IntroductionPlatelet plug formation at the site of vascular injury is initiated by von Willebrand factor (VWF) interacting with the subendothelial matrix, followed by its binding to platelet glycoprotein (GP) Ib 1,2 and subsequent platelet activation and aggregation. VWF is synthesized by endothelial cells and megakaryocytes, 3,4 and one of its main particular features is a polymer structure ranging in size from 500 000 to more than 20 million Dalton, 5 the largest forms being hemostatically the most efficient. 6 A broad range of values characterizes plasma VWF levels, which average around 10 g/mL. Acquired and inherited factors both modulate plasma VWF levels, and twin studies have demonstrated that 66% of all variations in plasma VWF are genetically determined, while 30% of them depend on ABO blood group, 7 O blood group individuals having plasma VWF levels 25% lower than non-O subjects. 8 ABO group genotyping shows that O 1 O 1 subjects have the lowest VWF levels, and non-O group individuals heterozygous for the O 1 allele have significantly lower VWF levels than AA, AB, or BB subjects. 9,10 Glycosylation accounts for 19% of VWF by weight, and ABO determinants identified on the N-linked oligosaccharide chains are part of this glycosylation process. 11,12 ABO groups are added to the N-linked glycan chains of VWF in the post-Golgi compartment of endothelial cells before VWF secretion, albeit with the variable contribution of the endothelial cells from different vascular beds. 13 The carbohydrate moiety plays an important part in VWF polymerization and function, 14 and also affects the liver-mediated clearance of VWF. In animal models, the removal of sialic acid has been shown to induce an increase in VWF clearance, 15 and the half-life of VWF is halved in mice characterized by an aberrantly glycosylated VWF (due to the absence of the enzyme ST3Gal-IV). 16 Moreover, recombinant VWF, lacking in carbohydrate, is cleared from the circulation faster than its glycosylated counterpart, 16 and posttranslational changes in VWF induced by Galgt2, aberrantly expressed in endothelial cells, lead to a 20-fold increase in VWF clearance. 17 ABO group determinants may also regulate the susceptibility of VWF to the proteolytic action of ADAMTS13, proteolysis being faster in the case of the O blood group. 18 A different susceptibility to cleavage by ADAMTS13 may thus be one of the ways in which ABO group affects VWF removal from the circulation and consequent VWF levels.Although the mechanisms behind ABO blood group and VWF levels have yet to be fully clarified, it has been clearly demonstrated that the effects are mediated by the ABO antigen structures on the N-linked oligosaccharide chains of circulating VWF, and particularly by H antigen expression. 19 Understanding these mechanisms is of clinical relevance: non-O individuals have been shown to carry a significantly greater risk of venous thromboembolism, ischemic heart disease, and peripheral vascular disease, 21-23 while the O blood group is much more common in von Willebra...

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [ 3 H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.M itochondria contain two separate potential pathways to provide dTTP for mitochondrial (mt) DNA replication ( Fig. 1): (i) deoxynucleotide transporters in the membrane introduce nucleotides from the cytosol (1, 2), and (ii) thymidine kinase 2 (TK2) in the mt matrix phosphorylates thymidine to dTMP (3-6), which is further phosphorylated by nucleotide kinases to dTTP. Preliminary evidence for a third pathway via an intra-mt ribonucleotide reductase (7) has not been followed up, nor could we confirm it. We do not further consider it here.The first pathway in Fig. 1 relies mainly on de novo synthesis of deoxyribonucleoside diphosphates by ribonucleotide reductase in the cytosol (8) and to a minor extent on the activity of the cytosolic thymidine kinase 1 (TK1) (9). Both enzymes are active only during the S phase of the cell cycle (10, 11). The first pathway is therefore absent from terminally differentiated cells. The second pathway in Fig. 1 uses thymidine imported from the extracellular milieu and is active also outside S phase because TK2 is not cell-cycle regulated. Regulation of this pathway may occur by an intra-mt substrate cycle involving TK2 and 5Ј(3Ј)-deoxyribonucleotidase 2 (dNT-2) (12), similar to the cytosolic substrate cycle between TK1 and deoxyribonucleotidase 1 (13). Several genetic diseases affecting mtDNA replication arise from malfunction of enzymes in either mt pathway (14, 15). Also, the genetic loss of the cytosolic thymidine phosphorylase results in mutatio...

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Three cytosolic and one plasma membrane-bound 5-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5-and 2(3)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease. Mitochondrial DNA synthesis occurs throughout the whole cell cycle, independent of nuclear DNA replication (1). It is catalyzed by a separate DNA polymerase that uses distinct 2Ј-deoxyribonucleoside 5Ј-triphosphate (dNTP) pools (2, 3), sequestered from cytosolic dNTPs by the mitochondrial membranes. What is the origin of mitochondrial dNTPs and how are pool sizes regulated? Despite considerable efforts, a mitochondrial ribonucleotide reductase has not been found, suggesting import of dNTPs or deoxyribonucleosides from the cytosol into mitochondria. dNTPs are synthesized by the cytosolic ribonucleotide reductase and can be imported directly by a permease of the mitochondrial membrane (4). Deoxyribonucleosides are derived from the extracellular fluid or by catabolism of dNTPs and, after import into mitochondria, phosphorylated by specific intramitochondrial deoxyribonucleoside kinases (5)(6)(7)(8).But what regulates the size of intramitochondrial dNTP pools? This is an important question, because it is well established for cytosolic dNTPs that pool imbalance is genotoxic (9) and can cause specific diseases. Thus, in some cases of hereditary severe immune deficiency, the accumulation of dATP (10, 11) or dGTP (12, 13) in the cytosol of blood cells results in the apoptotic destruction of B and͞or T cells. In a different autosomal recessive disease, neurogastrointestinal encephalomyopat...