Date of Award
Doctor of Philosophy (PhD)
Lisa K. Jennings, Ph.D.
Marshall Elam, M.D., Ph.D. Eldon E. Geisert, Ph.D. Roderick T. Hori, Ph.D. Susan Senogles, Ph.D.
platelets, CD9, tetraspanin, integrin, aggregations, confocal
CD9, a member of the tetraspanin family, is highly expressed on platelets (50,000-80,000 copies per platelet). Tetraspanins have been implicated in modulation of integrin function, and it is hypothesized that CD9 will modulate GPIIb-IIIa, the major platelet integrin.
The association between CD9 and GPIIb-IIIa was analyzed using immunoprecipitations and confocal microscopy. These two proteins were found to associate with each other, particularly at areas of platelet-platelet contact and at the periphery. Confocal analysis revealed CD9 localization was most intense at platelet-platelet contact, as well as in platelet filopodia and lamellipodia, but there is a lack of CD9 at areas of platelet-matrix contact. Co-localization with F-actin decreased as platelets progressed through the stages of spreading.
In order to analyze CD9 contributions to platelet function, a Fab fragment was generated from mAb7, an antibody which binds with high affinity to the large extracellular loop of CD9. Fab fragments were used to avoid complications of bound antibody crosslinking CD9 with the platelet FcγRII receptor. Confocal analysis of platelet spreading indicated that CD9 perturbation by mFab7 resulted in increased platelet spreading on a variety of matrices, although platelet adhesion was unaffected on all matrices except fibrin. Platelet-platelet interactions, including aggregation and disaggregation, were studied using light transmission aggregometry. CD9 perturbation increased the extent of platelet aggregation in response to threshold levels of agonist, with diminished effect with more potent agonists such as TRAP (Thrombin Receptor Agonist Peptide). CD9 perturbation also diminished, but did not completely inhibit, platelet disaggregation induced by eptifibatide. Interestingly, CD9 perturbation did not alter platelet-mediated clot retraction of platelets in PRP (Platelet rich plasma). In response to CD9 alteration by mFab7, both increased fibrinogen binding and platelet activation (as evidenced by CD63 and P-selectin expression) were elicited in the presence of threshold ADP levels. Both responses were dependent on presence of low-level agonist, as CD9 perturbation in the absence of agonist yielded no effect.
Cytoskeletal isolation revealed that CD9 perturbation results in increased incorporation of actin and other cytoskeletal molecules into the platelet core cytoskeleton. Immunoprecipitations results showed increased CD63 incorporation into the tetraspanin web, although the level of CD9 coprecipitation with GPIIb-IIIa was unaffected. A preliminary analysis of tyrosine phosphorylation, particularly Akt, did not reveal alterations on tyrosine phosphorylation of signaling molecules.
These results suggest that CD9 functions as a scaffold, organizing molecules into specific domains on the platelet surface. It appears to function as a “gatekeeper” for GPIIb-IIIa activation, particularly modulating activation and aggregate stability at low levels of agonist.
Hill, Sarah Kathleen , "The Tetraspanin CD9 Localizes to Platelet-Platelet Contacts and Regulates Thrombus Stability" (2008). Theses and Dissertations (ETD). Paper 350. http://dx.doi.org/10.21007/etd.cghs.2008.0138.