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.

Quantifying Residual Rivaroxaban Plasma Concentration after Antagonization with Andexanet Alfa: A Difficult Task in Routine Clinical Practice.

We describe the case of a 38-year-old man with a history of chronic portal vein thrombosis who presented with abdominal pain after a transjugular intrahepatic portosystemic shunt procedure. Under anticoagulation therapy with rivaroxaban, he experienced active splenic bleeding, leading to hemodynamic instability. Emergency interventions, including andexanet alfa and nanoparticle administration, successfully stopped the bleeding. However, routine tests showed persistently high rivaroxaban levels despite reversal with andexanet alfa. This case report shows that next to standard anti-Xa activity assay, high-performance liquid chromatography is as well unreliable in this regard. In contrast, viscoelastic tests might better serve as indicators of the efficacy of the reversal. The availability of modified anti-Xa tests is urgently needed, to monitor the effects of andexanet alfa reversal.

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.

Successful management of refractory immune-mediated thrombotic thrombocytopenic purpura during pregnancy and delivery using the anti-VWF nanobody caplacizumab.

Pregnancy is a potential trigger of acute thrombotic thrombocytopenic purpura (TTP). The management of pregnancy-associated immune-mediated TTP (iTTP) can be challenging, especially when it is refractory to standard treatment. Caplacizumab, a nanobody to von Willebrand factor (VWF) blocking its A1 domain, is a valuable new therapeutic option. Its use is, however, not approved during pregnancy and breastfeeding. We describe the successful off-label administration of caplacizumab during pregnancy and delivery in a patient with refractory iTTP. The favourable outcome without significant thrombotic or haemorrhagic complications indicates that caplacizumab may be an effective and safe treatment option in refractory iTTP during pregnancy.

Challenges in Coagulation Management in Neurosurgical Diseases: A Scoping Review, Development, and Implementation of Coagulation Management Strategies.

Bleeding and thromboembolic (TE) complications in neurosurgical diseases have a detrimental impact on clinical outcomes. The aim of this study is to provide a scoping review of the available literature and address challenges and knowledge gaps in the management of coagulation disorders in neurosurgical diseases. Additionally, we introduce a novel research project that seeks to reduce coagulation disorder-associated complications in neurosurgical patients. The risk of bleeding after elective craniotomy is about 3%, and higher (14-33%) in other indications, such as trauma and intracranial hemorrhage. In spinal surgery, the incidence of postoperative clinically relevant bleeding is approximately 0.5-1.4%. The risk for TE complications in intracranial pathologies ranges from 3 to 20%, whereas in spinal surgery it is around 7%. These findings highlight a relevant problem in neurosurgical diseases and current guidelines do not adequately address individual circumstances. The multidisciplinary COagulation MAnagement in Neurosurgical Diseases (COMAND) project has been developed to tackle this challenge by devising an individualized coagulation management strategy for patients with neurosurgical diseases. Importantly, this project is designed to ensure that these management strategies can be readily implemented into healthcare practices of different types and with sustainable integration.

Recommendations for the use of andexanet alfa in the management of bleeding in patients on oral factor Xa inhibitors in Switzerland: Guideline from the Working Party Hemostasis of the Swiss Society of Hematology

Anticoagulants are essential in preventing and treating thrombosis. Unfortunately, their use is accompanied by an enhanced risk of bleeding. Since the introduction of direct oral anticoagulants (DOACs), the risk of major bleeding has been reduced but not eliminated. Major bleeding events related to the use of factor Xa inhibitors can be challenging to manage. In recent years, four-factor prothrombin complex concentrates have been used in patients with severe bleeding taking oral direct factor Xa inhibitors (apixaban, edoxaban and rivaroxaban). Andexanet alfa (OndexxyaTM, AstraZeneca AG) is a specially designed recombinant version of human factor Xa that acts as a decoy receptor to reverse the effects of factor Xa inhibitors. Since 2 December 2020, andexanet alfa has been used in Switzerland for adult patients receiving apixaban or rivaroxaban when reversal of anticoagulation is required because of life-threatening or uncontrolled bleeding. However, the use of andexanet alfa remains a challenge owing to its cost, the reported thrombotic complications and the fact that its efficacy mainly relates to intracranial haemorrhage. Moreover, the use of nonspecific reversal agents together with andexanet alfa is controversial. The present recommendations on the use of andexanet alfa in the management of bleeding in patients on factor Xa inhibitors in Switzerland were developed by a group of Swiss experts from the Working Party Hemostasis of the Swiss Society of Hematology. These recommendations aim to provide support to clinicians in their decision-making in the management of patients with major bleeding receiving factor Xa inhibitors.

