

Human fibrinogen is a biomaterial used in surgical tissue sealants, scaffolding for tissue engineering, and wound healing. Here we report on the post-translational structure and functionality of recombinant human FI (rFI) made at commodity levels in the milk of transgenic dairy cows. Relative to plasma-derived fibrinogen (pdFI), rFI predominantly contained a simplified, neutral carbohydrate structure and >4-fold higher levels of the γ'-chain transcriptional variant that has been reported to bind thrombin and Factor XIII. In spite of these differences, rFI and pdFI were kinetically similar with respect to the thrombin-catalyzed formation of protofibrils and Factor XIIIa-mediated formation of cross-linked fibrin polymer. However, electron microscopy showed rFI produced fibrin with much thicker fibers with less branching than pdFI. In vivo studies in a swine liver transection model showed that, relative to pdFI, rFI made a denser, more strongly wound-adherent fibrin clot that more rapidly established hemostasis.

Oxidation Inhibits Iron-Induced Blood Coagulation

Blood coagulation under physiological conditions is activated by thrombin, which converts soluble plasma fibrinogen (FBG) into an insoluble clot. The structure of the enzymatically-generated clot is very characteristic being composed of thick fibrin fibers susceptible to the fibrinolytic degradation. However, in chronic degenerative diseases, such as atherosclerosis, diabetes mellitus, cancer, and neurological disorders, fibrin clots are very different forming dense matted deposits (DMD) that are not effectively removed and thus create a condition known as thrombosis. We have recently shown that trivalent iron (ferric ions) generates hydroxyl radicals, which subsequently convert FBG into abnormal fibrin clots in the form of DMDs. A characteristic feature of DMDs is their remarkable and permanent resistance to the enzymatic degradation. Therefore, in order to prevent thrombotic incidences in the degenerative diseases it is essential to inhibit the iron-induced generation of hydroxyl radicals. This can be achieved by the pretreatment with a direct free radical scavenger (e.g. salicylate), and as shown in this paper by the treatment with oxidizing agents such as hydrogen peroxide, methylene blue, and sodium selenite. Although the actual mechanism of this phenomenon is not yet known, it is possible that hydroxyl radicals are neutralized by their conversion to the molecular oxygen and water, thus inhibiting the formation of dense matted fibrin deposits in human blood.

Differences in fibrin fiber diameters in healthy individuals and thromboembolic ischemic stroke patients

Cerebrovascular disease is one of the leading causes of death and the cause of long-term adult disability. An important characteristic of thromboembolic ischemic stroke is a prothrombotic or hypercoagulable state and altered fibrin clot structure, whereas a resistance to fibrinolysis is also present. An expansive fibrin network is created when adding thrombin, and in stroke, the network appears thickened, netted and matted, compared with that of healthy individuals. Although this is clearly visible in micrographs of patients, there is a need to quantify the changes. The current study, therefore, investigates fibrin fiber diameters in stroke patients and compares it to healthy individuals. The fiber diameters were measured in nanometres, with University of Texas Health Science Center at San Antonio (UTHSCSA) Image Tool. A total of 100 measurements were done for each of the 12 patients in the healthy control group, and the same number of measurements was done for 12 stroke patients. These measurements were statistically analysed with NCSS 2007, using a significance level of 0.05. Normality was assessed with the Shapiro-Wilk W test and the thickest and thinnest fiber of each individual in the two groups was quantified and differences between groups were assessed with the Student's t-test. Results showed that there is a statistical difference in fibrin fiber thickness during thromboembolic ischemic stroke. We conclude that the changed coagulation and hemostasis, typically associated with stroke, causes a statistically relevant change in fibrin thickness, and that this netted and matted network is more resistant to lyses.

