-
Journal of Thrombosis and Haemostasis :... Dec 2018Essentials It is unclear whether there are differences between von Willebrand factor (VWF) activity assays. We compared the four most used VWF activity assays in 661 von... (Comparative Study)
Comparative Study
Essentials It is unclear whether there are differences between von Willebrand factor (VWF) activity assays. We compared the four most used VWF activity assays in 661 von Willebrand disease (VWD) patients. All assays correlated excellently, but a discrepant classification was seen in 20% of patients. Differences between VWF activity assays have a large impact on the classification of VWD. SUMMARY: Background Measuring the ability of von Willebrand factor (VWF) to bind to platelets is crucial for the diagnosis and classification of von Willebrand disease (VWD). Several assays that measure this VWF activity using different principles are available, but the clinical relevance of different assay principles is unclear. Objective To compare the four most widely used VWF activity assays in a large VWD patient population. Methods We measured VWF:RCo (ristocetin to activate VWF + whole platelets), VWF:GPIbR (ristocetin + platelet glycoprotein Ib receptor [GPIb] fragments), VWF:GPIbM (gain-of-function GPIb fragments that bind VWF spontaneously without ristocetin) and VWF:Ab (monoclonal antibody directed against the GPIb binding epitope of VWF to mimic platelets) in 661 VWD patients from the nationwide 'Willebrand in the Netherlands' (WiN) Study. Results All assays correlated excellently (Pearson r > 0.9), but discrepant results led to a different classification for up to one-fifth of VWD patients. VWF:RCo was not sensitive enough to classify 18% of patients and misclassified half of genotypic 2B VWD patients, especially those with p.Arg1306Trp. VWF:GPIbR was more sensitive, accurately classified the vast majority of patients, and was unaffected by the p.Asp1472His variant that causes artificially low VWF:RCo. VWF:GPIbM was the most precise assay but misclassified over a quarter of genotypic 2A, 2B and 3 patients. VWF:Ab, often not considered an actual VWF activity assay, performed at least equally to the other assays with regard to accurate VWD classification. Conclusion Although the different VWF activity assays are often considered similar, differences between assays have a large impact on the classification of VWD.
Topics: Adult; Biomarkers; Blood Platelets; Cross-Sectional Studies; Female; Hematologic Tests; Humans; Male; Middle Aged; Netherlands; Predictive Value of Tests; Protein Binding; Reproducibility of Results; von Willebrand Diseases; von Willebrand Factor
PubMed: 30358069
DOI: 10.1111/jth.14319 -
Biomedical Reports Jan 2017Platelet-derived polyphosphate has previously been indicated to induce coagulation. However, industrially synthesized polyphosphate has been found to have different...
Platelet-derived polyphosphate has previously been indicated to induce coagulation. However, industrially synthesized polyphosphate has been found to have different effects from those of the platelet-derived form. The present study investigated whether synthetic sodium polyphosphate inhibits coagulation using routine coagulation tests and thromboelastography. Synthetic polyphosphate was found to inhibit adenosine diphosphate-, epinephrine-, arachidonic acid-, ristocetin-, thrombin-, oxytocin- and pituitrin-induced platelet aggregation. The effects of synthetic polyphosphate in clotting inhibition were revealed by the analysis of clotting factor activity and platelet aggregation tests. Synthetic polyphosphate may inhibit platelet aggregation by reducing platelet calcium levels, as indicated by the results of flow cytometric analysis and high-throughput fluorescent screening. Furthermore, analysis of thromboxane (TX)B2 by ELISA indicated that synthetic polyphosphate reduces platelet aggregation by inhibiting the TXA2 signaling pathway. In conclusion, synthetic polyphosphate inhibits clotting factor activity and endogenous coagulation by reducing the levels of calcium ions and TXA2 to curb platelet aggregation.
PubMed: 28123708
DOI: 10.3892/br.2016.816 -
Haemophilia : the Official Journal of... May 2014The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory... (Review)
Review
The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.
Topics: Blood Coagulation Tests; Genetic Testing; Humans; Ristocetin; von Willebrand Diseases; von Willebrand Factor
PubMed: 24762278
DOI: 10.1111/hae.12410 -
ACS Infectious Diseases Jun 2020MurG (uridine diphosphate--acetylglucosamine/-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol -acetylglucosamine transferase) is an essential bacterial...
