Extracellular protein disulfide isomerase (PDI) is required for platelet thrombus formation and fibrin generation after arteriolar wall injury in live mice. PDI is secreted from platelets and endothelial cells on cellular activation, but the mechanism of capture of secreted PDI within the injured vasculature is unknown. We establish that, like the endothelial β3 integrin αVβ3, the platelet integrin αIIbβ3 binds PDI. PDI also binds to recombinant β3. Using intravital microscopy, we demonstrate that PDI accumulation at the site of laserinduced arteriolar wall injury is markedly reduced in β3-null (β3-/-) mice, and neither a platelet thrombus nor fibrin is generated at the vessel injury site. The absence of fibrin after vascular injury in β3-/- mice is because of the absence of extracellular PDI. To evaluate the relative importance of endothelial αVβ3 versus platelet αIIbβ 3 or αVβ3, we performed reciprocal bone marrow transplants on wildtype and β3-/- mice. PDI accumulation and platelet thrombus formation were markedly decreased after vessel injury in wild-type mice transplanted with β3-/-bone marrow or in β3-/- mice transplanted with wild-type bone marrow. These results indicate that both endothelial and platelet β3 integrins contribute to extracellular PDI binding at the vascular injury site.