Purpose Women with Systemic Lupus Erythematosus (SLE) and/or Antiphospholipid Syndrome (APS) have an increased risk of pregnancy complications. This study explored their perspectives on pregnancy, pregnancy complications, and medication use during pregnancy.Methods In collaboration with the Dutch National Patient Association for Lupus Erythematosus, an online questionnaire was developed and distributed among female members. The questionnaire presented eight potential pregnancy complication scenarios. Respondents indicated whether they found the standard medication regime sufficient and their willingness to take additional medication. They also assessed which side effects of biological therapies would discourage additional treatment. The survey ran from November 18, 2023, to March 19, 2024.Major findings Of 71 respondents (mean age 39), 42% had APS, 31% had SLE, and 27% had both. A majority (79%) would endure pregnancy complications, such as SLE flare or pre-eclampsia, if it meant a live-born baby. Willingness to use additional medication varied by scenario (51%-89%) depending on the specific complication at risk. Most side effects associated with the use of biologicals were deemed acceptable by the majority of respondents. However, a slight majority indicated they would refrain from additional medication if it led to an increased risk of infections such as urinary tract infections or pneumonia.Conclusion Most women would accept adverse obstetric outcomes or maternal complications as long as pregnancy results in a live birth. The majority of respondents consistently expressed a desire for additional medication to reduce the risk of complications during pregnancy, regardless of the specific complication at hand.

BACKGROUND:Intravenous thrombolysis (IVT) using recombinant tissue plasminogen activator prior to endovascular thrombectomy treatment (EVT) failed to improve treatment effect in acute ischemic stroke (AIS) patients compared with EVT alone. OBJECTIVES:We investigated whether primary and secondary hemostasis biomarkers are associated with the effect of intravenous thrombolytics on clinical and radiological outcomes after EVT. METHODS:In the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands (MR CLEAN)-NO IV, AIS patients were randomized to receive IVT plus EVT or EVT alone. We measured hemostatic biomarkers before and 24 hours postreperfusion to determine changes in biomarkers and the association of the biomarkers with short term stroke severity on National Institutes of Health Stroke Scale score, long-term functional outcome (modified Rankin scale [mRS] score), post-EVT extended Thrombolysis in Cerebral Infarction score, and final infarct size. RESULTS:This substudy included 214 of the 539 AIS patients who underwent IVT + EVT (n = 108/266) or EVT alone (n = 106/273). In the EVT group, low soluble glycoprotein VI (sGPVI) and high factor (F)VIII levels before treatment were associated with severe National Institutes of Health Stroke Scale score at 24 hours and poor mRS score at 90 days posttreatment, respectively. Also, in this group, sGPVI levels 24 hours after treatment were negatively associated with final infarct size. In the IVT + EVT group, high fibrinogen before treatment was associated with good extended Thrombolysis in Cerebral Infarction score, and low a disintegrin and metalloprotease with thrombospondin motif repeats 13 activity 24 hours posttreatment was associated with an unfavorable mRS score at 90 days. CONCLUSION:Our findings suggest that patients with high FVIII and fibrinogen and low sGPVI levels might be the most suitable candidates for IVT + EVT and that patients with low a disintegrin and metalloprotease with thrombospondin motif repeats 13 activity might be suitable for EVT alone.

Background For acute ischemic stroke due to intracranial large-vessel occlusion in the anterior circulation, guidelines recommend treatment with intravenous thrombolysis (IVT) with recombinant tissue plasminogen activator and endovascular thrombectomy (EVT). We investigated whether plasma fibrinolysis biomarkers were associated with the treatment effect of EVT, with or without IVT with recombinant tissue plasminogen activator, and their potential implications for clinical and radiological outcomes.Methods In this post hoc analysis of MR CLEAN-NO IV, we measured fibrinolytic biomarkers (tissue plasminogen activator, global clot lysis time, and D-dimer) before and 24 hours post reperfusion and assessed their associations with patients' clinical and radiological outcomes based on National Institutes of Health Stroke Scale score, modified Rankin Scale (mRS) score, post-EVT extended thrombolysis in cerebral infarction score, and final infarct size. To quantify these associations, we used linear and logistic regression.Results Blood was collected from 214 of 536 included patients, who received IVT+EVT (N = 108) or EVT alone (N = 106). In the IVT+EVT group, D-dimer levels 24 hours after treatment were higher and clot lysis time lower than in the EVT group, indicating more fibrinolysis. Pearson correlation showed that high D-dimer levels before and 24 hours after EVT were correlated with unfavorable long-term functional outcomes (mRS at 90 days), and D-dimer levels 24 hours after EVT linked to large infarct size. High tissue plasminogen activator levels after IVT+EVT were correlated with successful reperfusion. However, regression analysis adjusted for confounders showed no associations between fibrinolytic biomarkers and clinical or radiological outcomes.Conclusion None of the fibrinolysis biomarkers were independently associated with outcomes in adjusted regression analysis, failing to support their use as predictors for treatment decisions or therapeutic effectiveness. However, exploratory analyses suggested that higher tissue plasminogen activator levels after IVT+EVT correlated with successful reperfusion, whereas elevated D-dimer levels were linked to unfavorable outcomes and larger infarct size post-EVT. Larger studies are needed to clarify their role in stroke treatment with IVT and/or EVT.Clinical Trial Registration This study is a substudy of the MR CLEAN-NO IV trial, which is registered at the ISRCTN registry (ISRCTN80619088, https://www.isrctn.com/ISRCTN80619088).

