Babesia bovis (argentina): analysis of fibrinogen-like proteins during infection

Fibrinogen and fibrinogen-like proteins (FLP) were isolated from plasma and serum of cattle acutely infected with Babesia bovis. The sizes and chain structures of these proteins were examined and clotting assays performed. The results indicated that the blood was in a hypercoagulable state due mainly to enhanced production of hydrogen bonded fibrin and offset partly by slight inhibition of chain cross-linking. The latter appeared due to a Factor XIII inhibitor. Reduction of A alpha chains of plasma FLP was not evident, nor could lower molecular weight remnants be regularly detected strongly suggesting that fibrin(ogen) lysis rarely occurred. Similarly the size and chain structure of the majority of noncoagulable FLP of serum was consistent with their being the product of coagulation and not fibrinolysis. Only in heavily infected splenectomized cattle were products from lysed cross-linked fibrin detected and these constituted only about 3% of total serum FLP.     

Detection of soluble fibrin monomer complexes. Comparison of a haemagglutination assay with the ethanol gelation test

Hypercoagulability and disseminated intravascular coagulation (DIC) are characterized by the presence of circulating fibrin monomer complexes in plasma. In 342 patients with possible DIC fibrin monomers, fibrinogen, Reptilase Time, antithrombin III and other coagulation parameters were determined at frequent intervals. Testing of soluble fibrin monomer complexes was performed using a sensitive and reliable hemagglutation assay with red cells sensitized by fibrin monomers (FM-Test) and the ethanol gelation test (EGT). Method comparison regarding the influence of fibrinogen levels and fibrin degradation products shows that high fibrinogen levels lead to false-positive results with EGT. The same effect is observed for fibrin degradation products and EGT whereas no influence of fibrinogen level and fibrin degradation products on the FM-Test occurs. It is well-known that during DIC AT III level decreases caused by proteolytic activity. In this study it could be shown that fibrin monomer increases parallel to the decrease of AT III. The same effect does not occur due to fibrin degradation products.     

The plasma cryofibrinogen of calves infected with Oesophagostomum radiatum

A cryofibrinogen complex was detected in the plasma of cattle infected with Oesophagostomum radiatum. Its formation coincided with the development of hypofibrinogenaemia and it consisted basically of fibrinogen and soluble noncross-linked fibrin in both monomer and high molecular forms. Also present in the complex, albeit in trace amounts, were cold insoluble globulin, TgG, and plasminogen. It is suggested that the complex results from thrombin activation in vivo and its role is to maintain fibrin in solution thus preventing its deposition in the vascular system.     

Fibrinogen and fibrin interaction with concanavalin a dimer and its influence on coagulation

Concanavalin A dimer interacts with fibrinogen and soluble fibrin at pH 5.2 Analysis of the binding data shows that there are in both cases four binding sites per molecule and that the dissociation constant does not change by removal of fibrinopeptides A and B. Ultracentrifugal studies shows that no aggregates of fibrinogen or fibrin are formed through concanavalin A binding and that up to four molecules of concanavalin A dimer can be bind to one molecule of fibrinogen or fibrin. These results imply that the four carbohydrate chains in the molecule are accessible to concanavalin A dimer. There is a diminution in the coagulation of fibrinogen by thrombin at low relative lectin concentrations and an increase at high concentrations. However, the lectin always favours the aggregation of fibrin monomers and does not have any inhibitory effect on the release of fibrinopeptides. We conclude that the electric charge in the neighbourhood of the carbohydrate in both chains, Bβ and γ plays an important role in the attraction between monomeric fibrin and fibrinogen-monomeric fibrin. The different effect of concanavalin A on the coagulation, depending on the relative concentration of the lectin, would be the result of the screening of this electric charge favouring either the interaction of fibrinogen-monomeric fibrin or the polymerization of monomeric fibrin.     

The Susceptibility of the Higher Primates to Piroplasms

SYNOPSIS. The chimpanzee is shown to behave like man to infection with Babesia divergens or bovis : the intact animal is totally resistant, whereas the splenectomized animal develops a fulminating infection accompanied by blackwater. The splenectomized rhesus monkey reacts in the same way also, but splenectomized rabbits are insusceptible. In the chimpanzee the typical accolé position of the " divergens " organisms (as seen in cattle) is absent, but occurs in the rhesus. It is suggested that latent piroplasmosis in man may exist on a large scale in rural populations in infected localities.     

