Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia.

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13 vs 0.73±0.14; P<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14+ positively-selected human macrophages [mean phagocytic index, 6.81 (range, 4.75-9.86) vs 6.01 (range, 5.00-6.98); P=0.954]. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation [mean platelet survival at 300 minutes, 40% (range, 27-55) vs 35% (16-46); P=0.025]. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.

Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates.

Platelet transfusion has become essential therapy in modern medicine. Although the clinical advantage of platelet transfusion has been well established, adverse reactions upon transfusion, especially transmission of bacterial infection, still represent a major challenge. While bacterial contamination is favored by the storage of platelets at room temperature, cold storage may represent a solution for this important clinical issue. In this study, we aimed to clarify whether plasma has protective or detrimental effects on cold-stored platelets. We investigated the impact of different residual plasma contents in apheresis-derived platelet concentrates, stored at 4°C or room temperature, on platelet function and survival. We found that platelets stored at 4°C have higher expression of apoptosis marker compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model. While cold-induced apoptosis was independent of the residual plasma concentration, cold storage was associated with better adhesive properties and higher response to activators. Interestingly, delta (δ) granule-related functions, such as ADP-mediated aggregation and CD63 release, were better preserved at 4°C, especially in 100% plasma. An extended study to assess cold-stored platelet concentrates produced under standard care Good Manufacturing Practice conditions showed that platelet function, metabolism and integrity were better compared to those stored at room temperature. Taken together, our results show that residual plasma concentration does not have a cardinal impact on the cold storage lesions of apheresis-derived platelet concentrates and indicate that the current generation of additive solutions represent suitable substitutes for plasma to store platelets at 4°C.

Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations.

BACKGROUND: The NOD/SCID mouse model is a unique and sophisticated method to study the survival of human platelets (PLTs) in vivo. Meanwhile, several research groups adopted this model to analyze a wide range of PLT antibodies. Differences exist between the research groups regarding the method of PLT injection, the amount and route of antibody injection, and the preparation of blood samples collected from the animal, making it difficult to compare results between studies. STUDY DESIGN AND METHODS: We compared the survival of human PLTs infused into NOD/SCID mice via the tail vein or the retro-orbital plexus. The percentage of circulating human PLTs in the mouse circulation was determined by flow cytometry. Murine blood samples were prepared using two different methods: 1) direct fixation of whole blood samples and 2) isolation of PLTs by density gradient centrifugation. RESULTS: Recovery of human PLTs after tail vein injection was comparable to retro-orbital injection (13% vs. 11% of all circulating PLTs, p = 0.401). However, the survival rate of tail vein-infused PLTs was higher than that of retro-orbitally injected PLTs (median PLT survival after 5 hr 84% vs. 56%, p = 0.025). Moreover, we observed that determination of circulating human PLTs in directly fixed murine whole blood samples shows better reproducibility compared to the density gradient centrifugation method. CONCLUSIONS: Tail vein injection of human PLTs into the NOD/SCID mice is superior to retro-orbital injection in terms of human PLT survival. Direct fixation of whole blood samples allows better reproducibility of results compared to the density gradient centrifugation method.

Further insights into the anti-PF4/heparin IgM immune response.

Anti-platelet factor 4 (PF4)/heparin antibodies are not only the cause of heparin-induced thrombocytopenia but might also play a role in the antibacterial host defence. Recently, marginal zone (MZ) B cells were identified to be crucial for anti-PF4/heparin IgG antibody production in mice. Combining human studies and a murine model of polymicrobial sepsis we further characterised the far less investigated anti-PF4/heparin IgM immune response. We detected anti-PF4/heparin IgM antibodies in the sera of paediatric patients < 6 months of age after cardiac surgery and in sera of splenectomised mice subjected to polymicrobial sepsis. In addition, PF4/heparin-specific IgM B cells were not only found in murine spleen, but also in peritoneum and bone marrow upon in vitro stimulation. Together, this indicates involvement of additional B cell populations, as MZ B cells are not fully developed in humans until the second year of life and are restricted to the spleen in mice. Moreover, PF4/heparin-specific B cells were detected in human cord blood upon in vitro stimulation and PF4-/- mice produced anti-PF4/heparin IgM antibodies after polymicrobial sepsis. In conclusion, the anti-PF4/heparin IgM response is a potential innate immune reaction driven by a B cell population distinct from MZ B cells.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets.

Protamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 µg/ml ODSH: 75%, range 70-81% vs without ODSH: 49%, range 44-59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83%, range 77-93% vs without ODSH: 59%, range 29-61%, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations >16 µg/mL (p<0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 µg/ml ODSH: 53 ± 9, p<0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 µg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.