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Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules. This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C , which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty-acid -PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an 0-ester linkage.

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Isolated human syncytiotrophoblast microvillous plasma membranes (StMPM) have been examined by electron microscopy, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional PAGE (2D-PAGE), and immunoblots. Electron microscopy of StMPM pellets revealed populations of membrane-bounded vesicles that disrupted after treatment with the chaotrope 3M KCl for 16 hr; with increasing molarity of another chaotrope (NH4SCN), the vesicles became smaller and more homogeneous. NH4SCN treatment resulted in significant reduction on SDS and 2D-PAGE analysis of only one protein at 80kd, shown by immunoblotting to be transferrin; 3M KCl had little effect and appeared to be a poor chaotrope. Chromogenic silver staining of SDS-PAGE gels demonstrated over 50 StMPM-associated discrete protein components. Immunoblotting revealed transferrin (80kd), albumin (65kd), IgG heavy chain (56kd), and Gc protein (56kd). Alpha-2-macroglobulin (alpha 2M) was identified at 180kd and 95kd; the smaller component may be a proteolytic derivative indicating alpha 2M binding to a trophoblast surface protease. Numerous discrete protein dots, and groups of dots characteristic of charge heterogeneity of individual proteins, were observed on high resolution 2D-PAGE. The most intensely stained proteins were transferrin (80kd), albumin (65kd), placental-type alkaline phosphatase (66kd), and actin (46kd). This 2D-PAGE technique is a superior method for analyzing the trophoblast membrane proteins, and the system described will enable systematic mapping of these components.

Porcine factor VIII (FVIII; Hyate:C; Speywood Biopharm Ltd, UK) has been used since 1980 for the treatment both of patients with acquired haemophilia and those with congenital haemophilia and inhibitory antibodies. Each batch is extensively screened using cell-culture techniques to confirm the absence of viruses. The production process does not incorporate specific virucidal treatment steps, such as heat treatment or the addition of a solvent/detergent mixture. Low levels of porcine parvovirus were detected in some batches of the product in late 1996 and supply was suspended. In this retrospective study, sera from 81 recipients of porcine FVIII and 125 other volunteers were screened for evidence of antibodies against a range of porcine viruses: porcine parvovirus (PPV), encephalomyocarditis virus (EMCV), and porcine respiratory and reproductive syndrome virus (PRRSV). The 125 volunteer controls included subjects from six categories: healthy control subjects, pig abattoir personnel, personnel involved in the manufacture of porcine FVIII, recipients of porcine heparin, recipients of porcine insulin, and haemophiliacs treated only with human FVIII. No antibodies to PPV or PRRSV were detected in any subject. Four patients and two volunteers were found to have antibodies to EMCV, but this incidence is similar to that observed in the general population. In conclusion, there was no evidence of transmission of PPR or other marker porcine virus associated with the use of porcine FVIII concentrate (Hyate: C).