[Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide].

OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION: Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

Hyperadhesive von Willebrand Factor Promotes Extracellular Vesicle-Induced Angiogenesis: Implication for LVAD-Induced Bleeding.

Bleeding associated with left ventricular assist device (LVAD) implantation has been attributed to the loss of large von Willebrand factor (VWF) multimers to excessive cleavage by ADAMTS-13, but this mechanism is not fully supported by the current evidence. We analyzed VWF reactivity in longitudinal samples from LVAD patients and studied normal VWF and platelets exposed to high shear stress to show that VWF became hyperadhesive in LVAD patients to induce platelet microvesiculation. Platelet microvesicles activated endothelial cells, induced vascular permeability, and promoted angiogenesis in a VWF-dependent manner. Our findings suggest that LVAD-driven high shear stress primarily activates VWF, rather than inducing cleavage in the majority of patients.

vWFpp/ADAMTS13 ratio is a useful marker of postliver transplantation thrombotic microangiopathy: A pediatric case report.

vWFpp/ADAMTS13 ratio should be further studied as a useful marker for diagnosis of thrombotic microangiopathy postliver transplantation. Immunosuppressive regimen modification and plasma supplementation can lead to recovery.

The Plasma Levels of ADAMTS-13, von Willebrand Factor, VWFpp, and Fibrin-Related Markers in Patients With Systemic Sclerosis Having Thrombosis.

This study aimed to examine the hemostatic abnormalities in patients with systemic sclerosis (SSc) and the relationship between these abnormalities and thrombotic events (THEs), focusing on the difference in diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc (lcSSc). The plasma levels of ADAMTS-13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13), von Willebrand factor (VWF), VWF propeptide (VWFpp), d-dimer, and soluble fibrin (SF) were measured in 233 patients with SSc. The relationship between their levels and organ involvement, including THEs and interstitial lung disease (ILD), was evaluated. The plasma levels of VWF and VWFpp were significantly elevated and ADAMTS-13 activity was significantly decreased in patients with SSc compared to healthy participants. The VWFpp in dcSSc was significantly higher than in lcSSc. Twelve patients with SSc were complicated with acute THE, and 25 patients with SSc were complicated with past THE. The plasma levels of d-dimer and SF were significantly elevated in patients with SSc having THE. The plasma levels of VWF and VWFpp were significantly elevated in patients with SSc having ILD. The plasma levels of d-dimer were elevated in patients with SSc having other connective tissue diseases (CTDs). The plasma levels of ADAMTS-13 were significantly decreased and VWF, VWFpp, and SF were increased in patients with a d-dimer level of ≥1 µg/mL. Systemic sclerosis carries a high risk of THE, especially in patients with other CTDs. Plasma hemostasis-related markers are closely related to ILD and THE. These markers are important as markers of organ involvement as well as THE.

Evaluation of von Willebrand factor and von Willebrand factor propeptide in models of vascular endothelial cell activation, perturbation, and/or injury.

Pharmacologically, vasoactive agents targeting endothelial and/or smooth muscle cells (SMC) are known to cause acute drug-induced vascular injury (DIVI) and the resulting pathology is due to endothelial cell (EC) perturbation, activation, and/or injury. Alteration in EC structure and/or function may be a critical event in vascular injury and, therefore, evaluation of the circulatory kinetic profile and secretory pattern of EC-specific proteins such as VWF and VWFpp could serve as acute vascular injury biomarkers. In rat and dog models of DIVI, this profile was determined using pharmacologically diverse agents associated with functional stimulation/perturbation (DDAVP), pathological activation (lipopolysaccharide [LPS]/endotoxin), and structural damage (fenoldopam [FD], dopamine [DA], and potassium channel opener (PCO) ZD6169). In rats, FD caused moderate DIVI and time-related increase in plasma VWF levels ∼33% while in control rats VWF increased ∼5%. In dogs, VWF levels transiently increased ∼30% when there was morphologic evidence of DIVI by DA or ZD6169. However, in dogs, VWFpp increased >60-fold (LPS) and >6-fold (DDAVP), respectively. This was in comparison to smaller dynamic 1.38-fold (LPS) and 0.54-fold (DDAVP) increases seen in plasma VWF. Furthermore, DA was associated with a dose-dependent increase in plasma VWFpp. In summary, VWF and VWFpp can discriminate between physiological and pathological perturbation, activation, and injury to ECs.