The aim of this study is to investigate the association of neutrophil extracellular traps (NETs) markers with clinical and radiological outcomes in acute ischemic stroke (AIS) patients undergoing endovascular thrombectomy (EVT) and assess the effect of periprocedural heparin during EVT on NETs markers and their association with outcomes. From 198 AIS patients included in the MRCLEAN-MED trial, randomized to receive EVT with ( N = 104) or without ( N = 94) low-dose unfractionated heparin (5000 IU bolus followed by 500 IU/h for 6 h, n = 104), blood samples were collected at baseline, 1 h, and 24 h post-reperfusion. NETs markers (MPO-DNA, histone-DNA, citrullinated histone H3 [CitH3]) were measured in blood samples, and their associations with stroke severity (National Institutes of Health Stroke Scale [NIHSS] score at 24 h post-reperfusion), long-term functional outcome (modified Rankin Scale [mRS] score at 90-day), and final infarct size (5–7 days) were assessed in EVT and heparin + EVT–treated patients using logistic regression, linear regression, and Pearson’s correlation. Histone-DNA levels at 1 h post-heparin + EVT, but not EVT alone, were positively associated with final infarct size. Histone-DNA levels at 24 h post-heparin + EVT were negatively associated with infarct size mRS and NIHSS, while baseline CitH3 was positively correlated with NIHSS at 24 h post-EVT. Interaction analysis showed that the association between histone-DNA levels at 24 h and NIHSS at 24 h was different in the two treatment groups. No further associations were observed. At 1 h post-heparin + EVT, the histone-DNA levels were independently associated with larger infarct size, while at 24 h, histone-DNA linked to improved outcomes post-heparin + EVT and baseline-CitH3 to worse outcomes post-EVT, suggesting heparin may attenuate histone-DNA’s effect on outcome.

Current treatments for persistent or chronic immune thrombocytopenia (ITP) are limited by inadequate response, toxicity, and impaired quality of life. The Bruton tyrosine kinase inhibitor rilzabrutinib was evaluated to further characterize safety and durability of platelet response. LUNA2 Part B is a multicenter, phase 1/2 study in adults with ITP (≥ 3 months duration, platelet count < 30 × 109/L) who failed ≥ 1 ITP therapy (NCT03395210, EudraCT 2017-004012-19). Oral rilzabrutinib 400 mg bid was given over 24 weeks, with optional long-term extension (LTE). Primary endpoints were safety and platelet counts ≥ 50 × 109/L on ≥ 8 of the last 12 weeks of main treatment without rescue medication. From 22 March2018 to 31 January2023, 26 patients were enrolled. Patients had baseline median platelet count 13 × 109/L, ITP duration 10.3 years, and six prior ITP therapies (46% splenectomized). Nine (35%) patients achieved the primary endpoint. Platelet counts ≥ 50 × 109/L or ≥ 30 × 109/L and doubling from baseline without rescue therapy were sustained for a mean 9.3 weeks. 11 (42%) LTE-eligible patients were ongoing with median LTE platelet > 80 × 109/L. Three (12%) patients received rescue medication during main treatment, none in LTE. Clinically meaningful improvements were observed in fatigue and women's health. With a median treatment duration of 167 days (main treatment), 16 (62%) patients had ≥ 1 treatment-related adverse event (AE), mainly grade 1, including diarrhea (35%), headache (23%), and nausea (15%). There was no treatment-related grade ≥ 2 bleeding/thrombotic events/infections, serious AE, or death. Rilzabrutinib continues to demonstrate durable platelet responses with favorable safety profile in previously treated ITP patients. Trial Registration: NCT03395210, EudraCT 2017-004012-19.

