Gelatin zymograms of cord blood and mature platelets from healthy individuals. Human platelets from peripheral blood and cord blood samples were obtained from the Blood Transfusion Center and the Gynecology Unit of the Hospital of Urbino. Umbilical cord blood samples were collected immediately after delivery from women with uncomplicated healthy pregnancies; peripheral blood samples were drawn from healthy volunteers. Washed platelets were freshly isolated according to CD45⁺ leukocytes depletion and lysis procedure detailed in Cecchetti et al¹  (40mM Tris-HCl, 0.3M NaCl, 1mM EDTA, 1mM Na₃VO₄, 1mM NaF, NaN₃ 0.05%, NP-40 1%, pH 7.4). After centrifugation (20 000g, 4°C for 20 minutes) to remove cellular debris, the supernates of MMP standards from whole cord blood and the cleared platelet lysates were analyzed through Western blotting (using monoclonal antibody against MMP-2 and MMP-9) and gelatin zymography.^5,10  Sample aliquots (containing 250 μg of total protein) were analyzed on 7.5% polyacrylamide gels containing 2 g/L gelatin 90 Bloom Type A from porcine skin. (A) Western blots of pro- and complexed forms of MMP-9, and proMMP-2 (lanes 1 and 2, respectively) recognized as gelatinases circulating in human cord blood from healthy subjects. Lane standard, cord blood lysates used as calibrator. (B) In lane 1, the MMP gelatinolytic activities in mature platelet lysates. All MMP forms activated by 1mM APMA are separated in lane 2, whereas the residual gelatinolytic activity of APMA-activated MMP in platelet lysates after the treatment with 1mM EDTA and orthovanadate is shown in lane 3. Lane standard, cord blood gelatinases.^5,10  Molecular masses (kDa) are indicated.