Involvement Of Protein Kinase A And Calpain In Platelet Sema4D Shedding

Axon guidance molecule Sema4D (Semaphoring4D) has been proved to be expressed on platelets, and supports thrombus formation by amplifying Syk activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRy complex, where in a contact-dependent manner. We have shown that Sema4D exodomain can be cleaved by the metalloprotease ADAM17and produces a120kDa exodomain fragment that retains biological activity. And we also identified a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets. But our further study in here found that there are other regulatory mechanisms exist.First, we explored whether protein kinase A (PKA) is involved in the regulation of Sema4D exodomain shedding. PKA is a cAMP-dependent protein kinase. Activation of PKA has a negative feedback on agonists induced platelet activation and maintain platelets in a resting state to allow free circulation. Inhibition of PKA could results in metalloproteinase-dependent platelet GPIba shedding. To explore whether PKA involved in Sema4D exodomain shedding, we used PKA inhibitor H89and PKA activator forskolin to perform a series of experiments, and the results showed that inhibition of PKA induces metalloproteinase-dependent Sema4D exodomain shedding, activation of PKA could inhibits agonists induced Sema4D exodomain shedding. Further studies showed that inhibition of PKA induced Sema4D exodomain shedding was caused by enhancing ADAM17activity without dissociation of calmodulin from Sema4D.At the same time, we noticed that Sema4D undergoes intracellular domain cleavage as well since the28-kDa fragment disappears in some cases. However, the detail mechanism of the intracellular proteolytic cleavage of Sema4D has not been defined. As the ability of a protein to bind calmodulin confers a strong likelihood that this protein is a substrate for calpain. So, calmodulin binding to Sema4D give us an inspiration that Sema4D intracellular domain may be cleaved by an intracellular Ca2+-dependent cysteine protease calpain. Here we investigated the potential role of calpain in regulating Sema4D intracellular proteolytic cleavage. Our results showed that1) the intracellular cleavage of Sema4D was mediated by non-physiological agonists, such as W7, A23187and dibucaine;2) W7, A23187and dibucaine significantly enhanced calpain activity, which enable calpain to cleave Sema4D intracellular domain proteolytically;3) calpain in regulation of Sema4D intracellular cleavage may occur independently of ADAM17;4) Sema4D intracellular cleavage also involved in platelet activation and apoptosis induced by A23187.Therefore, our data reveal some new regulation mechanisms for platelet Sema4D extracellular and intracellular cleavage and that may replenish Sema4D shedding related intracellular protein, signaling pathways and related diseases.