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

The Location of the Carboxy-Terminal Region of γ Chains in Fibrinogen and Fibrin D Domains

Elongated fibrinogen molecules are comprised of two outer “D” domains, each connected through a “coiled-coil” region to the central “E” domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of γ chains in each D domain (the γXL site) by incorporating intermolecular ɛ-(γ-glutamyl)lysine bonds between amine donor γ406 lysine of one γ chain and a glutamine acceptor at γ398 or γ399 of another. Several lines of evidence show that crosslinked γ chains extend “transversely” between the strands of each fibril, but other data suggest instead that crosslinked γ chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the γXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled γ398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal γ chains in fibrinogen or fibrin are oriented toward the central domain and indicate that γXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking

The Relationship Between the Fibrinogen D Domain Self-Association/Cross-Linking Site (gammaXL) and the Fibrinogen Dusart Abnormality (Aalpha R554C-albumin): Clues to Thrombophilia in the Dusart Syndrome

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ( gammaXL ). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH-terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 \u3e I-9 \u3e 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal alphaC region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ( Dusart Syndrome ), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen alphaC regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the Dusart Syndrome

Radiotherapy for tumors of the stomach and gastroesophageal junction - a review of its role in multimodal therapy

There is broad consensus on surgical resection being the backbone of curative therapy of gastric- and gastroesophageal junction carcinoma. Nevertheless, details on therapeutic approaches in addition to surgery, such as chemotherapy, radiotherapy or radiochemotherapy are discussed controversially; especially whether external beam radiotherapy should be applied in addition to chemotherapy and surgery is debated in both entities and differs widely between regions and centers. Early landmark trials such as the Intergroup-0116 and the MAGIC trial must be interpreted in the context of potentially insufficient lymph node resection. Despite shortcomings of both trials, benefits on overall survival by radiochemotherapy and adjuvant chemotherapy were confirmed in populations of D2-resected gastric cancer patients by Asian trials. Recent results on junctional carcinoma patients strongly suggest a survival benefit of neoadjuvant radiochemotherapy in curatively resectable patients. An effect of chemotherapy in the perioperative setting as given in the MAGIC study has been confirmed by the ACCORD07 trial for junctional carcinomas; however both the studies by Stahl et al. and the excellent outcome in the CROSS trial as compared to all other therapeutic approaches indicate a superiority of neoadjuvant radiochemotherapy as compared to perioperative chemotherapy in junctional carcinoma patients. Surgery alone without neoadjuvant or perioperative therapy is considered suboptimal in patients with locally advanced disease. In gastric carcinoma patients, perioperative chemotherapy has not been compared to adjuvant radiochemotherapy in a randomized setting. Nevertheless, the results of the recently published ARTIST trial and the Chinese data by Zhu and coworkers, indicate a superiority of adjuvant radiochemotherapy as compared to adjuvant chemotherapy in terms of disease free survival in Asian patients with advanced gastric carcinoma. The ongoing CRITICS trial is supposed to provide reliable conclusions about which therapy should be preferred in Western patients with gastric carcinoma. If radiotherapy is performed, modern approaches such as intensity-modulated radiotherapy and image guidance should be applied, as these methods reduce dose to organs at risk and provide a more homogenous coverage of planning target volumes

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases

Nanofibrous Scaffolds Incorporating PDGF-BB Microspheres Induce Chemokine Expression and Tissue Neogenesis In Vivo

Platelet-derived growth factor (PDGF) exerts multiple cellular effects that stimulate wound repair in multiple tissues. However, a major obstacle for its successful clinical application is the delivery system, which ultimately controls the in vivo release rate of PDGF. Polylactic-co-glycolic acid (PLGA) microspheres (MS) in nanofibrous scaffolds (NFS) have been shown to control the release of rhPDGF-BB in vitro. In order to investigate the effects of rhPDGF-BB release from MS in NFS on gene expression and enhancement of soft tissue engineering, rhPDGF-BB was incorporated into differing molecular weight (MW) polymeric MS. By controlling the MW of the MS over a range of 6.5 KDa–64 KDa, release rates of PDGF can be regulated over periods of weeks to months in vitro. The NFS-MS scaffolds were divided into multiple groups based on MS release characteristics and PDGF concentration ranging from 2.5–25.0 µg and evaluated in vivo in a soft tissue wound repair model in the dorsa of rats. At 3, 7, 14 and 21 days post-implantation, the scaffold implants were harvested followed by assessments of cell penetration, vasculogenesis and tissue neogenesis. Gene expression profiles using cDNA microarrays were performed on the PDGF-releasing NFS. The percentage of tissue invasion into MS-containing NFS at 7 days was higher in the PDGF groups when compared to controls. Blood vessel number in the HMW groups containing either 2.5 or 25 µg PDGF was increased above those of other groups at 7d (p<0.01). Results from cDNA array showed that PDGF strongly enhanced in vivo gene expression of the CXC chemokine family members such as CXCL1, CXCL2 and CXCL5. Thus, sustained release of rhPDGF-BB, controlled by slow-releasing MS associated with the NFS delivery system, enhanced cell migration and angiogenesis in vivo, and may be related to an induced expression of chemokine-related genes. This approach offers a technology to accurately control growth factor release to promote soft tissue engineering in vivo