There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKC␣, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Gö 6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKC␣ but not PKC␤ was activated, occurring in an Syk-and phospholipase C-dependent manner. Syk and PKC␣ associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKC␣ and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKC␣ is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKC␣, consistent with a lack of effect of PKC␣ inhibition on Syk activity. PKC␣ also associates with Src; although in contrast to interaction with Syk, PKC␣ activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Gö 6976. Therefore, there is a complex interplay between PKC␣, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.

The d spacing of the 355 reflexion in silicon has been compared with the d spacing of the 800 reflexion in germanium using pseudo non-dispersive multiple-beam X-ray diffractometry with Mo Kal radiation. This technique gives the ratio of the two lattice parameters without the need for a precise knowledge of the X-ray wavelength. Symmetric transmission geometry was used to eliminate the refractive index correction. The results were: d(800 Ge) _ 1.0002348 (+ 0.0000006) at 22.5 °C d(355 Si) and d(800 Ge) _ 1.0002458 ( + 0-0000016) at 25 °C. d(355 Si)By using the known lattice parameter of silicon obtained by X-ray and optical interferometry it was found that the lattice parameter of germanium at 25 °C is 5.6579060+ 0.0000092/~.

Platelet adhesion to vascular subendothelial proteins at the site of blood vessel injury is critical for initiating haemostasis. Collagen is a major matrix protein that binds plasma von Willebrand factor (vWF) when the endothelium becomes damaged and therefore in vivo platelets are likely to encounter both of these agonists simultaneously, through glycoprotein VI (GPVI) and alpha2beta1 receptors for collagen and GPIb-V-IX and alphaIIbbeta3 receptors for vWF. We hypothesised a potentiatory role for vWF that would synergise with collagen leading to functional activation and show this to be the case for platelet aggregation, 5-HT secretion and calcium responses. Synergy between these two agonists is likely to involve receptors GPVI and GPIb-V-IX, for collagen and vWF, respectively, since 5-HT secretion in response to collagen is potentiated by vWF in the presence of either EGTA or EDTA, which prevent binding to integrins alphaIIbbeta3 (EGTA) or both alphaIIbbeta3 and alpha2beta1 (EDTA). In addition, vWF is also able to potentiate 5-HT secretion responses to collagen-related peptide, confirming that GPVI is able to support synergy with vWF. These findings are important in that they reveal a novel role for vWF in platelet activation as a potentiator of platelet activation by collagen.

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