![Induction of TF activity in response to IgG immune complexes. (A) Induction of TF by HIT ICs. THP-1 cells were incubated with buffer, PF4 (10 µg/mL), or PF4 plus KKO (50 µg/mL) for 3 hours in these and all experiments that follow unless indicated otherwise. The cells were washed free of unbound ICs and incubated overnight, and TF expression by the cell suspension was measured by the generation of FXa activity. Data from 5 independent experiments (mean ± standard error of the mean [SEM]) are shown as fold increase in the maximal velocity of FXa generation relative to cells incubated with PF4 alone, designated 1. (B). Subcellular distribution of TF activity induced by HIT ICs. THP-1 cells were incubated with PF4 and KKO, and total cell-associated TF activity in the cell suspension was determined as in panel A (cells). Aliquots from these same cell suspensions were centrifuged at 1200 g for 10 minutes, and the supernatants were collected (Sup 1). Aliquots from Sup 1 were centrifuged at 21 000 g for 20 minutes and again collected (Sup 2). TF activity in the 3 fractions was measured concurrently. Data shown are mean ± SEM of 4 independent experiments relative to cells stimulated by PF4 (10 μg/mL) alone. (C) Effect of anti-TF antibody (α-TF Ab) on FXa activity induced by HIT ICs. THP-1 cells were stimulated with PF4 and KKO in the presence of IgG α-TF Ab or control IgG (Con Ab 20 µg/mL each), and cell-associated TF activity was measured as in panel A. Data from 5 independent experiments (mean ± SEM) are shown relative to cells stimulated by PF4 (10 μg/mL) alone. (D) Effect of α-TFPI-α antibody on FXa activity induced by HIT ICs. THP-1 cells were stimulated with PF4 and KKO in the presence (+) or absence (−) of α-TFPI Ab (50 µg/mL) for 24 hours, and FXa activity was measured as in panel A. Data from 5 independent experiments (mean ± SEM) are shown relative to cells stimulated by PF4 (10 μg/mL) alone. †P = .001 by Student t test with Welch’s correction; $P = .005 by 1-way analysis of variance (ANOVA) with Sidak’s multiple comparison test; &P = .0178, #P = .0001, and ***P < .0001 by 1-way ANOVA with Sidak’s multiple comparison test; @P = .0468 by Student t test with Welch’s correction.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/135/23/10.1182_blood.2019001133/2/bloodbld2019001133f1.png?Expires=1720825986&Signature=la-sXGFOvbg5zZ8N7tSXsL8I662g6I~R3T~MYKurgc5Mwtq-XBX7ct7vn-RrDRWYZwPh2eUadb-3FcZlhddUYuFrCbmIQ4D2GEEciPJ1ieGPzUwYoabyh-KdaoBfhJOGAsqJ0Ac4rk8fo3btAPn88KMjAOC1qTVjm1PTmHSKyK~elt33xuW7MpQ8At0OtIZ-STgtQosuJgvOaaSqwhbrjk48~nQj2-an~ceUS0WFmVd4hv3BGxqhXkaWOM~53gq5yPxL7mlapWu4QAzxQ9M9asShhhTJbUE3s7AV4cImKqeleIFWDzpERrWIRj5DhM1oZv7npxkuncuAhuCfSfo7nQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Induction of TF activity in response to IgG immune complexes. (A) Induction of TF by HIT ICs. THP-1 cells were incubated with buffer, PF4 (10 µg/mL), or PF4 plus KKO (50 µg/mL) for 3 hours in these and all experiments that follow unless indicated otherwise. The cells were washed free of unbound ICs and incubated overnight, and TF expression by the cell suspension was measured by the generation of FXa activity. Data from 5 independent experiments (mean ± standard error of the mean [SEM]) are shown as fold increase in the maximal velocity of FXa generation relative to cells incubated with PF4 alone, designated 1. (B). Subcellular distribution of TF activity induced by HIT ICs. THP-1 cells were incubated with PF4 and KKO, and total cell-associated TF activity in the cell suspension was determined as in panel A (cells). Aliquots from these same cell suspensions were centrifuged at 1200 g for 10 minutes, and the supernatants were collected (Sup 1). Aliquots from Sup 1 were centrifuged at 21 000 g for 20 minutes and again collected (Sup 2). TF activity in the 3 fractions was measured concurrently. Data shown are mean ± SEM of 4 independent experiments relative to cells stimulated by PF4 (10 μg/mL) alone. (C) Effect of anti-TF antibody (α-TF Ab) on FXa activity induced by HIT ICs. THP-1 cells were stimulated with PF4 and KKO in the presence of IgG α-TF Ab or control IgG (Con Ab 20 µg/mL each), and cell-associated TF activity was measured as in panel A. Data from 5 independent experiments (mean ± SEM) are shown relative to cells stimulated by PF4 (10 μg/mL) alone. (D) Effect of α-TFPI-α antibody on FXa activity induced by HIT ICs. THP-1 cells were stimulated with PF4 and KKO in the presence (+) or absence (−) of α-TFPI Ab (50 µg/mL) for 24 hours, and FXa activity was measured as in panel A. Data from 5 independent experiments (mean ± SEM) are shown relative to cells stimulated by PF4 (10 μg/mL) alone. †P = .001 by Student t test with Welch’s correction; $P = .005 by 1-way analysis of variance (ANOVA) with Sidak’s multiple comparison test; &P = .0178, #P = .0001, and ***P < .0001 by 1-way ANOVA with Sidak’s multiple comparison test; @P = .0468 by Student t test with Welch’s correction.