sepharose and Thrombocytopenia

Patients with immune heparin-induced thrombocytopenia (HIT) possess antibodies that bind to a complex of platelet factor 4 (PF4) and heparin. We observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, we are able to provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd = 7-30 nM) and polystyrene adsorbed PF4 alone (Kd = 20-70 nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. We conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and therefore most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (suboptimal) promotes recognition of this epitope.

Topics: Aged; Antibodies; Antibody Affinity; Antibody Specificity; Antigen-Antibody Reactions; Blood Platelets; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Heparin; Humans; Immunoglobulin G; Platelet Factor 4; Sepharose; Thrombocytopenia

Patients with the heparin-induced thrombocytopenia syndrome (HIT) have heparin-associated antibodies (HAb+), which, in the presence of heparin, are responsible for platelet activation and aggregation. This study addressed the questions: (1) are the antibodies specific for heparin; and (2) how do the antibodies cause platelet aggregation?. Plasmas from 79 patients with HIT were divided into seven plasma samples: HAb+ plasma sample 1 (24 pooled plasmas); HAb+ plasma sample 2 (50 pooled plasmas); and HAb+ plasma samples 3 through 7 (individual plasmas). Normal patient plasmas were used as controls (HAb-).. All seven HAb+ plasma samples caused platelet aggregation (PLA) in the presence of heparin and formed a precipitation line with heparin in gel immunodiffusion plates (HAb- plasmas did neither). The HAb+ plasma samples reacted with heparin, as determined by immunoprecipitation in sodium dodecylsulfate-polyacrylamide gel, with the production of a band at 50 kd (no band with HAb- plasmas). The plasma samples 1 and 2 were passed over heparin sepharose beads three times; the unabsorbed plasmas produced 3+ PLA, the first effluent produced 2+ PLA, and the second and third effluents produced no PLA. The heparin sepharose beads stained 3+, 2+, and 1+, after the respective passages, with fluorescein-labeled goat sera containing anti-human immunoglobulin G antibody. HAb+ plasma samples were digested with pepsin to separate the F(ab')2 fragments from the Fc fragments. The F(ab')2 fragments reacted with heparin as determined by immunoprecipitation in sodium dodecylsulfate-polyacrylamide gel with the production of a band at 25 kd, but did not cause PLA in the presence of heparin.. Patients with HIT have heparin-specific antibodies that react with heparin in a classic F(ab')2 reaction and require the Fc fragment for platelet aggregation.

Topics: Absorption; Antibodies; Electrophoresis, Polyacrylamide Gel; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Heparin; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Platelet Activation; Platelet Aggregation; Precipitin Tests; Sepharose; Sodium Dodecyl Sulfate; Syndrome; Thrombocytopenia

A 46-year-old female who died as a result of thrombocytopenia associated with multiple arterial occlusions and septicemia while on heparin therapy was found to have a platelet-aggregating factor present in several plasma samples and in a sample of serum. This factor was subsequently shown to be an IgG with aggregating properties toward normal platelets that were enhanced by, but not dependent on, the presence of heparin. Further studies showed that heparin was unlikely to have acted as a hapten in initiating the IgG production but that its role was significant in aggravating the ensuing arterial thrombosis. The necessity of substitution of heparin with alternative anticoagulant/antithrombotic therapy to avoid the worst sequelae of this potentially catastrophic syndrome is discussed.

Topics: Blood Coagulation Factors; Chromatography, Affinity; Female; Heparin; Humans; Immunoglobulin G; Middle Aged; Platelet Activating Factor; Platelet Aggregation; Sepharose; Thrombocytopenia; Thrombosis

Topics: Blood Platelets; Humans; Immunoglobulin G; Immunologic Techniques; Platelet Count; Polysaccharides; Sepharose; Staphylococcal Protein A; Thrombocytopenia