Basal Ganglia versus Peripheral Infarcts: Predictive Value of Early Fiber Alterations.

BACKGROUND AND PURPOSE: Impairment of fiber integrity of the corticospinal tract in the subacute and chronic phases after ischemic stroke has been linked to poor motor outcome. The aim of the study was an assessment of fiber integrity in the acute poststroke phase and an evaluation of its association with the clinical course dependent on the infarction pattern (subtypes: peripheral versus basal ganglia infarction). MATERIALS AND METHODS: All patients who underwent mechanical recanalization of a large-vessel occlusion in the anterior circulation and postinterventional DTI were included (n = 165). The fractional anisotropy index of the patient-specific corticospinal tract within the posterior limb of the internal capsule was correlated to clinical parameters (NIHSS scores/mRS at 90 days), and the interaction of stroke subtype (peripheral infarcts versus basal ganglia infarction) was tested in a moderation analysis. RESULTS: The fractional anisotropy index was reduced in the acute poststroke phase with a correlation to clinical presentation, especially in case of peripheral infarcts (eg, with the NIHSS motor subscore: r = -0.4, P < .001). This correlation was absent for basal ganglia infarction (r = -0.008, P > .05). There was a significant association between the fractional anisotropy index and clinical outcome (mRS after 90 days, P < .01), which is moderated by stroke subtype with significant effects only for peripheral infarcts. CONCLUSIONS: Corticospinal tract abnormalities can be observed in the early stage after mechanical recanalization and have prognostic capacity. This finding increases the clinical value of early DTI imaging parameters. Because the effects observed were limited to peripheral infarcts, further and longitudinal evaluation of fiber integrities within basal ganglia infarction is required.

A short history of thrombectomy - Procedure and success analysis of different endovascular stroke treatment techniques.

BACKGROUND: The historical development of interventional stroke treatment shows a wide variation of different techniques and materials used. Thus, the question of the present work is whether the technical and procedural differences of thrombectomy techniques lead to different technical and clinical results. METHODS AND RESULTS: Analysis of a mixed retrospective/prospective database of all endovascular treated patients with an occlusion of the Carotid-T or M1 segment of the MCA at a single comprehensive stroke center since 2008. Patients were classified regarding the technical approach used. Six hundred sixty-eight patients were available for the final analysis. Reperfusion rates ranged between 56% and 100% depending on the technical approach. The use of balloon guide catheters and most recently the establishment of combination techniques using balloon guide catheters, aspiration catheters and stent retrievers have shown a further significant increase in the rates of successful recanalization, full recanalization and first-pass recanalization. Additionally, the technical development of interventional techniques has led to a subsequent drop in complications, embolization into previously unaffected territories in particular. CONCLUSION: Technical success of MT has improved substantially over the past decade owing to improved materials and procedural innovations. Combination techniques including flow modulation have emerged to be the most effective approach and should be considered as a standard of care.Level of evidence: Level 3, retrospective study.

Thrombus Histology of Basilar Artery Occlusions : Are There Differences to the Anterior Circulation?

BACKGROUND: For patients with acute vessel occlusions of the anterior circulation histopathology of retrieved cerebral thrombi has been reported to be associated to stroke etiology. Due to the relatively small incidence of posterior circulation stroke, exclusive histopathologic analyses are missing for this subgroup. The aim of the study was to investigate thrombus histology for patients with basilar artery occlusions and uncover differences to anterior circulation clots with respect to underlying etiology. METHODS: A total of 59 basilar thrombi were collected during intracranial mechanical recanalization and quantitatively analyzed in terms of their relative fractions of the main constituents, e.g. fibrin/platelets (F/P), red (RBC) and white blood cells (WBC). Data were compared to histopathological analyses of 122 thrombi of the anterior circulation with respect to underlying pathogenesis. RESULTS: The composition of basilar thrombi differed significantly to thrombi of the anterior circulation with an overall higher RBC amount (median fraction in % (interquartile range):0.48 (0.37-0.69) vs. 0.37 (0.28-0.50), p < 0.001) and lower F/P count (0.45 (0.21-0.58) vs. 0.57 (0.44-0.66), p < 0.001). Basilar thrombi composition did not differ between the different etiological stroke subgroups. CONCLUSION: The results depict a differing thrombus composition of basilar thrombi in comparison to anterior circulation clots with an overall higher amount of RBC. This may reflect different pathophysiologic processes between anterior and posterior circulation thrombogenesis, e.g. a larger proportion of appositional thrombus growth in the posterior circulation.

Na+-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

Microstructural Integrity of Salvaged Penumbra after Mechanical Thrombectomy.