Background:Life-long vitamin K antagonist (VKA) therapy is recommended as a standard of care in antiphospholipid syndrome (APS) patients with thrombosis. Concerns have been raised about the validity of international normalized ratio (INR) measurements in lupus anticoagulant (LA)-positive APS patients because LA may interfere with phospholipid-dependent coagulation tests and could elevate INR measurements. Objectives:Here, we aimed to determine the interference of antigen-specific monoclonal and isolated patient antibodies with LA activity on INR measurements. Methods:Pooled normal plasma and control plasma from patients on VKA (without LA) were incubated with monoclonal and isolated patient immunoglobulin G antiprothrombin and anti-beta-2-glycoprotein I antibodies that express LA activity. INR was determined before and after addition using 3 laboratory assays (Owren STA-Hepato Prest, Quick STA-NeoPTimal, and Quick STA-Neoplastine R) and 1 point-of-care test device (CoaguChek Pro II). Results:Antiprothrombin and anti-beta-2-glycoprotein I antibodies with LA activity interfered with recombinant human thromboplastin reagents (Quick STA-Neoplastine R and CoaguChek Pro II), particularly when added to plasma of VKA-treated controls. This effect was most evident on point-of-care test INR measurements, while the recombinant Quick reagent exhibited a lesser degree of interference. In contrast, tissue-derived thromboplastin reagents (Owren STA-Hepato Prest and Quick STA-NeoPTimal) remained largely unaffected by these antibodies, both in pooled normal plasma and VKA anticoagulated control plasma. Among these reagents, the Owren INR reagent exhibited the lowest sensitivity to the influence of LA antibodies. This observed difference in sensitivity is independent of the plasma dilution factor or the presence of factor V or fibrinogen in Owren reagent. Conclusion:INR reagents that utilize recombinant human thromboplastin are more sensitive to the presence of monoclonal and patient-derived antibodies with LA activity. Consequently, APS patients positive for LA should be monitored using tissue-derived thromboplastin reagents, given its reduced susceptibility to interference by LA-causing antibodies.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by enhanced platelet destruction and impaired platelet production, due to a loss of immune tolerance that leads to targeting of platelets and megakaryocytes by glycoprotein-autoantibodies and/or cytotoxic T cells. There is a high degree of heterogeneity in ITP patients signified by unpredictable disease trajectories and treatment responses. Initial studies in humans have identified intestinal microbiota perturbance in ITP. Recently, gut microbial perturbance has been linked to other autoimmune diseases. Based on these findings, we hypothesize that intestinal microbiota may influence ITP pathophysiology through several mechanisms, including induction of platelet-autoantibody production, increasing complement-dependent platelet cytotoxicity, disturbing T cell homeostasis, impairing megakaryocyte function, and increasing platelet-desialylation and -clearance. The pathophysiological heterogeneity of ITP may, at least in part, be attributed to a perturbed intestinal microbiota. Therefore, a better understanding of intestinal microbiota in ITP may result in a more personalized therapeutic approach.

Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibodymediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and a predisposition to bleeding. The ongoing, global phase 1/2 study showed that rilzabrutinib, a Bruton tyrosine kinase inhibitor specifically developed to treat autoimmune disorders, could be an efficacious and well-tolerated treatment for ITP. Clinical activity, durability of response, and safety were evaluated in 16 responding patients who continued rilzabrutinib 400 mg twice daily in the long-term extension (LTE) study. At LTE entry, the median platelet count was 87 x 10(9)/L in all patients, 68 x 10(9)/L in those who had rilzabrutinib monotherapy (n = 5), and 156 x 10(9)/L in patients who received concomitant ITP medication (thrombopoietin-receptor agonists and/or corticosteroids, n = 11). At a median duration of treatment of 478 days (range, 303-764), 11 of 16 patients (69%) continued to receive rilzabrutinib. A platelet count of >= 50 x 109/L was reported in 93% of patients for more than half of their monthly visits. The median percentage of LTE weeks with platelet counts >= 30 x 10(9)/L and >= 50 x 10(9)/L was 100% and 88%, respectively. Five patients discontinued concomitant ITP therapy and maintained median platelet counts of 106 x 10(9)/L at 3 to 6 months after stopping concomitant ITP therapy. Adverse events related to treatment were grade 1 or 2 and transient, with no bleeding, thrombotic, or serious adverse events. With continued rilzabrutinib treatment in the LTE, platelet responses were durable and stable over time with no new safety signals.

