Effect of different antibodies and inhibitors on platelet adhesion and [Ca2+]i elevations in platelets interacting with immobilized acid-soluble type I collagen under flow
| Condition . | Concentration . | Firmly adherent platelets, % . | [Ca2+]i elevations, % of control . | |
|---|---|---|---|---|
| α-like . | γ-like . | |||
| Anti-GP VI | 50 μg/ml | 14 ± 6* | 79 ± 11† | 0 |
| Anti-αIIbβ3 | 100 μg/ml | 88 ± 15 | 101 ± 22 | 105 ± 20 |
| Wortmannin (PI 3-K inhibitor) | 100 nM | 50 ± 6† | 0 | 0 |
| LY294002 (PI 3-K inhibitor) | 50 μM | 52 ± 11† | 0 | 0 |
| U73122 (phospholipase C inhibitor) | 21 μM | 10 ± 3* | 0 | 0 |
| PG E1 (increases cAMP) | 0.28 μM | 10 ± 2* | 0 | 0 |
| ASA (cyclooxygenase-1 inhibitor) | 400 μM | 81 ± 10 | 81 ± 6† | 87 ± 15 |
| MRS 2216+AR-C69931 (ADP receptors inhibitors) | 12.5 + 5 μM | 82 ± 9 | 102 ± 16 | 89 ± 14 |
| PP2 (Src-family kinases inhibitor) | 50 μM | 14 ± 5* | 0 | 0 |
| PP3 (Non-inhibitory PP2 analog) | 50 μM | 78 ± 12 | 91 ± 5 | 127 ± 26 |
| 8-Br-cGMP (cGMP analog) | 80 μM | 14 ± 5* | 0 | 0 |
| EGTA (Non membrane-permeable Ca2+ chelator) | 10 mM | 85 ± 14 | 110 ± 18 | 0 |
| BAPTA-AM (Membrane-permeable Ca2+ chelator) | 80 μM | 43 ± 16† | 0 | 0 |
| Condition . | Concentration . | Firmly adherent platelets, % . | [Ca2+]i elevations, % of control . | |
|---|---|---|---|---|
| α-like . | γ-like . | |||
| Anti-GP VI | 50 μg/ml | 14 ± 6* | 79 ± 11† | 0 |
| Anti-αIIbβ3 | 100 μg/ml | 88 ± 15 | 101 ± 22 | 105 ± 20 |
| Wortmannin (PI 3-K inhibitor) | 100 nM | 50 ± 6† | 0 | 0 |
| LY294002 (PI 3-K inhibitor) | 50 μM | 52 ± 11† | 0 | 0 |
| U73122 (phospholipase C inhibitor) | 21 μM | 10 ± 3* | 0 | 0 |
| PG E1 (increases cAMP) | 0.28 μM | 10 ± 2* | 0 | 0 |
| ASA (cyclooxygenase-1 inhibitor) | 400 μM | 81 ± 10 | 81 ± 6† | 87 ± 15 |
| MRS 2216+AR-C69931 (ADP receptors inhibitors) | 12.5 + 5 μM | 82 ± 9 | 102 ± 16 | 89 ± 14 |
| PP2 (Src-family kinases inhibitor) | 50 μM | 14 ± 5* | 0 | 0 |
| PP3 (Non-inhibitory PP2 analog) | 50 μM | 78 ± 12 | 91 ± 5 | 127 ± 26 |
| 8-Br-cGMP (cGMP analog) | 80 μM | 14 ± 5* | 0 | 0 |
| EGTA (Non membrane-permeable Ca2+ chelator) | 10 mM | 85 ± 14 | 110 ± 18 | 0 |
| BAPTA-AM (Membrane-permeable Ca2+ chelator) | 80 μM | 43 ± 16† | 0 | 0 |
Blood cell suspensions were prepared as described in the legend to Figure 1 and perfused over immobilized acid-soluble type I collagen at the wall shear rate of 250 s−1 without or with prior incubation for 10 minutes at 37°C with the indicated reagents. The frequency of platelets exhibiting different types of [Ca2+]i peaks was calculated as a fraction of the total number of platelets analyzed in the field of view during a 3-minute observation period and normalized to the corresponding value measured in untreated (control) platelets. Firmly adherent platelets were defined as those whose centroid was displaced by < 1 cell diameter over 30 seconds, and are reported here as percentage of all the platelets present in the field of view during the same observation period. All measurements were performed after perfusion for at least 120 seconds. In control experiments, 75% ± 11% of platelets analyzed established firm adhesion to collagen, 42% ± 7% exhibited α-like Ca2+ peaks, and 34% ± 6% exhibited γ-like peaks. All results are the mean ± 95% confidence intervals of measurements obtained analyzing 180 to 250 platelets in each of at least 3 separate experiments.
P < .01.
P < .05.