Evidence for a Second Type of Fibril Branch Point in Fibrin Polymer Networks, the Trimolecular Junction

Fibrin molecules polymerize to double-stranded fibrils by intermolecular end-to-middle domain pairing of complementary polymerization sites, accompanied by fibril branching to form a clot network. Mass/length measurements on scanning transmission electron microscopic images of fibrils comprising branch points showed two types of junctions. Tetramolecular junctions occur when two fibrils converge, creating a third branch with twice the mass/length of its constituents. Newly recognized trimolecular junctions have three fibril branches of equal mass/length, and occur when an extraneous fibrin molecule initiates branching in a propagating fibril by bridging across two unpaired complementary polymerization sites. When trimolecular junctions predominate, clots exhibit nearly perfect elasticity

Studies on the Basis for the Properties of Fibrin Produced from Fibrinogen-Containing γ′ Chains

Human fibrinogen 1 is homodimeric with respect to its γ chains (`γA-γA\u27), whereas fibrinogen 2 molecules each contain one γA (γA1-411V) and one γ\u27 chain, which differ by containing a unique C-terminal sequence from γ\u27408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the γ\u27 chains. Second, cross-linking of fibrin γ chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to γ\u27 chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to γ\u27 chains

Studies on the Ultrastructure of Fibrin Lacking Fibrinopeptide B (β-Fibrin)

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a “coarse” matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, “fine” fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles

Mouse models of neurodegenerative disease: preclinical imaging and neurovascular component.

Neurodegenerative diseases represent great challenges for basic science and clinical medicine because of their prevalence, pathologies, lack of mechanism-based treatments, and impacts on individuals. Translational research might contribute to the study of neurodegenerative diseases. The mouse has become a key model for studying disease mechanisms that might recapitulate in part some aspects of the corresponding human diseases. Neurode- generative disorders are very complicated and multifacto- rial. This has to be taken in account when testing drugs. Most of the drugs screening in mice are very di cult to be interpretated and often useless. Mouse models could be condiderated a ‘pathway models’, rather than as models for the whole complicated construct that makes a human disease. Non-invasive in vivo imaging in mice has gained increasing interest in preclinical research in the last years thanks to the availability of high-resolution single-photon emission computed tomography (SPECT), positron emission tomography (PET), high eld Magnetic resonance, Optical Imaging scanners and of highly speci c contrast agents. Behavioral test are useful tool to characterize di erent ani- mal models of neurodegenerative pathology. Furthermore, many authors have observed vascular pathological features associated to the di erent neurodegenerative disorders. Aim of this review is to focus on the di erent existing animal models of neurodegenerative disorders, describe behavioral tests and preclinical imaging techniques used for diagnose and describe the vascular pathological features associated to these diseases

The Polymerization and Thrombin-Binding Properties of Des-(Bβ1-42)-Fibrin

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains (“A” and “B,” respectively) in the central portion of the molecule, and exposure of a noncatalytic “secondary” thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the Bβ 1-42 sequence (“des-(Bβ 1-42)-fibrinogen”), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by Bβ 1-42 in the polymerization of α-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (μ, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(Bβ 1-42)-α-fibrin, thus indicating that the Bβ 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(Bβ 1-42)-α-fibrin polymerized even more slowly than thrombin-activated des-(Bβ 1-42)-α-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent α-fibrin monomers. Thrombin-activated des-(Bβ 1-42)-α-fibrin polymerized more rapidly at low ionic strength (μ, 0.04) than did native α,β-fibrin, a reversal of their behavior at physiological ionic strength (μ, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(Bβ 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native α,β-fibrin at low ionic strength, coupled with weak thrombin binding to des-(Bβ 1-42)-α-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(Bβ 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B15-42 sequence in fibrin plays a major role in forming or providing this site

The Polymerization of Fibrinogen Dusart (Aα 554 Arg→Cys) After Removal of Carboxy Terminal Regions of the Aα-Chains

