Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamicexpressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant AD-AMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model. IntroductionThe large multimeric glycoprotein VWF is critical to normal hemostasis through mediating platelet-subendothelial interactions as well as binding to platelets to support their aggregation at the site of endothelial damage. The disease phenotype of type 1 VWD is a mild to moderate quantitative reduction of supposedly functionally normal VWF, with plasma VWF levels between 5% and 50% of normal. 1 This disease can be caused by a wide array of defects including defective RNA or protein synthesis, premature protein degradation before cellular release, ineffective secretion, rapid plasma clearance, or a mutation that results in a null allele. 2 R1205H, the Vicenza mutation, has a relatively severe type 1 phenotype that involves accelerated VWF clearance. Often occurring with a second VWF variation, M740I, the Vicenza mutation shows a significant reduction in VWF antigen (VWF:Ag) to ϳ 0.15 U/mL, VWF Ristocetin Cofactor Activity (VWF:RCo) ϳ 0.20 U/mL, and Factor VIII levels Ͻ 0.30 U/mL, but maintains normal platelet VWF levels and function. [3][4][5][6] Patient bleeding scores, a marker of VWD severity, range between 2-17 (n ϭ 18), with a mean of 8 (bleeding score Ն 4 is positive). 1,7-9 Accelerated clearance of the mutant protein has been demonstrated via desmopressin (DDAVP) studies 3 and human recombinant protein infusion in the VWF knockout mouse, 10 as well as through high VWFpp/ VWF:Ag ratios, with observed ratios of 10 or greater for this indirect measurement of VWF clearance from the plasma. 4 R1205H VWF also often displays an increase in high molecular weight multimers along with occasional alteration in the typical multimer triplet band pattern, [3][4][5]11 and has been attributed to the rapid clearance of the protein and thus red...

Chronic kidney disease (CKD) patients are commonly treated with vitamin D analogs, such as calcitriol. Recent epidemiologic evidence revealed a significant interaction between vitamin D and magnesium, since an inverse relationship between vitamin D levels and mortality mainly occurs in patients with a high magnesium intake. The aim of the study was to assess the mechanisms involved by determining whether magnesium alone or combined with calcitriol treatments differentially impacts vascular calcification (VC) in male Sprague-Dawley rats with adenine-induced CKD. Treatment with moderate doses of calcitriol (80 mg/kg) suppressed parathyroid hormone to near or slightly below control levels. Given alone, this dose of calcitriol increased the prevalence of VC; however, when magnesium was given in combination, the severity of calcification was attenuated in the abdominal aorta (51% reduction), iliac (44%), and carotid arteries (46%) compared with CKD controls. The decreases in vascular calcium content were associated with a 20-50% increase in vascular magnesium. Calcitriol treatment alone significantly decreased TRPM7 protein (↓ to ∼11%), whereas the combination treatment increased both mRNA (1.7Â) and protein (6.8Â) expression compared with calcitriol alone. In summary, calcitriol increased VC in certain conditions, but magnesium prevented the reduction in TRPM7 and reduced the severity of VC, thereby increasing the bioavailable magnesium in the vascular microenvironment. These findings suggest that modifying the adverse effect profile of calcitriol with magnesium may be a plausible approach to benefiting the increasing number of CKD patients being prescribed calcitriol.

Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIb␣ binding. These "gain of function" mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant highmolecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8-to 10-week-old C57Bl6 VWF ؊/؊ mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride-induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chlorideinduced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD. IntroductionType 2B von Willebrand disease (2B VWD) is a qualitative variant of VWD, in which there is an increased affinity of the mutant von Willebrand factor (VWF) for platelet glycoprotein Ib␣ (GPIb␣). 1 Inherited in an autosomal-dominant manner, it arises as a result of missense mutations clustered within exon 28 of the VWF gene, the region that encodes the VWF A1 protein domain involved in the binding of VWF to GPIb␣. 1,2 The gain-of-function phenotype appears to arise through the destabilization of the A1 domain, mimicking the structural changes seen when immobilized VWF is activated through shear stress and allowing the binding of VWF to GPIb␣ in the absence of vascular injury. 3,4 The bleeding phenotype seen in 2B VWD patients probably arises through a multifactorial mechanism: (1) a decrease in plasma high-molecular-weight (HMW) multimers, (2) the occurrence of thrombocytopenia, and (3) the inability of platelets to interact with immobilized VWF at the site of vascular damage. 3 The thrombocytopenia and decrease in HMW multimers arise as a result of increased clearance of both platelets and VWF. 3 In addition, 2B VWF is more susceptible to ADAMTS13-mediated cleavage. 5 The VWF mutation database lists more than 50 reports of 24 different mutations leading to 2B VWD. 6 Of these, the mutations R1306W, V1316M, and R1341Q are the most common, having been reported 10, 9, and 7 times, respectively. 6 The recent study of Federici et al 7 of a cohort of 67 2B VWD patients showed a heterogeneous clinical presentation, dependent on the VWF A1 domain mutation. Of the 11 mutations present in this cohort, the V1316M mutation resulted in the most significant thro...

Patients with chronic kidney disease (CKD) have a markedly increased risk for developing cardiovascular disease. Nontraditional risk factors, such as increased phosphate retention, increased serum fibroblast growth factor 23 (FGF-23), and deficiencies in vitamins D and K metabolism, likely play key roles in the development of vascular calcification during CKD progression. Calcitriol [1,25-(OH)-D] is a key transcriptional regulator of matrix Gla protein, a vitamin K-dependent protein that inhibits vascular calcification. We hypothesized that calcitriol treatment would inhibit the development of vascular calcification and this inhibition would be dependent on vitamin K status in a rat model of CKD. Rats were treated with dietary adenine (0.25%) to induce CKD, with either 0, 20, or 80 ng/kg of calcitriol with low or high dietary vitamin K1 (0.2 or 100 mg/kg) for 7 weeks. Calcitriol at both lower (20 ng/kg) and moderate (80 ng/kg) doses increased the severity of vascular calcification, and contrary to our hypothesis this was not significantly improved by high dietary vitamin K1. Calcitriol had a dose-dependent effect on: 1) lowering serum parathyroid hormone, 2) increasing serum calcium, and 3) increasing serum FGF-23. Calcitriol treatment significantly increased aortic expression of the calcification genes and These data also implicate impaired vitamin D catabolism in CKD, which may contribute to the development of calcitriol toxicity and increased vascular calcification. The present findings demonstrate that in an adenine-induced rat model of CKD calcitriol treatment at doses as low as 20 ng/kg can increase the severity of vascular calcification regardless of vitamin K status.

Pathogenic accumulation of calcium (Ca) and phosphate (PO ) in vasculature is a sentinel of advancing cardiovascular disease in chronic kidney disease (CKD). This study sought to characterize acute distribution patterns of radiolabeled PO and Ca in cardiovascular tissues of rats with CKD (0.25% dietary adenine). The disposition of PO and Ca was assessed in blood and 36 tissues after a 10-minute intravenous infusion of one of the following: (i) PO pulse + tracer PO ; (ii) PO pulse + tracer Ca; or (iii) saline + tracer Ca in CKD and non-CKD animals. After the infusion, PO in blood was elevated (2.3× at 10 minutes, 3.5× at 30 minutes, p < 0.05) in CKD compared with non-CKD. In contrast, there was no difference in clearance of Ca from the blood. Compared with controls, CKD rats had a markedly increased PO incorporation in several tissues (skeletal muscle, 7.8×; heart, 5.5×), but accrual was most pronounced in the vasculature (24.8×). There was a significant, but smaller, increase in Ca accrual in the vasculature of CKD rats (1.25×), particularly in the calcified rat, in response to the acute phosphate load. Based on the pattern of tissue uptake of PO and Ca, this study revealed that an increase in circulating PO is an important stimulus for the accumulation of these minerals in vascular tissue in CKD. This response is further enhanced when vascular calcification is also present. The finding of enhanced vascular mineral deposition in response to an acute PO pulse provides evidence of significant tissue-specific susceptibility to calcification. © 2018 American Society for Bone and Mineral Research.