| Platelets are small cells (2-3 mum) that respond to vascular injury by halting blood flow. For decades, studies characterizing the mechanisms by which platelets acutely plug damaged vessels have dominated the literature. However, it has become increasing clear that platelets are multifunctional cells that modulate a variety of biological responses. One recently identified function of platelets involves the synthesis of proteins that regulate both acute and chronic inflammation. Activated platelets synthesize proteins by regulated translation of constitutively expressed mRNAs that they receive from megakaryocytes, their parent cell. The underlying hypothesis of my dissertation is that in addition to transferring mRNAs to platelets, megakaryocytes also deliver a functional spliceosome to platelets during thrombopoiesis that controls pre-mRNA splicing and signal-dependent protein synthesis.; In order to address this question, I first developed and characterized a human stem cell model of megakaryocyte differentiation and proplatelet formation. I found that stem cell-derived megakaryocytes display platelet biomarkers and following adherence to specific extracellular matrices that are expressed in human bone marrow, the differentiated cells develop proplatelet extensions. Next, I characterized the spliceosome in this model and found that megakaryocytes transfer mRNA processing components to platelets during thrombopoiesis. Additional studies in mature, human platelets revealed that platelets possess essential spliceosome components and pre-mRNA templates. Moreover, in response to outside-in signals platelets splice pre-mRNAs into mature messages that they subsequently translate into bioactive protein.; While cytoplasmic splicing may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells. In addition, signal-dependent splicing identifies a new, novel function of platelets that regulates their biological phenotype. Interruption of signal-dependent splicing in platelets may be a powerful therapeutic tool in the future that is used to treat diseases of disordered coagulation and inflammation. |
