Proteomic profiling for biomarker discovery in heparin-induced thrombocytopenia.

New analytical techniques can assess hundreds of proteins simultaneously with high sensitivity, facilitating the observation of their complex interplay and role in disease mechanisms. We hypothesized that proteomic profiling targeting proteins involved in thrombus formation, inflammation, and the immune response would identify potentially new biomarkers for heparin-induced thrombocytopenia (HIT). Four existing panels of the Olink proximity extension assay covering 356 proteins involved in thrombus formation, inflammation, and immune response were applied to randomly selected patients with suspected HIT (confirmed HIT, n=32; HIT ruled-out, n=38; positive heparin/PF4 [H/PF4] antibodies, n=28). The relative difference in protein concentration was analyzed using a linear regression model adjusted for sex and age. To confirm the test results, soluble P-selectin was determined using ELISA in above mentioned patients and an additional second dataset (n=49). HIT was defined as a positive heparin-induced platelet aggregation test (HIPA; washed platelet assay). Among 98 patients of the primary dataset, the median 4Ts score was 5 in patients with HIT, 4 in patients with positive heparin/PF4 antibodies, and 3 in patients without HIT. The median OD of a polyspecific heparin/PF4 ELISA was 3.0, 0.9, and 0.3, respectively. Soluble P-selectin remained statistically significant after multiple test adjustments. The area under the receiver-operating-characteristics-curve was 0.81 for Olink and 0.8 for ELISA. Future studies shall assess the diagnostic and prognostic value of soluble P-selectin in the management of HIT.

Indocyanine Green-Labeled Platelets for Survival and Recovery Studies.

Introduction: Before being implemented in daily clinical routine, new production strategies for platelet concentrates (PCs) must be validated for their efficacy. Besides in vitro testing, the establishment of new methods requires the labeling of platelets for in vivo studies of platelets' survival and recovery. Indocyanine green (ICG) is a Food and Drug Administration-approved near-infrared (NIR) fluorescent dye for diagnostic use in vivo, suitable for non-radioactive direct cell labeling of platelets. Methods: Platelets from PCs in storage solutions with different plasma concentrations were labeled with ICG up to concentrations of 200 µm. Whole blood (WB) was used as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. The impact of labeling processes was assessed by the quantification of CD62P expression and PAC-1 binding as platelet function markers. Platelet aggregation was analyzed by light transmission aggregometry. ICG-labeling efficiency and stability of platelets were determined by flow cytometry. Results: Platelets from PCs could be successfully labeled with 10 µm ICG after 1 and 4 days of storage. The best labeling efficiency of 99.8% ± 0.1% (immediately after labeling) and 81% ± 6.2% (after 24 h incubation with WB) was achieved by plasma replacement by 100% platelet additive solution for the labeling process. Since the washing process slightly impaired platelet function, ICG labeling itself did not affect platelets. Immediately after the ICG-labeling process, plasma was re-added, resulting in a recovered platelet function. Conclusion: We developed a Good Manufacturing Practice compatible protocol for ICG fluorescent platelet labeling suitable for survival and recovery studies in vivo as a non-radioactive labeling alternative.

We developed a simple, reproducible method according to Good Manufacturing Practice guidelines for labeling of platelets from platelet concentrates (PCs) with indocyanine green (ICG) as a non-radioactive alternative. PCs are medicinal drugs that are transfused to prevent or treat bleeding. They consist of the blood cells' platelets which are responsible for clotting processes in the body. Manufacturing procedures of PCs are continuously refined, and for in vivo testing, these platelets have to be labeled to track and to distinguish them from proband's or patient's own cells. Radioactive labeling, for a long time the gold standard for cell labeling, is no longer accepted. ICG is a fluorescent dye approved by the drug authorities and already used for diagnostic purposes in humans. We used ICG to label platelets from PCs. With our method, we achieved a labeling efficiency of 99.8%. We used whole blood (WB) as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. After the addition of ICG-labeled platelets to WB, the labeling efficiency decreased to 81% after 24 h. However, we were still able to distinguish the ICG-labeled platelets from the WB platelets. We could also show that platelet function was not impaired by the labeling processes. The good tolerance of ICG indicates a short path to clinical application in healthy volunteers and investigations of novel PC-manufacturing procedures. As a read-out system, flow cytometry systems equipped with NIR lasers and filters could offer the possibility of rapid visualization, cell tracking, re-isolation, and ex vivo studies.

Accuracy of Diagnosing Heparin-Induced Thrombocytopenia.