BACKGROUND AND PURPOSE: There are sparse data on the microstructural integrity of salvaged penumbral tissue after mechanical thrombectomy of large-vessel occlusions. The aim of the study was to analyze possible microstructural alteration in the penumbra and their association with clinical symptoms as well as angiographic reperfusion success in patients undergoing mechanical thrombectomy. MATERIALS AND METHODS: All patients who underwent mechanical thrombectomy for large-vessel occlusions in the anterior circulation and who received an admission CT perfusion together with postinterventional DTIs were included (n = 65). Angiographic reperfusion success by means of modified Thrombolysis in Cerebral Infarction (mTICI) scale and clinical outcome were recorded. Microstructural integrity was assessed by DTI evaluating the mean diffusivity index within the salvaged gray matter of the former penumbra. RESULTS: The mean diffusivity index was higher in completely recanalized patients (mTICI 3: -0.001 ± 0.034 versus mTICI <3: -0.030 ± 0.055, P = .03). There was a positive correlation between the mean diffusivity index and NIHSS score improvement (r = 0.49, P = .003) and the mean diffusivity index was associated with midterm functional outcome (r = -0.37, P = .04) after adjustment for confounders. In mediation analysis, the mean diffusivity index and infarction growth mediated the association between reperfusion success and clinical outcomes. CONCLUSIONS: The macroscopic salvaged penumbra included areas of microstructural integrity changes, most likely related to the initial hypoperfusion. These abnormalities were found early after mechanical thrombectomy, were dependent on angiographic results, and correlated with the clinical outcome. When confirmed, these findings prompt the evaluation of therapies for protection of the penumbral tissue integrity.

Creating high-quality radiology reports in foreign languages through multilingual structured reporting.

OBJECTIVES: Globalization and migration are increasing the demand for reports in different languages. We aimed to examine if structured reports created by non-German-speaking radiologists with multilingual templates show significant differences in quality to structured reports and free-text reports by German native speakers. METHODS: We used structured templates that allow radiologists to report in their mother tongue and then switch the report language to German or English automatically using proprietary software. German- and English-speaking radiology residents created structured reports in both German and English with these templates. Reports for three different exam types were created (intensive care chest x-ray, shoulder x-ray specifically for degenerative processes, and CT pulmonary angiogram for pulmonary embolism). The report quality of automatically translated German structured reports by English-speaking radiologists and German structured reports by German radiologists was then evaluated by German clinicians with a standardized questionnaire. The questionnaire was designed to assess attributes including content, comprehensibility, clinical consequences, and overall quality. RESULTS: Structured reports by English-speaking radiologists that were automatically translated into German and German structured reports by German radiologists both received very high or high overall quality ratings in the majority of cases, showing no significant differences in quality. Likewise, no significant differences were observed between the two report types regarding comprehensibility and clinical consequences. Structured reports by German radiologists received significantly better ratings for overall quality and comprehensibility compared to free-text reports by German radiologists. CONCLUSIONS: Multilingual structured reporting templates may serve as a feasible tool for creating high-quality radiology reports in foreign languages. KEY POINTS: • Multilingualism in structured reporting templates can be a useful tool for creating high-quality radiology reports in foreign languages. • German reports created with multilingual structured reporting templates by English-speaking radiologists and German structured reports by German radiologists exhibit no significant differences in overall report quality. • Multilingual structured reporting templates can help radiologists overcome communication barriers and facilitate teleradiology.

Aeshnid dragonfly larvae as bioindicators of methylmercury contamination in aquatic systems impacted by elevated sulfate loading.

Methylmercury (MeHg) levels in dragonfly larvae and water were measured over two years in aquatic systems impacted to varying degrees by sulfate releases related to iron mining activity. This study examined the impact of elevated sulfate loads on MeHg concentrations and tested the use of MeHg in dragonfly larvae as an indicator of MeHg levels in a range of aquatic systems including 16 river/stream sites and two lakes. MeHg concentrations in aeshnid dragonfly larvae were positively correlated (R(2) = 0.46, p < 0.01) to peak MeHg concentrations in the dissolved phase for the combined years of 2012 and 2013. This relation was strong in 2012 (R(2) = 0.85, p < 0.01), but showed no correlation in 2013 (R(2) = 0.02, p > 0.05). MeHg in dragonfly larvae were not elevated at the highest sulfate sites, but rather the reverse was generally observed. Record rainfall events in 2012 and above average rainfall in 2013 likely delivered the majority of Hg and MeHg to these systems via interflow and activated groundwater flow through reduced sediments. As a result, the impacts of elevated sulfate releases due to mining activities were not apparent in these systems where little of the sulfate is reduced. Lower bioaccumulation factors for MeHg in aeshnid dragonfly larvae were observed with increasing dissolved organic carbon (DOC) concentrations. This finding is consistent with previous studies showing that MeHg in high DOC systems is less bioavailable; an equilibrium model shows that more MeHg being associated with DOC rather than algae at the base of the food chain readily explains the lower bioaccumulation factors.

