Functional assays of expressed recombinant VWF proteins
| Expressed VWF type . | Platelet binding (% AVW-1 bound) . | Collagen binding . | FVIII binding . | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Ristocetin . | % WT . | Botrocetin . | % WT . | No agonist . | Amount bound (mU/mL) . | % WT . | Amount rFVIII bound (μU) . | % WT . | |
| WT-VWF | 60.1 | 100 | 68.7 | 100 | 2.9 | 5.18 | 100 | 338.8 | 100 |
| Tyr87Ser-VWF | 10.1 | 16.8 | 66.8 | 97.2 | 2.8 | 1.17 | 22.6 | 124.1 | 36.6 |
| Mock | 6.4 | 10.6 | 5.8 | 8.4 | 1.9 | 0.07 | 1.4 | 14.7 | 4.3 |
| Arg854Gln-VWF (2N) | — | — | — | — | — | — | — | 88.5 | 26.1 |
| Expressed VWF type . | Platelet binding (% AVW-1 bound) . | Collagen binding . | FVIII binding . | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Ristocetin . | % WT . | Botrocetin . | % WT . | No agonist . | Amount bound (mU/mL) . | % WT . | Amount rFVIII bound (μU) . | % WT . | |
| WT-VWF | 60.1 | 100 | 68.7 | 100 | 2.9 | 5.18 | 100 | 338.8 | 100 |
| Tyr87Ser-VWF | 10.1 | 16.8 | 66.8 | 97.2 | 2.8 | 1.17 | 22.6 | 124.1 | 36.6 |
| Mock | 6.4 | 10.6 | 5.8 | 8.4 | 1.9 | 0.07 | 1.4 | 14.7 | 4.3 |
| Arg854Gln-VWF (2N) | — | — | — | — | — | — | — | 88.5 | 26.1 |
AII of the transfected VWF samples listed in the first column were analyzed simultaneously. While Tyr87Ser-VWF displayed normal botrocetin-induced platelet binding (97% of WT-VWF), this mutant Tyr87Ser-VWF had reduced ristocetin-induced platelet binding (17% of WT-VWF). VWF binding to collagen type III was also markedly reduced by mutant (dimeric) VWF. The FVIII binding of expressed mutant Tyr87Ser was compared with WT-VWF, Arg854Gln-VWF (type 2N VWD), and mock-conditioned media and found to be reduced.