5. When platelets are stimulated with thrombin, they change shape and begin to s

Question

5. When platelets are stimulated with thrombin, they change shape and begin to secrete ATP and serotonin which trigger other platelets to contribute to the formation of a clot. Platelets contain high levels of two protein kinases: PKC, which initiates serotonin release, and myosin light-chain kinase (MLCK), which mediates the change in morphology. After thrombin exposure, several protein substrates are phosphorylated, including the light chain of myosin and a protein called MARCKS. When platelets are treated with a calcium ionophore, the myosin light chain is phosphorylated but not MARCKS. When platelets are treated with phorbol ester (an analog to diacylglycerol), MARCKS is phosphorylated but not myosin light chain. The data are shown below. In the absence of thrombin, serotonin release depends on both the phorbol ester and the calcium ionophore 100 100 with ionophore 80-without ionophore 60 E 60 40 20 20 0 10 20 10 20 phorbol ester (ug/ml PMA) phorbol ester (ug/ml PMA) a) Based on these experimental observations, describe the normal sequence of molecular events following thrombin exposure that lead to both phosphorylation events. (Don't do a lot of research. Just interpret the data.) Indicate how the ionophore and the phorbol ester mimic normal signaling events. (10 points) b) Hypothesize why serotonin release requires both the calcium ionophore and the phorbol ester. Design an experiment to test your hypothesis. (20 points)

Explanation / Answer

1. When myosine like chain kinase cells were exposed to thrombin it lead to increase in calcium ion level. Pre exposure to thrombin lead to mono and di phosphorylation of myosine like chain kinase cells. permeabilized platelets, as in the intact cells, release of serotonin was associated with the Ca2+-dependent phosphorylation of 47 000 and 20 000 Da polypeptides.

2. When an experiment was performed with TPA and calcium iron Ionophore it was observed that activation of protein kinase C is important for release of serotonin. Cyclic amp and cyclic GMP inhibit activation of protein kinase C by prohibition of signal dependent breakdown of inositol phospholipid.

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