• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to be used as a protein substrate for ADAMTS13. >50% of rhvWF-A2 is cleaved as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Asp1498-Val1665, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  20 kDa

• SDS-PAGE

  18 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 2 mM CaCl₂, 0.01% (w/v) Brij-35, pH 8.5

• Recombinant Human ADAMTS13 (rhADAMTS13) (Catalog # )

• Recombinant Human vWF?A2 (rhvWF?A2) (Catalog # 2764-WF)

• SDS-PAGE/Silverstain Reagents or equivalent

1. Dilute rhvWF-A2 to 200 μg/mL in Assay Buffer.

2. Dilute rhADAMTS13 to 20 μg/mL in Assay Buffer.

3. Mix 25 μL of 200 μg/mL rhvWF-A2 with 25 μL of diluted rhADAMTS13. Prepare two Blanks by mixing 25 μL of 200 μg/mL rhvWF-A2 with 25 μL of Assay Buffer. 

4. Incubate the mixture for 2 hours at 37 °C. Incubate one blank at 37 °C for 2 hours while keeping the other Blank at -20 °C.

5. Stop the reaction by adding 15 μL of the reaction mixture to 15 μL of SDS-PAGE sample buffer. Also combine 15 μL of the blanks with 15 μL SDS-PAGE sample buffer.

6. Analyze the cleavage by SDS PAGE (Load 20 μL per lane) followed by silver staining (1 μg rhvWF-A2 per lane).

7. The cleavage products can also be analyzed by Western blot, loading 0.1 μg/lane of rhvWF-A2 and using one of R&D Systems antibodies ( or ).

Per Reaction:

• rhADAMTS13: 10 μg/mL (0.5 μg)

• rhvWF-A2: 100 μg/mL (5 μg)

Background: vWF-A2

von Willebrand Factor (vWF) is a large, multimeric glycoprotein made by endothelial cells and megakaryocytes. The pre-pro-vWF protein contains 2813 amino acids (aa), which consists of 22 aa signal peptide, 741 aa propeptide and mature vWF monomer of 2050 aa (1?4). The pro-vWF undergoes dimerization in the endoplasmic reticulum (ER) through C-terminal “cysteine-knot” (CK) domain. The pro-vWF dimmers are transported to Golgi and form multimers by forming disulfide bond in amino?terminal region of the mature form. The proteolytic processing of pro-region also occurs in Golgi. The matured vWF is stored in Weibel-Pallade bodies in endothelial cells and granules in megakaryocytes and platelets. The unusually-large vWF (ulvWF) multimers released from cells are very efficient in binding to platelets to form thrombus. The population of these highly active ulvWF multimers is controlled by a specific protease, ADAMTS13, which cleaves between residues Tyr1605 and Met1606 in the A2 domain of vWF. In the plasma, vWF appears as a series of large and intermediate multimers with molecular masses from several thousand to 500 kDa. vWF also performs hemostatic functions (3?5). In a high shear-stressed environment, vWF undergoes conformational change to expose a binding site for glycoprotein Ib alpha. As a result, vWF facilitates aggregation of platelets. In addition to platelet binding, vWF binds coagulation factor VIII to increase the lifetime of FVIII in plasma. The purified recombinant human vWF-A2 contains the A2 domain of vWF.

• References:

1. Sadler, J. E. (1998) Annu. Rev. Biochem. 67:395.

2. Ruggeri, Z. M. (2003) Cur. Opin. Hemat. 10:142.

3. Michiels, J. J. et al. (2006) Clin. Appl. Thromb. Hemost. 12:397.

4. Groot, E. et al. (2007) Cur. Opin. Hemat. 14:284.

5. Lenting, P. J. et al. (2007) J. Thromb. Haemos. 5:1353.

• Long Name:

  von Willebrand Factor A2 Domain

• Entrez Gene IDs:

  7450 (Human); 22371 (Mouse); 116669 (Rat)

• Alternate Names:

  F8VWF; VWD; VWF von Willebrand factor; VWF; vWFA2; vWF-A2