David H. Farrell

Complete Sequence of Virulence Plasmid pJM1 from the Marine Fish Pathogen Vibrio anguillarum Strain 775

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.

Elevated plasma fibrinogen γ′ concentration is associated with myocardial infarction: Effects of variation in fibrinogen genes and environmental factors

Summary.  Background:  Fibrinogen γ′, a fibrinogen γ‐chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease.

Fibrinogen γ′ chain binds thrombin exosite II

Summary.  A high‐affinity thrombin‐binding site in an alternately processed fibrinogen variant, the γA/γ′ isoform, is characterized in this report. The binding site has been shown to be situated between γ′ 414 and 427, and Tyr418 and 422 in this part of the γ′ chain are known to be sulfated. A synthetic peptide corresponding to the γ′ chain carboxyl terminus is shown to bind thrombin with a Kd = 0.63 ± 0.16 µmol L−1. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the γ′ peptide via exosite II. Thus, thrombin binding to the γ′ chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin‐bound thrombin can retain enzymatic activity, and why fibrin‐bound thrombin is heparin‐resistant.

Influence of γ′ Fibrinogen Splice Variant on Fibrin Physical Properties and Fibrinolysis Rate

Objective—A splice variant of fibrinogen, &ggr;′, has an altered C-terminal sequence in its gamma chain. This &ggr;A/&ggr;′ fibrin is more resistant to lysis than &ggr;A/&ggr;A fibrin. Whether the physical properties of &ggr;′ and &ggr;A fibrin may account for the difference in their fibrinolysis rate remains to be established. Methods and Results—Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G′, in dyne/cm2), were measured after clotting &ggr;A and &ggr;′ fibrinogens (1 mg/mL). &ggr;′/&ggr;′ fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than &ggr;A/&ggr;A fibrin (12±2 fiber/10−3nm3 versus 16±2 fiber/10−3nm3 and 274±38 nm versus 257±41 nm for &ggr;′/&ggr;′ and &ggr;A/&ggr;A fibrin, respectively; P =NS). This resulted in a 20% increase of the permeability constant (6.9±1.7 10−9 cm2 versus 5.5±1.9 10−9 cm2, respectively; P =NS). Unexpectedly, &ggr;′ fibrin was found to be 3-times stiffer than &ggr;A fibrin (72.6±2.6 dyne/cm2 versus 25.1±2.3 dyne/cm2; P <0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate. Conclusions—Fibrinolysis resistance that arises from the presence of &ggr;A/&ggr;′ fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.

A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron.

The angR locus in Vibrio anguillarum encodes a trans-acting transcriptional activator which modulates several Fe2(+)-regulated loci in the anguibactin biosynthesis gene cluster. In this paper, the complete nucleotide (nt) sequence of the angR gene and deduced amino acid (aa) sequence of the AngR protein are presented. A region upstream from the angR gene is shown to have similarity with Fe2(+)-regulated operators in Escherichia coli which bind the Fur protein. The involvement of a Fur-like regulator is supported by transcription analysis which show that angR itself is Fe2(+)-regulated. The aa sequence of the AngR protein predicts a helix-turn-helix motif which shows striking homology with prokaryotic DNA-binding proteins, particularly the lambda and P22 Cro proteins. In addition, there are two 18-nt regions, upstream from the angR gene, which show similarity with the OR1 and OR2 operators of P22 cro. These regions overlap with, respectively, the -35, -10 region and the putative Fur-binding region upstream from angR. These results suggest that AngR may be a DNA-binding protein which modulates Fe2(+)-regulated transcription and is itself Fe2(+)-regulated at the transcriptional level.

Pathophysiologic roles of the fibrinogen gamma chain

Purpose of reviewFibrinogen binds through its γ chains to cell surface receptors, growth factors, and coagulation factors to perform its key roles in fibrin clot formation, platelet aggregation, and wound healing. However, these binding interactions can also contribute to pathophysiologic processes, including inflammation and thrombosis. This review summarizes the latest findings on the role of the fibrinogen γ chain in these processes, and illustrates the potential for therapeutic intervention. Recent findingsNovel γ chain epitopes that bind platelet integrin αIIbβ3 and leukocyte integrin αMβ2 have been characterized, leading to the revision of former dogma regarding the processes of platelet aggregation, clot retraction, inflammation, and thrombosis. A series of studies has shown that the γ chain serves as a depot for fibroblast growth factor-2 (FGF-2), which is likely to play an important role in wound healing. Inhibition of γ chain function with the monoclonal antibody 7E9 has been shown to interfere with multiple fibrinogen activities, including factor XIIIa crosslinking, platelet adhesion, and platelet-mediated clot retraction. The role of the enigmatic variant fibrinogen γ′ chain has also become clearer. Studies have shown that γ′ chain binding to thrombin and factor XIII results in clots that are mechanically stiffer and resistant to fibrinolysis, which may explain the association between γA/γ′ fibrinogen levels and cardiovascular disease. SummaryThe identification of new interactions with γ chains has revealed novel targets for the treatment of inflammation and thrombosis. In addition, several exciting studies have shown new functions for the variant γ′ chain that may contribute to cardiovascular disease.

