Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH.
Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG).
Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs.
The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.
© 2021 The Authors. Transfusion published by Wiley Periodicals LLC. on behalf of AABB.

Platelets release platelet-derived extracellular vesicles (PDEVs) upon activation - in a process that is regulated by generation of reactive oxygen species (ROS). Platelet NADPH oxidase-1 (Nox-1) contributes to ROS generation and thrombus formation downstream of the collagen receptor GPVI.
We aimed to investigate whether PDEVs contain Nox-1 and whether this is relevant for PDEV-induced platelet activation.
PDEVs were isolated through serial centrifugation after platelet activation with thrombin receptor agonist TRAP-6 (activated PDEVs) or in the absence of agonist (resting PDEVs). The physical properties of PDEVs were analyzed through nanoparticle tracking analysis. Nox-1 levels, fibrinogen binding and P-selectin exposure were measured using flow cytometry, and protein levels quantified by immunoblot analysis. ROS were quantified using DCF fluorescence and electron paramagnetic resonance.
Nox-1 was found to be increased on the platelet outer membrane upon activation and was present in PDEVs. PDEVs induced platelet activation, while co-addition of GPVI agonist collagen-related peptide (CRP) did not potentiate this response. PDEVs were shown to be able to generate superoxide in a process at least partially mediated by Nox-1, while Nox-1 inhibition with ML171 (also known as 2-APT) did not influence PDEV production. Finally, inhibition of Nox-1 abrogated PDEV-mediated platelet activation.
PDEVs are able to generate superoxide, bind to and activate platelets in a process mediated by Nox-1. These data provide novel mechanisms by which Nox-1 potentiates platelet responses, thus proposing Nox-1 inhibition as a feasible strategy to treat and prevent thrombotic diseases.
Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

Essentials Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin. The interaction between NPH-insulin, anti-PRT/heparin antibodies and platelets was investigated. Anti-PRT/heparin antibodies activate platelets in presence of NPH-insulin dependent on heparin. Cross-reactivity seems to have no major effect on the clinical outcome of medical patients.
Background Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin, a commonly used therapeutic agent for diabetes mellitus. Immunization against PRT/heparin complexes is common in diabetic patients. Objectives To investigate the impact of NPH-insulin on the interaction between anti-PRT/heparin antibodies and platelets. Methods The interaction between NPH-insulin and anti-PRT/heparin antibodies was tested using in-house enzyme immunoassays. The ability of anti-PRT/heparin antibodies to activate platelets in the presence of NPH-insulin (and heparin) was investigated using flow cytometry. Results Twenty-one out of 80 sera containing anti-PRT/heparin IgG showed binding to NPH-insulin. Anti-PRT/heparin IgG from immunized patients bound to platelets in the presence of NPH-insulin, but not in the presence of native insulin. Anti-PRT/heparin antibodies induced P-selectin expression in the presence of NPH-insulin in a heparin-dependent way (median mean fluorescence intensity in the presence of NPH-insulin: 55, 95% confidence interval [CI] 18.7-100.5 vs. NPH-insulin and heparin: 204, 95% CI 106.5-372.8). The clinical relevance of platelet-activating anti-PRT/heparin antibodies was assessed by investigating a multicenter study cohort of 332 acutely ill medical patients who received heparin. None of the 21 patients with anti-PRT/heparin IgG developed thrombocytopenia or thromboembolic complications. Conclusions Anti-PRT/heparin antibodies activate platelets in the presence of NPH-insulin in a heparin-dependent way. However, results from our preliminary study indicate no major impact of these antibodies on the clinical outcome in medical patients receiving heparin, particularly on thromboembolic complications.
© 2016 International Society on Thrombosis and Haemostasis.

Severe injury often results in substantial bleeding and mortality. Injury provokes cellular activation and release of extracellular vesicles. Circulating microvesicles (MVs) are predominantly platelet-derived and highly procoagulant. They support hemostasis and vascular function. The roles of MVs in survival after severe injury are largely unknown. We hypothesized that altered MV phenotypes would be associated with transfusion requirements and poor outcomes.
This single-centre study was approved by the Institutional Review Board. The study cohort consisted of patients with major trauma requiring blood product transfusion and 26 healthy controls. Plasma samples for MVs were collected upon admission to the emergency department (n=169) and post-resuscitation (n=42), and analysed by flow cytometry for MV counts and cellular origin: platelet (PMV), erythrocyte (RMV), leukocyte (LMV), endothelial (EMV), tissue factor (TFMV), and annexin V (AVMV). Twenty-four hour mortality is the outcome measurement used to classify survivors versus non-survivors. Data were compared over time and analysed with demographic and clinical data.
The median age was 34 (IQR 23, 51), 72% were male, Injury Severity Score was 29 (IQR 19, 36), and 24 h mortality was 13%. MV levels and phenotypes differed between patients and controls. Elevated admission EMVs were found both in survivors (409/µL) and non-survivors (393/µL) compared to controls (23/µL, p<0.001) and persisted over time. Admission levels of PMV, AVMV, RMV, and TFMV were significantly lower in patients who died compared to survivors, but were not independently associated with the 24 h mortality rate. Patients with low MV levels at admission received the most blood products within the first 24 h. AVMV and PMV levels either increased over time or stabilized in survivors but decreased in non-survivors, resulting in significantly lower levels at intensive care unit admission in non-survivors (1,048 vs. 1,880 AVMV/µL, p<0.00004 and 1,245 PMP/µL vs. 1,866 PMP/µL, p=0.003).
Severe injury results in endothelial activation and altered MV phenotypes. Significant differences in specific MV phenotypes or changes over time were associated with blood product requirements and the 24 h mortality rate.

Platelet inactivation technologies (PITs) have been shown to increase platelet storage lesions (PSLs). This study investigates amotosalen/ultraviolet (UV)A- and riboflavin/UVB-induced platelet (PLT) lesions in vitro. Particular attention is given to the effect of UVB alone on PLTs.
Buffy coat-derived PLT concentrates (PCs) were treated with amotosalen/UVA, riboflavin/UVB, or UVB alone and compared to untreated PCs throughout storage. In vitro PLT function was assessed by blood gas and metabolite analyses, flow cytometry-based assays (CD62P, JC-1, annexin V, PAC-1), hypotonic shock response, and static adhesion to fibrinogen-coated wells.
In our experimental conditions, riboflavin/UVB-treated PCs showed the most pronounced differences compared to untreated and amotosalen/UVA-treated PCs. The riboflavin/UVB treatment led to a significant increase of anaerobic glycolysis rate despite functional mitochondria, a significant increase of CD62P on Day 2, and a decrease of JC-1 aggregates and increase of annexin V on Day 7. The expression of active GPIIbIIIa (PAC-1) and the adhesion to fibrinogen was significantly increased from Day 2 of storage in riboflavin/UVB-treated PCs. Importantly, we showed that these lesions were caused by the UVB radiation alone, independently of the presence of riboflavin.
The amotosalen/UVA-treated PCs confirmed previously published results with a slight increase of PSLs compared to untreated PCs. Riboflavin/UVB-treated PCs present significant in vitro PSLs compared to untreated PCs. These lesions are caused by the UVB radiation alone and probably involve the generation of reactive oxygen species. The impact of these observations on clinical use must be investigated.
© 2015 AABB.