Von Willebrand factor and the thrombophilia of severe COVID-19: in situ evidence from autopsies.

Background: COVID-19 is accompanied by a hypercoagulable state and characterized by microvascular and macrovascular thrombotic complications. In plasma samples from patients with COVID-19, von Willebrand factor (VWF) levels are highly elevated and predictive of adverse outcomes, especially mortality. Yet, VWF is usually not included in routine coagulation analyses, and histologic evidence of its involvement in thrombus formation is lacking. Objectives: To determine whether VWF, an acute-phase protein, is a bystander, ie, a biomarker of endothelial dysfunction, or a causal factor in the pathogenesis of COVID-19. Methods: We compared autopsy samples from 28 patients with lethal COVID-19 to those from matched controls and systematically assessed for VWF and platelets by immunohistochemistry. The control group comprised 24 lungs, 23 lymph nodes, and 9 hearts and did not differ significantly from the COVID-19 group in age, sex, body mass index (BMI), blood group, or anticoagulant use. Results: In lungs, assessed for platelets by immunohistochemistry for CD42b, microthrombi were more frequent in patients with COVID-19 (10/28 [36%] vs 2/24 [8%]; P = .02). A completely normal pattern of VWF was rare in both groups. Accentuated endothelial staining was found in controls, while VWF-rich thrombi were only found in patients with COVID-19 (11/28 [39%] vs 0/24 [0%], respectively; P < .01), as were NETosis thrombi enriched with VWF (7/28 [25%] vs 0/24 [0%], respectively; P < .01). Forty-six percent of the patients with COVID-19 had VWF-rich thrombi, NETosis thrombi, or both. Trends were also seen in pulmonary draining lymph nodes (7/20 [35%] vs 4/24 [17%]; P = .147), where the overall presence of VWF was very high. Conclusion: We provide in situ evidence of VWF-rich thrombi, likely attributable to COVID-19, and suggest that VWF may be a therapeutic target in severe COVID-19.

Determination of Anti-Xa Inhibitor Plasma Concentrations Using a Universal Edoxaban Calibrator.

A universal calibrator for the determination of all anti-Xa inhibitors would support laboratory processes. We aimed to test the clinical performance of an anti-Xa assay utilizing a universal edoxaban calibrator to determine clinically relevant concentrations of all anti-Xa inhibitors. Following a pilot study, we enrolled 553 consecutive patients taking rivaroxaban, edoxaban, or apixaban from nine study centers in a prospective cross-sectional study. The Technochrom® anti-Xa assay was conducted using the Technoview® edoxaban calibrator. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), anti-Xa inhibitor drug concentrations were determined. Sensitivities and specificities to detect three clinically relevant drug concentrations (30 µgL-1, 50 µgL-1, 100 µgL-1) were determined. Overall, 300 patients treated with rivaroxaban, 221 with apixaban, and 32 with edoxaban were included. The overall correlation coefficient (rs) was 0.95 (95% CI 0.94, 0.96). An area under the receiver operating characteristic curve of 0.96 for 30 µgL-1, 0.98 for 50 µgL-1, and 0.99 for 100 µgL-1 was found. The sensitivities were 92.3% (95% CI 89.2, 94.6), 92.7% (89.4, 95.1), and 94.8% (91.1, 97.0), respectively (specificities 82.2%, 93.7%, and 94.4%). In conclusion, the clinical performance of a universal, edoxaban-calibrated anti-Xa assay was solid and most drug concentrations were predicted correctly.

Complication rates of peripherally inserted central catheters vs implanted ports in patients receiving systemic anticancer therapy: A retrospective cohort study.

