DEMONSTRATION OF PLATELET-DERIVED MICROVESICLES IN BLOOD FROM PATIENTS WITH ACTIVATED COAGULATION AND FIBRINOLYSIS USING A FILTRATION TECHNIQUE AND WESTERN BLOTTING

Platelet vesiculation in vitro is correlated to platelet activation. I t was therefore considered of interest to see if microvesicles (MV) ar e present in blood in clinical situations associated with platelet act ivation in vivo. Patients with both activated coagulation and fibrinol ysis, implying that thrombin has been generated, suit such a purpose. Thus, the aim of this study was to investigate whether microvesicles c ould be detected in patients with activated coagulation and fibrinolys is, as diagnosed by the presence of soluble fibrin (positive ethanol g elation tests) and positive tests for fibrin degradation products (FDP ). Platelet-rich plasma was prepared from citrated blood from patients (n = 22) and healthy controls (n = 32) matched as to age and sex. The intact platelets were removed from plasma by centrifugation. Any MV p resent were isolated from the platelet-free plasma by a filtration pro cedure, washed, solubilized in Triton X-100 and subjected to SDS-PAGE with Western blotting using a MAb against GPIIb alpha as an indicator of the presence of microvesicles. All of the 22 patients showed the pr esence of microvesicles detectable by the content of GPIIb alpha, wher eas this could be observed in only 4 out of the 32 normal controls and then in small or trace amounts only. The presence of microvesicles am ong cell-derived material in the plasma of two of the patients was als o confirmed by electron microscopy. To the best of our knowledge this is the first report on the presence of microvesicles in plasma from pa tients with both activated coagulation and fibrinolysis. Thus, the iso lation of the microvesicles by the filtration technique allowed studie s of the chemical composition of the microvesicles, and demonstrated t he presence of microvesicles in situations where platelets are expecte d to have been activated in vivo and a tendency to thrombosis exists.