The kinetics of thrombin- and SFLLRN-induced aggregation of human platelets in flow through tubes

The kinetics of aggregation of human platelets activated by alpha-thrombin (0.17-0.35 nM) and the hexapeptide SFLLRN (2-10 microM) was studied in plasma-free washed cell suspensions undergoing Poiseuille flow at 37 degrees C using a previously described double infusion technique. Platelet-rich Tyrodes, prepared from venous blood by multiple centrifugation, and agonist were rapidly mixed in a small chamber and the suspension flowed through various lengths of 1.19 and 0.76 mm diameter polyethylene tubing at mean transit times t from 0.2 to 43 s and mean tube shear rates = 41.9, 335, and 1335 s-1. Effluent was collected in 0.5% glutaraldehyde and single cells and aggregates in the volume range 1-10(5) micron 3 counted and sized using an aperture impedance counter. The rate and extent of aggregation with thrombin increased with increasing [thrombin] and , and although characterized by a small initial lag time, exhibited a very rapid growth of aggregates to macroscopic size, >> 10(5) micron 3, at low and moderate shear rates. With SFLLRN, the initial lag times were appreciably longer, but subsequently aggregates also rapidly grew to macroscopic size. We hypothesize that the initial lag time is due to the time required for sufficient secretion and surface organization of ligands such as vWF (known to be released by the platelet) to occur, in order for cross-bridging of the GPIIb-IIIa receptors on adjacent platelets to take place. It appears that thrombin, which, at the low concentrations used, primarily activates the platelet via binding to the GPIb alpha receptor, can more rapidly facilitate secretion of the ligand than SFLLRN, which activates the cell via binding to the seven transmembrane domain receptor.