Introduction: Rilzabrutinib is a potent oral, reversible Bruton tyrosine kinase inhibitor that can treat hematological autoimmune diseases through multiple putative mechanisms of action: (1) inhibition of B-cell activation, (2) interruption of antibody-coated cell phagocytosis by FcϒR in spleen and liver, and (3) induce sustained anti-inflammatory effects (Langrish J Immunol 2021). Preliminary evidence showed that rilzabrutinib treatment resulted in rapid and durable platelet responses with a favorable safety profile in previously treated patients with immune thrombocytopenia (ITP) as studied in part A of a phase 1/2 clinical study (LUNA 2; Kuter N Engl J Med 2022). This abstract summarizes the results of part B that focused on the durability of response with rilzabrutinib in relapsed ITP patients. Methods: Part B of the multicenter, open-label, phase 1/2 study evaluated the efficacy and safety of rilzabrutinib 400 mg bid in patients with relapsed ITP (NCT03395210). Adult patients aged 18-80 y were eligible with ≥2 baseline platelet counts <30x10 9/L no less than 7 days apart in the 15 days before the first dose. Eligible patients were required to have a past response (achievement of platelet count ≥50x10 9/L) to intravenous immunoglobulin (IVIg)/anti-D or corticosteroid (CS) that was not sustained and failed ≥1 other ITP therapy (that was not IVIg or CS). Stable doses of concomitant CS/thrombopoietin receptor agonists (TPO-RA) were allowed with rilzabrutinib. The primary endpoints for part B were safety and durable platelet response defined as platelet counts ≥50x10 9/L on ≥8 of the last 12 weeks of rilzabrutinib without rescue medication. Patients completing 24 weeks of rilzabrutinib with platelet counts ≥50x10 9/L or ≥30x10 9/L and doubling from baseline in ≥4 of the last 8 weeks of treatment without rescue medication could continue rilzabrutinib in the long-term extension (LTE) period. Results: At baseline, 26 enrolled patients had a median age of 57 y (range, 20-75), 62% were female, and median baseline platelet count was 13x10 9/L (range, 2-24x10 9/L). Patients had a median duration of ITP of 10.3 y (range, 0.7-48.2) and had received a median of 6 prior unique ITP therapies (range, 3-19; 46% splenectomy). Seventeen patients (65%) received concomitant non-rescue CS and/or TPO-RA. Nine patients (35%; 95% CI, 17%-56%) achieved the primary endpoint of durable platelet response. Approximately 25% of patients achieved platelet counts ≥50x10 9/L by day 15 of rilzabrutinib treatment (Figure 1A). In 16 patients who achieved platelet counts ≥50x10 9/L, median time to first platelet count ≥50x10 9/L was 15 days (range, 7-134). Median platelet counts for all patients (responders and non-responders) increased over time, exceeding the platelet count thresholds of 30x10 9/L at day 57 and 50x10 9/L at day 120 (Figure 1B). The mean number of weeks with platelet counts ≥50x10 9/L and/or ≥30x10 9/L and doubling from baseline was both 9.3 weeks (SD, 10.1). Three patients (12%) received rescue medication in the main treatment period. Fifteen patients (58%) completed 24 weeks of rilzabrutinib and 11 (42%) entered the LTE. Over the main treatment period, the median duration of treatment was 167 days (range, 7-169). Sixteen patients (62%) had ≥1 related treatment-emergent adverse event (AE), including 35% diarrhea, 23% headache, and 15% nausea. Most AEs were grade 1 or 2; there was 1 treatment-related AE of grade 3 blood creatinine phosphokinase increase. There was no treatment-related grade ≥2 bleeding/thrombotic events or infections, serious AEs, or deaths. Conclusion: Part B study results were consistent with part A. Rilzabrutinib demonstrated rapid, stable, and durable platelet responses in patients with relapsed ITP, with a favorable safety profile in part B.

A State of the Art lecture titled "coagulation biomarkers for ischemic stroke" was presented at the International Society on Thrombosis and Haemostasis (ISTH) Congress in 2022. Ischemic stroke (IS) is a common disease with major morbidity and mortality. It is a challenge to determine which patients are at risk for IS or have poor clinical outcome after IS. An imbalance of coagulation markers may contribute to the progression and prognosis of IS. Therefore, we now discuss studies on the association of selected coagulation biomarkers from the hemostasis, inflammation, and immunothrombosis systems with the risk of IS, stroke severity at the acute phase, and clinical outcome after treatment. We report on coagulation biomarker-induced risk of IS, stroke severity, and outcomes following IS derived from prospective population studies, case-control studies, and acute-phase IS studies. We found indications that many coagulation and inflammation biomarkers are associated with IS, but it is early to conclude that any of these biomarkers can be applied in a therapeutic setting to predict patients at risk of IS, stroke severity at the acute phase, and clinical outcome after treatment. The strongest evidence for a role in IS was found for beta-thromboglobulin, von Willebrand factor, factor VIII, fibrinogen, thrombin-activatable fibrinolysis inhibitor, D-dimer, and neutrophil extracellular traps, and therefore, they are promising candidates. Further research and validation in large-size populations using well-defined study designs are warranted. Finally, we provide a selection of recent data relevant to this subject that was presented at the 2022 ISTH Congress.

