![Studies on platelet uptake of albumin, fibrinogen and IgG in FPDMM. (A) Uptake of albumin, fibrinogen, and IgG by platelets from a father and daughter with FPDMM and a healthy control donor was assessed by flow cytometry. Washed platelets (1 × 106/mL) from all donors were incubated with Cy3-albumin (10 μg/mL), Cy3-fibrinogen (50 μg/mL), or Cy3-IgG (20 μg/mL) for 15 to 30 minutes at 37°C. Platelets were fixed and analyzed using flow cytometry and expressed as mean fluorescent intensity (MFI). (B) Immunofluorescence studies of platelet α-granule PF4 in patients with FPDMM. Platelets from the patients and the healthy control donor, immobilized on fibronectin-coated coverslips and fixed with 2% paraformaldehyde, were incubated with FITC-labeled anti-PF4 monoclonal antibody and images were taken using a Nikon E1000 microscope and quantified by ImageJ (v1.47; National Institutes Health). Corrected total cell immunofluorescence (CTCF) is shown (mean ± standard error of the mean [SEM]). P values shown are for comparisons by Student t test. (C) Albumin uptake in platelets from patients with FPDMM immobilized on fibronectin and assessed using immunofluorescence microscopy. Platelets from the father, daughter, and the healthy control donor, immobilized on fibronectin-coated coverslips, incubated with albumin Alexa Fluor 488 (30 μg/mL) (red) for the indicated time at 37°C and fixed. Images were taken with Epifluorescence microscope (EVOS FL Autoimaging) to evaluate internalized albumin. The panel (below) shows the CTCF (mean ± SEM) of albumin uptake in platelets from the patients and a control donor. P values shown are for comparisons by Student t test. (D) Fibrinogen uptake in platelets from patients with FPDMM immobilized on fibronectin and assessed using immunofluorescence microscopy. Platelets from the father and daughter with FPDMM and the healthy control donor were immobilized on fibronectin-coated coverslips and incubated with fibrinogen Alexa 546 (50 μg/mL) (red) for the indicated time at 37°C. Images were analyzed as described above. Platelets are outlined in yellow. Many platelets from the father were devoid of fibrinogen with a population showing substantial uptake. Daughter’s platelets appeared similar to control. The panel (below) shows changes in CTCF of fibrinogen uptake in platelets from FPDMM patients and control donor. P values are for comparisons made by the Student t test.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/8/7/10.1182_bloodadvances.2023011098/2/blooda_adv-2023-011098-gr1.jpeg?Expires=1724971439&Signature=Dk~qMlTQd2ZVJ55w-J8nB70Nlh2TaoMmHlZfldystMFWARJOADiGrQG63NmxU4JAmE7-pTurp7Wf3L2Jfpvz4b0C55PGqKPSq45KK6C3qEez7qWN4Yq5AskvanX82v2d3PWkNvE~0oVoMAt8GAKQMi0q2IdfN3uIi-QTMXZZxmsFhmZ44P3ZZgVMpDD~PN2N4M5MY-UNVy1MUXqDb2xc~IMR~A-qEj~ewEaFe~BE8uI62BcQGGGQY0Pc-c6g~gqKZqbjCQK2r-pZbHgKp0ZNSMqZ6WJY51BtlYJO0qzR9k05PSC3uQVZsbjayYTbUNfgvWTceYuQAb1UhzgxEOsuUg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Studies on platelet uptake of albumin, fibrinogen and IgG in FPDMM. (A) Uptake of albumin, fibrinogen, and IgG by platelets from a father and daughter with FPDMM and a healthy control donor was assessed by flow cytometry. Washed platelets (1 × 106/mL) from all donors were incubated with Cy3-albumin (10 μg/mL), Cy3-fibrinogen (50 μg/mL), or Cy3-IgG (20 μg/mL) for 15 to 30 minutes at 37°C. Platelets were fixed and analyzed using flow cytometry and expressed as mean fluorescent intensity (MFI). (B) Immunofluorescence studies of platelet α-granule PF4 in patients with FPDMM. Platelets from the patients and the healthy control donor, immobilized on fibronectin-coated coverslips and fixed with 2% paraformaldehyde, were incubated with FITC-labeled anti-PF4 monoclonal antibody and images were taken using a Nikon E1000 microscope and quantified by ImageJ (v1.47; National Institutes Health). Corrected total cell immunofluorescence (CTCF) is shown (mean ± standard error of the mean [SEM]). P values shown are for comparisons by Student t test. (C) Albumin uptake in platelets from patients with FPDMM immobilized on fibronectin and assessed using immunofluorescence microscopy. Platelets from the father, daughter, and the healthy control donor, immobilized on fibronectin-coated coverslips, incubated with albumin Alexa Fluor 488 (30 μg/mL) (red) for the indicated time at 37°C and fixed. Images were taken with Epifluorescence microscope (EVOS FL Autoimaging) to evaluate internalized albumin. The panel (below) shows the CTCF (mean ± SEM) of albumin uptake in platelets from the patients and a control donor. P values shown are for comparisons by Student t test. (D) Fibrinogen uptake in platelets from patients with FPDMM immobilized on fibronectin and assessed using immunofluorescence microscopy. Platelets from the father and daughter with FPDMM and the healthy control donor were immobilized on fibronectin-coated coverslips and incubated with fibrinogen Alexa 546 (50 μg/mL) (red) for the indicated time at 37°C. Images were analyzed as described above. Platelets are outlined in yellow. Many platelets from the father were devoid of fibrinogen with a population showing substantial uptake. Daughter’s platelets appeared similar to control. The panel (below) shows changes in CTCF of fibrinogen uptake in platelets from FPDMM patients and control donor. P values are for comparisons made by the Student t test.
