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The paper deals with the three-dimensional model of fibrin(ogen) molecule and fibrin fibril, based on the electron microscopy data on fibrin(ogen) molecule and on fibrin fibril structure, data on symmetry of fibrinogen primary structure, on domain structure of molecule, on location of sites of polymerization and on the data on polymerization mechanism of fibrin.
Fibrinogen Caracas I is a dysfibrinogenemia with a mild bleeding diathesis and a defective wound healing. We have characterized this abnormal fibrinogen using transmission electron microscopy (TEM) in combination with turbidity and permeation studies. Turbidometric and permeability analysis showed that the abnormal fibrin had a significantly decreased mass:length ratio and fiber diameter. In addition, the permeability studies of plasma fibrin clots showed that the gel porosity of the abnormal fibrinogen was reduced. Images of the abnormal fibrin structure obtained using TEM showed that the fibers were thinner, much less branched and less ordered than normal fibers. Diminished fibrin fiber diameter and reduced fibrin gel porosity have been taken as hallmarks of thrombophilic dysfibrinogenemias. The results of the present study show that these features are not necessarily predictive of thrombophilia. Further studies performed on a larger number of dysfibrinogenemias need to be conducted in order to establish the implications of these parameters on the clinical outcome.
Effects of divalent cations on fibrinogen and its reaction with thrombin were re-examined and correlated to improve definition of mechanisms underlying the acceleration of clot formation by the ions. The rate of release of fibrinopeptides from fibrinogen was not affected by any of the specific ions studied, but increased rates of fibrin monomer polymerization were obtained with all but magnesium ions. Maximal acceleration of monomer polymerization was observed with the divalent ions at concentration of 2.5-5 mM, and no added specific ion effects were observed with much higher levels. The acceleratory ions were found to have a corresponding effect on the solubility of fibrinogen, as judged from acceleration of its precipitation at low temperature. Although magnesium ions had no effect on fibrin monomer polymerization, they did have a low ranking effect on cryoprecipitation. These data confirm that the principal effect of divalent cations in the fibrinogen-fibrin transformation is to accelerate fibrin monomer polymerization, and suggest that the acceleration is associated with effects on the solubility of fibrinogen.
Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E. Surface Plasmon Resonance (SPR) technology, employed to further evaluate the epitopes in fibrin, showed that MAb 1H10 had a higher affinity for fragment-E (KD = 8.04 x 10(-9) M) than MAb 5F3 (KD = 1.13 x 10(-8) M). Individual association and dissociation rate constants of 7.97 x 10(5) M-1s-1 and 3.97 x 10(-3)s-1, respectively, for MAb 1HAb 1H10, and 5.16 x 10(5) M-1s-1 and 3.62 x 10(-3)s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However individual isolated polypeptide chains of fibrinogen fragment E (E-A alpha, E-B beta, E-gamma) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures. Furthermore, SPR pair-wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E. These distinct epitopes appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.
We measured the rate of lethality and abscess formation in rats that underwent intraperitoneal implantation of fibrin clots contaminated with either Escherichia coli or Bacteroides fragilis alone or in combination, to determine whether the two organisms together would produce a synergistic infection. Ten-day mortality produced by 10(9) colony-forming units (CFU) of E coli was 33.3%. Encapsulated B fragilis led to 3.3% mortality. Escherichia coli (5 X 10(8) CFU) plus B fragilis (5 X 10(8) CFU) led to a sharp increase both in the rate and final ten-day mortality (80.0%). Eighty percent of the rats that received E coli (10(9) CFU within fibrin clots) had abscesses determined on the basis of grossly purulent material. All animals that received B fragilis and survived ten days contained abscesses. Synergy between E coli and B fragilis was noted to occur only when 5 X 10(8) CFU of each organism was present within the fibrin clot. Lower numbers did not produce significant synergy compared with controls that received either E coli or B fragilis. Quantitation of the number of organisms present at 24 hours within contaminated fibrin clots demonstrated a similar amount of growth of both organisms, either when added alone or in combination as copathogens.
