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Abstract: The G haplotype is a group of co-inherited single nucleotide variants in the F5 gene that reduce venous thromboembolism (VTE) risk. Although 7% of the population is homozygous for the G haplotype (F5-G/G), the underlying mechanism of VTE protection is poorly understood. Using RNA sequencing data from 4651 blood donors in the INTERVAL study, we detected a rare excision event at the factor V (FV)-short splice sites in 5% of F5-G/Gs carriers as compared with 2.16% of homozygotes for the F5 reference sequence (F5-ref; P = .003). Highly elevated (∼10-fold) FV-short, a FV isoform that lacks most of the B-domain, has been linked with increased tissue factor inhibitor α (TFPIα) levels in rare hemorrhagic diathesis, including East Texas bleeding disorder. To ascertain whether the enhanced FV-short splicing seen in F5-G/G INTERVAL participants translated to increased plasma FV-short levels, we analyzed plasma samples from 7 F5-G/G and 13 F5-ref individuals in a recall-by-genotype study. A ∼2.2-fold higher amount of FV-short was found in a plasma pool from F5-G/G participants when compared with the pool of F5-refs (P = .029), but there was no difference in the total FV levels. Although no significant difference in TFPI levels were found, F5-G/Gs showed a ∼1.4-fold TFPI-dependent increase in lag time to thrombin generation than F5-refs (P = .0085). Finally, in an analysis of 117 699 UK Biobank participants, we discovered that, although being protective against VTE, the G haplotype also confers an increase in bleeding episodes (P = .011). Our study provides evidence that the effect of the common G haplotype is mediated by the FV-short/TFPI pathway.

Transcatheter aortic valve implantation (TAVI) has become the mainstay for high-risk or inoperable patients with symptomatic aortic valve stenosis, and research regarding the use of transcatheter valves in intermediate or low-risk patients is currently ongoing. The aim of this article is to provide comprehensive insight into the anesthetic management of patients undergoing TAVI and to highlight possible gaps in the current knowledge. One important procedural characteristic that is imperative to consider is the type of anesthesia being used and its possible complications. Increasingly, experienced centers have changed from general anesthesia with endotracheal intubation to local anesthesia with sedation, especially when the transfemoral access route is used for TAVI. There is still debate regarding what type of anesthesia should be used in the procedure, and the lack of randomized data makes it even more challenging for the operators.

Background: The sensitivity of the platelet function analyzer (PFA-100) was shown to be high for the detection of von Willebrand disease (VWD), but limited for platelet function defects (PFD). In patients with bleeding disorder of unknown cause (BDUC), however, data on the usefulness of PFA-100 is lacking., Methods: PFA-100 closure times (CTs) were measured with collagen-epinephrine [PFA (EPI)] and collagen-adenosine diphosphate [PFA (ADP)] cartridges in 818 patients with mild bleeding disorders from the Vienna Bleeding Biobank. Patients on anticoagulation or antiplatelet therapy or thrombocytopenic patients were not included., Results: Only 2% of the 532 BDUC patients had prolonged CTs in PFA (EPI) and PFA (ADP), and 64% in either PFA (EPI) or PFA (ADP). In total, 34% of BDUC patients did not have prolonged CTs in PFA (EPI) or PFA (ADP). These rates were similar to patients with coagulation factor deficiencies (n=27). The rate of pathological CTs was significantly higher in patients with VWD (n=79) and, although less pronounced, in PFD (n=180). In 15/18 (83%) VWD patients with VWF:Ag and/or VWF:RCo levels <30 IU/dL, the PFA-100 was prolonged in both cartridges. No association of the PFA-100 with the bleeding severity was observed in BDUC patients. However, prolonged CTs were associated with higher age, lower hematocrit, lower VWF:Ag or VWF:RCo levels, lower platelet counts, and higher fibrinogen levels in BDUC patients., Conclusion: We could not confirm a diagnostic utility for the PFA-100 in MBD patients, and specifically BDUC. No association between PFA-100 results and bleeding severity was observed in BDUC patients., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In patients with mild-to-moderate bleeding disorders (MBD), von Willebrand