[Fax Survey to Elucidate the Information Needs of General Practitioners in Lower Saxony Regarding the Topic of Medical Implants]. / Faxumfrage zum Informationsbedarf niedersächsischer Hausärzte zum Thema "Medizinische Implantate".

Background: Medial implants help a multitude of patients to gain more health, mobility and thus, quality of life. In collaboration with a still growing expectation of life especially, i.e., within Western industrial countries, this has led to an increasing use of implants over the last years. However, although biomechanical characteristics of modern implant materials have improved considerably, one big challenge still exists - the implant-associated infection. Early diagnostic and therapeutic interventions could clearly mitigate this issue, but are general practitioners sufficiently informed regarding this topic? Material and Methods: In March 2013 and in close cooperation with the Lower Saxony association of general practitioners, we initiated a survey to elucidate the information demands of general practitioners regarding the topic of medical implants. A total of 939 members of the association were contacted via fax and 101 (10.8 %) responded. Based on the obtained data, we then evaluated which topics are most interesting for this group of medical professionals. Results: The survey clearly indicates that general practitioners request more general implant-related data, e.g., type and specification of an implant as well as its location within the individual patient and contact addresses of the implanting hospital, but also want more specific information regarding diagnostic and therapeutic strategies in the case of implant-associated complications. Conclusion: The present article reports in detail on the conducted fax survey and shows some initial strategies as to how the identified challenges might be faced.

Primary haemostasis: newer insights.

At the same time as biophysical and omics approaches are drilling deeper into the molecular details of platelets and other blood cells, as well as their receptors and mechanisms of regulation, there is also an increasing awareness of the functional overlap between human vascular systems. Together, these studies are redefining the intricate networks linking haemostasis and thrombosis with inflammation, infectious disease, cancer/metastasis and other vascular pathophysiology. The focus of this state-of-the-art review is some of the newer advances relevant to primary haemostasis. Of particular interest, platelet-specific primary adhesion-signalling receptors and associated activation pathways control platelet function in flowing blood and provide molecular links to other systems. Platelet glycoprotein (GP)Ibα of the GPIb-IX-V complex and GPVI not only initiate platelet aggregation and thrombus formation by primary interactions with von Willebrand factor and collagen, respectively, but are also involved in coagulation, leucocyte engagement, bacterial or viral interactions, and are relevant as potential risk markers in a range of human diseases. Understanding these systems in unprecedented detail promises significant advances in evaluation of individual risk, in new diagnostic or therapeutic possibilities and in monitoring the response to drugs or other treatment.

[Cardiac myopathy due to overt hypothyroidism]. / Kardiale Myopathie bei manifester Hypothyreose.

HISTORY AND ADMISSION FINDINGS: A 51-year-old man presented with progressive tiredness, proximal muscle weakness, hair loss and weight gain for months. The patient showed mild pretibial myxedema and dry skin. Laboratory findings revealed strongly elevated cardiac enzymes as well as marked hypothyroidism. INVESTIGATIONS: The electrocardiogram, echocardiography, abdominal sonography and chest X-ray were unremarkable. Thyroid ultrasound demonstrated features of Hashimoto thyroiditis. TREATMENT AND COURSE: The findings supported the diagnosis of an overt hypothyroidism with myxedema and rhabdomyolysis. After starting levothyroxine and volume substitution laboratory parameters and clinical condition slowly normalized. CONCLUSION: Severe overt hypothyroidism may rarely present primarily as myopathy with myositis and cardiac involvement.

The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation.

