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Vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) have been recently described as rare complications following vaccination against SARS-CoV-2 with vector vaccines. We report a case of a young woman who presented with VITT and cerebral CVST 7 days following vaccination with ChAdOx1 nCov-19 (AstraZeneca). While the initial MRI was considered void of pathological findings, MRI 3 days later revealed extensive CVST of the transversal and sigmoidal sinus with intracerebral haemorrhage. Diagnostic tests including a platelet-factor-4-induced platelet activation assay confirmed the diagnosis of VITT. Treatment with intravenous immunoglobulins and argatroban resulted in a normalisation of platelet counts and remission of CVST.
Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.
Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.
Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates
(2022)
Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.
Background: Hyperthyroidism is known to induce a hypercoagulable state. It stimulates plasma levels of procoagulative factors and reduces fibrinolytic activity. So far most of the data have been derived from patients with endogenous hyperthyroidism with a wide variability in the underlying pathogenesis and severity of the disease. Objectives: In this study we experimentally induced thyrotoxicosis in healthy volunteers to explore the effects of thyroxine excess on the plasma proteome. Using a shotgun proteomics approach, the abundance of plasma proteins was monitored before, during and after thyrotoxicosis. Methods: Sixteen healthy male subjects were sampled at baseline, 4 and 8 weeks under 250 µg/day thyroxine p.o., as well as 4 and 8 weeks after stopping the application. Plasma proteins were analyzed after depletion of 6 high-abundance proteins (MARS6) by LC-ESI-MS/MS mass spectrometry. Mass spectrometric raw data were processed using a label-free, intensity-based workflow. Subsequently, the linear dependence between protein abundances and fT<sub>4</sub> levels were calculated using a Pearson correlation. Results: All subjects developed biochemical thyrotoxicosis, and this effect was reversed within the first 4 weeks of follow-up. None of the volunteers noticed any subjective symptoms. Levels of 10 proteins involved in the coagulation cascade specifically correlated with fT<sub>4</sub>, supporting an influence of thyroid hormone levels on blood coagulation even at nonpathological levels. Conclusions: The results suggest that experimental thyrotoxicosis exerts selective and specific thyroxine-induced effects on coagulation markers. Our study design allows assessment of thyroid hormone effects on plasma protein levels without secondary effects of other diseases or therapies.
Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.
Background: We assessed the effect of the uniform donor questionnaire (UDQ) on deferral rates in first-time and repeat donors. We focused on the introduced question about unprotected sexual contact with a new partner. Another goal was a stratified comparison of the deferral rates of the donor questionnaire (DQ) and UDQ. Methods: Data on donors and deferrals using the DQ and UDQ were collected at four blood establishments. The comparison included a 2-year period by questionnaire version. For the comparison of the questionnaires, an adjusted multinomial logistic regression was performed. Results: The analysis included 260,848 donations. First-time (FTD) and repeat donations (RD) showed higher deferral rates with the UDQ (FTD +5.4%, RD +1.4%). Deferral due to a new partner was 3.0% in first-time and 0.4% in repeat donors. The majority of these occurred in the youngest age groups. The most frequent deferral criterion was ‘disease' (5.1%). Conclusion: The regression revealed stronger predictors for deferral than the questionnaire version. Especially younger age carried a higher and independent risk for deferral. The additional deferrals of mainly young first-time donors due to a new sexual partner may identify those donors with potential heterosexual risk behavior who would otherwise not be identified.
Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intraindividual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by using differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolor flow cytometry was used to characterize resting and stimulated platelet subpopulations, and platelet age was determined based on RNA and HLA-I labeling. Signal transduction was analyzed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Compared with small platelets, large platelets adhered faster to collagen under flow and formed larger thrombi. Among the large platelets, a highly reactive juvenile platelet subpopulation was identified with high glycoprotein VI (GPVI) expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content, and increased platelet reactivity. There was a stronger difference in Akt phosphorylation and activation upon collagen stimulation between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population, explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage, and immune thrombocytopenia.
Background
Signs of an inflammatory process have been described in major depression.
Methods
In a double-blind, randomized study of celecoxib or placebo add-on to reboxetine in 40 depressed patients, celecoxib treatment has beneficial effects. In order to evaluate the tryptophan/kynurenine metabolism and to identify predictors for remission, tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and quinolinic acid (QUIN) were estimated in the serum of 32 patients before and after treatment and in a group of 20 healthy controls.
Results
KYN levels were significantly lower in patients (p = 0.008), and the QUIN/KYN ratios were significantly higher (p = 0.028). At baseline, the higher KYN/TRP ratio was predictive for remission during celecoxib add-on treatment (p = 0.04) as well as for remission in the overall patient group (p = 0.01). In the placebo group, remitters showed a higher KYNA/QUIN ratio (p = 0.032). In the overall group, remitters showed lower KYNA/KYN (p = 0.035) and QUIN/KYN (p = 0.011) ratios. The lower the formation of downstream metabolites, especially QUIN, the better the treatment outcome.
Conclusion
The high KYN/TRP ratio predicted remission after treatment with celecoxib in this small sample of depressed patients. Eventually, the KYN/TRP ratio might be a marker for those patients, which benefit from an additional anti-inflammatory treatment.