Real-time analysis of [Ca⁺⁺]_i during platelet translocation and aggregate formation on immobilized VWF.

Platelets loaded with Fluo-3 AM (2 × 10⁷/mL) were suspended with washed erythrocytes in homologous plasma and perfused over immobilized VWF for 3 minutes at a shear rate of 1500 seconds⁻¹. Panel A shows an example of aggregate formation. At 0 second, platelet 1 appears in the optical field; by 10 seconds, it has moved in the direction of flow by approximately 20 μm; at 20 seconds, it has moved by an additional few millimeters; and at 30 seconds, it is in the same position and 2 new platelets (2 and 3) are attached in close proximity, forming a small aggregate. Panel B shows [Ca⁺⁺]_i and instant velocity of platelets 1, 2, and 3. The translocation of platelet 1 occurs mostly during a few seconds of relatively rapid movement, coincident with the appearance of transient [Ca⁺⁺]_i peaks (α/β); a higher and longer lasting increase in [Ca⁺⁺]_i (γ) develops while the platelet is stationary. Cytosolic Ca⁺⁺ oscillations also appear when platelets 2 and 3 arrest on the surface, without a clear sequence from α/β to γ. Panel C, captured between 60 and 63 seconds after the appearance of platelet 1 in the field, shows the long-lasting synchronous increase of [Ca⁺⁺]_i in platelets forming a large aggregate. The 3-dimensional diagrams below each image show the measurement of [Ca⁺⁺]_i in all platelets in the field.