BACKGROUND: Activation of the platelet-specific collagen receptor, glycoprotein (GP) VI, induces intracellular reactive oxygen species (ROS) production; however the relevance of ROS to GPVI-mediated platelet responses remains unclear. OBJECTIVE: The objective of this study was to explore the role of the ROS-producing NADPH oxidase (Nox)1 and 2 complexes in GPVI-dependent platelet activation and collagen-induced thrombus formation. METHODS AND RESULTS: ROS production was measured by quantitating changes in the oxidation-sensitive dye, H2DCF-DA, following platelet activation with the GPVI-specific agonist, collagen related peptide (CRP). Using a pharmacological inhibitor specific for Nox1, 2-acetylphenothiazine (ML171), and Nox2 deficient mice, we show that Nox1 is the key Nox homolog regulating GPVI-dependent ROS production. Nox1, but not Nox2, was essential for CRP-dependent thromboxane (Tx)A2 production, which was mediated in part through p38 MAPK signaling; while neither Nox1 nor Nox2 was significantly involved in regulating CRP-induced platelet aggregation/integrin αIIbß3 activation, platelet spreading, or dense granule and α-granule release (ATP release and P-selectin surface expression, respectively). Ex-vivo perfusion analysis of mouse whole blood revealed that both Nox1 and Nox2 were involved in collagen-mediated thrombus formation at arterial shear. CONCLUSION: Together these results demonstrate a novel role for Nox1 in regulating GPVI-induced ROS production, which is essential for optimal p38 activation and subsequent TxA2 production, providing an explanation for reduced thrombus formation following Nox1 inhibition.

Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site.

BACKGROUND: Ectodomain shedding of glycoprotein Ibα (GPIbα), a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. AIM: To achieve substrate-specific inhibition of GPIbα shedding. METHODS: Development of monoclonal antibodies that directly bind the sequence around the GPIbα shedding cleavage site and inhibit GPIbα shedding by blocking ADAM17 access to the cleavage site. RESULTS: Six anti-GPIbα monoclonal antibodies with varying binding affinities were obtained. The prototypic clone, designated 5G6, and its monomeric Fab fragment bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. The clone 5G6 showed similar inhibitory potency as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation. CONCLUSIONS: The clone 5G6 specifically inhibits GPIbα shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.

[Photodynamic therapy of bladder cancer. A new option]. / Die photodynamische Therapie des Harnblasenkarzinoms. Eine neue Option.

BACKGROUND: Bladder cancer (BCa) is the third most common tumor in Germany. Currently, resection therapy for superficial BCa (Ta, CIS) includes photodynamic diagnostics (PDD) using HEXVIX® for improved assessment of tumor spread. Trials using these photosensitizers for photodynamic therapy (PDT) showed only limited success. Especially low tissue penetration due to short-wave excitation was a limiting factor. METHODS: This study which was funded by the German Research Foundation (DFG) examined the feasibility of the novel photosensitizer tetrahydroporphyrin-tetratosylate (THPTS) for PDT in a rat bladder cancer model. RESULTS: As THPTS is very effectively excitable at a near infrared wavelength of 760 nm it is within the so-called phototherapeutic window and allows tissue penetration of up to 15 mm. Thus THPTS can also be used for PDT of larger, solid tumors as was previously demonstrated for other tumor entities. Therefore, effective treatment of even muscle-invasive bladder cancer (≥T2) may become an option using THPTS. In this current study the effectiveness and safety of THPTS-PDT was examined in an orthotopic bladder cancer rat model.

[Syncope--algorithms for emergency medicine]. / Synkope--Algorithmen in der Notfallmedizin.

Syncope is a common symptom in the emergency department. While most causes are benign and self-limiting not requiring extensive in-hospital evaluation, others are potentially severe. The optimal evaluation of patients with syncope follows a risk-adapted diagnostic algorithm in order to exclude life-threatening conditions and to identify those with high risk for further deterioration, such as structural heart diseases requiring further diagnostic evaluation. Low risk patients can be discharged without further extensive diagnostic work-up. This article presents an algorithm for the diagnostics of syncope in accordance with current guidelines.

ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways.

BACKGROUND: Ligation of the platelet-specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide-dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. OBJECTIVES: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor on platelets, FcγRIIa. METHODS AND RESULTS: Using an H(2) DCF-DA-based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen-related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet-rich plasma from 14 healthy donors displayed little inter-individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15-20 min. The Syk inhibitor BAY61-3606, which blocks ITAM-dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. CONCLUSIONS: Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation.

TNF receptor-associated factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets.

BACKGROUND: Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb-IX-V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear; however, the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown. OBJECTIVE: The objective of this study was to identify novel binding partners for GPIb-IX-V and GPVI that could provide a potential link between redox pathways and early platelet signaling events. METHODS AND RESULTS: Using protein array analysis and affinity-binding assays, we demonstrated that the orphan TNF receptor-associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbß and GPVI. TRAF4, p47(phox) [of the NADPH oxidase (Nox2) enzyme complex] and other redox relevant signaling proteins such as Hic-5, co-immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP-TRAF4 or MBP-p47(phox) fusion proteins specifically pull-down GPIb/GPVI. GPIb- or GPVI-selective agonists induce phosphorylation of the TRAF4-associated proteins, Hic-5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition. CONCLUSION: These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb-IX-V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2-dependent redox pathways.

