RICM analysis of activation-independent platelet aggregation. (A-C) Perfusion of blood containing PPACK and PG E₁ over immobilized VWF (see legend to Figure 2). (A) Upper panel. Initial adhesion of a single platelet. Lower panel. A limited area of firm attachment holds the platelet in contact with the surface while stretching occurs under the effect of hydrodynamic force (bottom panel). Wall shear rate = 22 000 s^–1. Images from Video S6, part 1. (B) Top panel: a stretched platelet (labeled 1) is shown 4 seconds after the initial adhesion, and an arrowhead indicates the point of surface attachment. Other platelets, labeled 2 to 5, are attached to platelet 1, but connecting links are not discernible in this focal plane. Second panel: Between 4 and 7 seconds, the aggregate has been stretched downstream, and platelet 5 has changed position. Third panel: Between 7 and 42 seconds, platelet 2 has been stretched considerably; arrowheads highlight the long link with platelet 1. Other platelets attach transiently to stretched platelets 1 and 2, which are stationary on the surface. Bottom panel: Between 42 and 48 seconds, numerous platelets attach to the “backbone” formed by stretched platelets 1 and 2, resulting in a larger aggregate. Wall shear rate = 20 000 s^–1. Images from Video S6, part 2. (C) View of the interplatelet contacts in an activation-independent aggregate. Top panel: The links between platelets are not uniformly discernible on the surface. Bottom panel: The stretched platelets that form the “backbone” of the aggregate (arrowheads) are visible in a focal plane above the surface. Wall shear rate = 23 000 s^–1. Images from Video S6, part 3. (D) Perfusion of blood containing PPACK but no PG E₁. Top panel: Fluorescence image of platelets firmly attached to immobilized VWF. Shear rate = 16 000 s^–1. Bottom panel: RICM image. The adherent platelets are spread and in close contact with the surface, thus appear uniformly dark. Shear rate = 24 000 s^–1. Images from Video S7.