While implanted port catheters ("PORTs") have historically been the standard device for intravenous systemic anticancer therapy, the use of peripherally inserted central catheters (PICCs) has increased continuously and reliable catheter selection guidelines are lacking. We compare complication rates of PORTs and PICCs in cancer treatment in a retrospective study of 3365 patients with both solid organ (n = 2612) and hematologic (n = 753) malignancies, between 2001 and 2021. 26.4% (n = 890) of all patients were treated via PICCs and 73.6% (2475) via PORTs. 20.7% (578) experienced a major catheter-related complication with a higher rate in PICCs than in PORTs (23.5% vs 14.9%, P < .001). Among major complications, infections and mechanical complications were more common in PICCs than in PORTs (11.9% vs 6.4%, P = .001, 7.3% vs 4.2%, P = .002), whereas the rate of thrombosis was similar (3.4% vs 3.0%, P = .9). While PORTs had a higher rate of periprocedural complications (2.7% vs 1.1%, P < .05), PICCs overall complication rate exceeded PORTs within 3 days from implantation. Median follow-up was 49 (PICC) and 60 weeks (PORT). PORTs are safer and therefore should be preferred in this setting regardless of catheter dwell time.

Limited concordance of heparin/platelet factor 4 antibody assays for the diagnosis of heparin-induced thrombocytopenia: an analysis of the TORADI-HIT study.

BACKGROUND: Anecdotal reports suggest that the correlation between heparin/platelet factor 4 (PF4) antibody assays for the diagnosis of heparin-induced thrombocytopenia (HIT) is limited. OBJECTIVES: To investigate the correlation between widely used assays and examine possible factors contributing to variability. METHODS: This is a large, prospective cohort study with 10 participating tertiary care hospitals including 1393 patients with suspected HIT in clinical practice. HIT was defined by a positive heparin-induced platelet activation (HIPA) assay (washed platelet reference standard test). Three different immunoassays were used to measure heparin/PF4 antibodies: chemiluminescent immunoassay, enzyme-linked immunosorbent assay, and particle gel immunoassay. Various factors that could influence the assays were examined: sex (male or female), age (<65 years or ≥65 years), unfractionated heparin exposure, presence of thrombosis, cardiovascular surgery, and intensive care unit. Spearman's correlation coefficients were calculated. Z-scores and diagnostic odds ratios were determined in the aforementioned subgroups of patients. RESULTS: Among 1393 patients, 119 were classified as HIT-positive (prevalence, 8.5%). The median 4Ts score was 5 (IQR, 4-6) in patients with HIT compared with 3 (IQR, 2-4) in patients without HIT. Correlations (rs) between immunoassays were weak (0.53-0.65). Inconsistencies between immunoassays could not be explained by further analyses of z-scored test results and diagnostic odds ratios in subgroups of patients. CONCLUSION: The correlation between widely used heparin/PF4 antibody assays was weak, and key factors could not explain this variability. Standardization of immunoassays is requested to improve comparability.

Indications and Outcomes of Patients Receiving Therapeutic Plasma Exchange under Critical Care Conditions: A Retrospective Eleven-Year Single-Center Study at a Tertiary Care Center.

Background: Therapeutic plasma exchange (TPE) is frequently performed in critical care settings for heterogenous indications. However, specific intensive care unit (ICU) data regarding TPE indications, patient characteristics and technical details are sparse. Methods: We performed a retrospective, single-center study using data from January 2010 until August 2021 for patients treated with TPE in an ICU setting at the University Hospital Zurich. Data collected included patient characteristics and outcomes, ICU-specific parameters, as well as apheresis-specific technical parameters and complications. Results: We identified n = 105 patients receiving n = 408 TPEs for n = 24 indications during the study period. The most common was thrombotic microangiopathies (TMA) (38%), transplant-associated complications (16.3%) and vasculitis (14%). One-third of indications (35.2%) could not be classified according to ASFA. Anaphylaxis was the most common TPE-related complication (6.7%), while bleeding complications were rare (1%). The median duration of ICU stay was 8 ± 14 days. Ventilator support, renal replacement therapy or vasopressors were required in 59 (56.2%), 26 (24.8%), and 35 (33.3%) patients, respectively, and 6 (5.7%) patients required extracorporeal membrane oxygenation. The overall hospital survival rate was 88.6%. Conclusion: Our study provides valuable real-world data on heterogenous TPE indications for patients in the ICU setting, potentially supporting decision-making.