The tannic acid-phosphomolybdic acid-Levanol (Supranol) Fast Cyanine 5RN (TP-L) procedure for staining muscle cells and blood platelets was used because, with this method, proteins of the myosin-fibrin group should be selectively stained. However, in human blood and blood plasma clots and in vivo thrombi, fibrin was not stained. Blood platelets probably due to their content of contractile proteins were very well stained. Apparent fibrin staining in human autopsy thrombi may be due to the staining of disintegrated platelets and the absorbance of fibrin by stained hemoglobin. Problems encountered using Nuclear Fast Red as the nuclear stain were solved by changing the dye concentration or by using a differentiating agent. Myosin staining by the TP-L method depended on the pH of the tannic-acid solution used. Raising the pH to 7.4-8.0 changed the staining result, and collagen fibers were then stained.
A new spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-tPA), specific for the quantitation of tissue plasminogen activators, is described. The method is based on (1) the high-affinity binding (Kp = 1.4 +/- 2 nM) of tPA to a solid-phase fibrin network constructed by thrombin proteolysis of fibrinogen covalently coupled to polyglutaraldehyde-activated polyvinyl chloride microtiter plates, and (2) the subsequent development of PA activity by the fibrin-tPA complex and its measurement with a coupled assay using a chromogenic substrate highly selective for plasmin. Conditions were chosen such that the rate of para-nitroaniline release from the substrate is directly proportional to the concentration of tPA. The support is able to isolate tPA from the bulk of proteins present in any biological fluid allowing the assay to specifically detect tPA activity (range: 0.01 to 50 IU/ml) even in the presence of other activators, proteases, and inhibitors. Since the assay is done in a well-defined reaction mixture (the fibrin-tPA complex, plasminogen, and the synthetic substrate), kinetics studies using pure or crude tPA can be performed. Standard curves (rate measurement and endpoint methods) were made using the international standard (preparation 83/517) for tPA.
An electrophoretic modification of the conventional fibrin autography that can be used for the detection of plasminogen activators (urokinase type and tissue type) and fibrin-degrading enzymes in complex biological fluids is described. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins and the substrate plasminogen are transferred electrophoretically into the fibrin indicator gel, resulting in an efficient transfer of proteinases as well as high resolution and contrast of fibrinolytic zones caused by plasminogen activator activity. Picogram amounts of human urokinase type plasminogen activator (about 0.002 International Unit) are still detectable. The technique is also applicable to reversed fibrin autography for plasminogen activator inhibitors.
Thrombus formation in the rabbit carotid artery has been studied kinetically in vivo using a minimally invasive technique utilising radioisotopes. Clamping of the carotid artery for 5 min resulted in the simultaneous accumulation of platelets and fibrin at the site of injury over the next 45 min. Under the electron microscope the response was seen to range from platelet monolayer adhesion to mural thrombus formation with fibrin deposition. In animals rendered thrombocytopenic, fibrin deposition was impaired during the first 15-20 minutes after injury. Basic coagulation times and fibrinogen concentration were within normal limits. In addition the injured vessels in these animals accumulated more radiolabelled albumin, but not erythrocytes, than injured vessels in control animals. The results may imply a role for platelets in the enhancement of fibrin deposition during the early part of the response to injury and in contributing to the maintenance of normal permeability following vessel injury.
Polymeric fibrin is a strong enhancer of the activation of plasminogen by t-PA. At least two types of sites are involved in this enhancement i.e. a site within A alpha-(148-160), and a site within gamma-(311-379). These sites are not accessible in fibrinogen, but are exposed upon conversion of fibrinogen to fibrin. This explains why fibrinogen has no rate-enhancing properties, and helps to explain the effects of fibrin. Fibrin with its ordered structure appears to exert its rate-enhancing effect by presenting the above sites for interaction with t-PA and plasminogen; thus concentrating and correctly orienting these two reactants on its surface and inducing conformational changes which lead to higher catalytic efficiencies.
