Competition of tPA Binding to VSMCs
| Competitor . | Concentration . | Plasmin Generation (% of control) . | |
|---|---|---|---|
| . | . | High-Affinity Site . | Low-Affinity Site . |
| None | — | 100 | 100 |
| Ser478→Ala tPA | 1 μmol/L | 13 (±1) | ND |
| aPMSF-uPA | 1 μmol/L | 107 (±9) | 130 (±15) |
| PMSF-plasmin | 1 μmol/L | 98 (±6) | ND |
| RAP | 50 nmol/L | 130 (±15) | 125 (±17) |
| Anti-annexin II | 100 μg/mL | 97 (±7) | ND |
| Mannose | 25 mmol/L | 96 (±5) | 97 (±9) |
| Heparin | 50 μg/mL | 225 (±20)3-150 | 180 (±25) |
| Heparinase | 1 U/mL3-151 | 113 (±10) | 95 (±15) |
| Competitor . | Concentration . | Plasmin Generation (% of control) . | |
|---|---|---|---|
| . | . | High-Affinity Site . | Low-Affinity Site . |
| None | — | 100 | 100 |
| Ser478→Ala tPA | 1 μmol/L | 13 (±1) | ND |
| aPMSF-uPA | 1 μmol/L | 107 (±9) | 130 (±15) |
| PMSF-plasmin | 1 μmol/L | 98 (±6) | ND |
| RAP | 50 nmol/L | 130 (±15) | 125 (±17) |
| Anti-annexin II | 100 μg/mL | 97 (±7) | ND |
| Mannose | 25 mmol/L | 96 (±5) | 97 (±9) |
| Heparin | 50 μg/mL | 225 (±20)3-150 | 180 (±25) |
| Heparinase | 1 U/mL3-151 | 113 (±10) | 95 (±15) |
The effect of various potential competitors on tPA binding to the high- and low-affinity sites on VSMCs was determined at tPA concentrations of 10 nmol/L and 1 μmol/L and assay of plasmin generation in the absence and presence of 6-AHA, respectively. The binding of tPA was competed by preincubation of cells with the competitors for 10 minutes before further incubation with tPA as described in the legend to Fig 5. Data shown (mean ± SD of triplicate determinations) are expressed relative to the activity of tPA bound in the absence of competitor under the two experimental conditions.
Abbreviation: ND, not determined.
The apparent increase in tPA activity in the presence of heparin appears to be due to a direct potentiation of tPA activity as reported by others39 and was also observed if heparin was included in the plasminogen activation assay rather than in the preincubation with tPA.
Preincubation with heparinase for 60 minutes before washing and further incubation with tPA.