Arp2 activity is required for actin polymerization and shape change during activation of the human platelet

Human blood platelets prevent bleeding and thus preserve life by aggregating to form a clot. Aggregation requires platelet activation and shape change, which depends on the accurate regulation of actin dynamics. The molecular mechanism of actin dynamics regulation is exquisitely complex and yet to be explored.;My doctoral research focuses on Arp2, which belongs to a highly conserved eukaryotic protein superfamily, the Arp (a&barbelow;ctin-r&barbelow;elated p&barbelow;roteins) superfamily. Arp2 exists as one of the seven subunits of the Arp2/3 complex, which is hypothesized to nucleate actin filaments inside cells.;The first goal of my project is to locate Arp2 precisely within the actin structure of stimulated cells. I transfected GFP-tagged Arp2 into the cultured fibroblasts. I determined the location of endogenous Arp2 in cultured fibroblasts, and in spread human platelets. Arp2 was found 1∼10mum inside the leading edge of lamellipodia in both cell types.;The second goal is to test the function of Arp2 in actin polymerization. I designed a new pyrene-actin polymerization assay using permeabilized platelets to measure actin polymerization during platelet activation. I found that the increase in actin polymerization is largely due to Arp2/3 activity.;The third goal is to examine directly the role of Arp2 in platelet shape change during activation. I designed a protocol that enables the introduction of antibodies into human platelets without activating them. The introduction of affinity purified alphaArp2 antibodies into platelet freezes the platelets in their first stage of shape change: rounding. Platelets form "blebs" which suggests that Arp2 may also play a role in maintaining the normal actin filament structure. Both of the effects were dependent on the concentration of alphaArp2. My data shows that Arp2 is necessary for the shape change in activated platelets.;I propose a model of the function of Arp2: following thrombin activation, Arp2 increases the de novo actin nucleation, which causes the formation of actin-based protrusions, filopodia and lamellipodia, and therefore provides the molecular basis for the platelet shape change.