The six ploypeptide chains of normal fibrinogen are covalently linked by interchain disulphide bonds, and there are no free sulphhydryl groups. Fibrinogen Dusart is a congenital fibrinogen variant in which Aα 554 Arg is replaced by Cys; albumin is disulphide linked to these fibrinogen molecules, possibly at Aα 554 Cys. Functionally, Dusart fibrinogen displays markedly abnormal fibrin polymerization, characterized by delayed lateral fibril association and matrix fibre bundles that are thinner than normal fibrin bundles. These observations are consistent with experiments suggesting that the carboxy terminal region of the Aα-chain contains a polymerization domain that participates in lateral fibril associations. In order to investigate the location and the effect of albumin binding to Dusart fibrinogen, we examined the fibrinogen by electron microscopy, and compared the polymerization and ultrastructure of fibrin prepared from normal fibrinogen containing intact Aα-chains (fraction I-2) or plasmin degraded fibrinogen molecules lacking carboxy terminal regions of Aα-chains (fraction I-9D), with fibrin prepared from Dusart fraction I-2 and I-9D. Most bound albumin was released from Dusart fibrinogen by plasmin degradation involving the Aα-chains. Nevertheless, we were able to visualize albumin molecules remaining covalently bound to Dusart I-9D as well as to Dusart I-2 fibrinogen, as distinct globular domains situated near the fibrinogen D domain. The presence of albumin in these fractions was confirmed by Western blotting using anti-albumin. Dusart fibrin polymerized much more slowly than normal I-2, as previously reported, whereas polymerization of Dusart I-9D fibrin was faster than Dusart I-2 and nearly the same as normal I-9D fibrin. The ultrastructure of the Dusart I-9D fibrin matrix was indistinguishable from that of normal I-9D; both contained intensely striated thick fibres. In contrast, Dusart I-2 fibrin had thinner, more highly branched fibres with few striations. These results indicate; (1) most albumin bound to fibrinogen Dusart is removed by release of the carboxyl-terminal region of the Aα-chain, suggesting that at least 90% of the albumin is bound at Aα 554 Cys; (2) removal of the carboxyl-terminal region of the fibrinogen (I-9D) normalizes its polymerization properties relative to normal I-9D

Fibrinogen Naples I (Bβ A68T) Nonsubstrate Thrombin-Binding Capacities

Fibrinogen Naples I (Bβ A68T) is characterized by defective thrombin binding and fibrinopeptide cleavage at the fibrinogen substrate site in the E domain. We evaluated the fibrinogen of three homozygotic members of this kindred (II.1, II.2, II.3) who have displayed thrombophilic phenotypes and two heterozygotic subjects (I.1, I.2) who were asymptomatic. Electron microscopy of Naples I fibrin networks showed relatively wide fiber bundles, probably due to slowed fibrin assembly secondary to delayed fibrinopeptide release. We evaluated 125I-thrombin binding to the fibrin from subjects I.1, I.2, II.1, and II.2 by Scatchard analysis with emphasis on the high-affinity site in the D domain of fibrin(ogen) molecules containing a γ chain variant termed γ′. Homozygotic subjects II.1 and II.2 showed virtually absent low-affinity binding, consistent with the Bβ A68T mutation, whereas heterozygotes I.1 and I.2 showed only moderately reduced low-affinity binding. The homozygotes also showed impaired high-affinity thrombin binding, whereas that of the heterozygotes was nearly the same as normal. Genomic sequencing of the γ′ coding sequence (I.2, II.2), ELISA measurements of two γ′ chain epitopes (L2B, γ′409–412, and IF10, γ′417–427) (I.2, II.1, II.2, II.3), and mass spectrometry of Naples I fibrinogen (II.2) showed no differences from normal, thus indicating that there were no abnormal structural modifications of the γ′ chain residues in Naples I fibrinogen. However, thrombin reportedly utilizes both of its available exosites for binding to high- and low-affinity sites on normal fibrin, suggesting that binding is cooperative. Thus, reduced high-affinity thrombin binding to homozygotic Naples I fibrin may be related to the absence of low-affinity binding sites

Dynamic interaction between fetal adversity and a genetic score reflecting dopamine function on developmental outcomes at 36 months

Background: Fetal adversity, evidenced by poor fetal growth for instance, is associated with increasedrisk for several diseases later in life. Classical cut-offs to characterize small (SGA) and largefor gestational age (LGA) newborns are used to define long term vulnerability. We aimed atexploring the possible dynamism of different birth weight cut-offs in defining vulnerability indevelopmental outcomes (through the Bayley Scales of Infant and Toddler Development),using the example of a gene vs. fetal adversity interaction considering gene choices basedon functional relevance to the studied outcome. [...