MurG (uridine diphosphate--acetylglucosamine/-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol -acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes the -acetylglucosamine (GlcNAc) transformation of lipid I to lipid II during peptidoglycan biosynthesis. Park's nucleotide has been a convenient biochemical tool to study the function of MraY (phospho-MurNAc-(pentapeptide) translocase) and MurG; however, no fluorescent probe has been developed to differentiate individual processes in the biotransformation of Park's nucleotide to lipid II via lipid I. Herein, we report a robust assay of MurG using either the membrane fraction of a strain or a thermostable MraY and MurG of as enzyme sources, along with Park's nucleotide or Park's nucleotide--C-dansylthiourea and uridine diphosphate (UDP)-GlcN-C-FITC as acceptor and donor substrates. Identification of both the MraY and MurG products can be performed simultaneously by HPLC in dual UV mode. Conveniently, the generated lipid II fluorescent analogue can also be quantitated via UV-Vis spectrometry without the separation of the unreacted lipid I derivative. The microplate-based assay reported here is amenable to high-throughput MurG screening. A preliminary screening of a collection of small molecules has demonstrated the robustness of the assays and resulted in rediscovery of ristocetin A as a strong antimycobacterial MurG and MraY inhibitor.
Topics: Anti-Bacterial Agents; Fluorescence; Glycosyltransferases; Transferases
PubMed: 31769280
DOI: 10.1021/acsinfecdis.9b00242 -
The Journal of Antibiotics Nov 1982Ristocetin-ps-aglycone obtained by acid hydrolysis of ristocetin A, has a substantially greater antimicrobial activity against Gram-positive bacteria than the parent...
Ristocetin-ps-aglycone obtained by acid hydrolysis of ristocetin A, has a substantially greater antimicrobial activity against Gram-positive bacteria than the parent compound. None of the substances are active against Gram-negative bacteria, yeast or fungi. The ps-aglycone is several times more toxic than ristocetin A when administered intravenously. Both substances are well-tolerated when given subcutaneously, intraperitoneally and perorally. Both ristocetin A and the ps-aglycone have a very low absorption after oral administration. Plasma levels following intravenous administration of ristocetin A and the ps-aglycone are comparable, with both showing a rapid decline during the first 60 minutes followed by a somewhat slower elimination. The aggregating properties of the ps-aglycone could not be determined due to its low solubility at neutral pH.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Fungi; Hydrogen-Ion Concentration; Hydrolysis; Lethal Dose 50; Male; Mice; Platelet Aggregation; Rats; Rats, Inbred Strains; Ristocetin
PubMed: 7161195
DOI: 10.7164/antibiotics.35.1561 -
American Journal of Hematology Feb 2015Bernard-Soulier syndrome (BSS) is a rare severe autosomal recessive bleeding disorder. To date heterozygous carriers of BSS mutations have not been shown to have...
Bernard-Soulier syndrome (BSS) is a rare severe autosomal recessive bleeding disorder. To date heterozygous carriers of BSS mutations have not been shown to have bleeding symptoms. We assessed bleeding using a semi-quantitative questionnaire, platelet parameters, PFA-100 closure times, ristocetin response, GP Ib/IX expression and VWF antigen in 14 BSS patients, 30 heterozygote carriers for related mutations and 29 controls. Eight mutations in GP1BA, GP1BB or GP9 were identified including four previously unknown pathogenic mutations. Subjects with BSS reported markedly more mucocutaneous bleeding than controls. Increased bleeding was also observed in heterozygotes. Compared to controls, patients with BSS had lower optical platelet counts (P < 0.001), CD61-platelet counts (P < 0.001) and higher mean platelet volume (17.7 vs. 7.8 fL, P < 0.001) and ristocetin response and closure times were unmeasurable. Heterozygotes had higher MPV (9.7 fL, P < 0.001) and lower platelet counts (P < 0.001) than controls but response to ristocetin and closure times were normal. The VWF was elevated in both BSS and in heterozygotes (P = 0.005). We conclude that heterozygotes for BSS mutations have lower platelet counts than controls and show a bleeding phenotype albeit much milder than in BSS. Both patients with BSS and heterozygote carriers of pathogenic mutations have raised VWF.
Topics: Adolescent; Adult; Aged; Bernard-Soulier Syndrome; Blood Coagulation; Blood Platelets; Child; Coagulants; Female; Gene Expression; Heterozygote; Homozygote; Humans; Male; Middle Aged; Mutation; Phenotype; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; Severity of Illness Index; Surveys and Questionnaires; von Willebrand Factor
PubMed: 25370924
DOI: 10.1002/ajh.23891 -
EJHaem Aug 2022A previously healthy 33-year-old female presented with a large hematoma over her right knee after kneeling. She was found to have pancytopenia and massive splenomegaly....
