Scar formation in the nervous system begins within hours after traumatic injury and is characterized primarily by reactive astrocytes depositing proteoglycans that inhibit regeneration. A fundamental question in CNS repair has been the identity of the initial molecular mediator that triggers glial scar formation. Here we show that the blood protein fibrinogen, which leaks into the CNS immediately after blood-brain barrier (BBB) disruption or vascular damage, serves as an early signal for the induction of glial scar formation via the TGF-␤/Smad signaling pathway. Our studies revealed that fibrinogen is a carrier of latent TGF-␤ and induces phosphorylation of Smad2 in astrocytes that leads to inhibition of neurite outgrowth. Consistent with these findings, genetic or pharmacologic depletion of fibrinogen in mice reduces active TGF-␤, Smad2 phosphorylation, glial cell activation, and neurocan deposition after cortical injury. Furthermore, stereotactic injection of fibrinogen into the mouse cortex is sufficient to induce astrogliosis. Inhibition of the TGF-␤ receptor pathway abolishes the fibrinogen-induced effects on glial scar formation in vivo and in vitro. These results identify fibrinogen as a primary astrocyte activation signal, provide evidence that deposition of inhibitory proteoglycans is induced by a blood protein that leaks in the CNS after vasculature rupture, and point to TGF-␤ as a molecular link between vascular permeability and scar formation.

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The polymerization of fibrin occurs primarily through interactions between N-terminal A-and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a-and b-holes in the-and-modules. Of the potential knob-hole interactions-A:a, B:b, A:b, and B:a-the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers-based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the D364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mi-metic peptide, the (15-66) 2 fragment containing 2 B-knobs, and a monoclonal anti-body against the 15-21 sequence. The interaction of desB-fibrin with fragment D containing a-and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer. (Blood. 2007;109:130-138)

Integrin alphavbeta3 is specifically but transiently expressed on the tips of capillary sprouts as they invade the fibrin clot during angiogenesis of cutaneous wound repair. Specific blocking of alphavbeta3 function inhibits granulation tissue formation in cutaneous wounds. The mechanisms of regulation of alphavbeta3 expression on human dermal microvascular endothelial cells, however, have not been fully delineated. As alphavbeta3 was highly expressed on capillary sprouts in 5 d wounds rich in fibrin, but was almost undetectable on blood vessels in 7 d wounds rich in collagen, we hypothesized that the extracellular matrix environment could regulate human dermal micro- vascular endothelial cell alphavbeta3 expression. To address this, human dermal microvascular endothelial cells were cultured on surfaces coated with collagen, fibronectin, and gelatin, and mRNA levels of integrin alphav/beta3 were determined. Compared with human dermal microvascular endothelial cells on collagen, mRNA levels of alphav/beta3 were higher in human dermal microvascular endothelial cells on fibronectin and on gelatin. To simulate the in vivo environment better, human dermal microvascular endothelial cells cultured on collagen were overlaid by fibrin or collagen gels prior to assessment of alphav/beta3 mRNA levels. alphav/beta3 mRNA levels were higher in human dermal microvascular endothelial cells surrounded by a three-dimensional fibrin gel compared with a collagen gel, whether angiogenic factors were present or absent. As modulation of mRNA stability is a potential regulatory mechanism for integrin expression, integrin subunit mRNA stability was assessed. beta3 mRNA decayed much faster than alphav, alpha2, and beta1 mRNA. Three-dimensional fibrin gels enhanced alphav/beta3 mRNA stability compared with collagen gels. We propose that the provisional matrix molecules in the wound clot regulate angiogenesis associated with cutaneous wound repair through their modulation of integrin receptor expression.