Platelet transfusion effectiveness is measured by the corrected count increment (CCI), which is the difference between posttransfusion and pretransfusion platelet counts, corrected for body surface area and platelet dose.1 The CCI can be determined at any time posttransfusion but is usually measured after either one or 24 h. Platelet refractoriness is a consistently poor CCI and is usually defined as a one hour CCI below 5.0 or a 24 h CCI below 2.5 on two consecutive occasions.1 Many different patient- and transfusion product-related factors have been associated with lower CCI.2-4 However, transfusion practice has changed over the years, which has likely also changed the relative importance of some of these associations. For example, AB0-incompatibility was consistently shown to lower the CCI, which resulted in the standard practice to give AB0-matched PCs. The majority of previous studies on this subject were specifically on patients with hematologic malignancy, and some were on selected subpopulations of critically ill patients. We aimed to find predictors of lower CCI in patients that received a prophylactic platelet transfusion using PACER trial data. The PACER trial was a multicenter randomized controlled trial on severely thrombocytopenic patients at the hematology ward and the intensive care unit (ICU), in which patients were randomized to receive either no prophylactic platelet transfusion or one unit of prophylactic platelet transfusion prior to central venous catheter (CVC) placement, with catheter-related bleeding as primary outcome.5 The PACER trial was approved by the Amsterdam UMC, University of Amsterdam, medical ethics committee; study conduct was overseen by a data safety and monitoring board; and the trial was registered in the Dutch Trial Register (NL5534). For this analysis, patients in the transfusion arm that did not receive any further platelet transfusions in the 24 h following CVC placement were selected. There were no restrictions in the type of platelet concentrate (PC) transfused, although the Dutch standard during the study period consisted of five-donor pooled buffy coat platelets. Apheresis and/or human leukocyte antigen-matched PCs were transfused whenever indicated. PCs were stored in 100% plasma until June 2018 and in 35% plasma and 65% platelet additive solution (PAS) E from July 2018 onwards. The maximum PC storage time was 7 days. Pretransfusion platelet counts were obtained at most 24 h before transfusion, and posttransfusion platelet counts were obtained 24 h after transfusion to determine the CCI.1 A list of possible CCI predictors was compiled based on a literature search, and the 14 most important potential predictors were selected, limited by sample size and availability of data. Patient-related variables included age, gender, BMI, presence of allo-antibodies, bone marrow depression as primary cause of thrombocytopenia, disseminated intravascular coagulation (DIC), liver failure, renal failure, fever, sepsis, and corticosteroid use. Product-related variables included PC storage length, PC irradiation, and PC storage medium. Bone marrow depression was considered the primary cause of thrombocytopenia if the decrease in platelet count coincided either with disease progression of a hematologic malignancy or with the initiation of chemotherapy. DIC was considered present when the International Society on Thrombosis and Haemostasis DIC score was 5 or higher. Liver failure was defined as a Child-Pugh C classification. Renal failure was considered present when the estimated glomerular filtration rate was below 15 mL/min/1.73m2 and/or renal replacement therapy was provided. Fever was defined as a temperature of 38.5°C or higher, and sepsis was defined according to the Sepsis-2 criteria. The primary analysis was a linear regression model with variable selection using augmented backward elimination. Estimates were adjusted using parameter-wise shrinkage. To assess model stability and bias and variance inflation caused by the variable selection procedure, a bootstrap sensitivity analysis was performed. To account for missing data, a multiple imputation sensitivity analysis was performed. We included 131 transfusion episodes in the complete case primary analysis and an additional 35 in the multiple imputation sensitivity analysis (Table S1). In the primary analysis, sepsis, bone marrow depression, and DIC were the only significant patient-related predictors of a lower CCI (Tables 1 and S2). PC irradiation was the only significant product-related predictor of a lower CCI. Sepsis was associated with a mean (95% CI) lower CCI of 13.5 (6.7–20.2), bone marrow depression was associated with a mean (95% CI) lower CCI of 10.0 (3.4–16.6), DIC was associated with a mean (95% CI) lower CCI of 11.2 (4.2–18.2), and PC irradiation was associated with a mean (95% CI) lower CCI of 5.6 (1.0–10.2). Renal failure was included in the final model but was no longer significant after parameter estimate shrinkage, with a mean (95% CI) lower CCI of 5.1 (−1.6–11.9). The bootstrap sensitivity analysis arrived at the same predictors and comparable parameter estimates as the primary analysis (Tables 1 and S3). In the multiple imputation sensitivity analysis, renal failure was also a significant predictor of lower CCI (Tables 1 and S4). Moreover, presence of allo-antibodies, storage length, and storage medium were selected in three, four, and seven imputed datasets, respectively, but not in the final model. Parameter estimates were similar to the primary analysis and bootstrap sensitivity analysis. Additional details of both the primary and sensitivity analyses, and pre and posttransfusion platelet counts (Table S5) are provided as online supplement. In this prespecified, prospective, observational sub-study of the PACER trial, we found evidence for an association with lower CCI of sepsis, DIC, bone marrow depression, and PC irradiation. These associations held up after thorough model diagnostics. We found ambiguous results for the association between CCI and renal failure, presence of allo-antibodies and PC storage length and medium, and no evidence for an association with age, gender, BMI, liver failure, corticosteroid use, or fever. Previous studies on predictors of CCI were mostly in patients with hematologic malignancy or in a subset of critically ill patients, but not in a combination of both. We were able to identify bone marrow depression as negative predictor of CCI by including a combination of patients with and without bone marrow depression. Moreover, the use of thorough model diagnostics allowed us to be both more certain about the most significant predictors of CCI and provide a nuanced view on the remaining variables included. The fact that the parameter estimates for all selected predictors were similar in both the presumably unbiased global model, the selected model, the bootstrap model, and the multiple imputation model provides extra validity to our results. Although it is recommended to always determine posttransfusion platelet counts, in clinical practice, often prophylactic platelet transfusions before invasive procedures are given without follow-up measurement. Our data may be helpful to select patients that are more likely to have a lower platelet count increment after platelet transfusion, both in clinical practice and for further research. In general, in patients with characteristics known to be associated with a lower platelet count increment, a follow-up platelet count should be measured at all times. There are several limitations to the observations presented here. First, we were unable to account for all previously identified predictors of CCI. This was in part due to the limited sample size, which restricted the number of variables that we were able to include in model building. Another limiting factor was the type of data included in our database. For instance, we did not collect extensive data on co-medication and were thus unable to account for specific antimicrobial medications like amphotericin B and vancomycin. Also, we did not collect data on fluid balance, nor did we account for bleeding. Platelet loss through bleeding and hemodilution or -concentration could have influenced CCI. Because we selected patients that did not receive further platelet transfusions in the 24 h following the initial transfusion, clinically significant bleeding seems not to be a likely explanation for the lower CCI seen in some of our patients. However, we cannot account for differences in fluid balance. Especially patients with sepsis are likely to have large shifts in fluid balance, which is usually positive during early treatment and negative later on. Second, because the proportion of patients with allo-antibodies and the proportion of patients with liver failure were low, our study was underpowered to draw strong conclusions on their relationship with CCI. Third, this was an observational sub-study, which precludes any conclusions on causality. Finally, our results are based on observations from a specific subset of hematology ward and ICU patients requiring CVC placement. While patients at the hematology ward and in the ICU are among those most likely to require platelet transfusions, there are likely to be some differences in baseline characteristics between patients that do and do not require CVC placement, leading to selection bias. On the other hand, previous studies on predictors of CCI did not focus on platelet transfusion prior to invasive procedures. Our results should be more applicable to this specific population, which is representative of many thrombocytopenic patients seen in real-life practice. In conclusion, we identified predictors of 24-h CCI in a prospective sub-study of the PACER trial. The study population included severely thrombocytopenic hematology ward and ICU patients requiring CVC placement for which prophylactic platelet transfusions were given in one arm of the study. Significant predictors of a lower 24-h CCI included sepsis, DIC, bone marrow depression, and PC irradiation. The data will be available on reasonable request; please contact [email protected]. Data S1. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Multiple studies have reported immune thrombocytopenia (ITP) relapse following SARS-CoV-2 vaccination, however baseline ITP relapse rate and antibody response to vaccination are not known. Patients with ITP who received at least one of the first three SARS-CoV-2 vaccination doses were included in the study. One hundred and twenty-four patients met the inclusion criteria. Relapse rate was 4.2% following a first vaccine dose, 9.1% after a second and 2.9% after a third; baseline relapse rate was 7.6%. Ninety-four per cent of patients who received three vaccine doses developed a clinical antibody response. SARS-CoV-2 vaccination appears to be safe and effective in patients with ITP.

