The Regulation of Phosphorylation Events in Platelets

Abstract

Platelets play a vital role in processes of hemostasis and thrombosis under physiological and pathological conditions. Following vascular damage, platelets will accumulate and stably adhere to exposed subendothelial matrixes. The binding of platelet surface receptor Glycoprotein VI (GPVI) to exposed collagen initiates a signaling cascade, which culminates in platelet activation. Stimulation of GPVI pathways results in the generation of thromboxane and causes the platelets to secrete their granule contents. This generated thromboxane as well as constituents released from dense granules such as ADP, and serotonin, play an essential role in potentiating the platelet response through activation of other surface receptor mediated pathways. Importantly, downstream of all these separate pathways, kinases become activated and play a crucial role in phosphorylating their substrates to elicit critical cellular responses. Previously published studies have established the importance for myosin kinase in its role for phosphorylating the myosin light chain (MLC) downstream of ADP receptors. These studies have shown MLC phosphorylation occurs rapidly and is essential for shape change following the stimulation of ADP receptors. Technological advances in antibody development have resulted in the generation of commercially available phospho-specific antibodies for MLC phosphorylated on either threonine (Thr) 18 or serine (Ser) 19. These antibodies allowed us to revisit these prior studies and address whether phosphorylation on MLC (Ser) 19 would elicit one response while phosphorylation on (Thr) 18 may result in another functional response. Our result show, that MLC is phosphorylated rapidly on (Ser) 19 and plays an important role in shape change downstream of Gq pathways, while MLC (Thr) 18 phosphorylation occurs at a slower rate downstream of G12/13 pathways and contributes to platelet dense granule secretion. Protein kinase C's (PKC) are serine/threonine kinase, which become activated following the stimulation of many of the platelet surface receptors. PKCs are classified into three groups, classical (α, βI, βII, γ), novel (δ, ε, η, θ), and atypical (ζ, ι, λ, μ) based on their cofactor requirements for activation. The classical PKCs, which require diacylglycerol and calcium for their activation were investigated using the specific inhibitor Go6976. Much to our surprise, we demonstrated that downstream of GPVI pathways, Go6976 caused non-selective inhibition of Spleen tyrosine kinase (Syk) activity. This inhibition of Syk activity resulted in a concentration-dependent reduction in phosphorylation of downstream molecules Lat and PLCγ2 as well as platelet aggregation and secretion. Stimulation of surface receptors GPVI, CLEC-2, GPIb, and FcRIIa, all lead to the activation of tyrosine kinase pathways. The role for Syk in these pathways is essential and in the absence of its activity these pathways are completely shut down. We inadvertently discovered dextran sulfate (DxS) actives platelets. Our results show that DxS activates a Src-dependent pathway which does not utilize surface receptors GPVI, CLEC-2, GPIb, or FcRIIa. Platelets pretreated with Syk inhibitors OXSI-2 or Go6976 failed to cause αIIbβ3 activation in response to convulxin, however, platelets activated with DxS under the same conditions retained the ability to activate αIIbβ3. In response to DxS, platelet aggregation, intracellular calcium mobilization, and αIIbβ3 activation were significantly inhibited in platelets pre-treated with PI-3K inhibitors. Taken together these results for the first time establish a novel tyrosine kinase pathway in platelets that cause fibrinogen receptor activation in a PI-3K dependent manner without a role for Syk. In conclusion, we have evaluated the role of myosin light chain kinase, Syk, and PI-3 kinase downstream of platelet receptor-mediated pathways. We have examined the phosphorylation status of several of their effector molecules and have correlated these events with their functional responses in platelets. Here we have highlighted several roles for platelet kinases and their relative importance in regulating platelet functional outcomes.