ITP patients exhibit various clinical processes and responses to different treatments, indicating that different immune mechanisms may be responsible for the decrease in platelet count[31]. Multiple studies have established the crucial role of autoantibodies in the development of ITP, demonstrating that the main targets for these antibodies are GP IIb-IIIa and GPIb-IX. Goette et al[32] confirmed the occurrence of apoptosis in platelets from patients carrying anti-GPIIb-IIIa and anti-GPIb auto-antibodies. While the primary pathogenic mechanism through which auto-antibodies induce thrombocytopenia is phagocytosis of antibody-bound platelets by the reticuloendothelial system, complement activation-mediated lysis of antibody-bound platelets also appears to play a role in ITP[5].
Similarly to nucleated cells, platelet life span is controlled by an intrinsic apoptotic program. The main contributors to this process are the anti-apoptotic protein BcL-xL and the pro-apoptotic proteins Bak and Bax[33]. An imbalance between pro- and anti-apoptotic proteins leads to mitochondrial outer membrane permeabilization (MOMP), which is followed by collapse of the mitochondrial inner membrane potential (ΔΨm), cytochrome c being released into the cytoplasm, activation of caspase 3 and 9, externalization of phosphatidylserine (PS), and shedding of microparticles[34]. Previous studies have investigated the role of platelet apoptosis in the development of ITP. Platelet apoptosis was initially demonstrated in an animal model of ITP, where injection of antiGPIIb antibodies induced platelet apoptosis features, such as ΔΨm, PS exposure, and caspase activation, in murine platelets and that treatment with IVIg will inhibit platelet apoptosis thus potentially ameliorating ITP[35]. As for human ITP, evidence of platelet apoptosis, including activation of caspase 3, 8, and 9, with PS exposure, and with decreased ΔΨm, and demonstrated increased microparticle formation was observed in children with acute ITP, and this was improved by intravenous immunoglobulin infusion[36].
To test whether complement-independent plasma factors present in ITP could induce platelet apoptosis, complement was removed by purifying IgG. Apoptosis parameters such as mitochondrial membrane potential (ΔΨm)[28, 35, 37] and phosphatidylserine (PS) exposure[22, 28, 35, 38] in normal platelets after a 4-hour incubation with either ITP or healthy volunteers plasma purified IgG were tested using flow cytometry. A trend toward increased apoptosis was observed in normal platelets incubated with the purified IgG of ITP plasma positive for anti-GPIb/IX antibodies compared to those incubated with the purified IgG of ITP plasma positive for only anti-GPIIb/IIIa antibodies and anti-GPIb/IX and GPIIb/IIIa dual antibodies, as well as the healthy control. These findings suggest that platelet destruction in ITP caused by anti-GPIb/IX antibodies may occur through a complement-independent mechanism. However, further exploration is needed to validate this argument. It is worth noting that group III, which had both anti-GPIb/IX and GPIIb/IIIa double positive autoantibodies, did not show any significant differences compared to the healthy control group. We speculate that GPIIb/IIIa autoantibodies may inhibit the apoptosis induced by anti-GPIb/IX autoantibodies. Further experimental research is necessary to confirm this hypothesis.
As mentioned above, Nardi et al[22]found that HIV-1 antibodies can induce platelet particle formation through acting on platelet glycoprotein GPIIIa49-66, independent of complement, and the pathogenesis of this phenomenon is related to the production of reactive oxygen species (ROS). To examine whether anti-GPIb/IX-positive IgG can induce the platelet particle formation through a complement-independent pathway, the size and fluorescence intensity of PE-cy5-CD61-labeled platelets were detected using flow cytometry[22]. The results showed that purified IgG of plasma autoantibody-positive for GPIb/IX in ITP patients could promote the platelet particle formation. Therefore, our results also confirm the crucial role of anti-GPIb/IX antibodies in inducing platelet particle formation through a complement-independent pathway. However, the exact mechanism remains unclear and may also involve the action of anti-GPIb/IX antibodies on platelets and the induction of reactive oxygen species (ROS), which requires further investigation.
Anucleate platelets perform two fundamental processes, activation and apoptosis. Gyulkhandanyan et al. demonstrated that, depending on the triggering stimulus, platelets predominantly undergo ∆ψm depolarization-only, P-selectin exposure-only, or both responses.It suggests that a high level of platelet activation is not necessarily associated with platelet apoptosis, and that the apoptotic and activation responses may be concomitant, indicating that platelet apoptosis and activation are different phenomena driven by different mechanisms[39]. We investigated whether the platelet apoptosis induced by purified IgG of GPIb/IX antibody-positive plasma is a result of platelet activation. Flow cytometry was used to detect increased surface expression of P-selectin (CD62p) and CD63 on platelets treated with anti-GPIb/IX-positive and healthy control IgG. The results indicated that, although both groups had different levels of activation, there was no difference between the two groups in terms of platelet activation. This proves that the platelet apoptosis induced by purified IgG of GPIb/IX antibody-positive plasma is not associated with platelet activation, further indicating that the platelet particle formation is a cellular-level indicator of anti-GPIb/IX antibody action on platelets and subsequent platelet apoptosis.
Our research's shortcoming is that we only used traditional methods to detect platelet particle formation, consistent with the report by Michael Nardi and colleagues [22], that platelet particle formation implies not only a reduction in size but also a decrease in average fluorescence intensity. Current research has shown that calibrated beads are useful for standardizing the enumeration of platelet-derived microparticles using flow cytometry[40]. However, Gyulkhandanyan et al. believe that calibrated beads also do not allow differentiation between microparticles and small-size platelets. Gyulkhandanyan et al. used the term microparticles for definition of platelet-derived smaller particles which are characterized by FSC<101, contain the constitutive platelet marker GPIIbIIIa on their surface, and expose PS under appropriate stimulation[30].
Recent research has shown that sutimlimab, through selective blocking of C1s in the classical pathway, rapidly and effectively increases platelet count in patients with chronic ITP. Responder analysis revealed that 5 out of 12 patients (42%) achieved a durable response with sutimlimab monotherapy, despite previous unresponsiveness to TPO-RA[20]. Oral agents that inhibit C3 activation hold promise for future clinical trials in refractory ITP[21]. Our experimental data reveal that ITP patients with GPIb/IX autoantibodies can induce platelet apoptosis through complement-independent pathways. Further studies involving a larger population of ITP patients, with exploration of patient characteristics such as the presence of anti-GPIIb/IIIa and/or anti-GPIb/IX autoantibodies, may help identify potential predictors of clinical response to complement pathway inhibitors and evaluate the association between the specificity of antiplatelet autoantibodies and response to complement pathway inhibitors as a treatment.