Objective: Identify patient experience and preference towards thrombopoietin-receptor agonists (TPO-RAs) in treatment of immune thrombocytopenia (ITP) in the Netherlands.Methods: The Thrombopoietin-Receptor Agonist Patient experience (TRAPeze) survey used a discrete choice experiment (DCE) to elicit patient preferences and a patient burden survey (PBS) to evaluate the clinical and social impact of ITP. TRAPeze collected responses from 6th October to 19th November 2021.Results: Seventy-six respondents completed the DCE: treatment preference appeared to be driven by method of administration (odds ratio [OR] 4.33; 95% confidence interval [CI] 2.88-6.52), frequency of dosing (OR 2.33; 95% CI 1.86-2.92) and drug-food interactions (OR 1.91; 95% CI 1.54-2.37). Respondents preferred therapies delivered orally over subcutaneous injection (OR 4.22; 95% CI 2.76-6.46), dosed once weekly over once daily (OR 2.37; 95% CI 1.58-3.54) and without food restrictions over with restrictions (OR 1.90; 95% CI 1.52-2.38). Sixty-nine respondents completed the DCE and PBS (mean [range] age 53 [19-83] years, 65% female). Seven incomplete PBS responses were excluded from analysis. Respondents were currently, or most recently, receiving eltrombopag (n = 43) or romiplostim (n = 26), of which 30% (n = 21/69) had previously received another TPO-RA. Loss (29%, n = 6/21) and lack (29%, n = 6/21) of response were the most common reasons for switching TPO-RA. Only 28% (n = 18/65) of respondents felt their TPO-RA increased energy levels.Conclusion: Patients preferred therapies delivered orally, dosed less frequently and without food restrictions. QoL of ITP patients on TPO-RAs can be improved; the burden analyses presented can inform future efforts towards this.

Background: Von Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli. Objectives: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli. Methods: Platelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. Results: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 & mu;M PAR-1 ap) or glycoprotein VI (GPVI) (0.251 & mu;g/mL CRP-XL) signaling pathways caused a dose-dependent increase in alphagranule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alphagranules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 & mu;M PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules. Conclusion: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules.

