Whole-platelet homogenates from each subject were solubilized in denaturing buffer and boiled for 2 minutes. Aliquots with equal amounts of proteins (5 µg) were loaded on 12% polyacrylamide gels and subjected to 1-dimensional electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Patients' specimens were always run in duplicate or triplicate on the same gel with those of their respective controls. Proteins, thus resolved in bands, were electrophoretically transferred onto Hybond polyvinylidene difluoride membrane (Amersham International, Buckinghamshire, England) for 90 minutes at 100 V. Blots were then preincubated for 1 hour in Tris buffered saline-Tween 20 buffer, pH 7.6 (Tris hydrochloride, 25 mmol/L; sodium chloride, 137 mmol/L; and 0.1% Tween 20), containing 1% bovine serum albumin (blocking buffer). All incubations were performed at room temperature. After a thorough washing with TBS-T, the blots were incubated in blocking buffer for 90 minutes with monoclonal antibodies against the regulatory type I (Transduction Laboratories, Lexington, Ky; dilution 1:500), the regulatory type II (Biomol, Plymouth Meeting, Pa; dilution 1:500), and the catalytic (Transduction Laboratories; dilution 1:500) subunits of PKA, Rap1 (Transduction Laboratories; dilution 1:2500), and actin (clone DC40, Sigma-Aldrich Corp, St Louis, Mo; dilution 1:3500). After being extensively washed with TBS-T, the blots were incubated with the second antibody, horseradish peroxidase–linked antimouse (Sigma-Aldrich Corp; dilution 1:1000), in blocking buffer for 1 hour. The labeled blots were then washed with TBS-T, and immunoreactivity was detected with the Western blot detection system (Enhanced Chemiluminescent, Amersham International), followed by exposure to film. Quantitation of the immunoblots was performed by densitometric scanning of the autoradiograms using an image analysis system (National Institutes of Health Image, Version 1.47, Bethesda, Md). Not all patients and controls had assays for the various antibodies because a satisfactory amount of platelet proteins was not obtained for each subject.