11 research outputs found
GDP/GTP exchange factor MADD drives activation and recruitment of secretory Rab GTPases to Weibel-Palade bodies.
von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B, and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3B, and Rab3D activation upon MADD silencing. Rab activation, but not binding, was dependent on the differentially expressed in normal and neoplastic cells (DENN) domain of MADD, indicating the potential existence of 2 Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70 tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through previous activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but it did reduce VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator of VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs
Quantitative super-resolution imaging of platelet degranulation reveals differential release of VWF and VWF propeptide from alpha-granules
Background: Von Willebrand factor (VWF) and the VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body (WPB) exocytosis in endothelial cells, however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3D super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo we have studied alpha-granule cargo release following platelet activation.
Aims: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli.
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: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap than SPARC or fibrinogen. Activation of PAR-1 or GPVI signaling caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of VWF+ alpha-granules were retained, even at the highest agonist concentration used (20 ÎĽM PAR-1 ap). In contrast, the fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whilst SPARC and fibrinogen were still detected in 60-70%. 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: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules
Variable impact on mortality of AIDS-defining events diagnosed during combination antiretroviral therapy: not all AIDS-defining conditions are created equal.
Abstract
Background—The extent to which mortality differs following individual acquired
immunodeficiency syndrome (AIDS)–defining events (ADEs) has not been assessed among
patients initiating combination antiretroviral therapy.
Methods—We analyzed data from 31,620 patients with no prior ADEs who started combination
antiretroviral therapy. Cox proportional hazards models were used to estimate mortality hazard
ratios for each ADE that occurred in >50 patients, after stratification by cohort and adjustment for
sex, HIV transmission group, number of anti-retroviral drugs initiated, regimen, age, date of
starting combination antiretroviral therapy, and CD4+ cell count and HIV RNA load at initiation
of combination antiretroviral therapy. ADEs that occurred in <50 patients were grouped together
to form a “rare ADEs” category.
Results—During a median follow-up period of 43 months (interquartile range, 19–70 months),
2880 ADEs were diagnosed in 2262 patients; 1146 patients died. The most common ADEs were
esophageal candidiasis (in 360 patients), Pneumocystis jiroveci pneumonia (320 patients), and
Kaposi sarcoma (308 patients). The greatest mortality hazard ratio was associated with non-
Hodgkin’s lymphoma (hazard ratio, 17.59; 95% confidence interval, 13.84–22.35) and progressive
multifocal leukoencephalopathy (hazard ratio, 10.0; 95% confidence interval, 6.70–14.92). Three
groups of ADEs were identified on the basis of the ranked hazard ratios with bootstrapped
confidence intervals: severe (non-Hodgkin’s lymphoma and progressive multifocal
leukoencephalopathy [hazard ratio, 7.26; 95% confidence interval, 5.55–9.48]), moderate
(cryptococcosis, cerebral toxoplasmosis, AIDS dementia complex, disseminated Mycobacterium
avium complex, and rare ADEs [hazard ratio, 2.35; 95% confidence interval, 1.76–3.13]), and
mild (all other ADEs [hazard ratio, 1.47; 95% confidence interval, 1.08–2.00]).
Conclusions—In the combination antiretroviral therapy era, mortality rates subsequent to an
ADE depend on the specific diagnosis. The proposed classification of ADEs may be useful in
clinical end point trials, prognostic studies, and patient management
Variable impact on mortality of AIDS-defining events diagnosed during combination antiretroviral therapy: not all AIDS-defining conditions are created equal
Abstract
Background—The extent to which mortality differs following individual acquired
immunodeficiency syndrome (AIDS)–defining events (ADEs) has not been assessed among
patients initiating combination antiretroviral therapy.
Methods—We analyzed data from 31,620 patients with no prior ADEs who started combination
antiretroviral therapy. Cox proportional hazards models were used to estimate mortality hazard
ratios for each ADE that occurred in >50 patients, after stratification by cohort and adjustment for
sex, HIV transmission group, number of anti-retroviral drugs initiated, regimen, age, date of
starting combination antiretroviral therapy, and CD4+ cell count and HIV RNA load at initiation
of combination antiretroviral therapy. ADEs that occurred in <50 patients were grouped together
to form a “rare ADEs” category.
Results—During a median follow-up period of 43 months (interquartile range, 19–70 months),
2880 ADEs were diagnosed in 2262 patients; 1146 patients died. The most common ADEs were
esophageal candidiasis (in 360 patients), Pneumocystis jiroveci pneumonia (320 patients), and
Kaposi sarcoma (308 patients). The greatest mortality hazard ratio was associated with non-
Hodgkin’s lymphoma (hazard ratio, 17.59; 95% confidence interval, 13.84–22.35) and progressive
multifocal leukoencephalopathy (hazard ratio, 10.0; 95% confidence interval, 6.70–14.92). Three
groups of ADEs were identified on the basis of the ranked hazard ratios with bootstrapped
confidence intervals: severe (non-Hodgkin’s lymphoma and progressive multifocal
leukoencephalopathy [hazard ratio, 7.26; 95% confidence interval, 5.55–9.48]), moderate
(cryptococcosis, cerebral toxoplasmosis, AIDS dementia complex, disseminated Mycobacterium
avium complex, and rare ADEs [hazard ratio, 2.35; 95% confidence interval, 1.76–3.13]), and
mild (all other ADEs [hazard ratio, 1.47; 95% confidence interval, 1.08–2.00]).
Conclusions—In the combination antiretroviral therapy era, mortality rates subsequent to an
ADE depend on the specific diagnosis. The proposed classification of ADEs may be useful in
clinical end point trials, prognostic studies, and patient management