Site-specific analysis of von Willebrand factor O-glycosylation.

BACKGROUND: O-glycosylation of von Willebrand factor (VWF) affects many of its functions; however, there is currently no information on the occupancy of the 10 putative O-glycosylation sites. OBJECTIVES: The aim of this study was the site-specific analysis of VWF O-glycosylation. METHODS: Tryptic VWF-O-glycopeptides were isolated by lectin affinity chromatography and/or by reverse-phase high-performance liquid chromatography. Subsequently, the purified glycopeptides were analyzed by glycosidase digestion and mass spectrometry. RESULTS: We found that all 10 predicted O-glycosylation sites in VWF are occupied. The majority of the glycan structures on all glycosylation sites is represented by disialyl core 1 O-glycan. The presence of core 2 O-glycan was also confirmed; interestingly, this structure was not evenly distributed among all 10 glycosylation sites. Analysis of the glycopeptides flanking the A1 domain revealed that generally more core-2-type O-glycan was present on the C-terminal Cluster 2 glycopeptide (encompassing T(1468) , T(1477) , S(1486) and T(1487) ) compared with the N-terminal Cluster 1 glycopeptide (encompassing T(1248) , T(1255) , T(1256) and S(1263) ). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T(2298) in the C1 domain. In addition, we identify sulfation of core 2 O-glycans and the presence of the rare Tn antigen. CONCLUSIONS: This is the first study to describe the qualitative and semi-quantitative distribution of O-glycan structures on all 10 O-glycosylation sites, which will provide a valuable starting point for further studies exploring the functional and structural implications of O-glycosylation in VWF.

Desmopressin treatment improves platelet function under flow in patients with postoperative bleeding.

BACKGROUND: Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. OBJECTIVES: To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. PATIENTS/METHODS: Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3 µg kg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. RESULTS: Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factor VIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116 IU mL(-1) (interquartile range [IQR] 102-154 IU mL(-1) ) to a median of 160 IU mL(-1) (IQR 126-187 IU mL(-1) ) (P = 0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. In vitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. CONCLUSIONS: Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.