Dysfibrinogenemia in childhood: two cases of congenital dysfibrinogens

A 2-year-old asymptomatic boy and his relatives were investigated for a suspected fibrinogen mutation after coagulation tests revealed a decreased functional fibrinogen level (family A). Eight-year-old and 1-year-old asymptomatic brothers were investigated for a suspected fibrinogen mutation after coagulation tests revealed a decreased functional fibrinogen level and prolonged thrombin time (family B). To identify whether genetic mutations were responsible for these dysfibrinogens, DNA extracted from the blood was analyzed. Fibrin polymerization and fibrinolysis were measured by a turbidimetric method at 450 nm. DNA analysis was performed by the Sanger method. Mass spectroscopy was performed on a Biflex IV mass spectrometer. DNA sequencing showed the heterozygous point mutation Aα Arg16His in the fibrinogen of family A and the heterozygous point mutation Aα Arg16Cys in the fibrinogen of family B. Kinetics of fibrinopeptide release, fibrinolysis, and fibrin polymerization were impaired in the carriers of the mutations in both families. Mass spectroscopy showed the presence of mutant fibrinogen chains in circulation. Scanning electron microscopy revealed thicker fibrin fibers, differing significantly from the normal control in both cases. Two cases of asymptomatic dysfibrinogenemias, found by routine coagulation testing, were genetically identified as new cases of fibrinogen variants Aα Arg16His and Aα Arg16Cys.

Unfavorably altered fibrin clot properties in patients with active rheumatoid arthritis

Objective Altered fibrin clot properties have been reported in cardiovascular diseases (CVD) and inflammatory states. Given increased prevalence of CVD in patients with rheumatoid arthritis (RA), we investigated whether fibrin characteristics are also altered in RA patients. Patients and methods We studied 46 consecutive RA patients versus 50 controls matched for age and gender. Ex vivo plasma clot permeability, turbidity, tissue-type plasminogen activator (tPA)-induced fibrinolysis, and scanning electron microscopy (SEM) images of clots were evaluated. Results Patients with RA had lower clot permeability, faster clot formation, higher maximum clot absorbancy indicating thicker fibrin fibers, maximum clot mass and prolonged fibrinolysis time than controls. Maximum rates of clot lysis were similar in both groups. SEM images showed formation of dense clots with many projections on fibrin fibers. Clot permeability inversely correlated with fibrinogen, tPA, plasminogen activator inhibitor-1 (PAI-1), CRP, platelet count, disease activity score (DAS28) and a marker of oxidative stress, 8-iso-prostaglandin F 2α (r from -0.44 to -0.79; all, p < 0.0001). Similar positive associations were found for clot lysis time (r 0.44 to 0.69; all, p < 0.01). Multiple regression analysis showed that fibrinogen was the only independent predictor of clot permeability (R² = 0.87, p < 0.0001) and lysis time (R² = 0.80, p < 0.003) in RA. Maximum D-dimer levels released from clots, maximum clot turbidity and the time of clot formation were predicted by PAI-1 (all, p < 0.05). Conclusion We showed unfavorably altered plasma fibrin clot structure and function in RA, which might contribute to an increased risk of thrombotic events in this disease.

Effect of Rivaroxaban, An Oral Direct Factor Xa Inhibitor, On Whole Blood Clot Permeation and Thrombolysis: Critical Role of Red Blood Cells.