Human factor XIa cleaves fibrinogen: effects on structure and function

Factor XIa, the enzymatic form of the factor XI zymogen, is generated as a result of factor XII-dependent surface activation in plasma. Factor XIa degrades high molecular weight kininogen, its cofactor for activation (which binds factor XIa to the surface), as well as cleaves and activates coagulation factor IX. In this report, we present evidence that factor XIa can also cleave fibrinogen and decrease the thrombin-catalyzed formation of the fibrin clot. Furthermore, the products of factor XIa-digested fibrinogen markedly inhibited the rate of polymerization of fibrin monomers. Factor XIa initially cleaved the A alpha-chain of fibrinogen and subsequently degraded the B beta-chain. However, the cleavage sites on both chains were distinct from those susceptible to thrombin. The gamma-chain was degraded only after prolonged incubation with factor XIa. Furthermore, the profile of fibrinogen proteolysis by factor XIa was distinctly different from that of plasmin-catalyzed fibrinogenolysis. Unlike plasmin, factor XIa was not able to cleave the NH2-terminus of the B beta-chain of fibrinogen. Moreover, factor XIa, unlike plasmin, failed to hydrolyze fibrin. Further study of the proteolytic digests of fibrinogen produced by factor XIa may give additional insight into the mechanism of polymerization of this protein.     

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.     

Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP)

Factor VII Activating Protease (FSAP) is a plasma protease affecting both coagulation and fibrinolysis. Although a role in hemostasis is still unclear, the identification of additional physiologic substrates will help to elucidate its role in this context. FSAP has been reported to cleave fibrinogen, but the functional consequences of this are not known. We have therefore undertaken this study to determine the implications of this cleavage for fibrin-clot formation and its lysis. Treatment of human fibrinogen with FSAP released an N-terminal peptide from the Bβ chain (Bβ1-53) and subsequently the fibrinopeptide B; within the Aα chain a partial truncation of the αC-region by multiple cleavages was seen. The truncated fibrinogen showed a delayed thrombin-catalyzed polymerization and formed fibrin clots of reduced turbidity, indicative of thinner fibrin fibers. Confocal laser scanning and scanning electron microscopy of these clots revealed a less coarse fibrin network with thinner fibers and a smaller pore size. A lower pore size was also seen in permeability studies. Unexpectedly, FSAP-treated fibrinogen or plasma exhibited a significantly faster tPA-driven lysis, which correlated exclusively with cleavage of fibrinogen and not with activation of plasminogen activators. Similar observations were also made in plasma after activation of endogenous zymogen FSAP, but not in plasma of carrier of the rare Marburg I single nucleotide polymorphism. In conclusion, altering fibrin clot properties by fibrinogenolysis is a novel function of FSAP in the vasculature, which facilitates clot lysis and may in vivo contribute to reduced fibrin deposition during thrombosis.     

STUDIES ON BLOOD COAGULATION : II. THE FORMATION OF FIBRIN FROM THROMBIN AND FIBRINOGEN

Although calcium is essential for the formation of thrombin, it can be recovered quantitatively from formed horse thrombin without affecting its coagulating activity. Citrate also has no significant effect. As stated in the text, this does not exclude the possibility that thrombin is actually a calcium compound present in minute concentration; but confirming the results of Hammarsten, it does show that fibrin cannot be a calcium-protein compound unless one assumes molecular weights for fibrinogen greater than 1,000,000. Although the available experimental data concerning the properties of thrombin, the kinetics of its reaction with fibrinogen, and the quantitative relationships between the two do not allow a definite decision as to whether thrombin is an enzyme analogous to rennin, or whether it combines with fibrinogen to form an insoluble compound, fibrin, the weight of evidence does favor the enzyme theory. A given quantity of thrombin can form at least 200 times its weight of fibrin, and in view of the crudeness of the preparation this ratio is probably many times greater. There is no apparent stoichiometric relationship between thrombin and fibrinogen, and thrombin does not disappear from a mixture of the two until the moment of coagulation; the quantity which then disappears is many times the minimal quantity necessary to form the amount of fibrin produced.     

