• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <0.01 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to inhibit Recombinant Human Coagulation Factor X (Catalog # ) cleavage of a fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH    ₂ (Catalog # ). The IC    ₅₀ value is<200 nM, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com  

• Source

  Chinese Hamster Ovary cell line, CHO-derived Leu22-Leu444, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Leu22

• Predicted Molecular Mass

  49 kDa

• SDS-PAGE

  60-70 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)

• Recombinant Human Serpin A10/ZPI (rhSerpin A10) (Catalog # 8115-SA)

• Recombinant Human Coagulation Factor X (rhFactor Xa) (Catalog # )

• Substrate:  Mca-RPKPVE-Nval-WRK(Dnp)-NH2 Fluorogenic MMP Substrate (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Prepare a curve of rhSerpin A10 in Assay Buffer. Make the following serial dilutions: 9000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 3.906 nM.  (Note:  High points may not be achievable due to the stock concentration of some lots.)

2. Dilute rhFactor Xa to 0.8 μg/mL in Assay Buffer.

3. Combine equal volumes of each point of the rhSerpin A10 curve with 0.8 μg/mL rhFactor Xa. Include an enzyme control containing equal volumes of Assay Buffer and rhFactor Xa.

4. Incubate reaction mixtures at room temperature for 30 minutes.

5. Dilute Substrate to 20 μM in Assay Buffer.

6. Load into a plate 50 μL of the diluted incubated mixtures, and start the reaction by adding 50 μL of Substrate.  Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate.

7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

8. Derive the 50% inhibiting concentration (IC₅₀) of rhSerpin A10 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.

9. The specific activity of rhFactor Xa at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rhSerpin A10: 2250, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91 and 0.977 nM.

• rhFactor Xa:  0.020 μg

• Substrate:  10 μM
  

Background: Serpin A10/ZPI

Protein Z-dependent Protease Inhibitor (ZPI), also known as SerpinA10 (SERine Proteinase INhibitor-clade A10) is a monomeric, secreted member of the A (or extracellular) clade within the serpin superfamily of protease inhibitors (1-4).  In general, members of this superfamily regulate multiple proteolytic cascades, and are particularly effective due to the fact that their inhibitory activities can be fine-tuned through the participation of discrete, non-serpin co-factors (4). Serpins are unusual in that they are one-time use, non-recyclable proteins whose native state is thermodynamically unstable. They act as non-physiologic substrates for enzymes that, once cleaved, form a covalent bond with the target enzyme, rendering it inactive (1, 2). ZPI is a hepatocyte-derived glycoprotein associated with the coagulation cascade (3, 5-7). Following vessel damage, underlying support collagen and fibroblasts are exposed to circulating plasma and platelets. This results in the activation of two coagulation pathways; an intrinsic pathway involving platelets, and an extrinsic pathway involving vascular stromal cells. Both pathways converge at the activation step for factor X, which converts prothrombin into thrombin, a prelude to the generation of fibrin. ZPI negatively regulates the activation state of two coagulation factor enzymes; factor XIa and factor Xa (5-7). Factor XI is unique to the intrinsic pathway, while factor X, as noted, is common to both pathways. Inhibition is most efficiently accomplished by ZPI binding to either factor Xa (with protein Z [PZ], calcium and phospholipids) or XIa (lacking non-heparin co?factors), precluding them from activating downstream zymogens. Binding is accompanied by serpin cleavage, but unlike a typical serpin, ZPI does not stay bound to enzyme; rather, it dissociates into a 4 kDa C-terminal fragment and a 68 kDa N-terminal sequence (6, 8, 9). Following dissociation, a second ZPI molecule is recruited, and the process repeated. In humans, the majority of ZPI circulates in a complex with PZ. PZ serves as an intermediary, bringing ZPI in contact with factor Xa or XIa on the surface of platelets (1, 10-12). Once cleaved, ZPI dissociates from PZ, and PZ is free to bind and present additional ZPI to Xa and XIa. Cell-surface heparan sulfate on endothelium is also reported to act as a scaffold for ZPI:Xa interactions (8, 13).  Human and mouse systems are not strictly analogous, as ZPI > PZ in human blood, while PZ>ZPI in mouse blood (11). Mature human ZPI is 423 amino acids (aa) in length (aa 22-444), and it contains a factor Xa cleavage site between Tyr408Ser409. Human and mouse ZPI share 71% aa sequence identity. Notably on SDS-PAGE, human ZPI runs about 72 kDa, while mouse and rat ZPI exhibit MWs of 91 kDa and 51 kDa, respectively (14).     

• References:

1. Chen, H. et al. (2013) Cardiovasc. Haematol. Disord. Drug Targets 13:99.

2. Law, R.H.P. et al. (2006) Genome Biol. 7:216.

3. Han, X. et al. (1999) Biochemistry 38:11073.

4. Corral, J. et al. (2007) Br. J. Haematol. 137:99.

5. Han, X. et al. (1998) Proc. Natl. Acad. Sci. USA 95:9250.

6. Han, X. et al. (2000) Blood 96:3049.

7. Broze, G.J. et al. (2001) Thromb. Haemost. 86:8.

8. Vasse, M. (2011) Hamostaseologie 31:155.

9. Huang, X. et al. (2012) Blood 120:1726.

10. Huang, X. et al. (2010) J. Biol. Chem. 285:20399.

11. Girard, T.J. et al. (2013) J. Thromb. Haemost. 11:375.

12. Wei, Z. et al. (2009) Blood 114:3662.

13. Huang, X. et al. (2011) J. Biol. Chem. 286:8740.

14. Zhang, J. & G.J. Broze (2001) Thromb. Haemost. 85:861.

• Long Name:

  Serpin Peptidase Inhibitor, Clade A, Member 10

• Entrez Gene IDs:

  51156 (Human); 217847 (Mouse); 171154 (Rat)

• Alternate Names:

  antitrypsin), member 10; member 10; PZI; PZIprotein Z-dependent protease inhibitor; serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase; Serpin A10; serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin); ZPI; ZPIPZ-dependent protease inhibitor