Abstract 1064Poster Board I-86 Introduction:Decreased fibrinolysis has been reported in venous thrombosis. Thrombus degradation depends on its structure: thicker fibrin fibers are permeable to blood flow and highly susceptible to fibrinolytic enzymes, while thinner fibers are poorly permeable to flow and are resistant to fibrinolysis. Thrombin concentration present at the time of gelation profoundly influences fibrin clot structure: decrease in thrombin generation leads to the formation of thick fibrin fibers and to a decrease in activation of thrombin-activated fibrinolysis inhibitor (TAFI). Rivaroxaban, an oral direct factor Xa inhibitor, is in late stage clinical development for the prevention and treatment of venous and arterial thrombosis. The objective of this study was to evaluate the effect of Rivaroxaban on whole blood (WB) clot structure and degradability by t-PA. Compared to plasma clots, WB clots might better represent the in vivo formed thrombi. Methods:1- Clots were formed by adding to WB or to corresponding plasma, low concentration of tissue factor and calcium in the presence or absence of Rivaroxaban at therapeutic concentrations (0.15 and 0.25 μg/ml). 2- Clot permeability was calculated by measuring the flow rate of liquid through the clot. It was expressed as Darcy constant. 3- Clot degradability was evaluated by D dimers generation during clot perfusion with plasminogen and tissue-type plasminogen activator (t-PA). Results:1- In the absence of Rivaroxaban, WB clots had a lower porosity than that of corresponding plasma clots: Darcy constant of WB clots was 3.1 –fold lower than that of plasma clots. This decreased porosity of WB clots leads to thrombolysis resistance by preventing access of fibrinolytic enzymes to fibrin network: D dimers generation in t-PA-perfused clots for 60 min was 38 -fold lower in WB clots compared to plasma clots. 2- Rivaroxaban increased the permeation rate of WB clots and thrombolysis by t-PA: the addition of Rivaroxaban at 0.15 μg/ml in WB (corresponding in fact to plasma concentration of 0.25 μg/ml), increased the Darcy constant by 5.5 –fold and the clot degradability in 60 min by 108 -fold. These effects of Rivaroxaban were higher in WB clots than in corresponding plasma clots, as Rivaroxaban at 0.25 μg/ml in plasma clots increased the Darcy constant by 2.5-fold and clot degradation by 9.6-fold. In the presence of Rivaroxaban, the Darcy constant and the degradability of WB clots and of plasma clots were nearly identical. 3- To explain the greater efficacy of Rivaroxaban on WB permeation constants and thrombolysis in comparison to plasma clots a) we tested the possibility for Rivaroxaban to reduce the entrapment of red blood cells (RBC) into the network of fibrin as RBC can be responsible for fibrin pore occlusion. This possibility was excluded since Rivaroxaban had no effect on clot permeation rate in clots formed by clotting purified fibrinogen with thrombin in the presence or in the absence of RBC (condition in which there is no generation of thrombin): RBC induced a 2.5 times decrease in permeation rate due to entrapment of RBC into fibrin network, regardless of presence or absence of Rivaroxaban. b) we analyzed the effect of RBC on thrombin generation and its modification by Rivaroxaban: the addition of 0.1 ml RBC diluted ½ to 0,2 ml plasma increased the thrombin generation (540 % of control without RBC). This is probably due to exposure of phosphatidyl serine at surface of RBC during thrombin generation. The increase in thrombin generation by RBC was reduced to 140 % in presence of Rivaroxaban at 0.15 μg/ml. This is explained by Rivaroxaban's inhibition of factor Xa bound to cells. Conclusion:Thrombin generation was greater in WB than in plasma, leading to a lower porosity and degradability of WB clots as compared to plasma clots. Rivaroxaban, by decreasing thrombin generation, increased clot permeability and degradability to the same level in WB clots and plasma clots. This property of Rivaroxaban may contribute to its antithrombotic effect.This study received a support from Bayer-Schering-Pharma France. Disclosures:No relevant conflicts of interest to declare.

Effects of Acetylsalicylic Acid on Increase of Fibrin Network Porosity and the Consequent Upregulation of Fibrinolysis

Our earlier study in vivo showed that a lower dose of acetylsalicylic acid (ASA) brought greater enhancement in fibrin gel permeability (Ks) than a higher dose. To assess whether this finding related to modifications of fibrinogen clotting property by ASA, purified fibrinogen was incubated with ASA and/or salicylic acid (SA). The fibrinogen product was examined. Fibrinogen "clotting time" was not affected. Shortening of fibrin clot "lysis time" paralleled the increase of fibrin network porosity demonstrated by measurements of liquid permeability (Ks), fibrin fiber thickness, and 3-dimensional microscopic image, in a low ASA concentration-dependent way. Ks levels were not altered by SA alone but significantly decreased in samples treated by both where the concentrations were low for ASA and high for SA. In conclusion, ASA at the concentrations used did not influence the rate of fibrinogen gelation by thrombin. However, assembly of fibrin monomers was most probably altered, leading to enhancement of fibrin fiber thickness. A looser network was constructed by the thicker fibrin fibers, which benefits fibrinolysis. According to the known mechanism that SA interferes with ASA in preventing acetylation of platelet's proteins, an explanation for the low ASA concentration-dependent effects on fibrin network structure may be that fewer molecules of SA-the hydrolytic product of ASA-are generated from lower doses of ASA, which block acetylation of fibrinogen to a smaller extent and thus more significantly impair fibrin formation.