Introduction: Patients with immune thrombocytopenia (ITP) have a high incidence of comorbid conditions, elevated risk for bleeding and thrombosis, and reduced health-related quality of life (HRQoL). Rilzabrutinib is a potent oral, reversible inhibitor of Bruton tyrosine kinase with the potential to modulate multiple immunological pathways. In part A of a phase 1/2 study (LUNA 2) of ITP patients, rilzabrutinib treatment led to rapid and durable platelet responses with a favorable safety profile. This abstract reports the effects of rilzabrutinib on bleeding and HRQoL on patients in part B of the LUNA 2 study. Methods: The multicenter, open-label, phase 1/2, part B study evaluated the efficacy and safety of rilzabrutinib 400 mg BID in relapsed ITP (NCT03395210). Adults aged 18-80 y were eligible with ≥2 baseline platelet counts <30x10 9/L no less than 7 days apart in the 15 days before the first dose. Eligible patients were required to have a past response (achievement of platelet count ≥50x10 9/L) to intravenous immunoglobulin (IVIg)/anti-D or corticosteroid (CS) that was not sustained, and have failed ≥1 other ITP therapy (not IVIg or CS). Stable doses of concomitant CS/thrombopoietin receptor agonists (TPO-RA) were allowed with rilzabrutinib. The primary endpoints were safety and durable platelet response defined as platelet counts ≥50x10 9/L on ≥8 of the last 12 weeks of rilzabrutinib without rescue medication. Change from baseline in the ITP Bleeding Scale (IBLS) score (0 none to 2 marked bleeding) was a secondary endpoint to assess bleeding across 9 anatomical sites (8 for male/postmenopausal women). An exploratory HRQoL endpoint evaluated the EuroQol-5 Dimensions 5-Level (EQ-5D-5L) + Visual Analog Scale (EQ-VAS) and ITP Patient Assessment Questionnaire (ITP-PAQ; scores 0 worst to 100 best HRQoL). Results: For 26 enrolled patients with data cut-off of 31Jan2023, 15 patients completed 24 weeks of treatment and 11 discontinued due to lack of response (n=5); adverse events (AEs, n=3); and n=1 each lack of response/AE, noncompliance, and erroneous enrollment. At baseline, patients had a median age of 57 y (range, 20-75) with median platelet count of 13x10 9/L (range, 2-24x10 9/L). Patients had a median duration of ITP of 10.3 y (range, 0.7-48.2) and had received a median of 6 prior unique ITP therapies (range, 3-19; 46% splenectomy). Prior ITP medication included CS (100%), TPO-RA (85%), immunosuppressants or IVIg (81%), and rituximab (50%). During the study, 17 patients (65%) received concomitant non-rescue CS and/or TPO-RA. The median compliance to rilzabrutinib for patients with 24 weeks of treatment was 99%. Nine patients (35%; 95% CI, 17-56%) achieved the primary endpoint of durable platelet response. Treatment-related AEs were mainly grade 1/2, with no treatment-related grade ≥2 bleeding/thrombotic events or infections, serious AEs, or deaths. At baseline, the IBLS average score across sites was a mean of 0.27 (SD, 0.27) and mean change from baseline at week 25 was -0.07 (SD, 0.12). The anatomical site with the highest proportion of patients having a baseline score of 2 was skin (n=7 per medical history, n=6 per physical examination), all of whom had a decrease at 25 weeks. At baseline, median EQ-VAS was 78 (range, 8-100) with a median change from baseline (N=26) to week 25 (n=15) of +7 (range, -5 to 77) indicating an improvement in HRQoL. Median changes in ITP-PAQ domain scores from baseline to week 25 (Figure 1) showed clinically meaningful improvement in women's reproductive health with a 21-point increase (range, 0-38; median baseline: 54); clinically meaningful improvement in fatigue/sleep, activity, and overall HRQoL scales with ~13-point increases (range, -23 to 75; median baseline: 53, 50, and 52, respectively); and improvement in psychological, symptoms, social activity, and bother-physical health domains with 4- to 8-point increases (range, -13 to 56; median baseline: 70, 60, 75, 69, respectively). Fear and work domains had high scores at baseline (≥90) with minimal changes over time. Conclusion: Overall, rilzabrutinib showed durable platelet responses, high compliance, and improvements on HRQoL measures in difficult to treat patients with relapsed ITP. There was no evidence of increased bleeding with rilzabrutinib. Clinically meaningful improvements in HRQoL were observed in multiple individual and overall HRQoL health domains following rilzabrutinib.

