Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers

Diagnostic criteria for hematopoietic stem cell transplant-associated microangiopathy : results of a consensus process by an International Working Group

There are no widely accepted criteria for the definition of hematopoietic stem cell transplant -associated microangiopathy (TAM). An International Working Group was formed to develop a consensus formulation of criteria for diagnosing clinically significant TAM.The participants proposed a list of candidate criteria, selected those considered necessary, and ranked those considered optional to identify a core set of criteria. Three obligatory criteria and four optional criteria that ranked highest formed a core set. In an appropriateness panel process, the participants scored the diagnosis of 16 patient profiles as appropriate or not appropriate for TAM. Using the experts' ratings on the patient profiles as a gold standard, the sensitivity and specificity of 24 candidate definitions of the disorder developed from the core set of criteria were evaluated. A nominal group technique was used to facilitate consensus formation. The definition of TAM with the highest score formed the finalThe Working Group proposes that the diagnosis of TAM requires fulfilment of all of the following criteria: (i)4% schistocytes in blood; (ii) de novo, prolonged or progressive thrombocytopenia (platelet count50 x 109/L or 50% or greater reduction from previous counts); (iii) sudden and persistent increase in lactate dehydrogenase concentration; (iv) decrease in hemoglobin concentration or increased transfusion requirement; and (v) decrease in serum haptoglobin. The sensitivity and specificity of this definition exceed 80%.The Working Group recommends that the presented criteria of TAM be adopted in clinical use, especially in scientific trials

Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings

<div><p>The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of <i>F8</i> and <i>VWF</i> are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express <i>AVPR2</i> (vasopressin V2 receptor gene) and that <i>AVPR2</i> mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.</p></div

Thrombosis following desmopressin for uremic bleeding

An elderly patient with evidence of atherosclerosis and uremic bleeding diathesis developed two foci of cerebral thrombosis immediately after an infusion of desmopressin (DDAVP). Because large molecular weight multimers of von Willebrand factor (vWF) have been demonstrated to cause platelet aggregation under conditions of elevated fluid shear stress as occurs in atherosclerotic vessels, we investigated his plasma vWF at the time of the event and compared it to baseline values obtained 2 weeks later. Unusually large vWF multimers induced by the DDAVP infusion were present and likely contributed to the thrombotic process. Consequently, we believe DDAVP should be given with greater caution to patients with atherosclerosis

FVIII in HUVEC WPBs does not overlap with β-actin or Factor H.

<p>HUVECs were fixed and treated with Triton-X prior to staining with mouse monoclonal anti-FVIII plus donkey anti-mouse IgG AF-488 (green). This was followed by staining either with goat anti-β-actin plus chicken anti-goat IgG AF-647 (red) or with goat anti-Factor H plus chicken anti-goat IgG AF-647 (red). Merged images and the corresponding intensity scatter plots are: (A and C) FVIII and β-actin at 60×, N = 4; (B and D) FVIII and β-actin at 100×, N = 4; (E and G) FVIII and Factor H at 60×, N = 3; and (F and H) FVIII and Factor H at 100×, N = 3. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.</p

Fluorescent images of stained fibroblasts.

<p>Fibroblasts were stained with antibodies to Fibroblast Surface Protein (FSP), FVIII, and VWF plus relevant secondary antibodies, or with secondary detection antibodies alone. Images at 60× from each channel plus the merged image are shown. Cell nuclei were detected with DAPI (blue). (A-C) Fibroblasts were fixed and cell surfaces were stained with mouse anti-FSP plus goat anti-mouse IgM AF-488 (green). Cells were fixed again (to retain surface antibodies), and then treated with Triton-X for internal staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). (D-F) Fibroblasts were fixed and treated with Triton-X for internal staining. Cells were stained with mouse anti-FVIII plus goat anti-mouse IgG AF-647 (red), followed by staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green). (G-I) Fixed and Triton-X-treated Fibroblasts were stained with only the secondary fluorescently labeled antibodies used in panels D-F for FVIII and VWF detection. Cells were stained with goat anti-mouse IgG AF-647 (red), and then with chicken anti-rabbit IgG AF-488 (green). Images are representative of 3 experiments.</p

Quantification of <i>F8</i>, <i>VWF</i> and <i>AVPR2</i> gene expression levels in GMVECs, HUVECs and fibroblasts (relative to GMVEC levels).

<p>RNA was extracted from untreated GMVECs, HUVECs and fibroblasts maintained in serum-free medium for 24 hours. The mRNA levels of <i>F8</i>, VWF, and <i>AVPR2</i> were quantified relative to the mRNA levels in GMVECs after normalization to <i>GAPDH</i> in each PCR analysis. Data were collected from 4–7 separate RNA extractions using each cell type.</p><p>^aIndicates down-fold expression levels; 0.64 = 1.5 down-fold, 0.20 = 5 down-fold, and 5.1 (10⁻⁵) = 19,607 down-fold.</p><p>Quantification of <i>F8</i>, <i>VWF</i> and <i>AVPR2</i> gene expression levels in GMVECs, HUVECs and fibroblasts (relative to GMVEC levels).</p