Ultrastructural Morphology of Platelets and Fibrin Networks of Lactating and Non-Pregnant Rabbits

Platelets and fibrin play an important role in the coagulation process where they are involved in the maintenance of haemostasis. Fibrin dysfunction is associated with the development of vascular complications, while proneness to the formation of tight and rigid fibrin networks is independently associated with thrombotic disease. Rabbits have long been used successfully as animal models, and are often the species of choice for models of antithrombotic efficacy. It was previously shown that rabbit and human platelet and fibrin morphology are very similar in ultrastructure and fibrin fibre thickness. It was also previously reported that the thin minor fibres forms a thick fine network cover over the major fibres during pregnancy. According to research, white blood cell counts also changes during pregnancy and stays changed for up to 6 weeks post-partum; where the number of neutrophils increased, and the number of lymphocytes, basophils and eosinophils decreased. Here, we show that the same ultrastructure and white blood cell count changes occur in lactating rabbits (4 weeks post-partum). We therefore suggest that a rabbit morphology model studying platelet and fibrin morphology can be used successfully, either to study the effect of pharmaceutical products to be used during lactation and pregnancy in humans, or used in veterinary research. Furthermore, the effects of pharmaceutical products on immunology and white blood cell counts can possibly also be used successfully.

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, lyophilized 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.

Thrombin generation and fibrin clot structure

Generation of a hemostatic clot requires thrombin-mediated conversion of fibrinogen to fibrin. Previous in vitro studies have demonstrated that the thrombin concentration present at the time of gelation profoundly influences fibrin clot structure. Clots formed in the presence of low thrombin concentrations are composed of thick fibrin fibers and are highly susceptible to fibrinolysis; while, clots formed in the presence of high thrombin concentrations are composed of thin fibers and are relatively resistant to fibrinolysis. While most studies of clot formation have been performed by adding a fixed amount of purified thrombin to fibrinogen, clot formation in vivo occurs in a context of continuous, dynamic changes in thrombin concentration. These changes depend on the local concentrations of pro- and anti-coagulants and cellular activities. Recent studies suggest that patterns of abnormal thrombin generation produce clots with altered fibrin structure and that these changes are associated with an increased risk of bleeding or thrombosis. Furthermore, it is likely that clot structure also contributes to cellular events during wound healing. These findings suggest that studies explicitly evaluating fibrin formation during in situ thrombin generation are warranted to explain and fully appreciate mechanisms of normal and abnormal fibrin clot formation in vivo.

Marked increase of fibrin gel permeability with very low dose ASA treatment

Previous data from our group show that acetylsalicylic acid (ASA), especially at low dose, alters the network structure of fibrin, rendering it more porous. The present study was performed to extend the dose-response curve for effects of ASA on fibrinogen clotting properties and to examine the variability of these effects during a 24-h dose interval. Fifteen healthy volunteers received ASA 37.5 mg daily (low dose) for 10 days and, after an interval of 2 weeks, 320 mg daily (medium dose) for 7 days, followed by a single bolus dose of 640 mg (high dose). The plasma fibrinogen concentrations were determined and the permeability of fibrin gels (Ks) was assayed with a recently modified flow measurement technique. Three-dimensional (3D) structure of the fibrin network was studied by confocal microscopy. ASA therapy did not influence fibrinogen concentrations. Compared to baseline, Ks levels were increased by 21% and 31% in samples during medium and high dose ASA treatment (p<0.01) and, even more markedly, by 44% (p<0.0001) with very low dose ASA treatment (p<0.01, compared to the higher doses). The effects of ASA on fibrin gel permeability were stable over a 24-h dose interval. During ASA treatment, thicker fibrin fibers and larger network pores with irregular structure were observed by confocal microscopy. Acetylation of lysine residues in the fibrinogen molecule may explain the alterations in its clotting property, resulting in altered fibrin gel permeability. The mechanism(s) behind the greater increase in fibrin gel permeability and alterations in 3D structure of the fibrin network observed, and why this phenomenon is more pronounced at low compared to intermediate or high ASA doses, deserve further investigations.