Background Platelet alpha-granules contain Von Willebrand factor (VWF), which is stored in eccentric alpha-granule nanodomains, and VWF propeptide (VWFpp). Differential release of VWF and VWFpp has been reported from endothelial cells. It is unclear if this also occurs during platelet alpha-granule exocytosis. We have recently developed a 3D super-resolution imaging workflow for quantification of platelet alpha-granule content based on Structured Illumination Microscopy (SIM). With this we can study alpha-granule cargo release following platelet activation in hundreds of platelets simultaneously. Aims To study release of VWF and VWFpp from alpha-granules using quantitative super-resolution microscopy. Methods Platelets were activated with PAR-1 activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, SPARC and fibrinogen were imaged using 3D-SIM, followed by semi-automated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. Results VWF+ and VWFpp+ structures overlapped nearly completely (∼90%) in resting platelets, implying they are stored in similar eccentric alpha-granule nanodomains. A subset of VWF+/VWFpp+-structures was released completely at 0.6 µM PAR-1 ap, but at higher concentration (20 µM) significantly more VWFpp (85.3±1.6%) was released than VWF (37.6±1.4%). Release of other cargo was intermediate at 20 µM (SPARC: 62.2±1.4%; fibrinogen: 51.9±2.9%), providing further evidence for differential cargo release. Similar results were obtained using CRP-XL. Anti-VWF nanobody was taken up by VWF+/VWFpp-structures and increased with stimulus strength, demonstrating these were post-exocytotic structures. Conclusions VWF and VWFpp are differentially released from alpha-granules. This may affect how platelet-derived VWF and VWFpp contribute to formation and stabilization of hemostatic clots. Key points VWFpp and VWF are localized in the same, eccentric alpha-granule subdomain in resting platelets and do not overlap with other alpha-granule cargo proteins such as fibrinogen VWFpp and VWF are differentially secreted from individual alpha-granules upon activation with platelet agonists PAR-1 activating peptide and collagen-related peptide

Von Willebrand disease (VWD) is a bleeding disorder caused by quantitative (type 1 or 3) or qualitative (type 2A/2B/2M/2N) defects of circulating von Willebrand factor (VWF). Circulating VWF levels not always fully explain bleeding phenotypes, suggesting a role for alternative factors, like platelets. Here, we investigated platelet factor 4 (PF4) in a large cohort of patients with VWD. PF4 levels were lower in type 2B and current bleeding phenotype was significantly associated with higher PF4 levels, particularly in type 1 VWD. Based on our findings we speculate that platelet degranulation and cargo release may play a role across VWD subtypes.

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.

Background: Platelets are small, enucleated cells with their primary physiological role to repair vascular damage (hemostasis) and initiate thrombus formation in response to vascular injury. Low platelet count (thrombocytopenia) is associated with increased bleeding risk and is caused by decreased platelet production and/or increased platelet clearance. Platelet transfusion is a routinely used lifesaving procedure to control or prevent bleeding in patients with thrombocytopenia or platelet dysfunction. During storage platelet function deteriorates significantly over time and regulation of platelet granular content is essential in maintaining a healthy platelet function. Currently no in vitro tests are available to predict platelet age, which may be used to diagnose the underlying cause of thrombocytopenia, or to determine storage time of platelet concentrates (PC). Using confocal and super-resolution microscopy we could quantify loss of platelet granule content during aging. In this study we used Artificial intelligence techniques (AIT) to derive exact platelet age from imaging data. Aim: To develop AIT-based algorithms that can determine platelet age from images of platelets stored in plasma-rich-plasma (PRP) and in platelets concentrates (PC) measured by confocal microscopy. Methods: Platelets were stored in PRP for 8 hours at room temperature. Platelets were isolated and stained with antibodies for alpha-tubulin, von Willebrand Factor (VWF), SPARC or alpha-tubulin and imaged at T=0, 4 and 8 hours using confocal microscopy. Additionally 3 single donor PC in plasma were stored for 10 days at standard blood bank conditions. Platelet samples were pooled and analyzed at day 1,2,3,6,7,8,9 and 10. We trained a neural network consisting of multiple convolutional layers to classify age (time after donation) of platelets that were stored in either PRP or PC. Results: During storage in PRP as well as of PC we used for each time point 1334 62µm x 62µm x 4µm 3D images, containing 50 to 150 platelets per image, for training. When staining with markers VWF, SPARC or alpha-tubulin alone as training set we could already classify mean platelet age up to 78%, 85%, 88% respectively. When combining all three markers a maximum accuracy of 95% for platelets stored in PRP up to 8 hours was reached (Figure 1). The additional effect of combining these three markers highlights that the model used information in the images synergistically. For PC, an accuracy of 90% was reached for classifying mean platelet age of platelets stored up to 8 days. The model could not be trained to discriminate between days 9 and 10 resulting in an overfitted model. However the model was able to distinguish day between day 1, 3, 6 and 10 (Figure 2). As negative control a model trained on a randomized sample was made. This control model was not able to assign a category correctly, indicating an actual difference in platelet characteristics during storage was detected. By using visualization of the weight-gradient (e.g. where in the images the model assigns the most weight) we additionally confirmed that the model used separate individual platelets to characterize the age of the complete platelet sample. Conclusion: Combining confocal platelet images with a convolutional neural network, we trained a model that could synergistically integrate different stainings and information of individual platelets to predict with >95% accuracy platelet age in vitro as well as in PC up to 10 days of storage. Additionally our model could distinguish "young" platelets (stored 1, 3 or 6 days) from "old" platelets (stored 10 days). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal