Alphanate

Manufactured by Grifols

Sourced in Spain

Alphanate is a plasma-derived factor VIII concentrate. It is used to treat and prevent bleeding episodes in patients with hemophilia A.

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Lab products found in correlation

Protein g sepharose chromatography, by GE Healthcare (1 mentions) Fanhdi, by Grifols (1 mentions) Humate p, by CSL Behring (1 mentions)

3 protocols using alphanate

Platelet Aggregation Assay Protocol

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Blood was collected from healthy volunteers recruited in accordance with the Georgia Tech Institutional Review Board. All subjects were older than 18 and had not taken any antiplatelet medications within 10 days prior to the blood draw. Subjects with known anemia, transmittable blood diseases, or bleeding disorders were excluded from the study. Blood was drawn into a 60 mL syringe containing 3.5 United States Pharmacopeia units per milliliter heparin using standard phlebotomy techniques. All controls occluded the channel in ∼240 s with ∼10% variability, implying a comparable soluble VWF (sVWF) level among all subjects. Immediately prior to testing, blood was diluted by adding phosphate-buffered saline or Alphanate (Grifols, S.A.) purified VWF solution at a ratio of 0.35 mL dilutant per 1 mL blood. Each 0.35 mL of Alphanate contains 95 IU VWF, raising the sVWF concentration to 20 times normal in 5 mL blood (normal concentration is estimated based on Casa et al³⁵). Dilution tests 0.1 times normal sVWF follows published protocol³⁵ without cell restoration as restoration does not affect SIPA under this concentration. Tests were run within 4 hours of collection.

Liu Z.L., Bresette C., Aidun C.K, & Ku D.N. (2022). SIPA in 10 milliseconds: VWF tentacles agglomerate and capture platelets under high shear. Blood Advances, 6(8), 2453-2465.

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FVIII and VWF Inhibitor Interactions

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The native VWF-complexed FVIII concentrates of plasma origin (pdFVIII/VWF) used in the study were Fanhdi® (Grifols, Barcelona, Spain) and Alphanate® (Grifols, Los Angeles, CA, USA). Since both products share an identical purification process, for assessments they were considered the same concentrate type. Both products contain an approximate 1:1 ratio between FVIII:C and VWF:RCo activities. The pdFVIII was a monoclonally purified product containing no, or very little, VWF 19 (link). The FVIII concentrates produced by a recombinant DNA technique (containing no VWF) were: a third generation full-length rFVIII, and a B-domain deleted rFVIII (BDD-rFVIII). The VWF was a commercially available plasma-derived VWF concentrate. The FVIII-deficient plasma (containing VWF) and normal pooled plasma were purchased from Diagnostic Grifols (Barcelona, Spain).
Inhibitor human IgG was purified from a commercial pool of haemophilic plasmas with inhibitors (Technoclone, Vienna, Austria) using protein G Sepharose chromatography (GE Healthcare, Uppsala, Sweden). Characterization of the pool performed in our laboratory showed the presence of antibodies against both light and heavy (A1–A2) chains. The Mab ESH-8, human anti-FVIII C2 domain antibody was obtained from American Diagnostica GbmH (Pfungstadt, Germany).

Bravo M.I., Da Rocha-Souto B., Grancha S, & Jorquera J.I. (2014). Native plasma-derived FVIII/VWF complex has lower sensitivity to FVIII inhibitors than the combination of isolated FVIII and VWF proteins. Impact on Bethesda assay titration of FVIII inhibitors. Haemophilia, 20(6), 905-911.

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Expression and Purification of Recombinant VWF Variants

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Wild type full-length VWF (amino acids M₁-K₂₈₁₃)
was expressed in HEK293 cells and cultured as described previously [56 (link), 57 (link)]. P1337L, V1316M and S1285F were inserted into full-length cDNA
of wild-type VWF within the mammalian expression vector pcDNA3 [57 (link)] by site-directed mutagenesis employing the
QuickChange kit (Stratagene). The VWF concentrates Wilate (Octapharma),
Koãte DVI (Bayer), Humate-P (CSL Behring) and Alphanate (Grifols) were
provided by the Mayo Clinic Special Coagulation Laboratory. All concentrates
were excessively dialyzed into PBS prior to usage. An SDS-PAGE gel of all four
concentrates can be found in the HXMS Supporting Information (Fig.S27). VWF was
quantified by fluorescence and the commercial REAADS antigen activity assay
(Diapharma¹) as
described in the LTMS
Supporting Information (Fig.S11).

Tischer A., Brehm M.A., Machha V.R., Moon-Tasson L., Benson L.M., Nelton K.J., Leger R.R., Obser T., Martinez-Vargas M., Whitten S.T., Chen D., Pruthi R.K., Bergen HR I.I.I., Cruz M.A., Schneppenheim R, & Auton M. (2019). Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease.. Journal of molecular biology, 432(2), 305-323.

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