To cite this article: Alb anez S, Ogiwara K, Michels A, Hopman W, Grabell J, James P, Lillicrap D. Aging and ABO blood type influence von Willebrand factor and factor VIII levels through interrelated mechanisms. J Thromb Haemost 2016; 14: 953-63. Essentialsvon Willebrand factor (VWF) and factor VIII (FVIII) levels are modulated by age and ABO status. The effect of aging and ABO blood type on VWF and FVIII was assessed in 207 normal individuals. Aging and ABO blood type showed combined and bidirectional influences on VWF and FVIII levels. Aging and ABO blood type influence VWF levels through both secretion and clearance mechanisms. Summary. Background:The effect of aging and ABO blood type on plasma levels of von Willebrand factor (VWF) and factor VIII (FVIII) have been widely reported; however, a comprehensive analysis of their combined effect has not been performed and the mechanisms responsible for the age-related changes have not been determined. Objectives: To assess the influence of aging and ABO blood type on VWF and FVIII levels, and to evaluate the contribution of VWF secretion and clearance to the age-related changes. Methods: A cross-sectional observational study was performed in a cohort of 207 normal individuals, whose levels of VWF, FVIII, VWF propeptide (VWFpp), VWFpp/VWF:Ag ratio and blood type A antigen content on VWF (A-VWF) were quantified. Results: Aging and ABO blood type exerted interrelated effects on VWF and FVIII plasma levels, because the age-related increase in both proteins was significantly higher in type non-O individuals (b = 0.011 vs. 0.005). This increase with age in non-O subjects drove the differences between blood types in VWF levels, as the mean difference increased from 0.13 U/mL in the young to 0.57 U/mL in the old. Moreover, A-VWF was associated with both VWF antigen (b = 0.29; 95% confidence interval [CI], 0.09, 0.50) and VWF clearance (b = À0.15; 95% CI, À0.25, À0.06). We also documented an effect of ABO blood type on VWF secretion with aging, as old individuals with blood type non-O showed higher levels of VWFpp (mean difference 0.29 U/mL). Conclusions: Aging and ABO blood type have an interrelated effect on VWF and FVIII levels, where the effect of one is significantly influenced by the presence of the other.

Essentials• Dysregulated DNA and histone release can promote pathological immunothrombosis.• Weibel-Palade bodies (WPBs) are sentinel-like organelles that respond to proinflammatory stimuli.• Histones induce WPB exocytosis in a caspase, calcium and charge-dependent mechanism.• A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis.Summary. Background: Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective: To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods:We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release in vitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions:Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

Haemophilia A is caused by mutations in the gene encoding coagulation factor VIII (FVIII). In severe Haemophilia A (sHA), two inversions are responsible for approximately 50% of the genetic alterations (intron 22 and intron 1 inversions). The other mutations are extremely diverse and each affected family generally has its own mutation. Our aim was to detect the genetic alterations present in the FVIII gene (F8) in 54 unrelated male patients with sHA in Venezuela. We initially detected the presence of the intron 22 inversion by performing inverse PCR, and the negative patients for this inversion were analysed for the intron 1 inversion by PCR. Patients negative for both inversions were analysed using Conformation Sensitive Gel Electrophoresis for mutations in all exons, promoter region and 3¢-UTR. sHA causative mutations were identified in 49 patients. Intron-22 and -1 inversions were detected in 41% and 0% of patients respectively. Besides these two mutations, 25 different mutations were identified, including nine nonsense, four small deletions, two small insertions, four missense, three splicing mutations and three large deletions. Seven novel mutations were identified, including two nonsense mutations, two small deletions, one small insertion, one missense mutation and one splicing mutation. Thirty one percent of the patients with identified mutations developed inhibitors against exogenous FVIII. This is the first report of F8 mutations in patients with sHA in Venezuela; the data from this study suggests that the spectrum of gene defects found in these patients is as heterogeneous as reported previously for other populations.