A novel highly porous silica and chitosan-based hemostatic dressing is superior to HemCon and gauze sponges.

BACKGROUND Hemostatic dressings have become increasingly popular as the optimal initial treatment for severe hemorrhage. The purpose of this study was to compare the hemostatic properties of a novel highly porous silica and chitosan-based dressing (TraumaStat) to HemCon, and gauze dressing in a severe groin injury model in swine. METHODS Thirty swine were blindly randomized to receive TraumaStat, HemCon, or standard gauze dressing for hemostatic control. A complex groin injury involving complete transaction of the femoral artery and vein was made. After 30 seconds of uncontrolled hemorrhage, the randomized dressing was applied and pressure was held for 5 minutes. Fluid resuscitation was initiated to achieve and maintain the baseline mean arterial pressure and the wound was inspected for bleeding. Failure of hemostasis was defined as pooling of blood outside of the wound. Animals were then monitored for 120 minutes and surviving animals were euthanized. RESULTS Blood loss before treatment was similar between groups (p > 0.1). TraumaStat had one failure, compared with five for gauze, and eight for HemCon (p = 0.005, TraumaStat vs. HemCon). TraumaStat significantly reduced median blood loss when compared with both HemCon and gauze (117 vs. 774 and 268 mL respectively, p < 0.05). At study conclusion, TraumaStat animals had a greater median hematocrit than both HemCon (24 vs. 19, p = 0.033), and gauze (24 vs. 19, p = 0.049) animals. Median volume of fluid resuscitation and mortality were not different between groups (p > 0.1). CONCLUSIONS TraumaStat was superior to HemCon and gauze dressings in controlling bleeding from a severe groin injury. TraumaStat may be a better hemostatic dressing for control of active hemorrhage than current standards of care.

Expression of Antisense to Integrin Subunit β3 Inhibits Microvascular Endothelial Cell Capillary Tube Formation in Fibrin

αvβ3antagonists are potent angiogenesis inhibitors, and several different classes of inhibitors have been developed, including monoclonal antibodies, synthetic peptides, and small organic molecules. However, each class of inhibitor works by the same principal, by blocking the binding of ligands to αvβ3. In an effort to develop an αvβ3 inhibitor that down-regulates the actual level of αvβ3, we developed an antisense strategy to inhibit αvβ3 expression in vitro. β3 antisense expressed in endothelial cells specifically down-regulated αvβ3 and inhibited capillary tube formation, with the extent of down-regulation correlating with the extent of tube formation inhibition. This inhibition was matrix-specific, since tube formation was not inhibited in Matrigel. These findings support the notion that αvβ3is required for an essential step of angiogenesis in fibrin, namely capillary tube formation. These results suggest that pseudogenetic inhibition of β3 integrins using antisense techniques may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.

The Role of γA/γ′ Fibrinogen in Plasma Factor XIII Activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, γA/γA fibrin, or a minor variant form, γA/γ′ fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, γA/γ′ fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin γA and γ′ chains and a significantly more rapid cross-linking of fibrin α chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, γA/γ′ fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.

Assessment of Genetic Determinants of the Association of γ′ Fibrinogen in Relation to Cardiovascular Disease

Objective—&ggr;′ fibrinogen is a newly emerging biomarker that is associated with cardiovascular disease (CVD). However, the genetic determinants of &ggr;′ fibrinogen levels are unknown. We therefore conducted a genome-wide association study on 3042 participants from the Framingham Heart Study Offspring Cohort. Methods and Results—A genome-wide association study with 2.5 million single-nucleotide polymorphisms (SNPs) was carried out for &ggr;′ fibrinogen levels from the cycle 7 examination. Fifty-four SNPs in or near the fibrinogen gene locus demonstrated genome-wide significance (P<5.0×10−8) for association with &ggr;′ fibrinogen levels. The top-signal SNP was rs7681423 (P=9.97×10−110) in the fibrinogen gene locus near FGG, which encodes the &ggr; chain. Conditional on the top SNP, the only other SNP that remained genome-wide significant was rs1049636. Associations between SNPs, &ggr;′ fibrinogen levels, and prevalent CVD events were examined using multiple logistic regression. &ggr;′ fibrinogen levels were associated with prevalent CVD (P=0.02), although the top 2 SNPs associated with &ggr;′ fibrinogen levels were not associated with CVD. These findings contrast those for total fibrinogen levels, which are associated with different genetic loci, particularly FGB, which encodes the B&bgr; chain. Conclusion—&ggr;′ fibrinogen is associated with prevalent CVD and with SNPs exclusively in and near the fibrinogen gene locus.