Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder initiated by antibodies to complexes between platelet factor 4 (PF4) and heparin. The risk of recurrent thromboembolism persists after heparin is cleared and platelet activation leading to release of PF4 has dissipated. We asked whether antigenic complexes between polyphosphates and PF4 released from activated platelets might intensify or sustain the prothrombotic phenotype of HIT. PF4 forms stable, ultralarge complexes with polyphosphates of various sizes, including those released from platelets, which are recognized by the HIT-like monoclonal KKO, an immunoglobulin G2bκ monoclonal heparin/PF4 binding antibody, and by human HIT antibodies. KKO helps to protect PF4/polyphosphate complexes from degradation by phosphatases. Complement is activated when HIT antibodies bind to PF4/polyphosphate complexes and PF4 reverses the inhibition of complement by polyphosphates. Polyphosphates and PF4 are stored primarily in separate granules in resting platelets, but they colocalize when the cells are activated. Platelets activated by subaggregating doses of thrombin receptor activating peptide release polyphosphates and PF4, which form antigenic complexes that allow KKO to further activate platelets in the absence of heparin and exogenous PF4. These studies suggest that thrombin- or immune complex-mediated release of endogenous antigenic PF4/polyphosphate complexes from platelets may augment the prothrombotic risk of HIT and perpetuate the risk of thrombosis after heparin has been discontinued.

Autoimmune heparin‐induced thrombocytopenia

“…Furthermore, binding of high concentrations of PF4 to platelets may also induce exposure of HIT antigen(s) in the absence of added polyanions . In this case, polyanions on the platelet surface probably augment the close proximity of PF4 . For the phenomenon of aHIT, this clearly shows that the antigens can be exposed by factors other than heparin.…”

A Platelet Factor 4-Dependent Platelet Activation Assay Facilitates Early Detection of Pathogenic Heparin-Induced Thrombocytopenia Antibodies

“…[3][4][5][6] Recent studies demonstrate that "pathogenic" (platelet-activating) HIT antibodies ABBREVIATIONS: ELISA = enzyme-linked immunosorbent assay; HIT = heparin-induced thrombocytopenia; OD = optical density; PEA = PF4-dependent P-selectin expression assay; PF4 = platelet factor 4; SRA = serotonin release assay; TPE = therapeutic plasma exchange; UFH = unfractionated heparin preferentially recognize PF4 in a complex with endogenous platelet surface glycosaminoglycans 7 and/or polyphosphates. 8 On the basis of these findings, we described a novel diagnostic test, the PF4-dependent P-selectin expression assay (PEA). 9,10 In the PEA, platelets pretreated with PF4 are incubated with samples from patients suspected of having HIT and platelet surface p-selectin expression is measured as a marker of platelet activation.…”

Platelet factor 4/heparin complexes present epitopes differently on solid-phase vs platelet surfaces

“…13 We hypothesized that KKO might bind to PF4/H complexes in the purified solid-phase system differently than PF4/H complexes coated on the platelet surface. It is well known that the epitope for anti-PF4/P antibodies is conformation sensitive, and additional polyanions on the platelet surface (such as chondroitin sulfate or polyphosphates) 14,15 might influence the conformation of PF4 and PF4/H complexes 15 or their three-dimensional presentation. To further investigate our hypothesis, we combined biophysical and immunohematological methods and directly compared the binding forces of KKO on a single-molecule level when it interacted with PF4 or PF4/H complexes immobilized either on a solid phase (purified system) or on platelet surfaces.…”