A previously healthy 33-year-old female presented with a large hematoma over her right knee after kneeling. She was found to have pancytopenia and massive splenomegaly. Von Willebrand Factor (VWF) antigen level was 0.38 units/ml, ristocetin cofactor activity 0.13 units/ml, and VWF multimeric distribution was normal. Bone marrow examination revealed an indolent B-cell lymphoma. Diagnosis was consistent with acquired von Willebrand syndrome as an autoimmune epiphenomenon of a lymphoma. Diagnostic and therapeutic splenectomy under hemostatic coverage was performed. VWF antigen levels and activities immediately normalized postoperatively and remained within the normal range several months later. Splenic pathology confirmed hairy cell leukemia with a BRAF mutation.
PubMed: 36051021
DOI: 10.1002/jha2.486 -
Organic & Biomolecular Chemistry Apr 2014Dalbavancin, a semi-synthetic glycopeptide with enhanced antibiotic activity compared to vancomycin and teicoplanin, binds to the C-terminal lysyl-d-alanyl-d-alanine...
Dalbavancin, a semi-synthetic glycopeptide with enhanced antibiotic activity compared to vancomycin and teicoplanin, binds to the C-terminal lysyl-d-alanyl-d-alanine subunit of Lipid II, inhibiting peptidoglycan biosynthesis. In this study, micro-calorimetry and electrospray ionization (ESI)-MS have been used to investigate the relationship between oligomerisation of dalbavancin and binding of a Lipid II peptide mimic, diacetyl-Lys-d-Ala-d-Ala (Ac2-Kaa). Dalbavancin dimerised strongly in an anti-cooperative manner with ligand-binding, as was the case for ristocetin A, but not for vancomycin and teicoplanin. Dalbavancin and ristocetin A both adopt an 'closed' conformation upon ligand binding, suggesting anti-cooperative dimerisation with ligand-binding may be a general feature of dalbavancin/ristocetin A-like glycopeptides. Understanding these effects may provide insight into design of novel dalbavancin derivatives with cooperative ligand-binding and dimerisation characteristics that could enhance antibiotic activity.
Topics: Anti-Bacterial Agents; Binding Sites; Calorimetry; Dimerization; Glycopeptides; Ligands; Models, Molecular; Molecular Conformation; Ristocetin; Solutions; Spectrometry, Mass, Electrospray Ionization; Teicoplanin
PubMed: 24608916
DOI: 10.1039/c3ob42428f -
Journal of Clinical Medicine Feb 2023Increased von Willebrand Factor (vWF) activity mediates platelet adhesion and might be a contributor to the development of thrombotic complications after surgery....
Effect of Supplemental Oxygen on von Willebrand Factor Activity and Ristocetin Cofactor Activity in Patients at Risk for Cardiovascular Complications Undergoing Moderate-to High-Risk Major Noncardiac Surgery-A Secondary Analysis of a Randomized Trial.
Increased von Willebrand Factor (vWF) activity mediates platelet adhesion and might be a contributor to the development of thrombotic complications after surgery. Although in vitro studies have shown that hyperoxia induces endovascular damage, the effect of perioperative supplemental oxygen as a possible trigger for increased vWF activity has not been investigated yet. We tested our primary hypothesis that the perioperative administration of 80% oxygen concentration increases postoperative vWF activity as compared to 30% oxygen concentration in patients at risk of cardiovascular complications undergoing major noncardiac surgery. A total of 260 patients were randomly assigned to receive 80% versus 30% oxygen throughout surgery and for two hours postoperatively. We assessed vWF activity and Ristocetin cofactor activity in all patients shortly before the induction of anesthesia, within two hours after surgery and on the first and third postoperative day. Patient characteristics were similar in both groups. We found no significant difference in vWF activity in the overall perioperative time course between both randomization groups. We observed significantly increased vWF activity in the overall study population throughout the postoperative time course. Perioperative supplemental oxygen showed no significant effect on postoperative vWF and Ristocetin cofactor activity in cardiac risk patients undergoing major noncardiac surgery. In conclusion, we found no significant influence of supplemental oxygen in patients undergoing major non-cardiac surgery on postoperative vWF activity and Ristocetin cofactor activity.
PubMed: 36769870
DOI: 10.3390/jcm12031222