Title: Unraveling the platelet-specific function of DCBLD2
Other Titles: Identificatie van de plaatjes specifieke functie van DCBLD2
Authors: Nuyttens, Benedicte
Issue Date: 5-Sep-2014
Abstract: Platelets are small anucleate cells or cell fragments circulating in the blood. They play important roles in various processes such as fetal development, inflammation and tumor metastasis. Their main function however remains the maintenance of hemostasis, this by preventing excessive blood loss after vascular injury. To better understand the complex mechanisms by which platelets fulfill their role in primary hemostasis, transcriptomics and proteomic studies were conducted. These studies revealed many new platelet proteins potentially involved in platelet function and thrombosis. Among them are transmembrane receptors such as the discoidin, CUB and LCCL domain containing 2 protein (DCBLD2) for which involvement in thrombus formation has been suggested in a zebrafish model. As today very little is known about the DCBLD2 receptor and even less is known about its role in thrombosis and hemostasis the scope of this project was to further characterize DCBLD2 and uncover its platelet-specific function. We first produced highly pure recombinant human and murine extracellular domain of DCBLD2 next used to immunize mice and generate anti-DCBLD2 monoclonal antibodies. Using one of the produced antibodies, 20D9, DCBLD2 can be detected on human platelets in flow cytometry and immunoprecipitated from the platelet membrane. These antibodies are further used as basic tools, necessary for the study of DCBLD2 on platelets.In the second part of this thesis we studied the role of DCBLD2 in thrombosis and hemostasis in vivo in knock-out mice and in vitro on both murine and human platelets. We show that genetic ablation of DCBLD2 in mice has no effect on hemostasis, thrombus stability or stroke outcome but enhances thrombus formation in a FeCl3-induced thrombosis model in the mesenteric artery. The absence of DCBLD2 or addition of an excess recombinant extracellular domain of DCBLD2, results in enhanced platelet aggregation in vitro which is accompanied by stronger platelet activation, detected as an increase in both alfaIIbß3 activation and alfa-granule secretion. Finally, upon platelet activation, DCBLD2 surface expression on human platelets is slightly decreased and its intracellular tail becomes phosphorylated. These data thus identify DCBLD2 as a receptor that down-regulates platelet activation and platelet-dependent thrombus formation, most prominently in an environment of low stimulation.Semaphorin 4B (Sema4B) has been shown to bind DCBLD2 in vitro and to regulate its surface expression by ubiquitination and proteasome-dependent degradation. A platelet proteome study, in addition, reported that Sema4B is shed from the surface of activating platelets, rendering Sema4B an interesting ligand candidate for DCBLD2 on platelets. In a third line of research we thus aimed to confirm our hypothesis that Sema4B is shed from activated platelets and binds DCBLD2. We indeed demonstrate binding of both recombinant proteins in vitro but are unable to detect Sema4B in platelets or shed from activated platelets. Moreover we find that Sema4B has no apparent effect on human platelet aggregation and although the DCBLD2 surface expression significantly decreases upon platelet activation, we demonstrate this is not associated with an increased ubiquitination of the receptor. Thus, our results are unable to confirm Sema4B as a ligand for DCBLD2 on platelets.In conclusion, this study reveals that DCBLD2 is a new platelet inhibitory receptor who plays a role in attenuating platelet activation and thrombus formation. However, further research is necessary as the ligand for DCBLD2 and the mechanism by which it transmits its inhibitory signals remain to be defined.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Chemistry, Campus Kulak Kortrijk
Chemistry - miscellaneous

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