Its activity was reduced by approximately 50% with an energy load of 30 W, which is 5-fold lower than the energy used for platelet lysis. Using the highenergy Masitinib VEGFR/PDGFR inhibitor sonication protocol employed in previous reported studies, we found that platelet PAI-1 activity was reduced approximately 90%. Taken together, with the activity rates observed in the present study, one would expect sonication to reduce platelet PAI-1 activity to 7�C8%, i.e. to similar levels as reported in previous studies. The magnitude of the reduction in PAI-1 activity was similar when freezing/thawing was used for platelet lysis. However, whereas the reduced activity by sonication was independent of whether tPA was added before or after lysis, the Bortezomib clinical trial underestimation of activity by freezing/thawing could partially be prevented by adding tPA before lysis. Another common procedure for platelet disruption is to use detergents such as Triton X-100. However, it has been shown that Triton X-100 decreases the half-life of active PAI-1 markedly, and 0.2% Triton X-100 decrease the functional half-life of PAI-1 to less than 1 minute at 37uC. Therefore, also with such protocols it is crucial to add tPA before lysis. Since addition of Triton X-100 is not physiological and may facilitate the binding of tPA and PAI-1, we investigated if Triton X-100 affected the results of the Western blot analysis. However, when Triton X-100 was added to the platelets lysed by sonication and freezing/thawing no such enhancement was observed. A potential concern in the present study could have been that the procedure we used in some way could have reactivated PAI-1 although it was in fact inactive in vivo. In vitro PAI-1 can be reactivated by denaturants such as SDS, guanidine HCl, and urea, and it has also been suggested that negatively charged phospholipids exposed on the surface of activated platelets could reactivate PAI-1. On the other hand, it has been reported that SDS may cause dissociation of the tPA-PAI-1 complex. To rule out the possibility that our results were due to reactivation and/or dissociation of the tPA-PAI-1 complex formed, we performed a series of experiments both with and without SDS in the loading buffer before electrophoresis. However, these studies showed no detectable differences in PAI-1 activity whether SDS was present or not. This is in agreement with a previous study reporting that the SDS-activatable form of PAI-1 might not be present in human platelets. How, then, could the activity of PAI-1 be preserved for such a prolonged period of time in the platelet? A potential mechanism has been suggested by Lang and Schleef, who showed that platelets possess a unique mechanism for stabilization of active PAI-1, by packaging together with other large a-granule proteins in a calcium-dependent manner. Active PAI-1 in plasma is stabilized by binding to vitronectin which has also been detected in platelet a-granules. However, some studies have failed to detect the vitronectin-PAI-1 complex in platelets and it is therefore controversial whether vitronectin is the stabilizing factor of PAI-1 in platelets. This issue remains to be evaluated. From a clinical perspective, there is compelling evidence that platelet-derived PAI-1 has an important physiologic and pathophysiologic role in making platelet-rich blood clots resistant to both endogenous and pharmacological thrombolysis. Despite this, most previous studies have reported activity levels of platelet PAI-1 that are probably far too low to explain its putative functional role.