A machine-learning model for reducing misdiagnosis in heparin-induced thrombocytopenia: A prospective, multicenter, observational study.

Background: Diagnosing heparin-induced thrombocytopenia (HIT) at the bedside remains challenging, exposing a significant number of patients at risk of delayed diagnosis or overtreatment. We hypothesized that machine-learning algorithms could be utilized to develop a more accurate and user-friendly diagnostic tool that integrates diverse clinical and laboratory information and accounts for complex interactions. Methods: We conducted a prospective cohort study including 1393 patients with suspected HIT between 2018 and 2021 from 10 study centers. Detailed clinical information and laboratory data were collected, and various immunoassays were conducted. The washed platelet heparin-induced platelet activation assay (HIPA) served as the reference standard. Findings: HIPA diagnosed HIT in 119 patients (prevalence 8.5%). The feature selection process in the training dataset (75% of patients) yielded the following predictor variables: (1) immunoassay test result, (2) platelet nadir, (3) unfractionated heparin use, (4) CRP, (5) timing of thrombocytopenia, and (6) other causes of thrombocytopenia. The best performing models were a support vector machine in case of the chemiluminescent immunoassay (CLIA) and the ELISA, as well as a gradient boosting machine in particle-gel immunoassay (PaGIA). In the validation dataset (25% of patients), the AUROC of all models was 0.99 (95% CI: 0.97, 1.00). Compared to the currently recommended diagnostic algorithm (4Ts score, immunoassay), the numbers of false-negative patients were reduced from 12 to 6 (-50.0%; ELISA), 9 to 3 (-66.7%, PaGIA) and 14 to 5 (-64.3%; CLIA). The numbers of false-positive individuals were reduced from 87 to 61 (-29.8%; ELISA), 200 to 63 (-68.5%; PaGIA) and increased from 50 to 63 (+29.0%) for the CLIA. Interpretation: Our user-friendly machine-learning algorithm for the diagnosis of HIT (https://toradi-hit.org) was substantially more accurate than the currently recommended diagnostic algorithm. It has the potential to reduce delayed diagnosis and overtreatment in clinical practice. Future studies shall validate this model in wider settings. Funding: Swiss National Science Foundation (SNSF), and International Society on Thrombosis and Haemostasis (ISTH).

Prothrombinase-Induced Clotting Time to Measure Drug Concentrations of Rivaroxaban, Apixaban, and Edoxaban in Clinical Practice: A Cross-Sectional Study.

Prothrombinase-induced clotting time (PiCT) is proposed as a rapid and inexpensive laboratory test to measure direct oral anticoagulant (DOAC) drug levels. In a prospective, multicenter cross-sectional study, including 851 patients, we aimed to study the accuracy of PiCT in determining rivaroxaban, apixaban, and edoxaban drug concentrations and assessed whether clinically relevant drug levels could be predicted correctly. Citrated plasma samples were collected, and the Pefakit® PiCT was utilized. Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure drug concentrations. Cut-off levels were established using receiver-operating characteristics curves. We calculated sensitivities and specificities with respect to clinically relevant drug concentrations. Spearman's correlation coefficient between PiCT and drug concentrations was 0.85 in the case of rivaroxaban (95% CI 0.82, 0.88), 0.66 for apixaban (95% CI 0.60, 0.71), and 0.78 for edoxaban (95% CI 0.65, 0.86). The sensitivity to detect clinically relevant drug concentrations was 85.1% in the case of 30 µg L-1 (95% CI 82.0, 87.7; specificity 77.9; 72.1, 82.7), 85.7% in the case of 50 µg L-1 (82.4, 88.4; specificity 77.3; 72.5, 81.5), and 85.1% in the case of 100 µg L-1 (80.9, 88.4; specificity 73.2%; 69.1, 76.9). In conclusion, the association of PiCT with DOAC concentrations was fair, and the majority of clinically relevant drug concentrations were correctly predicted.

