The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.

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Fibroblast growth factors are essential molecules for development. Here we characterize Fgfl7, a new member of the fibroblast growth factor (FGF) family. The Fgfl7 gene maps to mouse chromosome 14 and is highly conserved between mouse and human (93% identity). It exhibits 60% amino acid identity with Fgf8 and 50% identity with Fgf8. Both Fgf8 and Fgf17 have a similar structure and a similar pattern of alternative splicing in the 5' coding region. When expressed in 3T3 fibroblasts, mouse FGF17 is transforming, indicating that it can activate the 'c' splice form of either FGF receptor (FGFR) one or two. During midgestation embryogenesis, in situ hybridization analysis localized Fgf17 expression to specific sites in the midline structures of the forebrain, the midbrain-hindbrain junction, the developing skeleton and in developing arteries. Comparison to Fgf8 revealed a striking similarity in expression patterns, especially in the central nervous system (CNS), suggesting that both genes may be important for CNS development, although Fgf17 is expressed somewhat later than Fgf8. In the developing skeleton, both genes are expressed in costal cartilage while Fgf8 is preferentially expressed in long bones. In the developing great vessels Fgfl7 is preferentially expressed, suggesting that it may have a more prominent role in vascular growth.

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FGF-8 is a member of the family of fibroblast growth factors and is expressed during vertebrate embryo development. Eight potential FGF-8 isoforms are generated by alternative splicing in mice, several of which are expressed during embryogenesis in epithelial locations. The significance of the multiple isoforms is currently unknown. In this report, we investigate the expression patterns and the specificity of the FGF-8 isoforms for known fibroblast growth factor (FGF) receptors. RNAs for seven of the eight potential isoforms are present at multiple sites of embryonic Fgf8 expression. None of the FGF-8 isoforms exhibited activity when assayed with BaF3 cells expressing the "b" splice forms of FGF receptors 1-3, which are mostly expressed in epithelial tissues. Mesenchymally expressed "c" splice forms of FGF receptors 2 and 3 and FGF receptor 4 were activated by several FGF-8 isoforms. These findings are consistent with the hypothesis that the multiple FGF-8 isoforms are functionally redundant and function to signal in paracrine (epithelial to mesenchymal) contexts.The mammalian fibroblast growth factor (FGF) 1 family currently consists of structurally related polypeptides encoded by 10 genes (FGF-1-10) (reviewed in Ref. 1;. Four distinct genes code for high affinity transmembrane receptor tyrosine kinases (FGFR1-4) that bind FGF ligands and display varying patterns of expression (reviewed in Ref. 5). Alternative mRNA splicing generates isoforms of receptors 1-3 that exhibit unique ligand binding characteristics (6 -9). FGF receptor activation involves ligand binding and receptor dimerization, followed by transphosphorylation of the receptor and transduction of the signal into a biological response (5). FGF signal transduction has been implicated in development, wound healing, angiogenesis, and tumorigenesis (reviewed in Ref. 1). Germ line mutations of FGFs and FGFRs in mice (10 -15) and humans (16 -21) demonstrate the importance of FGF signaling in the process of development.FGF-8 was first identified as an androgen-induced growth factor secreted by the mouse mammary tumor cell line SC-3 (2). Subsequently, Fgf8 was identified as a Wnt1-cooperating protooncogene in murine mammary tumorigenesis (22). Fgf8 expression has been detected during murine and chicken embryogenesis in regions of outgrowth and patterning, including the primitive epiblast, the apical ectodermal ridge of the limb bud, the primitive streak, the tail bud, the facial primordia, and the midbrain-hindbrain junction (22-29). The murine and human genes encoding FGF-8 have been localized to mouse chromosome 19 and human chromosome 10q24 -26 (25, 26, 30, 31). The murine Fgf8 gene is unusual in the FGF family in that there are four exons (exons 1A-1D) equivalent to the usual exon 1 in other FGF genes (22,25,32). Alternative splicing of the four alternatively spliced exons results in potentially eight protein isoforms that differ at their amino termini and share a common carboxyl terminus encoded by exons 1D, 2, and 3 (22,25,32). Human FGF8 is similar ...