Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity.

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Serine/threonine kinases in signal transduction in response to thrombin in human platelets. Use of 17-hydroxywortmannin to discriminate signals.

Although the importance of protein kinases in platelet activation, particularly protein kinase C (PKC), is well established there remain many problems regarding the various phosphorylation cascades, the role of phosphatases and the importance of other serine/threonine and tyrosine kinases. A particular problem is the mechanism of activation of the fibrinogen receptor, GPIIb/IIIa, a critical step in aggregation. Although GPIIIa is phosphorylated (on threonine) neither the stoichiometry nor the minor changes on activation seem adequate to explain the response. Relatively unspecific inhibitors of PKC such as staurosporine prevent PO4 incorporation into most kinase substrates but only inhibit platelet aggregation partially. However, staurosporine does induce activation and then inhibits several renaturable serine/threonine kinases, probably via phosphatases. Staurosporine did not, however, inhibit the platelet Ca2+ signal in response to thrombin but rather enhanced it. 17-Hydroxywortmannin (HWT), a fungal metabolite, has been shown to inhibit respiratory burst in neutrophils and causes haemorrhages. It was recently reported to be a myosin light chain kinase (MLCK) inhibitor and to inhibit PKC only at much higher concentrations. In platelets, HWT inhibits aggregation and partially inhibits phosphorylation of myosin light chain and P47 in thrombin-activated platelets. It also allows the discrimination of an early and a late phase in the cytoplasmic Ca2+ signal since at lower concentrations it only inhibits the late phase. The late phase of ATP release was also inhibited in a dose-dependent manner. The activation of most of the renaturable serine/threonine kinases was also inhibited by HWT. These results support earlier conclusions that the early phase of the Ca2+ signal is phospholipase C dependent but indicate that other mechanisms must be responsible for the late phase. The relative specificity of HWT for MLCK might indicate that this has an unexpected major role in controlling these late phase reactions including activation of GPIIb/IIIa or its clustering. However, staurosporine completely inhibits phosphorylation of myosin light chain by its kinase (as well as other kinases) and has the opposite effect on Ca2+ signals. Clearly, the interactions and feed-back mechanisms between these kinases are very complex but the results suggest that phosphatases acting together with their complementary kinases should also be considered as important platelet activation regulators. P47, long considered a major PKC substrate, may also be phosphorylated by MLCK.