Effect of Epitope Specific Antibodies on Single Platelet Physiology with Implications for Immune Thrombocytopenia Purpura

Background: Platelets play a vital role in both hemostasis and thrombosis and dysfunction thereof may lead to uncontrolled bleeding and clotting. Consequently, patients with Immune Thrombocytopenia Purpura (ITP) exhibit extremely low platelet counts caused by enhanced platelet clearance and destruction due to platelet reactive antibodies. However, even though all patients have thrombocytopenia, only 20 percent of ITP patients develop major bleeding episodes, which cannot be reliably predicted by platelet count alone. While platelet auto-antibodies have been investigated previously, whether epitope-specific antibodies directly affect platelet function remains poorly understood. To that end, we explored the possible physiological impacts of antibodies on single platelet adhesion, spreading, morphology and activation. Because 70% of platelet reactive antibodies in ITP are directed toward the integrin GPIIb/IIIa, we leveraged well-characterized monoclonal antibodies toward GPIIb/IIIa to better understand their physiological effects of platelet-fibrinogen interactions via the assays described above. Overall, we found that antibodies toward GPIIb/IIIa alter the functionality of individual platelets on fibrinogen surfaces in an epitope dependent manner. Most notably, antibodies that bound to either the head or tail region of αIIb (MBC 290.5 and MBC 314.5) increased the percent of platelets expressing phosphatidylserine, when compared to the control and antibodies binding to the head or tail region of βIIIa (AP3, AP5, and Libs 2). However, the mean intensity of this expression was on average much weaker than the βIIIa antibodies. This suggesting differing functional consequences of platelets to various epitopes and in turn could help explain the differing effects antibodies could have in ITP.

Methods: Healthy donor platelets were diluted to 10 million/mL in Tyrode's modified HEPES buffer to ensure that single platelets were being measured and to reduce the number of platelet aggregates. These platelets were then incubated and adhered on 100 µg/mL human fibrinogen-coated coverslips for 2 hours in the presence of an antibody toward a selected epitope (Figure 1A). Adhered platelets were then stained with a cell membrane stain and Annexin V (PS exposure), fixed and then imaged with fluorescence microscopy. After imaging, thousands of platelets were then counted and analyzed. The antibody treated platelets were then normalized to the non-treated control.

Results: Our preliminary data indicates an epitope-specific effect of antibodies on platelet physiology at the single cell level. Using well-characterized antibodies to various epitopes of GPIIb/IIIa (Figure 1A), we found that when compared to the non-antibody treated control, MBC 290.5 and Libs 2 decreased platelet spreading area by 29% and 31% respectively. However, while MBC 290.5 did not alter platelet density (n/mm²) Libs 2 enhanced platelet adhesion by increasing platelet density by 85%. This indicates the possible decrease in functionality of platelets treated with MBC 290.5. Additionally, both MBC 290.5 and MBC 314.5 increased the percentage of platelets that exposed PS by 97% and 87%, respectively, while AP3 and Libs 2 decreased the percentage of PS-exposed platelets by 64% and 49% respectively. Interestingly, although there was a decrease in the percent PS-exposed platelets, the mean intensity of the platelets that were PS exposed was much greater than the control with an increase of 360% and 228% respectively (Figure 1B).

Conclusion: Although auto-antibodies cause platelet clearance, leading to low platelet counts, little is understood about the possible ramifications of antibodies on platelet behavior. Importantly, we show here that antibodies have a physiological consequence on platelets and different epitope-specific antibodies exhibit unique signatures for altering single platelet physiology, which could help explain how patients with ITP have varying clinical presentations.

Lam:Sanguina, Inc.: Current equity holder in private company.