RESUMEN
Antibodies to heparin-platelet factor 4 (PF4) complexes have been observed in patient with heparin-induced thrombocytopenia (HIT) syndrome. These antibodies may be of various isotypes and differ with respect to their ability to activate platelets/endothelial cells. This study determined the isotypes and functionality of antiheparin-platelet factor 4 (AHPF4) antibodies in 111 patients treated with heparin and clinically suspected for HIT. In this patient population, 50% had detectable AHPF4 cumulative IgA, IgG, and IgM (determined by enzyme-linked immunosorbent assay, ELISA), but only 35% was positive when tested with the (14)C-serotonin release assay (SRA). Using antihuman Ig specific for different isotypes, we found that 50% of the 111 samples was positive for IgG, 45% for IgM, and 37% for IgA. In 50 normal human serum (NHS) samples, only two were positive for IgG, but 33 were positive for IgM, indicating a potential humoral response to the heparin-PF4 complex prior to heparin administration. Patients that were ELISA(+) for AHPF4 antibody titer were subdivided into SRA-positive (+) and SRA-negative (-) groups. The SRA(+) group had a mean ELISA optical density (OD) for AHPF4 IgA/IgG/IgM of 2.1, while the SRA(-) group had a mean OD of 0.8 (P<.001). The SRA(+) group had greater mean OD values for all three individual isotypes. Using flow cytometry, we determined the ability of different patient samples to activate platelets. Samples that contained IgG and were SRA(+) activated platelets (as measured by microparticle generation and P-selectin expression) in the presence of therapeutic concentrations of heparin. NHS and samples containing IgA and/or IgM that were SRA(-) were not able to produce microparticles nor were they able to increase expression of P-selectin. Together, these data indicate that IgG is the principal mediator of platelet activation in patients with HIT, with IgA and IgM playing a less significant role in the pathophysiology of this syndrome.
Asunto(s)
Autoanticuerpos/inmunología , Heparina/efectos adversos , Isotipos de Inmunoglobulinas/inmunología , Factor Plaquetario 4/inmunología , Trombocitopenia/inducido químicamente , Reacciones Antígeno-Anticuerpo , Autoanticuerpos/sangre , Radioisótopos de Carbono , Estudios de Casos y Controles , Técnicas de Laboratorio Clínico/normas , Ensayo de Inmunoadsorción Enzimática , Heparina/inmunología , Heparina/uso terapéutico , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/sangre , Agregación Plaquetaria/inmunología , Prevalencia , Serotonina/metabolismo , Trombocitopenia/diagnóstico , Trombocitopenia/inmunologíaRESUMEN
The pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated via a heterogeneous group of heparin(s)-platelet factor 4 (H-PF4) complexes bound to their antibodies. These anti-H-PF4 (AHPF4) antibodies that are capable of binding to the FcgammaRIIA receptor [cluster of differentiation (CD) 32] on platelets, resulting in platelet activation, widely vary in their specific activities as platelet activation (functionality). Predisposing factors related to specific pathologic conditions may also contribute to the generation of these antibodies and their relative functionality during HIT syndrome. To understand this phenomenon, a sub-study was carried out in patients undergoing elective total hip and knee replacement surgery (ECHOS Study) and who were treated with unfractionated heparin (UFH) and a low-molecular-weight heparin (LMWH; Clivarin). Approximately 600 patients per arm [UFH=7,500 anti-Xa U twice a day (b.i.d.) subcutaneous (s.c.) and clivarin=4200 U once daily (o.d.) s.c.], age >40 years, received prophylactic treatment for a minimum of 11-14 days. Plasma samples were collected at pre-dose, days 2-4, days 11-14 and at follow-up 6-8 weeks after discharge and were analyzed for AHPF4 antibody titers. Functionality of the enzyme-linked immunosorbant assay (ELISA)-positive AHPF4 antibodies to cause platelet activation was tested by 14C-serotonin release assay (SRA). Both UFH and clivarin treatments in orthopedic surgical patients resulted in a progressive generation of AHPF4 antibodies. The relative prevalence/functionality of AHPF4 antibodies in clivarin arm was markedly lower (two- to threefold, p<0.001) as compared to UFH at each time point. Most of the samples in clivarin group were found to be SRA negative, suggesting the presence of AHPF4 antibodies that did not activate platelets (nonfunctional). Within the UFH arm, the relative prevalence/functionality of AHPF4 antibodies was much higher (p<0.002) in knee group compared to the corresponding hip group. This study, for the first time, reports on the elevated levels of AHPF4 antibodies generated by heparin associated with the pathogenesis of knee surgery. Clinical significance of the differential generation of HIT-associated antibodies remains unexplored at this time.