The role of P‐selectin in the immune destruction of platelets

Abstract

Summary. Antibody-mediated platelet destruction is a poorly understood process, although several lines of evidence suggest that Fcc receptor (FccR)-expressing splenic macrophages may be involved. In this study, chemiluminescence (CL) was used to measure the in vitro metabolic response of human monocytes to platelets sensitized with a human immunoglobulin (Ig)G1 recombinant antihuman platelet antigen-1a (anti-HPA-1a) antibody (B2G1; P-hrIgG1). CL responses were inhibited, but not abrogated, in the presence of 10 lg/ml human IgG or murine IgG2a, suggesting that FccRI was principally involved. Experiments to determine the effect of Fab fragments to FccRII found that CL responses to P-hrIgG1 were significantly enhanced, indicating that crosslinking of monocyte FccRII by plateletbound hIgG may modulate concomitant activation by FccRI. Several observations suggested that the CL responses to P-IgG were dependent on the activation of resting platelets during their co-culture with monocytes and their subsequent P-selectin-mediated adhesion. First, the magnitude of the CL response was related to the level of P-selectin expression following platelet activation with a-thrombin. Second, CL responses were inhibited in the presence of antibodies that block the binding of P-selectin to P-selectin glycoprotein ligand-1 but not when platelets were pretreated and then washed. Third, the addition of anti-HPA-1a to monocytes from HPA-1a-negative donors preincubated with HPA-1a-positive platelets resulted in rapid CL responses. Finally, PGI 2 inhibited the CL response to resting P-hrIgG1. Thus, evidence is presented that the interaction of human monocytes with P-hrIgG1 is mediated by FccRI, modulated via FccRII, and enhanced by the presence of P-selectin on the platelet membrane.

HPA‐1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia

“…The transfer of maternal HPA‐1a antibodies to the fetal compartment may therefore be reduced by an unpredictable degree because of absorption in the placenta. Equally, splenic destruction of IgG anti‐HPA‐1a‐sensitized PLTs may be facilitated by direct monocyte‐PLT interactions, 25 a phenomenon not described with RBCs.…”

“…Bioassay measuring monocyte chemiluminescence (CL) responses to antibody‐coated red blood cells (RBCs) has proved a useful guide to clinical management in hemolytic disease of the newborn (HDN) due to anti‐D 24 . A similar bioassay for anti‐HPA‐1a has been developed, 25 but its predictive value in FMAIT has not been examined. We therefore undertook a study to evaluate the predictive values of anti‐HPA‐1a potency and biological activity for fetal outcome.…”

Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia

“…The monocyte CL assay has been described before (53). Briefly, monocytes were prepared from pooled whole blood samples from 6 random donors (40), resuspended in HBSS, 20% RPMI (Sigma-Aldrich), 2% FCS (HBSS/RPMI/FCS) (3 × 10 8 /l), and left to incubate in white flatbottomed 96-well plates (Optiplate-TM 96; PerkinElmer) for 2 hours at 37°C in a humidified atmosphere of 5% CO2. Cryopreserved HPA-1a1b platelets were thawed and sensitized with the recombinant antibodies or the clinical samples.…”

“…However, in contrast to the complete absence of monocyte CL responses observed in the red cell studies, B2G1Δnab elicited a residual response in excess of that seen with a F(ab′) 2 anti-HPA-1a antibody that lacks a constant region altogether. This difference may be an in vitro artefact explained by P-selectin-mediated direct adhesion of platelets to monocytes, which enhances both the rate and, at low antibody concentrations, the magnitude of the monocyte CL response to anti-HPA-1a-sensitized platelets (40).…”

“…Residues of IgG1 responsible for the immunogenic Gm allotype were replaced by the non-immunogenic residues from IgG2 ( Figure 1) (39). Platelets sensitized with the wild-type IgG1 anti-HPA-1a described above have been shown to trigger monocyte chemiluminescence (CL) in an interaction mediated by FcγRI and FcγRII (40). It is therefore logical to hypothesize that the modified recombinant HPA-1a antibody (B2G1Δnab) could block binding of maternal HPA-1a antibodies to platelets, reduce monocyte activation, and ultimately prevent platelet destruction.…”