Antibody-mediated inhibition of α5 subunit decreases megakaryocyte numbers in vivo in JAK2V617F+ mice and in JAK2V617F+ patient samples. (A) Percentage and number of total CD41+ megakaryocytes in BM of WT C57BL/6J control (n = 10, 10- to 18-week-old males) and JAK2V617F+ mice (n = 11, 10- to 18-week-old males). Number of CD41+ megakaryocytes correspond to n = 3 animals from each group (14-week-old males). (B) Cell surface expression of α5 (HMα5-1) and β1 (Ha2/5) subunits on CD41+ BM megakaryocytes. Data for α5 subunit represents WT (n = 10, 10- to 18-week-old males) and JAK2V617F+ mice (n = 11, 10- to 18-week-old males); data for β1 subunit represent WT (n = 10, 12- to 14-week-old males) and JAK2V617F+ mice (n = 10, 12- to 14-week-old males). Note that y-axis does not start at 0. (C) Cell surface expression of extended conformation β1 subunit (9EG7) in CD41+ BM megakaryocytes from WT (n = 14, 18- to 25-week-old males and females) and JAK2V617F+ mice (n = 12, 18- to 25-week-old males and females). (D) Percentage out of total BM cells and number of MKP (Lin-c-Kit+Sca-1−CD41+CD150+) in BM of WT (n = 10, 19- to 25-week-old females) and JAK2V617F+ mice (n = 8, 19- to 25-week-old females). (E) Cell surface expression of α5 (HMα5-1) and β1 (Ha2/5) subunits in MKP from WT (n = 10, 19- to 25-week-old females) and JAK2V617F+ mice (n = 8, 19- to 25-week-old females). Note that y-axis does not start at 0 for β1 subunit. (F) Cell surface expression of 9EG7 in MKP from WT and JAK2V617F+ mice (n = 10 each, 18- to 21-week-old females). (A-F) Data are an aggregate of at least 3 independent experiments. (G) Representative data of flow cytometric analysis of α5 (HMα5-1), β1 (Ha2/5) subunits, and 9EG7 expression in MKP shown in panels D-F. (H) Schematic representation of 5H10-27 treatment protocol. JAK2V617F+ mice were injected with either rat IgG2aκ or 5H10-27. Black arrows indicate antibody injection; red arrows indicate date animals were analyzed. (I) Representative flow cytometric analysis of 5H10-27 antibody-labeled CD41+ cells in 5H10-27-injected animals. Cells collected from treated animals were stained ex vivo only with anti-rat IgG2aκ phycoerythrin (PE) secondary antibody (middle), or with 5H10-27 followed by anti-rat IgG2aκ PE (right). Percentage of (J) MKP and (K) CD41+ and CD42d+ cells in JAK2V617F+ animals treated with rat IgG2aκ (n = 7, 8- to 10-week-old females) or 5H10-27 (n = 5, 8- to 10-week-old females). Data represent aggregate from 3 independent experiments. (L) Mean fluorescence intensities of α5 (P1D6) and β1 (TS2/16) integrin subunits in megakaryocytes differentiated from HC and JAK2V617F+ PMF patients (n = 5 per group). (M) 2 × 105 megakaryocytes derived from HC and JAK2V617F+ PMF patients were plated on fibronectin-coated plates and incubated for 3 hours to allow for adhesion in the presence of an unrelated IgG or anti-α5 integrin antibody (SAM-1) with blocking function. Adherent megakaryocytes were counted and expressed as number of cells per field (n = 5 per group). (L-M) All 5 HC and PMF patients 1 through 5 were used in studies. (N) CD34+ cells from HC and JAK2V617F+ PMF patients were differentiated on fibronectin coated-plates in the presence of an unrelated IgG or anti-α5 integrin antibody (SAM-1) with blocking function for 13 days. Three of 5 HC and PMF patients 5 through 7 were used in studies. Normalized cell counts from 3 independent experiments are shown. Data are expressed as mean ± standard deviation; ns, not significant. *P < .05, **P < .01, ***P < .001, ****P < .0001.