Functional association of phosphoinositide-3-kinase with platelet glycoprotein Ibalpha, the major ligand-binding subunit of the glycoprotein Ib-IX-V complex.

BACKGROUND: The adhesion receptor glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet-mediated tumor metastasis. The cytoplasmic C-terminal domain of the ligand-binding GPIbalpha subunit contains binding sites for filamin (residues 561-572, critically Phe568/Trp570), 14-3-3zeta (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide-3-kinase (PI3-kinase) regulatory subunit, p85. OBJECTIVES: We previously showed that, as compared with wild-type receptor, deleting the contiguous sequence 580-590 or 591-610, but not upstream sequences, of GPIbalpha expressed as a GPIb-IX complex in Chinese hamster ovary cells inhibited VWF-dependent Akt phosphorylation, which is used as a read-out for PI3-kinase activity. Pulldown experiments using glutathione-S-transferase (GST)-p85 or GST-14-3-3zeta constructs, and competitive inhibitors of 14-3-3zeta binding, suggested an independent association of 14-3-3zeta and PI3-kinase with GPIbalpha. The objective of this study was to analyze a further panel of GPIbalpha deletion mutations within residues 580-610. RESULTS: We identified a novel deletion mutant, Delta591-595, that uniquely disrupts 14-3-3zeta binding but retains the functional p85/PI3-kinase association. Deletion of other sequences within the 580-610 region were less discriminatory, and either partially affected p85/PI3-kinase and 14-3-3zeta binding (Delta580-585, Delta586-590, Delta596-600, Delta601-605), or strongly inhibited binding of both proteins (Delta606-610). CONCLUSIONS: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14-3-3zeta in GPIb-dependent signaling or platelet functional studies involving truncation of the C-terminal residues in cell-based assays and mouse models. The Delta591-595 mutation provides another strategy for determining the function of GPIbalpha-associated 14-3-3zeta by selective disruption of 14-3-3zeta but not p85/PI3-kinase binding.

Presence of platelet-associated anti-glycoprotein (GP)VI autoantibodies and restoration of GPVI expression in patients with GPVI deficiency.

BACKGROUND: Glycoprotein (GP)VI deficiency is a rare platelet disorder with a mild bleeding tendency. However, its pathophysiology remains unclear. OBJECTIVES: We characterized a novel GPVI-deficient patient with immune thrombocytopenic purpura and searched for the presence of anti-GPVI autoantibodies in this and another patient with GPVI deficiency. METHODS AND RESULTS: A 12-year-old Japanese girl (case 1) with moderate thrombocytopenia and mild bleeding showed selectively impaired collagen-induced platelet aggregation. Flow cytometric analysis indicated that the patient had a defect in the expression of GPVI-FcRgamma. An eluate of her platelet-associated IgG contained anti-alpha(IIb)beta3 autoantibodies. Moreover, using GPVI-FcRgamma-transfected cells, we unexpectedly identified anti-GPVI antibodies against the soluble ectodomain of GPVI in the eluate, despite the patient's GPVI deficiency. In contrast, anti-GPVI antibodies were not detectable in her plasma. In another case of GPVI deficiency (case 2) without detectable plasma anti-GPVI antibodies, we again detected platelet-associated anti-GPVI antibodies. In a 2-year follow-up of case 1, the platelet count increased to within the normal range and the bleeding tendency improved. Interestingly, GPVI was again expressed on her platelets, in association with a decrease in the relative amount of anti-GPVI antibodies. CONCLUSIONS: This is the first demonstration of platelet-associated anti-GPVI antibodies in GPVI-deficient subjects, in one case with spontaneous restoration of GPVI expression. These results strongly suggest an autoimmune mechanism in GPVI deficiency.

TIRF microscopy evanescent field calibration using tilted fluorescent microtubules.

Total internal reflection fluorescence microscopy has become a powerful tool to study the dynamics of sub-cellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field, that is, the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Here, we present a novel method to quantitatively characterize the illumination in total internal reflection fluorescence microscopy using tilted, fluorescently labelled, microtubules. We find that the evanescent field is well described by a single exponential function, with a penetration depth close to theoretically predicted values. The use of in vitro reconstituted microtubules as nanoscale probes results in a minimal perturbation of the evanescent field; excitation light scattering is eliminated and the refractive index of the sample environment is unchanged. The presented method has the potential to provide a generic tool for in situ calibration of the evanescent field.