Biochemical and biophysical conditions for blood clot lysis.

We have studied how pharmacological dissolution of blood clots was affected by clot retraction, the mode of transport of fibrinolytic agents into the clot and the thickness of the composite fibrin fibers. Retracted clots were resistant to fibrinolysis in a milieu without dissolved plasminogen, because the amount of fibrin-bound plasminogen in retracted clots was insufficient for successful clot lysis. In plasma containing plasminogen, retracted clots were successfully lysed with fibrin-specific plasminogen activators, but not with non-fibrin-specific activators. Preincubation of retracted clots in plasma increased their plasminogen content as well as their sensitivity to fibrinolysis. The rate of lysis was increased up to 100-times when plasminogen activator and plasminogen were introduced into cylindrical clots by pressure-induced bulk flow in comparison with diffusion alone. The magnitude of the increase was similar in retracted and nonretracted clots, but the absolute rate of lysis was faster in non-retracted clots. The influence of fibrin fiber thickness on fibrinolysis was studied by atomic force microscopy. The time to complete lateral section of fibers did not differ between thick and thin composite fibers, and the rate of diameter reduction was faster in thick fibers than in thin ones. Taken together our results suggest that lysis of retracted clots proceeds in circular stages: (a) activation of bound plasminogen followed by partial degradation of fibrin, (b) opening of new plasminogen-binding sites on partly degraded fibrin, (c) binding of plasminogen to the new binding sites which enhances the susceptibility of clots to lysis. Lysis is accelerated by bulk flow of plasminogen activator and plasminogen into clots in comparison to diffusion alone. Fibrinolysis of thick composite fibrin fibers proceeds more efficiently than lysis of thin fibers.

Effect of zinc ions on fibrin network structure.

In addition to calcium, other physiologically important divalent cations (magnesium and zinc) are known to influence fibrin formation and structure. We have studied the effect of different concentrations (0-20 micromol/l) of zinc ions (Zn2+) in the absence and presence of calcium on the gel structure formed in purified fibrinogen-enzyme systems. For that purpose, we used turbidity measurement, liquid permeation and confocal three-dimensional microscopy of the gel as well as sodium dodecyl sulphate (SDS)-gel electrophoresis. The results of turbidity measurements indicated that the clotting time decreased with increasing concentrations of Zn2+. The fiber mass: length ratio (mu) values showed that the porosity of the gels increased in a concentration-dependent manner, i.e. at higher concentrations of Zn2+, larger pores with thicker fibrin fibers were formed. Three-dimensional microscopy data of the gels were in good agreement with the mu data. On SDS-gel electrophores of reduced fibrin, no cross-linking was observed in the presence of zinc ions only (without the addition of calcium ions), nor were D-D dimer bands observed in non-reduced plasmin digested fibrin samples in the presence of zinc ions only. The above results show that zinc changes the fibrin gel structure and that this effect appears to be independent of calcium.

Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells.