Importance: Heparin-induced thrombocytopenia (HIT) is a life-threatening condition that requires urgent diagnostic clarification. However, knowledge of the diagnostic utility of the recommended diagnostic tests is limited in clinical practice. Objective: To evaluate the current diagnostic practice for managing the suspicion of HIT. Design, Setting, and Participants: This prospective diagnostic study was conducted from January 2018 to May 2021 among consecutive patients with suspected HIT from 11 study centers in Switzerland, Germany, and the United States. Detailed clinical data and laboratory information were recorded. Platelet factor 4/heparin antibodies were quantified using an automated chemiluminescent immunoassay (CLIA). A washed-platelet heparin-induced platelet activation (HIPA) test was used as a reference standard to define HIT. Exposures: Suspicion of HIT. Main Outcomes and Measures: The primary outcome was the diagnostic accuracy of the 4Ts score, the CLIA, and the recommended algorithm serially combining both tests. Results: Of 1448 patients included between 2018 and 2021, 1318 were available for the current analysis (median [IQR] age, 67 [57-75] years; 849 [64.6%] male). HIPA was positive in 111 patients (prevalence, 8.4%). The most frequent setting was intensive care unit (487 [37.0%]) or cardiovascular surgery (434 [33.0%]). The 4Ts score was low risk in 625 patients (46.8%). By 2 × 2 table, the numbers of patients with false-negative results were 10 (9.0%; 4Ts score), 5 (4.5%; CLIA), and 15 (13.5%; recommended diagnostic algorithm). The numbers of patients with false-positive results were 592 (49.0%; 4Ts score), 73 (6.0%; CLIA), and 50 (4.1%; recommended diagnostic algorithm), respectively. Conclusions and Relevance: In this diagnostic study of patients suspected of having HIT, when the recommended diagnostic algorithm was used in clinical practice, antibody testing was required in half the patients. A substantial number of patients were, however, still misclassified, which could lead to delayed diagnosis or overtreatment. Development of improved diagnostic algorithms for HIT diagnosis should be pursued.

ISTH bleeding assessment tool and platelet function analyzer in children with mild inherited platelet function disorders.

OBJECTIVES: To evaluate the diagnostic performance of platelet function analyzer (PFA) and The International Society on Thrombosis and Hemostasis bleeding-assessment-tool (ISTH-BAT) in detecting mild inherited platelet function disorders (IPFDs) in children with suspected bleeding disorders. METHODS: Prospective single-center diagnostic study including consecutive patients <18 years with suspected bleeding disorder and performing a standardized workup for platelet function defects including ISTH-BAT, PFA, platelet aggregation testing, blood smear-based immunofluorescence, and next-generation sequencing-based genetic screening for IPFDs. RESULTS: We studied 97 patients, of which 34 von Willebrand disease (VWD, 22 type-1, 11 type-2), 29 IPFDs (including delta-/alpha-storage pool disease, Glanzmann thrombasthenia, Hermansky-Pudlak syndrome) and 34 with no diagnosis. In a model combining PFA-adenosine diphosphate (ADP), PFA-epinephrine (EPI), and ISTH-BAT overall performance to diagnose IPFDs was low with area under the curves of 0.56 (95% CI 0.44, 0.69) compared with 0.84 (95% CI 0.76, 0.92) for VWD. Correlation of PFA-EPI/-ADP and ISTH-BAT was low with 0.25/0.39 Spearman's correlation coefficients. PFA were significantly prolonged in patients with VWD and Glanzmann thrombasthenia. ISTH-BAT-scores were only positive in severe bleeding disorders, but not in children with mild IPFDs or VWD. CONCLUSION: Neither ISTH-BAT nor PFA or the combination of both help diagnosing mild IPFDs in children. PFA is suited to exclude severe IPFDs or VWD and is in this regard superior to ISTH-BAT in children.

Therapeutic strategies in FcγIIA receptor-dependent thrombosis and thromboinflammation as seen in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT).

INTRODUCTION: Fcγ-receptors (FcγR) are membrane receptors expressed on a variety of immune cells, specialized in recognition of the Fc part of immunoglobulin G (IgG) antibodies. FcγRIIA-dependent platelet activation in platelet factor 4 (PF4) antibody-related disorders have gained major attention, when these antibodies were identified as the cause of the adverse vaccination event termed vaccine-induced immune thrombocytopenia and thrombosis (VITT) during the COVID-19 vaccination campaign. With the recognition of anti-PF4 antibodies as cause for severe spontaneous and sometimes recurrent thromboses independent of vaccination, their clinical relevance extended far beyond heparin-induced thrombocytopenia (HIT) and VITT. AREAS COVERED: Patients developing these disorders show life-threatening thromboses, and the outcome is highly dependent on effective treatment. This narrative literature review summarizes treatment options for HIT and VITT that are currently available for clinical application and provides the perspective toward new developments. EXPERT OPINION: Nearly all these novel approaches are based on in vitro, preclinical observations, or case reports with only limited implementation in clinical practice. The therapeutic potential of these approaches still needs to be proven in larger cohort studies to ensure treatment efficacy and long-term patient safety.