Two markers of fibrin formation, fibrinopeptide A (FpA) and soluble fibrin (SF), were studied in order to elucidate whether they reflected intravascular fibrin formation in the same manner. Plasma was collected for measurements of FpA and SF in 34 patients during the course of colorectal surgery. Although both parametres reflected haemostatic activation postoperatively, covariation was neither observed preoperatively, nor on the second, third, and eighth postoperative days. At 3 months following surgery, however, FpA and SF was correlated. At that time, the activation of the coagulation system has ceased. The present results demonstrate a lack of correlation between these markers of fibrin formation during clotting activation. Quite different properties of the FpA and the SF molecules, different clearance rates and distribution volumes may explain the lack of correlation observed.
Accurate birefringence measurements show that fibrinogen orients to a small degree in high magnetic fields. This effect can be explained as due to the molecule having about 30% (by weight) alpha-helix oriented relatively parallel to the long axis. Birefringence measurements on fully oriented fibrin suggest that aligned alpha-helical content is less than that estimated for fibrinogen. But because of limitations in the analysis this difference must be viewed with caution. Highly oriented fibrin results when polymerization takes place slowly in a strong magnetic field. Low-angle neutron diffraction patterns from oriented fibrin made in the presence of EDTA, made in the presence of calcium, or stabilized with factor XIIIa are very similar, showing that the packing of the molecules within the fibers is the same or very similar in these different preparations. The induced magnetic birefringence was used to follow fibrin formation under conditions in which thrombin was rate limiting. The fiber network formed by approximately the gelation point constitutes a kind of matrix or frame that is largely built upon during the remaining approximately 85% of the reaction. After gelation the reaction is pseudo-first order.
A synthetic heptapeptide from the amino terminus of the beta chain in human fibrin was used as an antigen to produce monoclonal antibodies that bind to fibrin even in the presence of human fibrinogen at the concentration found in plasma. As expected, the antifibrin activity was inhibited by the peptide antigen but not by a control heptapeptide. In a chicken ex vivo circulatory model for fibrin detection, intravenously administered monoclonal antibodies bound to human fibrin-coated disks placed in an extracorporeal chamber. These findings may lead to better methods for identifying deep vein and coronary artery thrombi.
Cryopreserved human monocytes have been examined for their procoagulant and profibrinolytic capacities on lipopolysaccharide (LPS) stimulation, when given the possibility to act upon fibrinogen in heparinized plasma. As shown by the appearance of fibrinopeptide A, indicating thrombin action on the fibrinogen molecule, and by the appearance of D-Dimer, indicating the action of plasmin on fibrin, it is apparent that LPS-instructed monocytes give rise to fibrin which is subsequently lysed. Thus, delineation (fibrin formation) appears to be followed by fibrin removal (restitution).
Plasma levels of C-reactive protein (CRP, a marker of the reactant plasma protein component of the inflammatory response) and of fibrin D-dimer (a marker of cross-linked fibrin turnover) have each been associated in recent studies with the risk of future ischemic heart disease (IHD). Previous experimental studies have shown that fibrin degradation products, including D-dimer, have effects on inflammatory processes and acute-phase protein responses. In the Speedwell Prospective Study, we therefore measured CRP and D-dimer levels in stored plasma samples from 1690 men aged 49 to 67 years who were followed-up for incident IHD for an average of 75+/-4 months (mean+/-SD) and studied their associations with each other, with baseline and incident IHD, and with IHD risk factors. CRP and D-dimer levels were each associated with age, plasma fibrinogen, smoking habit, and baseline evidence of IHD. CRP was associated with D-dimer (r=0.21, P<0.00001). On univariate analyses, both CRP and D-dimer were associated with incident IHD. The incidence of IHD increased with CRP independently of the level of D-dimer (P=0.0002) and also increased with D-dimer independently of the level of CRP (P=0.048). In multivariate analyses, inclusion of D-dimer and conventional risk factors reduced the strength of the association between CRP and incident IHD; likewise, inclusion of CRP and conventional risk factors reduced the strength of the association between D-dimer and incident IHD. We conclude that although these respective markers of inflammation and fibrin turnover show modest association with each other in middle-aged men, they may have additive associations with risk of incident IHD. Further larger studies are required to test this hypothesis.
