Kinetic parameters of prothrombinase supported by activated platelets treated with RO318220
. | DMSO control . | . | RO318220, 20 μM . | . | RO318220, 50 μM . | . | RO318220, 100 μM . | . | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Agonist . | Maximum activity . | Kd app . | Maximum activity . | Kd app . | Maximum activity . | Kd app . | Maximum activity . | Kd app . | ||||
| No factor Va added | ||||||||||||
| IIa (0.2 U/mL) | 27.9 (0.95) | 0.11 (0.01) | 5.8 (0.44) | 0.14 (0.03) | 4.5 (0.53) | 0.38 (0.10) | — | — | ||||
| SFLLRN (50 μM) | 22.1 (0.72) | 0.12 (0.01) | 5.7 (0.31) | 0.10 (0.02) | 2.4 (0.10) | 0.09 (0.01) | — | — | ||||
| AYPGKF (1 mM) | 14.5 (0.65) | 0.15 (0.02) | — | — | 2.3 (0.17) | 0.11 (0.03) | — | — | ||||
| 2 nM factor Va added | ||||||||||||
| IIa (0.2 U/mL) | 62.3 (1.20) | 0.09 (0.006) | 38.4 (1.04) | 0.08 (0.008) | 51.6 (1.24) | 0.06 (0.006) | 185.3 (3.5) | 0.05 (0.004) | ||||
| SFLLRN (50 μM) | 83.8 (2.26) | 0.07 (0.007) | 54.9 (1.65) | 0.07 (0.007) | — | — | 197.8 (7.7) | 0.1 (0.01) | ||||
| SFLLRN (50 μM) | 57.4 (0.74) | 0.09 (0.004) | — | — | 36.1 (1.02) | 0.06 (0.007) | — | — | ||||
| AYPGKF (1 mM) | 47.9 (0.58) | 0.07 (0.003) | — | — | 52.7 (2.52) | 0.05 (0.01) | — | — | ||||
| 20 nM factor Va added | ||||||||||||
| IIa (0.2 U/mL) | 78.5 (1.74) | 0.04 (0.004) | 74.7 (0.72) | 0.03 (0.001) | — | — | — | — | ||||
| SFLLRN (50 μM) | 86.5 (1.48) | 0.03 (0.002) | 80.7 (1.91) | 0.03 (0.003) | — | — | — | — | ||||
. | DMSO control . | . | RO318220, 20 μM . | . | RO318220, 50 μM . | . | RO318220, 100 μM . | . | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Agonist . | Maximum activity . | Kd app . | Maximum activity . | Kd app . | Maximum activity . | Kd app . | Maximum activity . | Kd app . | ||||
| No factor Va added | ||||||||||||
| IIa (0.2 U/mL) | 27.9 (0.95) | 0.11 (0.01) | 5.8 (0.44) | 0.14 (0.03) | 4.5 (0.53) | 0.38 (0.10) | — | — | ||||
| SFLLRN (50 μM) | 22.1 (0.72) | 0.12 (0.01) | 5.7 (0.31) | 0.10 (0.02) | 2.4 (0.10) | 0.09 (0.01) | — | — | ||||
| AYPGKF (1 mM) | 14.5 (0.65) | 0.15 (0.02) | — | — | 2.3 (0.17) | 0.11 (0.03) | — | — | ||||
| 2 nM factor Va added | ||||||||||||
| IIa (0.2 U/mL) | 62.3 (1.20) | 0.09 (0.006) | 38.4 (1.04) | 0.08 (0.008) | 51.6 (1.24) | 0.06 (0.006) | 185.3 (3.5) | 0.05 (0.004) | ||||
| SFLLRN (50 μM) | 83.8 (2.26) | 0.07 (0.007) | 54.9 (1.65) | 0.07 (0.007) | — | — | 197.8 (7.7) | 0.1 (0.01) | ||||
| SFLLRN (50 μM) | 57.4 (0.74) | 0.09 (0.004) | — | — | 36.1 (1.02) | 0.06 (0.007) | — | — | ||||
| AYPGKF (1 mM) | 47.9 (0.58) | 0.07 (0.003) | — | — | 52.7 (2.52) | 0.05 (0.01) | — | — | ||||
| 20 nM factor Va added | ||||||||||||
| IIa (0.2 U/mL) | 78.5 (1.74) | 0.04 (0.004) | 74.7 (0.72) | 0.03 (0.001) | — | — | — | — | ||||
| SFLLRN (50 μM) | 86.5 (1.48) | 0.03 (0.002) | 80.7 (1.91) | 0.03 (0.003) | — | — | — | — | ||||
Gel-filtered, aspirin-treated platelets were incubated with DMSO or various concentrations of RO318220 before activation and assembly with prothrombinase components. Thrombin generation was measured and analyzed as described in “Materials and methods.” Mean maximum activity is measured as nM thrombin formed/min, mean Kd app (dissociation constants) in nM, and standard errors of the means are given in parentheses.
—indicates not assayed.