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

Background: Aging is associated with a state of hypercoagulability, as the result of increased concentrations of plasma coagulation proteins. Plasma levels of Factor VIII (FVIII) and von Willebrand factor (VWF) increase with age in humans, but the potential contribution of increases in gene expression with age has not been studied. These two proteins circulate in a non-covalent complex and are cleared together from plasma, hence, a reduction in the expression of their clearance receptors is also a possible pathogenetic explanation. In contrast, plasma levels of ADAMTS13 have been shown to be reduced in later life in humans, but again the mechanism responsible for this age-related pathophysiology is currently unknown. In this study, we utilized a mouse model in which age-related changes in plasma levels of FVIII, VWF and ADAMTS13 were initially documented. Here, we evaluated age-related changes in the gene expression of VWF, FVIII, ADAMTS13 and the clearance receptors low-density lipoprotein receptor-related protein 1 (LRP1), scavenger receptor class A member 5 (SCARA5) and Stabilin-2 (Stab2). Methods: Liver, spleen and lung samples were collected from normal C57BL/6 mice at 9- (n=10), 55- (n=8) and 97-weeks of age (n=15). Also, liver and spleen samples were collected at 3-weeks of age (n=5). Total mRNA was isolated from the tissues and gene expression analysis performed through qRT-PCR by a two-step relative quantification against mouse GAPDH. Expression of murine Factor IX (f9) and Protein C (proc) genes were also measured as positive and negative controls, as the developmental expression of these genes has been extensively studied. The 9-weeks old mice were used as a reference, and expression levels in this group were set as 1. Results were expressed as the fold change median and 95% CI from the 9 week standard group. Data was log10 transformed and compared with a Mann-Whitney test. Additionally, plasma levels of murine VWF, FVIII and ADAMTS13 were measured through ELISA, chromogenic assays and ELISA-based activity assays, respectively, in samples obtained at the same time-points examined for gene expression. Results: Levels of VWF in plasma showed significant increases with age (p<0.0001), reaching a 2-fold increase by 97-weeks. Expression levels increased gradually with age in all three tissues evaluated, reaching a 1.4-fold increase in the lungs (p=0.008), 1.8-fold in the spleen (p=0.01) and 10.3-fold in the liver (p<0.0001) of 97-weeks old mice. When FVIII plasma levels were measured, a similar age-related increase was observed (p<0.0001). Expression levels increased significantly with age in the lungs by 2-fold (1.53-2.68, p=0.002), but no specific age-related changes were observed in liver and spleen. Plasma levels of mouse ADAMTS13 activity showed an opposite pattern to what has been reported for the human protein, with an age-related increase (p<0.0001). When ADAMTS13 gene expression was analyzed in the liver, higher levels were observed in the 3-week old group [1.32 (1.25-1.41), p=0.04], but no significant changes in expression occurred at later time points. Finally, gene expression analysis of LRP1, SCARA5 and Stab2 genes was performed in liver and spleen, the two main organs involved in VWF/FVIII clearance. Expression of these three receptor genes was significantly reduced in both tissues at 3-weeks (<0.04 fold for all estimates). Expression of LRP1 in the liver was an exception to this pattern, with a level that was similar to the 9-week old mice [1.44 (0.96-2.17), p=0.77]. Interestingly, no Stab2 expression was detected in the liver at any point. With aging, no significant changes occurred in SCARA5 and LRP1 gene expression that could be associated with higher plasma levels of VWF/FVIII. However, splenic Stab2 expression significantly decreased with age, reaching a 0.18-fold (0.13-0.25, p=0.02) reduction in the 97-weeks old spleen samples. The positive control gene used (f9) showed no increases in expression with age [1.11 (1.00-1.23), p=0.60], possibly due to strain differences with reported studies, while the negative control gene proc showed no changes [0.87 (0.82-0.93), p=0.28], as expected. Conclusions: Changes in gene expression with increasing age appear to be contributing to the increases in VWF and FVIII plasma levels. Our studies have shown age-related increases in expression of the VWF and FVIII genes and reduced expression of the clearance receptor Stabilin-2. Disclosures No relevant conflicts of interest to declare.