Fibrin is a temporary matrix which not only covers a wound, but also provides a structure for invading cells during healing. Changes in the polymerization conditions before gelation of the clot affect the structure of fibrin and thus might influence the interaction with invading cells. Therefore we tested whether changes in the fibrin structure influence the formation of capillary-like tubular structures by human microvascular endothelial cells (hMVEC) in an in vitro angiogenesis model. Opaque [125I]fibrin structures prepared at pH 7.0, fibrin matrices at pH 7.4 and transparent [125I]fibrin structures prepared at pH 7.8 were neutralized (pH 7.4) before seeding hMVEC on top of them in confluent density. Endothelial cells were stimulated with a growth factor [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)165] and a cytokine [tumor necrosis factor (TNF)-alpha] to induce the u-PA/u-PA receptor-dependent formation of capillary-like tubular structures. The formation of these structures was quantified by determining the length of the invasive structures by image analysis and by measuring the accompanying [125I]fibrin degradation. Ingrowth of tubular structures proceeded at a faster rate in opaque matrices consisting of thick fibrin fibers as compared to transparent gels with fine fibrin fibers. The more rapid ingrowth of tubular structures in opaque fibrin gels induced by bFGF/TNF-alpha or VEGF165/TNF-alpha was accompanied by a larger extent of fibrin degradation. Both processes were inhibited by aprotinin and epsilon-aminocaproic acid indicating the involvement of plasmin. They were also inhibited by anti-u-PA or anti-u-PA receptor IgG, but not by anti-t-PA IgG, suggesting the involvement of cell-bound u-PA activity. However, in the opaque fibrin gels, the tubular structures dissolved upon prolonged incubation due to excessive fibrin degradation. Simulation of hMVEC with bFGF alone did not induce tubular structures, but ca used a high degree of t-PA- and plasmin-dependent fibrin lysis, and, after several days, a partial detachment of sheets of cells. Gradual inhibition of the excessive fibrin degradation by a series of aprotinin concentrations did not lead to tube formation in bFGF-treated cells. These data indicate that the formation and stability of tubular structures by hMVEC in fibrin is accompanied by controlled fibrinolysis and depends critically not only on cell-bound u-PA-dependent plasminogen activation, but also on the fibrin structure. Because the fibrin structure is largely influenced by the conditions in which fibrin has been polymerized, these conditions may have considerable impact on angiogenesis during wound healing and vascularization of tumour stroma.

Localization of an effective fibrin beta-chain polymerization site: implications for the polymerization mechanism.

To examine whether fibrin N-terminal Aalpha 17-23 and Bbeta 15-25 may contain high-affinity polymerization sites, GPRVVER and GHRPLDKKREE analogs were prepared, and their abilities to inhibit fibrin monomers from repolymerizing were compared in turbidity and clottability assays. Within Aalpha 17-23, GPR is the most active site (IC30 of 0.95-1.36 mM). Its extension into GPRVVER (IC30 of 1.75-2.3 mM) reduced activity. Within Bbeta 15-25, acyl-DKKREE (IC30 of 0.30-0.53 mM) can account for GHRPLDKKREE activity (IC30 of 0. 33-0.44 mM). Comparison of the assays showed that calcium, whose presence induces thick fibrin fibers, elicited a higher turbidity than clottability inhibition. Similarly, the lateral-association-promoting GHRP (IC30 of 1.25-1.43 mM) gave a high turbidity vs clottability inhibition ratio (137%). In contrast, low ratios were found for the linear-association-initiating GPR (73%) and for acyl-DKKREE (34%). Structure-activity correlation showed that fibrinogen-like acyl-GPRP and acyl-GHRP could inhibit D. E association at the millimolar range, but in a manner different from fibrin-related GPR peptides did, which required the NH2 as well as Arg presence. To explain Bbeta 20-25 masking, it is proposed that DKKREE in fibrinogen may engage in ionic and hydrogen bonds with KDSDW, the Aalpha 29-33 sequence implicated in thrombin binding. To explain acyl-GPRP and acyl-GHRP inhibition of D.E association, it is proposed that fibrinogen packing may be mediated by E domain association with alphaC (Aalpha 220-609) fragments of adjacent molecules, and by alphaC-alphaC association. A modified polymerization mechanism is deduced by taking into account fibrinogen N-terminal conformation as well as E domain binding to thrombin vs alphaC fragments. This model proposes the following. (1) Upon thrombin binding to fibrinogen KDSDW, DKKREE may become exposed. (2) Fibrinopeptide A cleavage further unmasks the NH2 and Arg group of GPR, leading to DKKREE and GPR initiation of polymerization. (3) The micromolar-effective thrombin-fibrin(ogen) binding may initiate a partial alphaC repulsion. Subsequent DKKREE and GPR binding to D domains of other fibrin(ogen) will lead to the formation of the trimer and bring additional molecules to fibrin N-terminal region, and the combined steric congestion may lead to a complete alphaC repulsion from the overcrowded E domain. (4) Repulsion of the large Aalpha 220-609 fragments may unmask multiple polymerization sites beyond the fibrin N-terminal region.

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.

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.

The effect of fibrin structure on fibrinolysis.

Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.