Planeación de la perforación del pozo

Following ligand binding, the conserved ITAM tyrosine residues are phosphorylated by Src family kinases (SFK). Fyn and Lyn are the major two Src kinases mediating FcR phosphorylation as shown using mutant mice and precipitation studies (Ezumi et al, 1998; Quek et al, 2000),although, significantly, a residual level of activation is seen in mice deficient in the two Src kinases indicating involvement of one or more other Src kinases (Quek et al, 2000). Both Fyn and Lyn have been shown to associate via their SH3 domains with a poly-proline region of the cytoplasmic tail of GPVI placing them

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proximal to their substrate. Further, mutation of this region results in a partial inhibition of GPVI mediated signalling (Suzuki-Inoue et al, 2002; Bori-Sanz et al, 2003; Schmaier

et al, 2009). The protein tyrosine phosphatase CD148 has also been shown to play a

role in regulating Fyn and Lyn downstream of GPVI by dephosphorylating an inhibitory tyrosine residue which, due to intermolecular binding, holds the SFK in an inactive conformation (Senis et al, 2009). GPVI also contains a calmodulin (CaM) binding site which has shown to bind calmodulin in resting cells but which promotes dissociation upon activation. The functional significance of this site in mediating platelet activation is unclear (Andrews et al, 2002; Locke et al, 2003).

SFK-mediated phosphorylation of the two ITAM tyrosines provides a docking site for the tandem SH2 domains of the Syk family of tyrosine kinases, namely Syk, which is widely expressed in haematopoietic cells, and ZAP-70 (-chain associated protein of 70 kDa), which is localised to T-cells and a sub-population of natural killer cells. Binding of Syk or ZAP-70 to a phosphorylated ITAM leads to activation through a combination of molecular re-organisation and phosphorylation by both SFKs and auto- phosphorylation (Futterer et al, 1998; Brdicka et al, 2005; Arias-Palomo et al, 2007; Deindl et al, 2007). Classically, SH2 domains have micromolar affinity for phosphotyrosine groups, but the interaction between the two ITAM tyrosines and the tandem SH2 domains of Syk has nanomolar affinity due to cooperativity (Grucza et al, 1999). Moreover, mutagenesis studies have shown that both of the conserved ITAM tyrosine residues and also both of the Syk SH2 domains are required for signalling thereby favouring a model in which Syk kinase binds to the dually phosphorylated ITAM (Kurosaki et al, 1995; Fruehling and Longnecker, 1997; Abtahian et al, 2006).

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Mouse platelets which are deficient in Syk are unresponsive to collagen confirming the critical role of the tyrosine kinase in platelet activation by collagen (Poole et al, 1997).

GPVI-mediated activation of Syk has been shown to mediate phosphorylation of a number of downstream targets including the membrane scaffolding protein LAT (linker for activation of T-cells), which has nine conserved tyrosine residues (Pasquet et al, 1999; Judd et al, 2002) (Figure 1.3). When phosphorylated, LAT has been shown to bind to a range of SH2 domain-containing proteins thereby forming a „signalosome‟ that plays a key role in mediating platelet activation by GPVI. Among the proteins that bind to LAT in platelets are the cytosolic adaptors proteins Grb2 (growth factor receptor bound protein 2) and Gads (Grb2 related adaptor protein downstream of Shc), and PLC (Asazuma et al, 2000). The adaptor protein SLP-76 (SH2 domain containing leukocyte protein of 76 kDa) is also recruited to the signalosome through binding to Gads and PLC (Liu et al, 1999; Yablonski et al, 2001) and is critical for activation of PLC (Clements et al, 1999; Gross et al, 1999; Judd et al, 2002). LAT also binds to PI- 3 kinase (phosphatidylinositol 3-kinase) in platelets(Gibbins et al, 1998). The product of PI 3-kinase, PIP, binds to the pleckstrin homology (PH) domain of PLC2 thereby

supporting its localisation to the membrane. The LAT-Gads-SLP-76 signalling triad is the focus of the work in Chapter 4 and is discussed in further detail later (see Figure 1.3).

The recruitment of PLC2 to the signalosome allows it to become activated as a consequence of phosphorylation by the Tec family kinases, Btk (Bruton‟s tyrosine

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kinase) and Tec, which also bind to PIP3 in the membrane (Quek et al, 1998; Atkinson et al, 2003), and by binding to SLP-76 (Gross et al, 1999). A role for both Btk and Tec

was shown using double-deficient mouse platelets which exhibited a much greater loss signalling through GPVI compared to the individual knock-out platelets (Atkinson et al, 2003). However, because of the interplay of proteins in the LAT signalosome, it is unclear whether additional kinases also mediate phosphorylation of PLC2, including Src family kinases.

The Vav family of guanine nucleotide exchange factors (GEF) have also been shown to be recruited to the LAT signalosome by binding to SLP-76, Btk/Tec and Syk, and to play a critical role in mediating PLC2 activation (Pearce et al, 2002; Pearce et al, 2004). In this pathway, Vav is believed to function as an adaptor rather than as a GEF for small G proteins, as mutation of the GEF domain does not inhibit TCR signalling (Kuhne et al, 2000). As with the SFKs and Btk/Tec kinases, there is redundancy between the Vav family members, as mice deficient in both Vav1 and Vav3 show defective GPVI signalling whereas mice deficient in Vav1, Vav2 or Vav3 alone, or a combination of both Vav1 and Vav2 show no obvious defect in GPVI signalling (Pearce

et al, 2002; Pearce et al, 2004).

Activation of PLC2 leads to hydrolysis of its substrate, phosphatidylinositol-4,5- bisphosphate (PIP2), to form the two second messengers, inositol-1,4,5-trisphosphate

(IP3) and 1,2-diacylglycerol (DAG), which release Ca2+ from intracellular stores and

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blockade in GPVI signalling, although this is not complete as mouse platelets also express a small amount of PLC1 (Suzuki-Inoue et al, 2003).

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