Fig. 2.
Fig. 2. Monocyte tissue factor induction by PF4/heparin antibodies. / (A) Induction of tissue factor by HITT plasma. Monocytes derived from peripheral blood were incubated in duplicate for 6 hours with HITT (♦) or control plasma (⋄) alone, HITT plasma + PF4 (▪), control plasma + PF4 (■), HITT plasma + heparin (●), control plasma + heparin (○), HITT plasma + PF4/heparin (▴), or control plasma + PF4/heparin (▵). After incubation with antigen and antibody, cells were washed 3 times with Tris-buffered saline (TBS) containing 0.1% bovine serum albumin (BSA) and incubated in buffer (TBS/BSA) containing 10 mM calcium chloride and recombinant Factor VIIa (2 nM) for 30 minutes. Plasma-derived Factor X (200 nM) was added to wells, and activation to Factor Xa was measured at designated time intervals by using chromogenic substrate S-2765. Data shown are representative of at least 2 independent experiments, and results are depicted as means and SD. Data shown for HIT patient are representative of 2 patients studied. In the presence of PF4, P < .05 by one-tailed t test for HITT versus control plasma at 10 minutes (∗) and 30 minutes (#). (B) Specificity of tissue factor induction by KKO. Monocytes derived from peripheral blood were incubated in triplicate with PF4 in the presence of serum-free media (SFM; ○), KKO (▴), isotype control (▵), or lipopolysaccharide (LPS; 100 ng/mL; ▪). Additional wells containing KKO were incubated with polyclonal rabbit anti–tissue factor antibody (♦) or preimmune rabbit control IgG (⋄). Cell-surface procoagulant activity was determined as described in (A) legend. Data shown are representative of at least 3 independent experiments, and results are depicted as means and SD. P < .05 by a 2-tailedt test for KKO versus KKO + antitissue factor antibody at 20 minutes (∗) and 30 minutes (#). (C) Time course of tissue factor expression in the presence of antibody or antigen. To determine the time course of cell-surface tissue factor induction by PF4/heparin antibodies, monocytes derived from peripheral blood were incubated in duplicate with serum-free media containing (SFM; ○), or SFM and lipopolysaccharide (LPS; 100 ng/mL; ▪), SFM and KKO (▴), or SFM and isotype control (⋄) in the presence of PF4 for varying time intervals (0, 3, 6, and 12 hours). Data shown are representative of 2 independent experiments, and results are depicted as means and SD. Cell-surface procoagulant activity induced by KKO was maximal at approximately 6 hours, suggesting de novo synthesis of tissue factor. No significant time-dependent increase in tissue factor activity was seen in the presence of SFM or isotype control.

Monocyte tissue factor induction by PF4/heparin antibodies.

(A) Induction of tissue factor by HITT plasma. Monocytes derived from peripheral blood were incubated in duplicate for 6 hours with HITT (♦) or control plasma (⋄) alone, HITT plasma + PF4 (▪), control plasma + PF4 (■), HITT plasma + heparin (●), control plasma + heparin (○), HITT plasma + PF4/heparin (▴), or control plasma + PF4/heparin (▵). After incubation with antigen and antibody, cells were washed 3 times with Tris-buffered saline (TBS) containing 0.1% bovine serum albumin (BSA) and incubated in buffer (TBS/BSA) containing 10 mM calcium chloride and recombinant Factor VIIa (2 nM) for 30 minutes. Plasma-derived Factor X (200 nM) was added to wells, and activation to Factor Xa was measured at designated time intervals by using chromogenic substrate S-2765. Data shown are representative of at least 2 independent experiments, and results are depicted as means and SD. Data shown for HIT patient are representative of 2 patients studied. In the presence of PF4, P < .05 by one-tailed t test for HITT versus control plasma at 10 minutes (∗) and 30 minutes (#). (B) Specificity of tissue factor induction by KKO. Monocytes derived from peripheral blood were incubated in triplicate with PF4 in the presence of serum-free media (SFM; ○), KKO (▴), isotype control (▵), or lipopolysaccharide (LPS; 100 ng/mL; ▪). Additional wells containing KKO were incubated with polyclonal rabbit anti–tissue factor antibody (♦) or preimmune rabbit control IgG (⋄). Cell-surface procoagulant activity was determined as described in (A) legend. Data shown are representative of at least 3 independent experiments, and results are depicted as means and SD. P < .05 by a 2-tailedt test for KKO versus KKO + antitissue factor antibody at 20 minutes (∗) and 30 minutes (#). (C) Time course of tissue factor expression in the presence of antibody or antigen. To determine the time course of cell-surface tissue factor induction by PF4/heparin antibodies, monocytes derived from peripheral blood were incubated in duplicate with serum-free media containing (SFM; ○), or SFM and lipopolysaccharide (LPS; 100 ng/mL; ▪), SFM and KKO (▴), or SFM and isotype control (⋄) in the presence of PF4 for varying time intervals (0, 3, 6, and 12 hours). Data shown are representative of 2 independent experiments, and results are depicted as means and SD. Cell-surface procoagulant activity induced by KKO was maximal at approximately 6 hours, suggesting de novo synthesis of tissue factor. No significant time-dependent increase in tissue factor activity was seen in the presence of SFM or isotype control.

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