Accuracy of a Single, Heparin-Calibrated Anti-Xa Assay for the Measurement of Rivaroxaban, Apixaban, and Edoxaban Drug Concentrations: A Prospective Cross-Sectional Study.

Background: Applying a single anti-Xa assay, calibrated to unfractionated heparin to measure rivaroxaban, apixaban, and edoxaban would simplify laboratory procedures and save healthcare costs. Aim: We hypothesized that a heparin-calibrated anti-Xa assay would accurately measure rivaroxaban, apixaban, and edoxaban drug concentrations and correctly predict clinically relevant drug levels. Methods: This analysis is part of the Simple-Xa study, a prospective multicenter cross-sectional study conducted in clinical practice. Patients treated with rivaroxaban, apixaban, or edoxaban were included. Anti-Xa activity was measured using the Siemens INNOVANCE® Heparin assay. Drug concentrations were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cut-off levels were determined in a derivation dataset (50% of patients) and sensitivities and specificities were calculated in a verification dataset (50% of patients). Results: Overall, 845 patients were available for analysis. Correlation coefficients (r s ) between the heparin-calibrated anti-Xa assay and drug concentrations were 0.97 (95% CI 0.97, 0.98) for rivaroxaban, 0.96 (0.96, 0.97) for apixaban, and 0.96 (0.94, 0.99) for edoxaban. The area under the receiver operating characteristics curve (ROC) was 0.99 for all clinically relevant drug concentrations. In the verification dataset, the sensitivity was 94.2% (95% CI 90.8-96.6) for 30 µg L-1, 95.8% (92.4-98.0) for 50 µg L-1, and 98.7% (95.5-99.9) for 100 µg L-1. Specificities were 86.3% (79.2-91.7), 89.8% (84.5-93.7), and 88.7% (84.2-92.2), respectively. Conclusion: In a large prospective study in clinical practice, a strong correlation of heparin-calibrated anti-Xa measurements with LC-MS/MS results was observed and clinically relevant drug concentrations were predicted correctly.

Liver infarctions as the first manifestation of antiphospholipid antibody syndrome in pregnancy: a case report.

BACKGROUND: The differential diagnosis of abdominal pain in pregnant women is broad. Liver diseases as the origin of abdominal pain in pregnancy are rare, and severe forms occur in less than 0.1% of pregnancies. Some disorders, such as hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome and preeclampsia, are unique to pregnancy, while others, such as antiphospholipid antibody syndrome, may manifest in pregnancy but have consequences beyond the current pregnancy. All of them require prompt identification and treatment. CASE PRESENTATION: A 27-year-old Caucasian woman who was 15+1 weeks pregnant reported to the emergency department twice due to stabbing right-upper-quadrant abdominal pain. Initial laboratory testing revealed mild leukocytosis and slightly elevated liver enzymes. On second presentation, the patient was febrile and had an increased C-reactive protein concentration. Over the course of the next days, nonhemolytic anemia and thrombocytopenia emerged with elevated liver enzymes. Coagulation studies also revealed a prolongation of activated partial thromboplastin time. Magnetic resonance imaging showed nonspecific alterations in the right liver lobe, possibly corresponding to infection or infarction. A hepatic viral infection was ruled out. At that time, the most likely diagnosis was cholangitis with liver abscess formation, and antibiotic therapy was started. Further worsening of the anemia and thrombocytopenia, development of proteinuria, together with a miscarriage on the fourth day of hospitalization resulted in the tentative diagnosis of (triple-positive) antiphospholipid antibody syndrome, which was confirmed 12 weeks after the initial investigation. Treatment consisted of prompt anticoagulation with heparin and later on with a vitamin K antagonist as well as high-dose glucocorticoid therapy. There was no need for intravenous immunoglobulin therapy or plasma exchange, although we suspected a catastrophic form of antiphospholipid antibody syndrome due to infarctions of the liver, placenta, and possibly kidneys (proteinuria). The outcome was favorable. CONCLUSION: We report a 27-year-old pregnant woman whose abdominal pain was caused by liver infarctions as the first manifestation of catastrophic antiphospholipid antibody syndrome. The antiphospholipid antibody syndrome was possibly secondary to hitherto clinically silent systemic lupus erythematosus since the antinuclear antibodies were increased later on. Hydroxychloroquine therapy was initiated to prevent antiphospholipid antibody syndrome recurrence in a future pregnancy.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbß3 rather than α5ß1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

Synthesis of coagulation factors during long-term ex situ liver perfusion.