High-resolution visualization of fibrinogen molecules and fibrin fibers with atomic force microscopy

We report an atomic force microscopy (AFM) study of fibrinogen molecules and fibrin fibers with resolution previously achieved only in few electron microscopy images. Not only are all objects triads, but the peripheral D regions are resolved into the two subdomains, apparently corresponding to the βC and γC domains. The conformational analysis of a large population of fibrinogen molecules on mica revealed the two most energetically favorable conformations characterized by bending angles of ～100 and 160 degrees. Computer modeling of the experimental images of fibrinogen molecules showed that the AFM patterns are in good agreement with the molecular dimensions and shapes detected by other methods. Imaging in different environments supports the expected hydration of the fibrinogen molecules in buffer, whereas imaging in humid air suggests the 2D spreading of fibrinogen on mica induced by an adsorbed water layer. Visualization of intact hydrated fibrin fibers showed cross-striations with an axial period of 24.0 ± 1.6 nm, in agreement with a pattern detected earlier with electron microscopy and small-angle X-ray diffraction. However, this order is clearly detected on the surface of thin fibers and becomes less discernible with the fiber's growth. This structural change is consistent with the proposal that thinner fibers are denser than thicker ones, that is, that the molecule packing decreases with the increasing of the fibers' diameter.     

Preparation of fibrin des-AA by thrombin

The active thrombin is formed in the blood stream when the blood coagulation system is activated. It attacks fibrinogen, splits off two fibrinopeptides A and fibrinogen is transformed into des-AA fibrin which is able to polymerize spontaneously forming protofibrils. At high thrombin concentration the enzyme splits off two fibrinopeptides B and des-AA fibrin units are transformed into des-AABB fibrin. These two forms of fibrin are widely used in the biological experiments. However des-AA fibrin is obtained usually from fibrinogen using the snake poisons (such as reptilase). Des-AA fibrin was obtained also by physiological enzyme thrombin, but that des-AA fibrin samples had the contamination of des-AABB fibrin. At the present paper we have described the method of the des-AA fibrin preparation by thrombin without any contamination of des-AABB fibrin.     

Viscoelasticity of fibrinogen solution and of blood during coagulation studied by a new damped oscillation rheometer

The behavior of a newly developed damped oscillation type rheometer was analyzed for fibrinogen solution and blood during coagulation. This rheometer consists of a cylindrical tube suspended from a torsion wire, that is filled with liquid to be tested. The logarithmic damping factor (LDF) during coagulation for blood and fibrinogen solution was obtained by this rheometer, which was closely related to the changes of viscosity and/or viscoelasticity of the blood sample. The slight increase of LDF prior to the rapid decrease was observed for blood. The increase of LDF would be reflected in the formation of the aggregation structure of red blood cells (rouleaux network) prior to the formation of fibrin network. The value of LDF for fibrinogen solution sharply increased and then decreased through a maximum value with the progress of coagulation, although the change of LDF was remarkably dependent on the fibrinogen concentration. The initial increase in LDF for fibrinogen solution was considered to be due to the formation of small clots in the solution. The decrease in LDF after attaining a maximum value is ascribed to the formation of fully developed fibrin network. The maximum value of LDF during coagulation for fibrinogen solution is higher than that for blood. The behavior was compared with that for non-biological fluids such as viscosity standard liquids and polyvinyl alcohol solution. From those data, it was concluded that the higher value of LDF than that for Newtonian liquids was due to the formation of aggregation structure or inhomogeneous fine clots in the liquid, which was accompanied with the appearance of the elasticity.     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin

Tissue-type plasminogen activator (t-PA) plays a central role in fibrinolysis in vivo. Although it is known to bind to fibrin, the dissociation constant (Kd) and number of moles bound per mole of fibrin monomer (n) have never been measured directly. In this study, the binding of both the one-chain form and the two-chain form of recombinant, human t-PA to fibrin was measured. Although more one-chain t-PA than two-chain t-PA is bound to fibrin, the Kd's and n's were within experimental error of each other. Significantly more t-PA is bound to clots made from fibrinogen which has been digested with plasmin than to clots made from intact fibrinogen. The additional binding was shown to be due to the formation of new set(s) of binding site(s) with dissociation constants that are 2-4 orders of magnitude tighter than the binding site present on clots made from intact fibrinogen. epsilon-Aminocaproic acid was capable of competing for the loose binding site present on both intact and degraded fibrin but had little effect on the binding of t-PA to the new site(s) formed by plasmin digestion. This increase in binding caused by plasmin-mediated proteolysis of fibrin suggests a possible mechanism for a positive regulation capable of accelerating fibrinolysis.     

