Factor X (FX) initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin (FII) which then presents FX to a specific, high-affinity site consisting of FVIIIa bound to a high-affinity, low-capacity receptor on activated platelets. We have demonstrated the localization of FX in lipid rafts and shown that FX-raft association requires Ca2+ and is enhanced by saturating concentrations of FVIIIa. Here we investigate FII-raft association and define the domains through which shared FX and FII sites are mediated on the surface of human platelets. Activated (thrombin receptor peptide, SFLLRN, 25 μM) gel-filtered platelets (3.5 x 108/ml) were incubated with 125I-FII or 125I-FX to determine direct platelet binding and then they were lysed with Triton-X100 (0.025-0.25%) followed by sucrose density gradient centrifugation. FII was localized to lipid rafts in SFLLRN stimulated (~25% total binding) but not to unactivated platelets. The optimal associations of FX and FII with lipid rafts required Ca2+ and were not affected by the presence of EGR-FIXa or FIX (45 nM). The association of FII with lipid rafts was completely abolished in the presence of FX (1.5 μM) whereas, Gla (des) FX was unable to compete with raft associated FII. Similarly, 125I-FII fragment 1 association with lipid rafts was inhibited by FX but not by Gla (des) FX. Prothrombin and FII fragment 1 (residues 1-155) were equipotent inhibitors of FX-raft association. FVIIIa (20 nM) had no effect on FII-raft association but significantly increased (~2-fold to ~45%) FX-raft association. In contrast, the presence of FVa (20 nM) had no effect on FX-raft association but significantly (~2-fold to ~45%) increased FII-raft association. The structural integrity of lipid rafts was completely disrupted by 10 mM methyl-β-cyclodextrin (MβCD), a known cholesterol depleting drug, which completely prevented FII or FX association with lipid rafts, and this removal was reversed by cholesterol repletion. Furthermore, MβCD (up to 40 mM) had no effect on the amount of FII or FX bound to activated platelets, thus suggesting that neither platelet activation by SFLLRN nor the exposure of FII receptors was affected by MβCD treatment. These experiments demonstrate the localization of a shared FX/FII site in lipid rafts and support the hypothesis that these interactions are mediated by the Gla-domains of FX and FII and are specific and essential for the assembly of F-X activating complex on the activated platelet membrane.