Thrombosis with thrombocytopenia syndrome (TTS) and vaccine-induced immune thrombocytopenia and thrombosis (VITT): Brighton Collaboration case definitions and guidelines for data collection, analysis, and presentation of immunisation safety data.

This is a revision of the online November 2021 Brighton thrombosis with thrombocytopenia syndrome (TTS) case definition and a new Brighton Collaboration case definition for vaccine-induced immune thrombocytopenia and thrombosis (VITT). These case definitions are intended for use in clinical trials and post-licensure pharmacovigilance activities to facilitate safety data comparability across multiple settings. They are not intended to guide clinical management. The case definitions were developed by a group of subject matter and Brighton Collaboration process experts as part of the Coalition for Epidemic Preparedness Innovations (CEPI)-funded Safety Platform for Evaluation of vACcines (SPEAC). The case definitions, each with defined levels of diagnostic certainty, are based on relevant published evidence and expert consensus and are accompanied by specific guidelines for TTS and VITT data collection and analysis. The document underwent peer review by a reference group of vaccine safety stakeholders and haematology experts to ensure case definition useability, applicability and scientific integrity.

Clinical Course and Management of Patients with Emergency Surgery Treated with Direct Oral Anticoagulants or Vitamin K Antagonists-Results of the German Prospective RADOA-Registry.

(1) Background: The clinical management of anticoagulated patients treated with direct oral anticoagulants (DOAC) or Vitamin K antagonists (VKA) needing emergency surgery is challenging. (2) Methods: The prospective German RADOA registry investigated treatment strategies in DOAC- or VKA-treated patients needing emergency surgery within 24 h after admission. Effectiveness was analysed by clinical endpoints including major bleeding. Primary observation endpoint was in hospital mortality until 30 days after admission. (3) Results: A total of 78 patients were included (DOAC: 44; VKA: 34). Median age was 76 years. Overall, 43% of the DOAC patients and 79% of the VKA patients were treated with prothrombin complex concentrates (PCC) (p = 0.002). Out of the DOAC patients, 30% received no hemostatic treatment compared to 3% (1/34) of the VKA patients (p = 0.002), and 7% of the DOAC patients and 21% of the VKA patients developed major or clinically relevant non-major bleeding at the surgical site (p = 0.093). In-hospital mortality was 13% with no significant difference between the two treatment groups (DOAC: 11%, VKA: 15%; p > 0.20). (4) Conclusions: The 30-day in-hospital mortality rate was comparable between both patient groups. VKA patients required significantly more hemostatic agents than DOAC patients in the peri- and postoperative surgery period.

Aggregates of nonmuscular myosin IIA in erythrocytes associate with GATA1- and GFI1B-related thrombocytopenia.

BACKGROUND: The transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. OBJECTIVES: To expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. METHODS: We assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. RESULTS: We investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 µm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. CONCLUSION: Aggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

Thrombotic anti-PF4 immune disorders: HIT, VITT, and beyond.

Antibodies against the chemokine platelet factor 4 (PF4) occur often, but only those that activate platelets induce severe prothrombotic disorders with associated thrombocytopenia. Heparin-induced thrombocytopenia (HIT) is the prototypic anti-PF4 disorder, mediated by strong activation of platelets through their FcγIIa (immunoglobulin G [IgG]) receptors (FcγRIIa). Concomitant pancellular activation (monocytes, neutrophils, endothelium) triggers thromboinflammation with a high risk for venous and arterial thrombosis. The classic concept of HIT is that anti-PF4/heparin IgG, recognizing antigen sites on (cationic) PF4 that form in the presence of (anionic) heparin, constitute the heparin-dependent antibodies that cause HIT. Accordingly, HIT is managed by anticoagulation with a nonheparin anticoagulant. In 2021, adenovirus vector COVID-19 vaccines triggered the rare adverse effect "vaccine-induced immune thrombotic thrombocytopenia" (VITT), also caused by anti-PF4 IgG. VITT is a predominantly heparin-independent platelet-activating disorder that requires both therapeutic-dose anticoagulation and inhibition of FcγRIIa-mediated platelet activation by high-dose intravenous immunoglobulin (IVIG). HIT and VITT antibodies bind to different epitopes on PF4; new immunoassays can differentiate between these distinct HIT-like and VITT-like antibodies. These studies indicate that (1) severe, atypical presentations of HIT ("autoimmune HIT") are associated with both HIT-like (heparin-dependent) and VITT-like (heparin-independent) anti-PF4 antibodies; (2) in some patients with severe acute (and sometimes chronic, recurrent) thrombosis, VITT-like antibodies can be identified independent of proximate heparin exposure or vaccination. We propose to classify anti-PF4 antibodies as type 1 (nonpathogenic, non- platelet activating), type 2 (heparin dependent, platelet activating), and type 3 (heparin independent, platelet activating). A key concept is that type 3 antibodies (autoimmune HIT, VITT) require anticoagulation plus an adjunct treatment, namely high-dose IVIG, to deescalate the severe anti-PF4 IgG-mediated hypercoagulability state.

