Binding and activation of monocytes by pf4/heparin specific antibodies
Since only a subset of patients with platelet activating auto-antibodies develop thrombosis in Heparin-Induced Thrombocytopenia and Thrombosis (HITT), it is presumed that other factors, in addition to platelet activation, contribute to thrombogenesis. To this end, we investigated the role of monocyte activation and tissue factor expression in HIT. As measured by flow cytometry, KKO, a murine monoclonal P+H specific antibody (Ab) that mimics human HIT Abs, but not an isotype control (1C), bound to U937, THP-1 and peripheral blood monocytes (PBMOs) in the presence of PF4 , but not heparin, or P+H [KKO+PF4, Mean Flouresence Intensity (MFI) : THP-1=395, U937=410, PBMOs= 171 v. IC+PF4. MFI: THP-1=23, U937=21, PBMOs= 20]. In cell culture studies, binding of KKO, but not 1C. results in monocyte activation. IL-8, a surrogate marker for monocyte activation, was increased in culture supernatants from U937 and THP-1 cells incubated with KKO+PF4 and HIT+PF4, but not with IC+PF4 or control sera+PF4. Representative data from experiments with U937 cells (+S.D.), assayed for IL-8 at 24 hrs in serum-free media (SFM) are shown below: IL-8 (pg/mL) None TNFa KKO 1C HIT sera CTRL sera SFM 85 (±5) 720(±85) 83(±7) 76(±2) 49(±2) 34(±8) SFM+ PF4 132(±14) 825(±70) 366(±11) 151(±ll) 235(±16) 17(±4) SFM+Hep 49(±7) 617(162) 52(±3) 59(±7) 19(±6) 12(±2) SFM+P/H 71(±ll) 848(±54) 79(±6) 111(±18) 24(±5) 37(±3) In the presence of PF4, binding of KKO to PBMOs, but not 1C, also leads to increased cell surface expression of tissue factor as measured by Factor Vila-catalyzed activation of Factor X. After a 6 hr incubation with KKO or 1C in the presence of PF4, heparin or P+H, cell surface tissue factor (TF) was significantly higher in cells incubated with KKO+PF4 (mOD/min: 173+7) v. IC+PF4 (mOD/min:55±ll). Factor Xa generation on cells incubated with both antibodies was reduced to background levels in the presence of anti-TF Ab. In conclusion, we demonstrate 1) that monocytes bind to P+H antibodies in the presence of PF4, 2) binding of P+H antibodies leads to increased cellular activation (IL-8) of monocytes and 3) binding and activation results in differences in functional tissue factor activity. We propose, that in susceptible individuals, monocyte activation and tissue factor expression may contribute to the hypercoagulabe state associated with HITT.
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