disease (VWD) is diagnosed at von Willebrand factor (VWF) levels ≤50 IU/dL. Although VWF levels are unstable, repeated testing for VWD diagnosis is not necessarily advised in recent guidelines., Objectives: To analyze the relevance of repeated VWF testing to diagnose VWD in patients with MBD., Methods: Data of 277 patients with MBD from the Vienna Bleeding Biobank with at least 2 separate assessments of VWF antigen (VWF:Ag) and activity (VWF:Act) were analyzed., Results: In repeated VWF measurements, 36 patients (13.0%) had "changing" VWF levels (≤/>50 IU/dL), 27 (9.7%) had persistent levels ≤50 IU/dL ("pathologic"), and 214 (77.3%) had levels >50 IU/dL ("normal"). Of the 36 changing patients, 22 (61%) were diagnosed with VWD at baseline, whereas the others only met VWD diagnostic criteria at repeated measurements. Using logistic regression, we estimated a probability of change of 26.4% (95% CI, 12.5-47.4) at baseline VWF levels of 30 IU/dL, 50.8% (95% CI, 35.6-65.8) at 50 IU/dL, 18.8% (95% CI, 12.3-27.6) at 60 IU/dL, and 1.2% (95% CI, 0.3-4.9) at 80 IU/dL. Baseline VWF was a strong predictor for changing status (Χ 2  = 49.9; P < .001), while age, sex, Vicenza score, and blood type O had limited added value (Χ 2  = 5.1; P = .278). Baseline VWF:Ag or VWF:Act cutoffs of 80 IU/dL had negative predictive values of 98.1% and 99.1% for changing status, respectively., Conclusion: Our data emphasize an overlap between patients with VWD and MBD with bleeding disorder of unknown cause and underline the need for repeated VWF testing, especially in patients with VWF levels <80 IU/dL., Competing Interests: Declaration of competing interest D.M. received honoraria for advisory board meetings from CSL Behring. D.K. received honoraria for advisory board meetings from CSL Behring. C.A. received honoraria from Bayer, CSL Behring, Novo Nordisk, Pfizer, Roche, Sobi, and Takeda for lectures and/or participation in advisory board meetings. I.P. has occasionally received honoraria from Bayer, CSL Behring, Novo-Nordisk, Pfizer, Roche, Sobi, and Takeda for lectures and advisory board meetings. J.G. received honoraria for lectures, advisory board meetings, and research funding for the Medical University of Vienna from CSL Behring, Novartis, Amgen, and Sobi. A.T., J.B., P.Q., and H.H. have no conflicts of interest to declare., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD., (© 2019 by The American Society of Hematology.)

Background: High platelet reactivity (HPR) under treatment with clopidogrel or aspirin is associated with adverse outcome. We aimed to investigate whether high platelet reactivity (HPR) to both aspirin and clopidogrel is a stronger predictor of adverse events compared to isolated HPR to clopidogrel or aspirin., Methods: In this prospective cohort study platelet reactivity to adenosine diphosphate (ADP) and arachidonic acid (AA) was assessed by Multiple Electrode Aggregometry (MEA) in 403 patients undergoing percutaneous coronary intervention. The rates of the composite of cardiac adverse events (acute coronary syndrome, stent thrombosis, stroke, death and revascularization) were recorded during 12-month follow-up., Results: The composite endpoint of cardiovascular adverse events occurred more often in patients with high platelet reactivity (HPR) to both agonists ADP and AA (37.5%) than in those with isolated HPR to ADP (33.3%), AA (25.6%) or without any HPR (18.6%; p=0.003). Classification tree analysis indicated that any HPR emerged as an independent predictor influencing outcome, which was associated with a 1.75 higher risk of cardiac adverse events (OR=1.75: 95%CI=1.1-2.9). Interestingly, the predictive value of HPR tended to be greater among patients with diabetes mellitus (OR=2.18; 95%CI=1.20-3.95). C-reactive protein and diabetes mellitus were independent predictors of high platelet reactivity to both agonists., Conclusions: Dual low responsiveness to clopidogrel and aspirin is a strong predictor of cardiac adverse events, especially in patients with diabetes mellitus, which underlines the need for personalized antiplatelet treatment., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Thrombin activates pore forming channel protein Orai1 resulting in store operated Ca(2+) entry (SOCE) with subsequent Ca(2+)-dependent release of platelet granules, activation of integrin αIIbβ3, adhesion, aggregation and thrombus formation. Platelets lack nuclei and are thus unable