A well-characterized in vitro model system composed of
thrombin-stimulated gel-filtered human platelets,
fibrin-(
ogen),
plasminogen, and recombinant
tissue plasminogen activator (rt-PA) was used to examine the relationship between platelet-
fibrin adhesive interactions and the lytic resistance of a platelet-rich
thrombus.
Laser light scattering kinetic experiments demonstrated that the
ligand-mimetic
peptide D-RGDW and an anti-alpha IIb beta 3
monoclonal antibody both inhibited clot retraction, but neither
integrin-targeted
reagent affected the overall delay in lysis of "bulk"
fibrin caused by
thrombin-stimulated platelets. However, lysis of the model platelet-rich
thrombus did proceed some 30% more quickly when treated with a
plasminogen activator inhibitor (PAI)-resistant
t-PA variant. Taken together, these results confirm that platelet-released
PAI-1 is a major determinant of global lytic resistance. Next events occurring during fibrinolysis in the unique microenvironment near the platelet surface were monitored by scanning electron microscopy and quantitative fluorescence microscopy. Scanning electron micrographs of the partially lysed model
thrombus in the presence of 200 mumol/L of
D-RGDW showed no platelet aggregates, and
fibrin was attached by fewer strands to the platelets. Quantitative fluorescence microscopy, using
fluorescein-labeled
fibrin, showed that
fibrin adherent to the surface of
thrombin-stimulated platelets lysed 20% to 50% more slowly than bulk
fibrin (monitored in parallel by
laser light scattering). Furthermore, this microspectroscopic technique showed that
D-RGDW reduced the quantity of platelet-bound
fibrin, and accelerated lysis near the platelet surface with both native rt-PA and the PAI-resistant variant. These observations suggest that the dense network of
fibrin bound to the platelet surface is protected from fibrinolysis by tissue-type
plasminogen activators. Further, uncoupling
fibrin from its platelet receptors uniquely hastens fibrinolysis at the cell/
fibrin interface.