The thrombomodulin-protein C system is essential for the maintenance of pregnancy

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.     

Characterization of the interaction of recombinant apolipoprotein(a) with modified fibrinogen surfaces and fibrin clots.

Elevated levels of lipoprotein(a) [Lp(a)] in plasma are a significant risk factor for the development of atherosclerotic disease, a property which may arise from the ability of this lipoprotein to inhibit fibrinolysis. In the present study we have quantitated the binding of recombinant forms of apolipoprotein(a) [17K and 12K r-apo(a); containing 8 and 3 copies, respectively, of the major repeat kringle sequence (kringle IV type 2)] to modified fibrinogen surfaces. Iodinated 17K and 12K r-apo(a) bound to immobilized thrombin-modified fibrinogen (i.e., fibrin) surfaces with similar affinities (Kd approximately 1.2-1.6 microM). The total concentration of binding sites (Bmax) present on the fibrin surface was approximately 4-fold greater for the 12K than for the 17K (Bmax values of 0.81 +/- 0.09 nM, and 0.20 +/- 0.01 nM respectively), suggesting that the total binding capacity on fibrin surfaces is reduced for larger apolipoprotein(a) (apo(a)) species. Interestingly, binding of apo(a) to intact fibrin was not detected as assessed by measurement of intrinsic fluorescence of free apo(a) present in the supernatants of sedimented fibrin clots. In other experiments, the total concentration apo(a) binding sites available on plasmin-modified fibrinogen surfaces was shown to be 13.5-fold higher than the number of sites available on unmodified fibrin surfaces (Bmax values of 2.7 +/- 0.3 nM and 0.20 +/- 0.01 nM respectively) while the affinity of apo(a) for these surfaces was similar. The increase in Bmax was correlated with plasmin-mediated exposure of C-terminal lysines since treatment of plasmin-modified fibrinogen surfaces with carboxypeptidase B produced a significant decrease in total binding signal as detected by ELISA (enzyme linked immunosorbent assay). Taken together, these data suggest that apo(a) binds to fibrin with poor affinity (low microM) and that the total concentration of apo(a) binding sites available on modified-fibrinogen surfaces is affected by both apo(a) isoform size and by the increased availability of C-terminal lysines on plasmin-degraded fibrinogen surfaces. However, the low affinity of apo(a) for fibrin indicates that Lp(a) may inhibit fibrinolysis through a mechanism distinct from binding to fibrin, such as binding to plasminogen.     

Interaction of fibrinogen and fibrin with protamine sulfate

Fibrinogen and fibrin sedimentation by different protamine sulphate preparations have been studied. Ionic strength and protamine sulphate concentration are found to influence the sedimentation reaction (paracoagulation). High sedimentation activity is inherent in protamine sulphate preparations with the lower electrophoretic mobility, that is with the higher molecular weight. The protamine sulphate reaction with fibrinogen and fibrin is of electrostatic character as the long polycationic chain of protamine is coupled with the negatively charged loci either of fibrin or of fibrinogen molecules, thus evoking aggregation. In this case the fibrin molecules being brought together favour the specific mutual binding due to the active sites of polymerization, specific fibres or gel being formed.     

Engineered Molecular Therapeutics Targeting Fibrin and the Coagulation System: a Biophysical Perspective

The coagulation cascade represents a sophisticated and highly choreographed series of molecular events taking place in the blood with important clinical implications. One key player in coagulation is fibrinogen, a highly abundant soluble blood protein that is processed by thrombin proteases at wound sites, triggering self-assembly of an insoluble protein hydrogel known as a fibrin clot. By forming the key protein component of blood clots, fibrin acts as a structural biomaterial with biophysical properties well suited to its role inhibiting fluid flow and maintaining hemostasis. Based on its clinical importance, fibrin is being investigated as a potentially valuable molecular target in the development of coagulation therapies. In this topical review, we summarize our current understanding of the coagulation cascade from a molecular, structural and biophysical perspective. We highlight single-molecule studies on proteins involved in blood coagulation and report on the current state of the art in directed evolution and molecular engineering of fibrin-targeted proteins and polymers for modulating coagulation. This biophysical overview will help acclimatize newcomers to the field and catalyze interdisciplinary work in biomolecular engineering toward the development of new therapies targeting fibrin and the coagulation system.     

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.     

Enhanced fibrinolysis by proteolysed coagulation factor Xa

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.