Gian Paolo Visentin

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.

Characterization of the humoral immune response in heparin-induced thrombocytopenia

Recent reports indicate that antibodies associated with heparin‐induced thrombocytopenia and thrombosis (HITP) are specific for complexes formed between heparin and the heparin‐binding, platelet alpha granule protein, platelet factor 4 (PF4). As with other disorders mediated by immune complexes (IC), the characteristics of the involved immunoglobulins could affect the ability of IC to cause symptoms. We therefore studied the class, subclass, and potency of antibodies specific for heparin:PF4 complexes formed by two groups of patients: one with severe thrombocytopenia, with or without thrombosis, and a positive serotonin release assay (SRA) (Group 1) and another with mild or absent thrombocytopenia, absence of thrombosis, and a negative SRA despite having formed antibodies reactive with heparin:PF4 complexes (Group 2).

Platelet Factor 4 Differentially Modulates CD4+CD25+ (Regulatory) versus CD4+CD25− (Nonregulatory) T Cells

Active suppression mediated by CD4+CD25+ T regulatory (Tr) cells plays an important role in the down-regulation of T cell responses to both foreign and self-Ags. Platelet factor 4 (PF4), a platelet-derived CXC chemokine, has been shown to strongly inhibit T cell proliferation as well as IFN-γ and IL-2 release by isolated T cells. In this report we show that human PF4 stimulates proliferation of the naturally anergic human CD4+CD25+ Tr cells while inhibiting proliferation of CD4+CD25− T cells. In coculture experiments we found that CD4+CD25+ Tr cells exposed to PF4 lose the ability to inhibit the proliferative response of CD4+CD25− T cells. Our findings suggest that human PF4, by inducing Tr cell proliferation while impairing Tr cell function, may play a previously unrecognized role in the regulation of human immune responses. Because platelets are the sole source of PF4 in the circulation, these findings may be relevant to the pathogenesis of certain immune-mediated disorders associated with platelet activation, such as heparin-induced thrombocytopenia and autoimmune thrombocytopenic purpura.

Immune complexes formed following the binding of anti–platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcgammaRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.

Heparin-induced thrombocytopenia and thrombosis.

Thrombocytopenia and thrombosis, recognized complications of heparin therapy, have long been thought to be antibody mediated. However, in vitro studies have failed to provide satisfactory explanations for platelet destruction and paradoxical thrombosis in patients with heparin sensitivity. Recently, several groups of investigators have shown that plasma from patients with heparin-induced thrombocytopenia and thrombosis contains IgG and IgM antibodies specific for complexes containing heparin and platelet factor 4, a heparin-binding protein normally contained in platelet alpha granules. These observations have provided new insights into the pathogenesis of this serious side-effect of heparin therapy and should point the way to improved diagnosis, prevention, and, possibly, treatment.