XylT (beta1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

Summary. Background: Clotting factor (F) VIII is an independent risk factor for primary and recurrent venous thromboembolism (VTE). The causes for high plasma FVIII levels are not fully understood, but an involvement of genetic factors has been demonstrated. A multifunctional endocytic receptor, lowdensity lipoprotein receptor-related protein 1 (LRP1), mediates cellular uptake and subsequent degradation of FVIII and may contribute to variations in FVIII levels. Objective: We assessed the association of a genetic variation of LRP1 (663C > T) with basal FVIII levels and the risk of venous thrombosis in a group of high-risk patients and in healthy controls. Patients and methods: One hundred and fifty-two patients with a history of recurrent VTE (median age 56 years, 47% women) were compared with 198 age-and sex-matched controls (median age 53 years, 50% women). The LRP1 663C > T genotype was analyzed by mutagenic separated polymerase chain reaction assay and heterozygosity was confirmed by sequence analysis. Results: LRP1 663C > T genotype distribution differed significantly between patients (663CC n ¼ 138, 663CT n ¼ 14) and controls (663CC n ¼ 190, 663CT n ¼ 8; P ¼ 0.048). In multivariable linear regression analysis including LRP1 663C > T, ABO blood group, von Willebrand factor antigen, C-reactive protein and age, LRP1 663CT was independently associated with FVIII activity (P ¼ 0.02). LRP1 663CT was also associated with increased odds for VTE following adjustment for blood group O, FV Leiden and the prothrombin variation 20210G > A in multivariate analysis (odds ratio 3.3, 95% CI 1.3-8.5). Conclusions: According to our data the LRP1 663C > T polymorphism influences plasma FVIII levels independently of blood group, C-reactive protein and von Willebrand factor and is significantly associated with the risk of VTE.

The angiotensin-converting enzyme (ACE) has been suggested to affect blood coagulation and fibrinolysis. Results from literature on the role of the frequent insertion/deletion (I/D) polymorphism in the ACE gene in venous thromboembolism (VTE) are controversial. Only limited data on ACE serum levels inVTE exist. We determined the ACE I/D polymorphism by genotyping and ACE serum levels by an enzymatic assay in 100 high-risk patients with objectively confirmed recurrentVTE and at least one event of an unprovoked deep venous thrombosis or pulmonary embolism. One hundred twenty-five age- and sex-matched healthy individuals served as controls. ACE genotype frequencies were not significantly different between patients (DD: 26.0%, ID: 52.0%, II: 22.0%) and controls (DD: 29.6%, ID: 44.8%, II: 25.6%; p = 0.56). Neither individuals with ACE DD genotype nor those with ACE ID genotype had a higher risk for VTE in comparison to those with ACE II genotype (odds ratio and [95% confidence interval]: 1.0 [0.5-2.1] and 1.4 [0.7-2.6], respectively). Serum ACE levels (U/l) did not differ between patients (median = 25.25, 25th -75th percentile: 20.20-33.70) and controls (24.20, 17.85-34.50, p = 0.49). In the total population involved in the study the ACE DD genotype (n = 63: 36.00 [26.40-43.00]) was associated with higher ACE levels than the ACE ID genotype (n = 108: 24.10 [19.80-31.48], p < 0.001) and the ACE II genotype (n = 54: 19.35 [15.00-22.95], p < 0.001). In conclusion, we found a significant association of the ACE I/D polymorphism with ACE serum levels. However, neither the serum levels nor the I/D genotype were associated with VTE.

Our data indicate that storage at 4 degrees C is accompanied by maintained mRNA levels. PLTs with intact mRNA levels and short reaction times in thrombelastography might be functionally superior to PLTs that are devoid of mRNA and show less augmented P-selectin surface expression. In therapeutic applications, that is, if PLTs are transfused to control acute bleeding, PLTs kept at 4 degrees C may be advantageous.

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