The lipophilic photosensitizer, merocyanine 540 (MC), has been proposed for use as an age reduce virus transmission by cellular blood components. in a previous study, we demonstrated the antiviral activity of this dye in the presence of light (450-600nm). The photoinactivation of lipid-containing viruses is probably mediated by reactive singlet oxygen. More importantly, platelets were highly susceptible to photodamage by MC as shown by significant morphological alterations, a disruption of the response to physiologic agonists, the spontaneous release of granule contents. Since other photosensitizers which have been proposed as antiviral agents for use with blood products are also singlet oxygen producers, we investigated further the Interaction of MC with the platelet membrane and the mechanism of platelet inactivation by MC and light. The binding of MC to platelets in the dark was measured at 550nm. Platelets in buffer containing 0, 0.35 or 5% BSA were treated with 0-24 micro-g/ml MC, washed and lysed with 1% Triton X-100 (TX-100). Increasing concentrations of BSA decreased MC binding to platelets, although 5% BSA did not completely block association of the dye with the platelet membrane. Most of the dye is associated with the lipid fraction of the membrane. Platelets labeled with (14)C-arachidonic acid (AA) were treated with MC then lysed. Pellets and supernatants were counted and (14)C-AA partitioned into the TX-l00-soluble fraction. Treatment of the supernatant with chloroform:methanol resulted in extraction of most of the dye into the lipid phase although a significant proportion remained In the protein layer. 14C-AA-labeled platelets were activated by treatment with 15 micro- g/ml MC and light and released 22% of the releasable AA to the medium versus 7.6% In the untreated platelets. Activation has been further illustrated by the immunoblot identification of PADGEM (platelet activation dependent granule-external membrane protein) in membranes of fully-treated platelets. In addition, membrane proteins analyzed by SDS-PAGE reveal proteolysis of actin-binding protein and talin, consistent with activation of platelets treated with dye + light. In an effort to identity MC binding proteins, membrane proteins isolated from control, MC-treated and MC + light-treated platelets were separated on Sepharose 4B (S4B) and identified by SDS-PAGE. Membranes from platelets treated with MC alone display broad but coincident MC and protein peaks. Membranes treated with MC + light exhibit a shift in the MC peak to a high molecular weight species suqgesting the formation of protein complexes.
