Publication Date:
2004-11-16
Description:
Heparin-induced thrombocytopenia (HIT) is a potentially devastating complication of heparin therapy. The condition is due to formation of antibodies to the heparin-platelet factor 4 (HPF4) complex that activate platelets and/or endothelial cells by binding to FcγIIa (CD32) receptors. It is not clear why only a sub-population of patients that form HPF4 antibodies also develops clinical HIT, and why only a fraction of those develop thrombosis. We hypothesized that some functionally active HPF4 antibodies (i.e. that activate platelets) may also activate leukocytes and endothelial cells by upregulating procoagulant activity; a property not shared by non-functional HPF4 antibodies. Citrated plasmas were obtained from patients clinically suspected of HIT. A commercially available ELISA (GTI, Brookfield, WI) combined with a 14C-serotonin release assay (SRA) was used to stratify patient plasmas into functional (F) and non-functional (NF) HPF4 antibody groups. Pooled normal plasma (NP) served as negative control. Some (n=20) GTI-ELISA positive samples (both F and NF) were also tested by flow cytometry using HPF4 coated microspheres (DiaMed-Caribbean, Inc., Miami, FL) for immunological antibody detection, and fluorescent annexin V binding to detect functional activity. The ability of HPF4 antibodies to cause platelet-monocyte aggregate (conjugate) formation was evaluated by two-color flow cytometry. The functional consequences of HPF4 antibody interaction with EaHy926 hybridoma endothelial cells were studied in the presence/absence of washed platelets and therapeutic heparin levels. Cell surface procoagulant activity (PCA) was measured by a single-stage clotting assay. Approximately 25% of ELISA positive plasmas were also positive by SRA. Although most ELISA positive samples were strongly positive in the microsphere flow cytometry assay (5–20 fold increase in mean fluorescence intensity [MFI] compared to NP) there was no direct correlation between the ELISA OD values and MFI. In the functional flow cytometric assay, not all the SRA positive samples were able to cause platelet activation. Platelet-monocyte aggregate formation in the presence of functional HPF4 antibodies was increased by 2–6 fold (MFI increase 50–300%). This was significantly higher than seen with the NF (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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