In vitro lysis of fibrin, as indicated by increased fibrinogen-fibrin-related antigen (FR-antigen) in serum is usually seen when whole blood, or plasma, or highly purified fibrinogen prepared by several different procedures is clotted and kept at temperatures above 0 degrees C. This increase is both time and temperature dependent, occurs despite the addition of various plasmin and cathepsin inhibitors, and is probably caused by thrombin evolved during clotting and/or added in vitro. In these experiments, the FR-antigen was measured by a sensitive, reproducible hemagglutination inhibition immunoassay adapted to the AutoAnalyzer. Serum from whole blood contained more than serum from plasma, and fibrin rather than fibrinogen proved to be essential for the in vitro lysis. The phenomenon was also caused by Arvin or Reptilase, suggesting that splitting of one or more arginine or lysine bonds in fibrin may be at least partially responsible. To obtain minimal levels of FR-antigen (< 0.5 mug/ml), plasma is clotted for 4 hr at 0 degrees C with 1.0-5.0 U/ml thrombin, CaCl(2) (0.0125 mole/liter), and epsilon aminocaproic acid (0.05 mole/liter). Slightly higher levels, probably adequate for clinical diagnosis, are obtained by 10-30 min clotting at room temperature. Since endogenous and/or exogenous thrombin is essential for the collection of serum FR-antigen, all the FR-antigen found in normal serum probably results from an irreducible amount of in vitro lysis rather than from continuous intravascular clotting and fibrinolysis.
The effect of variations in haematocrit (hct) on the retention of 51Cr-labelled platelets and 125I-labelled fibrinogen from normal, heparinized, human blood, circulated in an experimental "mini-Kiil" dialyzing system, was studied. Platelet retention (PR) on dialysis membranes and blood lines was found to increase with increasing hct. When only platelet rich plasma was circulated, a significantly lower PR was noted on the blood lines. Fibrin(ogen) deposition on the same surfaces showed a similar tendency. When 125I-labelled fibrin monomer was added to normal blood, prior to dialysis, fibrin deposition on the membranes and blood lines was of a much higher degree than in the control. However, PR was not significantly influenced by the presence or deposition of the fibrin monomer. Reduced platelet function, caused by incubation with acetylsalicylic acid prior to dialysis, led to less PR on the blood lines, but not on the membranes. The observed difference was, however, not statistically significant (p = 0.06). Pretreatment of platelets with prostaglandin E1, however, was able to reduce PR significantly both on dialysis membranes and blood lines.
Plasminogen activator inhibitor 2 (PAI-2) is a major product of activated human monocytes. Here we show that monocytes inhibited u-PA- but not t-PA-mediated fibrinolysis, by secreting PAI-2 into an overlying fibrin clot. Extracts of arterial and venous human thrombi were found to contain active PAI-2. PAI-2 was cross-linked to fibrin in a reaction catalyzed by two major transglutaminases (TG), tissue TG and factor XIII. The activity of PAI-2 was not affected by such cross-linking. Cross-linking of PAI-2 to fibrin was inhibited by Tridegin, a specific inhibitor of TG, and also by EDTA and iodoacetamide. The use of competitive peptides mimicking the loop between helices C and D of PAI-2 identified Gln 83 and 86 as residues important in cross-linking. This study defines a mechanism by which PAI-2 is localized to fibrin, where it acts as an effective inhibitor of u-PA-mediated fibrinolysis.