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A dimeric 140 kDa [(Fib-2).sub.2] fragment containing both Fib-2 regions are prepared by limited proteolysis in order to clarify the role of another pair of fibrin-binding regions, Fib-2, located at the disulfide-linked COOH-terminal ends of fibronectin. Experiments revealed about 30% inhibition of the Fib-2 mediated binding by increasing concentrations of Fib-1 fragment.

Details of the crystal structure of human thrombin in complex with the central part of human fibrin are revealed and results are presented of the molecular modeling of the fibrinopeptides A and B (fpA- and fpB-) containing portions of the Aalpha and Bbeta chains, in both fibrinogen and protofibrils. Analysis provides a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils at the second stage.

The NMR solution structure of the recombinant bovine fibrinogen alphaC-domain fragment, including the residues Aalpha374-538, which reveals a type of I' beta-hairpin and a collapsed hydrophobic cluster is reported. The low stability of the alphaC-domain provides a proper explanation for the previously observed intra- and intermolecular interactions of these domains in fibrinogen and fibrin.

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1[alpha] functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1[alpha] gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1[alpha], lacking the oxygen-sensitive degradation domain, namely HIF-1[alpha][DELTA]ODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1[alpha][DELTA]ODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-[A.sub.165] mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 [micro]g per wound in 30/[micro]l of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-[A.sub.165] protein (1.25 [micro]g per wound in 30 [micro]l of fibrin). However, the maturity of the vessels induced by HIF-1[alpha][DELTA]ODD was significantly higher than that induced by VEGF-[A.sub.165] protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis. gene delivery | nonviral vector | polycation | fibrin

The binding of [[alpha].sub.2]-antiplasmin ([[alpha].sub.2]AP) to fibrin (ogen) and its fragments are analyzed by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The noncovalent interaction of [[alpha].sub.2]-AP with fibrin has contributed to regulation of the initial stage of fibrinolysis and has provided proper orientation of the cross-linking sites to facilitate covalent cross-linking of [[alpha]-2]-AP to the fibrin clot.

Several recombinant fragments containing individual extracellular domains of VE-cadherin and several fragments corresponding to various fibrin(ogen) regions are prepared to test the interactions involved in fibrin(ogen) region/domains and localize the complementary fibrin-binding site in VE-cadherin. The experimental results confirmed that the fibrin-binding properties of the VE-cadherin are not influenced by the calcius ions that are required to maintain proper conformation and adhesive properties of the VE-cadherin.