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SummaryBackground-The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood.

Summary.  Background: A signaling pathway is difficult, if not impossible, to elucidate in platelets using only in vivo studies. Likewise, the physiological significance of signaling information obtained exclusively from in vitro observations is unknown. Therefore, both in vitro and in vivo experiments are required to establish the physiological significance of a signaling pathway. Objective: To evaluate the physiological significance of signaling data obtained from botrocetin (bt)/von Willebrand factor (VWF)‐stimulated washed platelets. Method: Stable thrombus formation in response to FeCl3‐induced injury of the mouse carotid artery was used to evaluate the physiological significance of signaling data obtained from bt/VWF‐stimulated washed platelets. Results: Syk, PLCγ2, Gαq and P2Y12, but not LAT, were found either to be required for or to affect stable thrombus formation. Prior in vitro studies had demonstrated that LAT is not required for bt/VWF‐induced platelet aggregation in the presence of exogenous fibrinogen. These data provide the first demonstration of the in vivo role for these signaling molecules in GPIb‐dependent/initiated signal transduction and are consistent with the signaling pathway deduced from in vitro studies of bt/VWF‐stimulated washed platelets using metabolic inhibitors and knockout mice. Conclusion: The broad agreement between the in vitro and the in vivo results establish that bt/VWF stimulation of washed platelets can provide physiologically significant glycoprotein Ib‐dependent/initiated signaling data.

A human phosphophoryn (PP) cDNA was previously cloned from immature root apex total RNA in our laboratory. This cDNA comprises 2,364 bp, encoding 788 amino acids. More than 80% of the sequences are arranged as (DSS)(n) (n = 1-16), DS, and NSS motifs. We hypothesize that the capability of PP to bind Ca(2+) and nucleate hydroxyapatite may depend on these repeated sequences. Two polypeptides were synthesized based on the human PP cDNA sequence to test the hypothesis. One polypeptide has the amino acid sequence DDPNSSDESNGNDD (synthetic polypeptide 1, SP1), which is from the N-terminal end of PP; the other polypeptide, DSKSDSSKSESDSS (synthetic polypeptide 2, SP2), is the PP repeated sequence motif. Phosphorylation of the polypeptides was accomplished by reacting them with adenosine triphosphate and casein kinases I and II. The ability of these molecules to cause mineralization was tested in a steady-state agarose gel system. The results show that phosphorylated SP2 (P-SP2) precipitated approximately 60% of the total Ca + PO(4) precipitated by PP. P-SP1 precipitated about 23% of that precipitated by PP and was similar to the amount precipitated in the control gel, that is, without added peptides. Transmission electron microscopy and X-ray diffraction analysis showed that the precipitate formed in the P-SP2-containing gel was hydroxyapatite. The capability of P-SP2 to nucleate Ca + PO(4) and precipitate hydroxyapatite is a result of the repeated sequence motif, which contains a high percentage of phosphorylated serine. This molecule could be used in the repair and regeneration of dental tissue.

In implantology it is known that fibronectin affects cell-substrate adhesion, consequently, the structure and composition of the initially adsorbed fibronectin layer to a large extent determines the biological response to a biomaterial implanted into the body. In this study we have used neutron reflectometry and quartz-crystal microbalance with dissipation to investigate the amount of fibronectin adsorbed, the layer density, thickness and structure of films adsorbed to polished silicon oxide surfaces. We have cultured MG63 osteoblast-like cells on surfaces coated and uncoated with fibronectin and monitored the cellular response to these surfaces. The results show that at fibronectin concentrations in the range 0.01 to 0.1 mg/ml a single highly hydrated layer of fibronectin approximately 40-50 Å in thickness adsorbs to a polished silicon oxide surface and is likely to correspond to one diffuse monolayer of fibronectin arranged side-on. Cells cultured on this fibronectin layer have dramatically different morphology and growth to those grown on bare surfaces. Using a model silicon oxide surface has enabled us to study the substrate/protein interface, together with the impact of a fibronectin layer on the cellular response using consistent experimental conditions across a unique set of experimental techniques.