Fig 1.
Generation of platelet specific GARP knockout mice.
GARP and P-selectin expression on 700 μM PAR4-AP (A) or 20 μg/mL CRP (B) activated murine platelets. Mean fluorescence intensity (MFI) of anti-GARP-APC and anti-P-selectin CD62P-PE are shown. (C) Genotypic analysis of genomic DNA from control C57BL/6, littermates and platelet specific cKO mice. Loxp is 181 bp (fl/fl); wt allele is 111 bp, Cre 250 bp. (D) Phenotypic characterization of littermates and cKO mice using flow cytometry. Platelets were labeled with anti-CD41-FITC and anti-GARP-APC and a histogram was made based on APC intensity of CD41+ cells: isotype control (black line), littermates (grey line) and cKO (black).
Table 1.
GARP cKO platelets are well formed and GARP cKO mice display unaltered hematological parameters.
Fig 2.
GARP expression is elevated upon platelet activation but does not affect outside-in and inside-out signaling.
MFI of P-selectin (anti-CD62P-PE) (A-B), JON/A (anti-GPIIbIIIa-PE) (C-D) and fibrinogen binding (E-F) to unactivated or 200 μM PAR4-AP (A-C-E) or 2.5 μg/mL CRP (B-D-F) activated platelets of littermates and cKO mice are measured using flowcytometry. Platelets were gated based on CD41 (anti-CD41-FITC) expression. Graphs show mean ± SD, n = 3. Washed platelets of littermates and cKO mice were allowed to spread on 100 μg/mL fibrinogen while activated with 200 μM PAR4-AP (G) or 2.5 μg/mL CRP (H) during 5, 10 or 30 min. Platelets are divided in different phases of activation (1) round, (2) only filopodia, (3) filopodia and lamellipodia, (4) fully spread. (I) Representative images of platelet spreading; scale bars represent 5 μm.
Fig 3.
GARP deficiency in platelets does not affect platelet aggregation.
Aggregation was induced in PRP of GARP+/+ and GARP cKO mice using 75, 125 or 250 μM PAR4-AP (A) and 0.75, 2.5 or 5 μg/mL CRP (C). Maximal light transmission was calculated and graphed of at least 3 individual experiments. Representative curves for 125 μM PAR4-AP (B) and 2.5 μg/mL CRP (D) induced aggregations in GARP+/+ (grey) and GARP cKO (black) are shown. (E) Clot retraction of PRP supplemented with erythrocytes was induced using 5 U/mL thrombin and 20 mM CaCl2 in GARP+/+ littermates and GARP cKO. Pictures were made every two minutes and clot area was analyzed using ImageJ. Representative pictures are shown on the right. Measured clot area is presented on the right; n = 3. (F) Whole blood of GARP cKO mice and GARP+/+ littermates was perfused over a Horm collagen (100 μg/mL) coated coverslip at a shear rate of 1600 s-1. Thrombus formation was visualized during 5 min and surface coverage was quantified using ImageJ; n = 3. Representative pictures of thrombus formation under flow are shown on the right; scale bars represent 50 μm.
Fig 4.
Unaltered bleeding time and thrombus formation in Pf4 specific GARP cKO mice.
(A) 2 mm of the tail tip was dissected and tail bleeding times were measured (n = 10). (B) Carotid artery was injured using topical application of 12% FeCl3 and blood flow was monitored using a flow probe until flow stopped due to the formation of an occlusive thrombus (n = 10). (C) Mesenteric arteries were injured using 10% FeCl3 and thrombus formation was followed using intravital microscopy until full occlusion was reached (n = 10). Representative pictures after 5, 8 and 10 min of mesenteric thrombus formation are shown on the right. Scale bars represent 50 μm. (D) The right middle cerebral artery of littermate mice (n = 14) and platelet specific GARP knockout mice (n = 12) was occluded during 60 min and reperfusion was allowed during 23 hours.
Fig 5.
Generation of endothelial specific GARP knockout mice.
(A) Phenotypic characterization of littermates and cKO mice using flow cytometry. Single cells were labeled with anti-CD45-PeCy7, anti-CD31-PE, anti-CD41-FITC and anti-GARP-APC and a histogram was made based on APC intensity of CD45-CD31+CD41- population. Isotype control (black line), littermates (grey line) and cKO (black). (B) Genotypic analysis of genomic DNA from control C57BL/6, littermates and endothelial specific cKO mice. Loxp is 181 bp (fl/fl); wt allele is 111 bp, Cre 450 bp.
Fig 6.
Unaltered bleeding time and thrombus formation in Tie2 specific GARP cKO mice.
(A) 2 mm of the tail tip was dissected and tail bleeding times were measured (controls: n = 11; cKO: n = 9). (B) Carotid artery was injured using topical application of 12% FeCl3 and blood flow was monitored using a flow probe until flow stopped due to the formation of an occlusive thrombus (controls: n = 10; cKO: n = 11). (C) Mesenteric arteries were injured using 10% FeCl3 and thrombus formation was followed using intravital microscopy until full occlusion was reached (controls: n = 10; cKO: n = 11). Representative pictures after 5, 8 and 10 min of mesenteric thrombus formation are shown on the right. Scale bars represent 50 μm. (D) The right middle cerebral artery of littermate mice (n = 10) and endothelium specific GARP knockout mice (n = 16) was occluded during 60 min and reperfusion was allowed during 23 hours.