Do patients with the heparin-induced thrombocytopenia syndrome have heparin-specific antibodies?
OBJECTIVE
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?


METHODS
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-).


RESULTS
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.


CONCLUSIONS
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.
Department of Surgery, University of Missouri Health Sciences Center, Columbia.
J G
JG
Adams
L J
LJ
Humphrey
X
X
Zhang
D
D
Silver
D000042:Absorption; D000906:Antibodies / Q000097:blood* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000009:adverse effects* / Q000276:immunology*; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000302:isolation & purification; D007141:Immunoglobulin Fc Fragments / Q000097:blood / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification; D015539:Platelet Activation / Q000276:immunology; D010974:Platelet Aggregation / Q000276:immunology*; D011233:Precipitin Tests; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate; D013577:Syndrome; D013921:Thrombocytopenia / Q000097:blood / Q000139:chemically induced*
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D012967:Sodium Dodecyl Sulfate; D006493:Heparin; D012685:Sepharose; D016650:Fluorescein-5-isothiocyanate