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure.

ABSTRACT: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

Vaccine-Induced Immune Thrombocytopenia and Thrombosis (VITT)-Insights from Clinical Cases, In Vitro Studies and Murine Models.

An effective worldwide vaccination campaign started and is still being carried out in the face of the coronavirus disease 2019 (COVID-19) pandemic. While vaccines are great tools to confront the pandemic, predominantly adenoviral vector-based vaccines can cause a rare severe adverse effect, termed vaccine-induced immune thrombocytopenia and thrombosis (VITT), in about 1 in 100,000 vaccinated individuals. VITT is diagnosed 5-30 days post-vaccination and clinically characterized by thrombocytopenia, strongly elevated D-dimer levels, platelet-activating anti-platelet factor 4 (PF4) antibodies and thrombosis, especially at atypical sites such as the cerebral venous sinus and/or splanchnic veins. There are striking similarities between heparin-induced thrombocytopenia (HIT) and VITT. Both are caused by anti-PF4 antibodies, causing platelet and leukocyte activation which results in massive thrombo-inflammation. However, it is still to be determined why PF4 becomes immunogenic in VITT and which constituent of the vaccine triggers the immune response. As VITT-like syndromes are increasingly reported in patients shortly after viral infections, direct virus-PF4 interactions might be most relevant. Here we summarize the current information and hypotheses on the pathogenesis of VITT and address in vivo models, especially murine models for further studies on VITT.

Laboratory and demographic predictors of functional assay positive status in suspected heparin-induced thrombocytopenia: A multicenter retrospective cohort study.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against platelet factor 4 (PF4) bound to heparin anticoagulants. A priori identification of patients at-risk for HIT remains elusive and a number of risk factors have been identified, but these associations and their effect sizes have limited validation in large cohorts of suspected HIT patients. The aim of this study was to investigate existing anti-PF4/heparin antibody thresholds and model the relationship of demographic variables and anti-PF4/heparin antibody levels with functional assay positivity across multiple institutions in the absence of detailed clinical data. In a large collection of suspected HIT patients (n = 8904), we tested for associations between laboratory and demographic variables and functional assay positive status as well as anti-PF4/heparin antibody levels. We also tested for correlation between IgG-specific and polyspecific (IgG/IgA/IgM) anti-PF4/heparin antibody values and their ability to predict functional assay positive status using area under the receiver operating characteristic (AUROC). Logistic regression identified increasing anti-PF4/heparin antibody OD levels (OR = 51.84 [37.27-74.34], p < 2.0 × 10-16) and female sex (OR = 1.47 [1.19-1.82], p = 3.5 × 10-4) as risk factors for positive functional assay in the largest cohort with consistent effect sizes in two other cohorts. In a subset of 1175 patients, polyspecific and IgG-specific anti-PF4/heparin antibody values were heterogeneous (mean coefficient of variation = 31.9 %), but strongly correlated (rho = 0.878; p < 2 × 10-16) with similar prediction of functional assay positivity (polyspecific AUROC = 0.976 and IgG-specific AUROC = 0.980). Thus, we recapitulate previously identified risk factors of functional assay positivity, providing precise effect sizes in a large observational population of suspected HIT patients. Our data reinforce the necessity of functional assay confirmation and suggest that, despite heterogeneity, polyspecific and IgG-specific anti-PF4/heparin antibody assays predict functional assay positive status similarly, even in the absence of 4Ts scores and detailed clinical data.

Whole genome analysis of plasma fibrinogen reveals population-differentiated genetic regulators with putative liver roles.