to modify protein abundance by transcriptional regulation. Nevertheless, they still contain pre-mRNA and mRNA and are thus able to express protein by stimulation of rapid translation. Platelet translation is sensitive to phosphoinositide-3-kinase (PI3K) and actin polymerization. The present study explored whether platelet activation via thrombin modifies Orai1 protein abundance. According to RT-PCR platelets contain pre-mRNA and mRNA encoding Orai1. Activation with thrombin (0.1 U/ml) results in a significant decline of pre-mRNA, which is, according to Western blotting and confocal microscopy, paralleled by a marked and statistically significant increase of Orai1 protein abundance. The increase of Orai1 protein abundance is insensitive to inhibition of transcription with actinomycin (4 μg/ml), but is significantly blunted by inhibition of translation with puromycin (100 nM) and by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 μM). In conclusion, activation of platelets stimulates the translational expression of Orai1, thus augmenting platelet Ca(2+) signaling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Glycopeptides, such as vancomycin, are powerful antibiotics against methicillin-resistant Staphylococcus aureus. Balhimycin, a glycopeptide antibiotic isolated from Amycolatopsis balhimycina, is similarly effective as vancomycin. Side effects of vancomycin include triggering of platelet apoptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulation of apoptosis may involve increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization and/or caspase activation. An effect of balhimycin on apoptosis has, however, never been reported. The present study thus tested whether balhimycin triggers platelet apoptosis. Human blood platelets were treated with balhimycin and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V-binding, cytosolic Ca(2+) activity from fluo-3AM fluorescence, ceramide formation utilizing antibodies, mitochondrial potential from DiOC6 fluorescence, and caspase-3 activity utilizing antibodies. As a result, a 30 min exposure to balhimycin significantly decreased cell volume (≥1 μg/ml), triggered annexin V binding (≥1 μg/ml), increased cytosolic Ca(2+) activity (≥1 μg/ml), stimulated ceramide formation (≥10 μg/ml), depolarized mitochondria (≥1 μg/ml) and activated caspase-3 (≥1 μg/ml). Cell membrane scrambling and caspase-3 activation were virtually abrogated by removal of extracellular Ca(2+). Cell membrane scrambling was not significantly blunted by pancaspase inhibition with zVAD-FMK (1μM). In conclusion, balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca(2+), but not on activation of caspases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Activation of platelets by thrombin opens pore forming channel protein Orai1 with subsequent store operated Ca(2+) entry (SOCE) and Ca(2+) dependent platelet granule release, integrin α(IIb)β(3) activation, adhesion, aggregation and thrombus formation. Orai1 and thus SOCE as well as platelet activation are up-regulated by the serum- and glucocorticoid-inducible kinase-1 (SGK1), which transcriptionally regulates Orai1 expression in megakaryocytes and thus determines Orai1 protein abundance in mature, circulating platelets. As platelets are devoid of nuclei, they are unable to modify protein abundance by regulation of transcription. However, they contain mRNA and thus could express novel protein by stimulation of protein translation. Translation is sensitive to actin polymerization and phosphoinositide-3-kinase (PI3K). Translational regulation of SGK1 expression has never been described before. The present study thus explored whether thrombin regulates SGK1 expression in platelets. As a result, according to RT-PCR mRNA encoding SGK1 is present in circulating platelets and significantly decreased by activation of platelets with thrombin (1 U/ml). The protein abundance of SGK1 is significantly enhanced by thrombin treatment, an effect significantly decreased by inhibition of translation with puromycin (100 nM) but not by inhibition of transcription with actinomycin (4 μg/ml). The increase of SGK1 protein abundance is blunted by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 μM), and by disruption of the cytoskeleton with cytochalasin B (1 μM). In conclusion, activation of platelets with thrombin stimulates the translation of SGK1., (Copyright © 2012 Elsevier Inc. All rights reserved.)