Thrombus formation is mediated through a Src kinase-dependent pathway but does not require activation of map kinase or cGMP-dependent kinases Stuart J.Marshall1, Yotis A. Senis2, Jocelyn M. Auger2, Gary Salmon2 and Steve P.Watson1,3 1Dept. of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K. 2Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, CT13 9NJ, U.K.3Division of Medical Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K

Print Abstract Search PubMed for: Marshall SJ Yotis AS Auger JM Salmon G Watson SP

Glycoprotein (GP) Ib-IX-V, the platelet receptor for von Willebrand factor (vWf), plays an important role in haemostasis by mediating platelet adhesion to exposed sub-endothelium at high shear and by promoting aggregate formation. Recently, we have reported that ristocetin-vWf stimulates GPIIb-IIIa-dependent aggregation through activation of Src family kinases (Marshall et al, 2003). Interestingly, Li et al., (2003) reported that this response is also dependent on protein kinase G (PKG). The present study was undertaken to investigate the role of cGMP in this tyrosine kinase-dependent pathway.

Platelet aggregation was investigated in platelet rich plasma (PRP) in response to subthreshold (1mg/ml) and submaximal (1.4mg/ml) concentrations of the antibiotic ristocetin which promotes binding of vWf to GPIb-IX-V. Platelet cGMP levels were measured using an ELISA.

The phosphodiesterase-5 inhibitor sildenafil and the NO donor glyco-SNAP-1 (G-SNAP) dose dependently inhibited aggregation induced by ristocetin (1.4 mg/ml) with maximal inhibition of 97.4 ± 0.4% (n=4) and 98.0 ± 0.9% (n=4) observed at 30 and 10µM, respectively. Furthermore, sildenafil (30µM) and G-SNAP (10µM) did not enhance aggregation to a subthreshold concentration of ristocetin. However, ristocetin stimulated aggregation was significantly attenuated by the Src kinase inhibitor PD173952 (Kraker et al, 2000). In addition, the specific PKG inhibitors Rp-8-pCPT-cGMPS (0.3mM) and Rp-8-Br-PET-cGMPS (0.3mM) had no apparent effect on aggregation to a submaximal concentration of ristocetin. Furthermore, both inhibitors potentiated the inhibitory action of G-SNAP at the higher concentration of 1 mM suggesting a PKG-independent mode of action. These data suggest that cGMP does not play a role in ristocetin-mediated aggregation. In support of this, ristocetin (1.4mg/ml) did not increase cGMP levels over basal levels. In contrast, sildenafil and G-SNAP promoted a significant rise in intracellular cGMP from a basal value of 9.9±3.6nmol/1x10⁷ platelets to 130.2±6.1 and 190±10.2 nmol/1x10⁷ platelets, respectively (n=4).

These results demonstrate that the phosphodiesterase-5 inhibitor sildenafil promotes a similar increase in cGMP to that induced by G-SNAP, but that cGMP and PKG do not play a role in mediating ristocetin-induced aggregation. The explanation for the opposing conclusions of those of Li et al., (2003) may be explained, in part, by a non-specific action of the two PKG inhibitors.

Marshall SJ, Asazuma N, Best D, Wonerow P, Salmon G, Andrews RK, Watson SP. (2002) Biochem J. 361, 297-305.
Li Z, Xi X, Gu M, Feil R, Ye RD, Eigenthaler M, Hofmann F, Du X. (2003) Cell. 112, 77-86.
Kraker AJ, Hartl BG, Amar AM, Barvian MR, Showalter HD, Moore CW. (2000) Biochem Pharmacol. 60, 885-898.