Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.Author Summary: Platelets are blood cells that, upon injury, trigger the blood to clot. Following blood vessel damage platelets encounter the extracellular matrix protein collagen to which they respond. They become activated, aggregating to form a major component of blood clots. The platelet collagen receptor GPVI stimulates platelet activation through a complex signalling pathway, and while many of the molecules involved in the activation of this pathway have been identified, their specific roles in determining the rate and extent of the exceptionally rapid platelet response have not been determined. Furthermore, while signalling proteins responsible for forward reactions are known, reverse or negative feedback elements are not well understood. Platelets also trigger thrombosis in diseased arteries, causing heart attacks and strokes, and therefore platelets, and particularly the GPVI signalling pathway, are therapeutic targets. To begin to understand the components in the GPVI signalling pathways that may represent tractable therapeutic targets we have developed a mathematical model of the key initiating events that occur upon stimulation of GPVI. In so doing, we have established the importance of a specific phosphatase-controlled negative feedback in determining the rate of initiation of platelet activation.