Robust viability assessment of grafts during normothermic liver perfusion is a prerequisite for organ use. Coagulation parameters are used commonly for liver assessment in patients. However, they are not yet included in viability assessment during ex situ perfusion. In this study, we analysed coagulation parameters during one week ex situ perfusion at 34℃. Eight discarded human livers were perfused with blood-based, heparinised perfusate for one week; perfusions in a further four livers were terminated on day 4 due to massive ongoing cell death. Coagulation parameters were well below the physiologic range at perfusion start. Physiologic levels were achieved within the first two perfusion days for factor V (68.5 ± 35.5%), factor VII (83.5 ± 26.2%), fibrinogen (2.1 ± 0.4 g/L) and antithrombin (107 ± 26.5%) in the livers perfused for one week. Despite the increased production of coagulation factors, INR was detectable only at 24h of perfusion (2.1 ± 0.3) and prolonged thereafter (INR > 9). The prolongation of INR was related to the high heparin level in the perfusate (anti-FXa > 3 U/mL). Intriguingly, livers with ongoing massive cell death also disclosed synthesis of factor V and improved INR. In summary, perfused livers were able to produce coagulation factors at a physiological level ex situ. We propose that single coagulation factor analysis is more reliable for assessing the synthetic function of perfused livers as compared to INR when using a heparinised perfusate.

Daratumumab for immune thrombotic thrombocytopenic purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic microangiopathy. It is caused by a severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, 13) deficiency due to circulating autoantibodies, and is associated with significant morbidity and mortality. Current treatment options include plasma exchange, immunosuppression, and caplacizumab. When remission is achieved, the risk of relapse is high, especially in patients with persistent ADAMTS13 deficiency. We report the eradication of persistent ADAMTS13 inhibitory autoantibodies and restoration of normal ADAMTS13 activity using the anti-CD38 antibody daratumumab in two patients with iTTP. One patient had a frequently relapsing course, and the other a treatment-refractory first episode. There were no relevant adverse drug reactions.

Structural analysis of receptors and actin polarity in platelet protrusions.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Automated Thrombin Generation Assay for Rivaroxaban, Apixaban, and Edoxaban Measurements.

Background: The thrombin generation assay (TG) is a promising approach to measure the degree of anticoagulation in patients treated with direct oral anticoagulants (DOAC). A strong association with plasma drug concentrations would be a meaningful argument for the potential use to monitor DOAC. Objectives: We aimed to study the correlation of TG with rivaroxaban, apixaban, and edoxaban drug concentrations in a large, prospective multicenter cross-sectional study. Methods: Five-hundred and fifty-nine patients were included in nine tertiary hospitals. The Technothrombin® TG was conducted in addition to an anti-Xa assay; LC-MS/MS was performed as the reference standard. Results: Correlation (rs) between thrombin generation measurements and drug concentrations was -0.72 for peak thrombin generation (95% confidence interval, CI, -0.77, -0.66), -0.55 for area under the curve (AUC; 95% CI -0.61, -0.48), and 0.80 for lag time (95% CI 0.75, 0.84). In contrast, rs was 0.96 with results of the anti-Xa activity (95% CI 0.95-0.97). Sensitivity with regard to the clinically relevant cut-off value of 50 µgL-1 was 49% in case of peak thrombin generation (95% CI, 44, 55), 29% in case of AUC (95% CI, 24, 34), and 64% in case of lag time (95% CI, 58, 69). Sensitivity of the anti-Xa assay was 95% (95% CI, 92, 97). Conclusions: The correlation of thrombin generation measurements with DOAC drug concentrations was weak, and clinically relevant drug levels were not predicted correctly. Our results do not support an application of TG in the monitoring of DOAC.