Depletion of all platelet integrins impacts hemostasis, thrombosis, and tumor metastasis

Figures

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  Figure 1. Platelet count, expression of major surface glycoproteins, and platelet function in PF4Cre-β1−/−/β3−/− mice (A) Platelet count and volume. The platelet count and volume were determined in PF4Cre (n = 28–30) and PF4Cre-β1−/−/β3−/− mice (n = 30). Data are from 6 independent experiments and were analyzed with an unpaired t test. (B) Glycoprotein surface expression. The surface expression of various glycoproteins on platelets in whole blood from PF4Cre (n = 3–6) and PF4Cre-β1−/−/β3−/− mice (n = 3–6) was evaluated using selective antibodies and flow cytometry. Data are from 2 independent experiments and were analyzed with an unpaired t test or a Mann-Whitney test. (C) Fibrinogen binding. Whole blood from PF4Cre (n = 6–9) and PF4Cre-β1−/−/β3−/− mice (n = 6–9) was stimulated for 10 min with ADP (2 μmol/L) or PAR-4 peptide (1 mmol/L) and the binding of fluorescein isothiocyanate (FITC)-fibrinogen was detected by flow cytometry. Data are from 3 independent experiments and were analyzed using a Mann-Whitney test. (D) P-selectin exposure. Whole blood from PF4Cre (n = 6–9) and PF4Cre-β1−/−/β3−/− mice (n = 6–9) was stimulated for 10 min with U46619 (2 μmol/L) or PAR-4 peptide (1 mmol/L) and…

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  Figure 2. PF4Cre-β1−/−/β3−/− mice are not able to form thrombosis and present an increased bleeding time (A and B) Thrombus formation in vitro. Whole blood from PF4Cre (n = 4) and PF4Cre-β1−/−/β3−/− mice (n = 4) was perfused at 300 s−1 through PDMS flow chambers coated with collagen (200 μg/mL) for 8 min. Platelet aggregation was visualized in random fields by DIC microscopy (A, scale bars, 50 μm) and the area of the thrombi was quantified (B). Data are from 2 independent experiments and were analyzed using a Mann-Whitney test. (C and D) Tail bleeding time. The tail of PF4Cre (n = 5) and PF4Cre-β1−/−/β3−/− mice (n = 5) was severed. The time required for the bleeding to stop was recorded (C) and the volume of blood lost was measured (D). Data are from 1 experiment and were analyzed with a Mantel-Cox test (C) or a Mann-Whitney test (D). (E) Carotid bleeding time. The carotid artery of PF4Cre (n = 4) and PF4Cre-β1−/−/β3−/− mice (n = 4) was punctured and the time required for the bleeding to stop was recorded. Data are from 1 experiment and were analyzed with a Mantel-Cox test. (F–H) FeCl3-induced thrombosis of the carotid artery. Thrombosis was triggered in PF4Cre (n = 4) and…

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  Figure 3. PF4Cre-β1−/−/β3−/− platelets display a reduced binding to TCs and promote primary tumor growth and metastatic growth less efficiently (A) Platelet-tumor cell interactions in vitro. Left: infographics of the experiment. Middle: representative confocal microscopy images (scale bars, 10 μm). Right: graph representing the number of platelet interactions per tumor cell for PF4Cre (n = 356 cells) and PF4Cre-β1−/−/β3−/− platelets (n = 288 cells). Data are from 1 experiment and were analyzed using a Mann-Whitney test after assessment of nonnormal distribution (Shapiro-Wilk test). (B) Orthotopic in vivo tumor model. Left: infographics of the experiment. Right: representative images of tumors in PF4Cre and PF4Cre-β1−/−/β3−/− mice at sacrifice. Scale bars, 1 cm. (C) Orthotopic in vivo tumor model. Left: longitudinal measurement of the tumor in vivo (using Caliper) in PF4Cre (n = 13) and PF4Cre-β1−/−/β3−/− mice (n = 12) during the experiment. The mean and range (dotted lines) are plotted. Data are from 2 independent experiments and were analyzed by two-way ANOVA. On the upper left, the numbers of mice with different tumor volumes were classified as indicated. Data are from 2 independent…