Figure 3.
Heparin and danaparoid, but not fondaparinux or argatroban, interfere with anti-PF4 Ab binding to PF4. (A) Sera from VITT patients with anti-PF4–IgG were tested in an in-house PF4 enzyme-linked immunoassay (ELISA). Sera were incubated on PF4 (25 µg/mL)-coated microplates. Where indicated, increasing concentrations (0.07, 0.13, 0.26, 0.52, 1.04, 2.08, 4.17, 8.33, 16.67, and 33.33) of heparin and danaparoid (both U/mL) or fondaparinux and argatroban (both µg/mL) were added as described previously.14 Heparin and danaparoid equally reduced the Ab binding to PF4. However, fondaparinux and argatroban had no effect. Each dot represents the mean value of VITT patient sera tested, and the numbers are reported in each graphic for each anticoagulant (n = 3-6). The signal of anti-PF4 Ab binding without anticoagulants was set to 100%. Data are shown as mean ± SEM. (B) Sera from VITT patients with anti-PF4–IgG were tested in an in-house PF4 ELISA to analyze the VITT–IgG/PF4 complex dissociation by anticoagulants. Patient sera were first incubated in PF4-coated microplates for 1 hour to facilitate VITT–IgG/PF4 binding and complex formation. Increasing amounts (0.07-33.33 U or µg/mL) of anticoagulants were added to VITT–IgG/PF4 complex containing microplates. While heparin and danaparoid potently disrupted the IgGs bound to PF4, no disruption was observed with fondaparinux and argatroban. Each dot represents the mean value of VITT patient sera tested, and the numbers are reported in each graphic for each anticoagulant (n = 3). The signal of anti-PF4 Ab binding without anticoagulants was set to 100%. Data are shown as mean ± SEM. (C) IgG binding to healthy washed platelets after incubation with sera from VITT patients in the presence of PF4 (25 µg/mL) or without PF4 (D) was assessed by flow cytometry and expressed as fold increase normalized to HCs. VITT patients showed significantly higher binding at the baseline in comparison with HCs, which was significantly inhibited by heparin and danaparoid. Fondaparinux and argatroban also showed slight displacement of IgG binding. Violin plots showing the distribution of the values were generated using Graphpad Prism 8. ns, not significant. *P < .05 and **P < .01.

Heparin and danaparoid, but not fondaparinux or argatroban, interfere with anti-PF4 Ab binding to PF4. (A) Sera from VITT patients with anti-PF4–IgG were tested in an in-house PF4 enzyme-linked immunoassay (ELISA). Sera were incubated on PF4 (25 µg/mL)-coated microplates. Where indicated, increasing concentrations (0.07, 0.13, 0.26, 0.52, 1.04, 2.08, 4.17, 8.33, 16.67, and 33.33) of heparin and danaparoid (both U/mL) or fondaparinux and argatroban (both µg/mL) were added as described previously.14 Heparin and danaparoid equally reduced the Ab binding to PF4. However, fondaparinux and argatroban had no effect. Each dot represents the mean value of VITT patient sera tested, and the numbers are reported in each graphic for each anticoagulant (n = 3-6). The signal of anti-PF4 Ab binding without anticoagulants was set to 100%. Data are shown as mean ± SEM. (B) Sera from VITT patients with anti-PF4–IgG were tested in an in-house PF4 ELISA to analyze the VITT–IgG/PF4 complex dissociation by anticoagulants. Patient sera were first incubated in PF4-coated microplates for 1 hour to facilitate VITT–IgG/PF4 binding and complex formation. Increasing amounts (0.07-33.33 U or µg/mL) of anticoagulants were added to VITT–IgG/PF4 complex containing microplates. While heparin and danaparoid potently disrupted the IgGs bound to PF4, no disruption was observed with fondaparinux and argatroban. Each dot represents the mean value of VITT patient sera tested, and the numbers are reported in each graphic for each anticoagulant (n = 3). The signal of anti-PF4 Ab binding without anticoagulants was set to 100%. Data are shown as mean ± SEM. (C) IgG binding to healthy washed platelets after incubation with sera from VITT patients in the presence of PF4 (25 µg/mL) or without PF4 (D) was assessed by flow cytometry and expressed as fold increase normalized to HCs. VITT patients showed significantly higher binding at the baseline in comparison with HCs, which was significantly inhibited by heparin and danaparoid. Fondaparinux and argatroban also showed slight displacement of IgG binding. Violin plots showing the distribution of the values were generated using Graphpad Prism 8. ns, not significant. *P < .05 and **P < .01.

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