Analysis of platelets in thrombocytopenia mice induced by subcutaneous 5A7 injection. (A) Schematic diagram of platelet staining for FACS analysis. In this experiment, AlexaFluor 488–labeled 5A7 was administered in vivo so it could be detected regardless of subcellular localization. The platelet population was gated using a Brilliant Violet (BV) 421–labeled anti-αIIb antibody. PE-Cy7 labeled anti-rat IgG was used to detect 5A7 bound to GPIbα on the platelet surface. DyLight649 anti-GPIbα antibody (Xia.G5) was used to stain GPIbα on the platelet surfaces. (B) MFI of platelet GPIbα and αIIb corrected for platelet size (FSC) and expressed relative to normal control. Total results of 2 independent experiments are shown (n = 10 in each group); ***P < .001 determined by Student t test. NS, not significant. (C) Platelet subpopulations identified by presence/absence of in vivo–administered AlexaFluor 488–labeled 5A7 and their subcellular location. Positive signals on the abscissa indicate the presence of 5A7 either inside or on the surface of platelets. Positive signals on the ordinate indicate the presence of 5A7 on the platelet surface. The criteria of the quadrant were determined by a control blood sample collected from an untreated mouse stained with PE-Cy7 labeled anti-rat IgG (left). Mean percentage and standard deviations were calculated for the 10 mice tested. (D) GPIbα expression on platelet surface was determined by staining with DyLight 649 anti-GPIbα antibody (Xia.G5) which does not compete with 5A7 for GPIbα binding. The MFI of each population is indicated as relative to normal controls. MFI of GPIbα corrected by platelet size (FSC) is shown in the bottom row of the table. Results are obtained from 10 mice. (E) Confocal analysis of platelets in Q2 (top) and Q3/Q4 (bottom) subpopulations. Bar represents 5 µm. (F) During the course of chronic thrombocytopenia induced by 5A7 subcutaneous administration every 3 days, the percentage of platelets negative for surface GPIbα expression was monitored by FACS.