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Lymphocytes from 18 patients with the Wiskott-Aldrich Syndrome (WAS) were examined by scanning electron microscopy (SEM). Most peripheral blood lymphocytes from normal individuals are covered with slender microvillus projections, but a large proportion of lymphocytes from WAS patients were found to be relatively devoid of microvilli. A lymphocyte morphology scoring system was developed to quantify the density of microvilli: Grade 4 classified those lymphocytes with >75% of the surface covered with microvilli with progressive decrements to grade 1, which were those without microvilli. The mean lymphocyte morphology score of eight normal individuals was 3.62 ± .22. The mean lymphocyte score of WAS patients was substantially lower (2.89 ± .27, P .001). In addition, WAS lymphocytes often were qualitatively abnormal, with short, blunted microvilli. These morphological criteria were used to diagnose WAS from the cord blood lymphocytes of one |at-risk” patient. Thus, WAS is the first primary immunodeficiency in which morphological abnormalities have been identified that can aid in diagnosis.

Detergent-insoluble, marginal microtubular band (MB) cytoskeletons were isolated from unstimulated blood platelets after pretreatment with glycerol or with Taxol. MB cytoskeletons retained the shape of intact platelets and behaved in suspension as coherent structural units. The major structural component was a continuous coil of long microtubule(s), often with granular/amorphous material present in the centre; few typical actin filaments were observed. The coiled microtubules often had an amorphous surface coating, but no discrete inter-microtubule bridges were seen. Tubulin and actin (identified by immunochemical staining) were major polypeptides. None of the minor (> 10) polypeptide components comigrated with high molecular weight microtubule-associated proteins in brain tubulin. A novel polypeptide, resolved by two-dimensional electrophoresis and designated IEF-51K, was present in MB cytoskeletons in amounts approximately equivalent to each of the tubulin polypeptides. Evidence suggests that IEF-51K is a distinct, previously undescribed component of the platelet cytoskeletal system.

Treatment of platelets (109cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane “ghosts” for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII-detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, <21.4 seconds or <25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)-electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue-and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohy-dride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnor-mal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti-human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 ± 0.6 (n = 26) as compared with 0.37 ± 0.34 (n = 26) for the abnormal PAT group (P <. 001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.