Quantifying Protein Tyrosine Phosphatase in Platelets

Platelets and Thrombosis: The Signaling Dynamics

Platelets, small fragments of megakaryocytes, are centrally involved in hemostasis and thrombosis. They prevent excessive blood loss, promote the appropriate immune response and subsequent wound repair, but they can also cause damage by clogging the blood vessels of the heart (heart attack) and brain (stroke)—top causes of death in Western countries.

Understanding the regulatory mechanisms governing platelet production and function has far-reaching implications in the treatment and prevention of thrombosis. Dr. Yotis Senis, a Professor at the Institute of Cardiovascular Sciences, University of Birmingham, UK, is particularly interested in how a family of enzymes called protein tyrosine phosphatases (PTPs), which dephosphorylate proteins that control platelet activation in response to vascular injury, regulate thrombus formation.

The Need for Quantitating Tyrosine Phosphorylation Changes

At the core of what Dr. Senis’s group is investigating is the question of how PTPs work in conjunction with protein-tyrosine kinases (PTKs) to regulate signaling pathways within platelets, hence, controlling their response to vascular injury, thrombosis and hemostasis.

To answer this question, analyzing the phosphorylation status of several protein groups of interest is routine in Dr. Senis’s lab. Tyrosine kinases Src, Csk and Syk, tyrosine phosphatases CD148, Shp1 and Shp2, ITIM-containing receptors G6b-B and TLT-1, ITAM-containing receptors GPVI-FcR γ-chain and CLEC-2, and integrins αIIbβ3 and α2β1 are all on the list! However, quantifying phosphorylation events by traditional Western blotting methods is only semi-quantitative, at best.

New-Found Confidence in Protein Tyrosine Phosphatase Data

With proper optimization by Zoltan Nagy, Ph.D., Postdoctoral Research Associate in the Senis lab, Wes™ provides Dr. Senis’s team with quantitative data using a minimal amount of their precious samples while also saving valuable time. “In many of our experiments, we are limited by the amount of lysate, and Wes reduces our sample requirements by orders of magnitude,” says Dr. Senis. Because detected phosphorylation events can be quantified with Wes, various signaling events downstream of ITAM, ITIM and integrin receptors important in platelet activation and function have been elucidated. Dr. Senis says using Wes has made his team much more confident about changes in protein expression and phosphorylation events. “[We] can start to model signaling networks,” he adds. Their work, which utilizes Wes to uncover cell-intrinsic mechanisms that prevent pathological thrombosis from occurring, has recently been published in Blood. Dr. Senis says he continues to be inspired by his family, noting that his two sons are his biggest personal achievement, by far.

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