Genetic studies have identified numerous regions associated with plasma fibrinogen levels in Europeans, yet missing heritability and limited inclusion of non-Europeans necessitates further studies with improved power and sensitivity. Compared with array-based genotyping, whole genome sequencing (WGS) data provides better coverage of the genome and better representation of non-European variants. To better understand the genetic landscape regulating plasma fibrinogen levels, we meta-analyzed WGS data from the NHLBI's Trans-Omics for Precision Medicine (TOPMed) program (n=32,572), with array-based genotype data from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium (n=131,340) imputed to the TOPMed or Haplotype Reference Consortium panel. We identified 18 loci that have not been identified in prior genetic studies of fibrinogen. Of these, four are driven by common variants of small effect with reported MAF at least 10% higher in African populations. Three ( SERPINA1, ZFP36L2 , and TLR10) signals contain predicted deleterious missense variants. Two loci, SOCS3 and HPN , each harbor two conditionally distinct, non-coding variants. The gene region encoding the protein chain subunits ( FGG;FGB;FGA ), contains 7 distinct signals, including one novel signal driven by rs28577061, a variant common (MAF=0.180) in African reference panels but extremely rare (MAF=0.008) in Europeans. Through phenome-wide association studies in the VA Million Veteran Program, we found associations between fibrinogen polygenic risk scores and thrombotic and inflammatory disease phenotypes, including an association with gout. Our findings demonstrate the utility of WGS to augment genetic discovery in diverse populations and offer new insights for putative mechanisms of fibrinogen regulation. Key Points: Largest and most diverse genetic study of plasma fibrinogen identifies 54 regions (18 novel), housing 69 conditionally distinct variants (20 novel).Sufficient power achieved to identify signal driven by African population variant.Links to (1) liver enzyme, blood cell and lipid genetic signals, (2) liver regulatory elements, and (3) thrombotic and inflammatory disease.

Long-term outcome in vaccine-induced immune thrombocytopenia and thrombosis.

BACKGROUND: Rapid diagnosis and treatment has improved outcome of patients with vaccine-induced immune thrombocytopenia and thrombosis (VITT). However, after the acute episode, many questions on long-term management of VITT remained unanswered. OBJECTIVES: To analyze, in patients with VITT, the long-term course of anti-platelet factor 4 (PF4) antibodies; clinical outcomes, including risk of recurrent thrombosis and/or thrombocytopenia; and the effects of new vaccinations. METHODS: 71 patients with serologically confirmed VITT in Germany were enrolled into a prospective longitudinal study and followed for a mean of 79 weeks from March 2021 to January 2023. The course of anti-PF4 antibodies was analyzed by consecutive anti-PF4/heparin immunoglobulin G enzyme-linked immunosorbent assay and PF4-enhanced platelet activation assay. RESULTS: Platelet-activating anti-PF4 antibodies became undetectable in 62 of 71 patients (87.3%; 95% CI, 77.6%-93.2%). In 6 patients (8.5%), platelet-activating anti-PF4 antibodies persisted for >18 months. Five of 71 patients (7.0%) showed recurrent episodes of thrombocytopenia and/or thrombosis; in 4 of them (80.0%), alternative explanations beside VITT were present. After further COVID-19 vaccination with a messenger RNA vaccine, no reactivation of platelet-activating anti-PF4 antibodies or new thrombosis was observed. No adverse events occurred in our patients subsequently vaccinated against influenza, tick-borne encephalitis, varicella, tetanus, diphtheria, pertussis, and polio. No new thrombosis occurred in the 24 patients (33.8%) who developed symptomatic SARS-CoV-2 infection following recovery from acute VITT. CONCLUSION: Once the acute episode of VITT has passed, patients appear to be at low risk for recurrent thrombosis and/or thrombocytopenia.

Gas plasma-induced platelet activation corresponds to reactive species profiles and lipid oxidation.

Surgical-induced hemostasis is a critical step in the closure of incisions, which is frequently achieved via electrocauterization and subsequent tissue necrotization. The latter is associated with postoperative complications. Recent in vivo work suggested reactive species-producing gas plasma technology as a pro-homeostatic agent acting via platelet activation. However, it remained elusive how platelet activation is linked to lipid and protein oxidation and the reactive species compositions. A direct relation between the reactive species composition and platelet activation was revealed by assessing the production of several reactive species and by using antioxidants. In addition, platelet lipidome and proteome analysis identified significantly regulated key lipids in the platelet activation pathway, such as diacylglycerols and phosphatidylinositol as well as oxylipins like thromboxanes. Lipid oxidation products mainly derived from phosphatidylethanolamine and phosphatidylserine species were observed at modest levels. In addition, oxidative post-translational modifications were identified on key proteins of the hemostasis machinery. This study provides new insights into oxidation-induced platelet activation in general and suggests a potential role of those processes in gas plasma-mediated hemostasis in particular.

Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.