Skip to main content
. 2017 Feb 22;1(7):417–428. doi: 10.1182/bloodadvances.2017004689

Figure 3.

Figure 3.

HEK293 cells expressing normal αIIbβ3 adhere to fibrinogen and D100 but do not adhere to immobilized fibrinogen fragment ‘D98,’ whereas adhesion does occur with HEK293 cells expressing constitutively active αIIbβ3 mutants; adhesion of αIIb(FF)β3-HEK cells to immobilized ‘D98’ is inhibited by function blocking antibodies to αIIbβ3, small-molecules inhibitors of αIIbβ3, and ‘D98,’ but not by an antibody to the fibrinogen γ-12 peptide or soluble fibrinogen. (A) HEK293 cells (2 × 103 cells/ µL; 50 µL) expressing normal αIIbβ3 were labeled with calcein and added to microtiter wells precoated with fibrinogen, D100, or ‘D98’ (10 µg/mL coating concentration) for 1 hour at 22°C (left). After washing, the fluorescence of adherent cells was measured. αIIbβ3-HEK cells adhered to immobilized fibrinogen and D100 but did not adhere to ‘D98.’ Data reported as mean ± SD. HEK293 cells expressing normal αIIbβ3, normal αVβ3, or constitutively active αIIbβ3 mutants were allowed to adhere to ‘D98’ (right). Whereas the cells expressing normal αIIbβ3 or αVβ3 did not adhere, HEK293 cells expressing any 1 of the 3 activating mutations [αIIb(FF), β3N339S, or β3Δ717] demonstrated substantial adhesion (n = 6; P < .01 compared with normal αIIbβ3). Data reported as mean ± SD. Net normalized adhesion is adhesion value (AFU) divided by the expression level (minus background) of each cell line. Expression levels were measured by mAb 10E5 binding and expressed as geometric mean fluorescence intensity (GMFI): normal αIIbβ3 69 ± 40 AFU; αIIb(FF)β3 31 ± 16 AFU; αIIbβ3(N339S) 58 ± 17 AFU; αIIbβ3(Δ717) 86 ± 40 AFU; normal αVβ3 (using mAb LM609) 187 ± 24 AFU. (B) Adhesion of HEK293 cells expressing the αIIb(FF)β3 mutant to ‘D98’ was tested in the presence of mAb’s 10E5 (anti-αIIb ; 20 µg/mL), 7E3 (anti-β3; 20 µg/mL), and mAb 7E9 (anti-fibrinogen γ-12: 20 µg/mL). 10E5 and 7E3 both inhibited adhesion (n = 7; P < .01 for each), whereas 7E9 did not (P = .30). (C) Adhesion of αIIb(FF)β3-HEK cells to immobilized ‘D98’ was also inhibited by the small-molecule inhibitors of αIIbβ3 RUC-4 (5 µM), eptifibatide (100 µM), and tirofiban (10 µM) (n = 3; P < .005 for each). (D) Soluble fibrinogen (1.5 mg/mL) did not inhibit the adhesion of αIIb(FF)β3-HEK cells to ‘D98’ (n = 3; P = .50), but soluble ‘D98’ (1.0 mg/mL) did inhibit the adhesion (n = 3; P = .02). Data reported as mean ± SD.