RIP3-Mediated Programmed Cell Necrosis in Platelets of Immune Thrombocytopenia Purpura

Introduction: Platelets are capable of distinct cell death pathways as they contain machinery for various processes including apoptosis and necrosis. Since the platelets of Immune Thrombocytopenia Purpura (ITP) follow macrophage-mediated abrupt clearance, we hypothesize that ITP platelets do not undergo routine apoptosis-induced platelet clearance but do follow a distinct pathway i.e. programmed cell necrosis before the clearance. In order to achieve our hypothesis, we studied the surface expressions and internal contents of ITP platelets.

Methods: To understand the existence of programmed cell neurosis in ITP platelets, we recruited 12 chronic ITP patients and 22 healthy individuals after obtaining their proper informed consent for this study. Samples were prepared following the standard protocols for the flow cytometry and protein studies. The status of mitochondrial membrane potential (⧍Ψm) of platelets was measured using JC-1 assay (BD bioscience) and the intracellular level of calcium in the platelets was measured using Fluo-4 NW Calcium assay. The Phosphatidyl Serine (PS) expression on the surface of the platelets was measured using Annexin V binding. The surface markers of activated platelets such as P-Selectin and αIIbβ3 were measured using CD62P and PAC-1 antibodies respectively. All the samples were processed on FACS Canto (BD Bioscience). In addition, western blot was carried out to measure the expressions of a necrotic protein, pRIP3 and pro-apoptotic protein (Bak). Alpha-tubulin was used as an internal control. A statistical analysis of Welch's t-test was applied with 95% confidence interval; P values less than 0.05 were considered significant.

Results: As observed by the Flow cytometry studies, the PS expression on the ITP platelets was significantly higher when compared to the control group [3.217% ± 1.199% (ITP) Vs 1.102% ± 0.176% (Control); p = 0.01]. However, there were no significant alterations in the surface markers of platelet activation (P-Selectin and αIIbβ3) on the ITP platelets. Interestingly, we have observed increased levels of calcium [685.75 ± 78.615 (ITP) Vs 498.773 ± 28.646 (Control); p = 0.032] and significant loss of mitochondrial membrane potential (⧍Ψm) [13.452% ± 4.778% (ITP) Vs 4.214% ± 0.985% (Control); p = 0.038] in the ITP platelets. The western blot revealed a significant increase of a necrotic protein, pRIP3. To exclude the possible role of apoptosis in ITP platelets, a pro-apoptotic protein i.e. Bak was also measured which remained unaltered when compared to control.

Conclusion: The cellular load of calcium and subsequent dysfunctional mitochondrial membrane are the key for necrosis. The ITP platelets exhibited increased intracellular calcium and decreased mitochondrial membrane potential as evidenced by the flow cytometry. Though the PS exposure on the ITP platelets was significantly increased, no significant alterations were observed for the platelet activation markers such as P-Selectin and αIIbβ3 on the surface of the ITP platelets. This suggest the existence of cellular changes without undergoing activation of platelets. The increased expression of a necrotic protein, pRI3 and unaltered pro-apoptotic protein, Bak indicate that the ITP platelets do undergo a distinct pathway of cell death which is programmed cell necrosis.

Ethical approval: The study was approved by the Institutional Human Ethics Committee (EC-514). Informed consents were obtained from all the participants before enrolling in this study.

Funding: DST-SERB (ID: EMR-000955).