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SummaryHuman umbilical vein endothelial cells (HUVEC) are inducible for tissue factor (TF) activity in culture. Based on experiments using ECGF (4–20 µg/ml) with heparin (90 µg/ml), we obtained the followingresults: 1) In confluent HUVEC cultures, ECGF had essentially no influence on the levels of inducible TF. 2) In growing HUVEC cultures, ECGF reduced the TF response shortly after seeding but full response was regained when cells were kept confluent for 2–3 days. 3) Although secondary cultures responded best to TF induction in the absence of ECGF, the response was essentially equal over at least 8 passages in the prcgcncc of ECGF 1) Of total cellular TF induced in HUVEC, about 25% was available on the surface, and less than 4% was released with theshed plasma membrane vesicles. The proportion of total TF a>ctivity available on the surface of mtact cells wasnot influenced by the presence of ECGF 5) T½, for the decay of TF activity induced was 8.3—9.5 h, whereas in HUVEC when protein synthesis was blocked afterIF induction a T½ of about 30 h was found.

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

More observations indicate that the hydrophilic protein glycocalicin found in soluble fractions after homogenization of platelets in the presence of calcium ions, exists in a different molecular form on the intact platelet. Thus, SDS-PAGE of SDS-dissolved whole platelets show glycocalicin to be derived from a more complex protein than the solubilized protein itself. Crossed immunoelectrophoresis of Triton x-100 extracts of platelets revealed anti-glycocalicin-reactive material existing as two components with different eloctrophoretic mobilities and with the fast-moving one corresponding to highly purified water-soluble glycocalicin. Crossed hydrophobic interaction immunoelectrophoresis confirmed the hydrophilic nature of this component, whereas the slow-moving component showed the characteristic retardation of an amphiphilic membrane protein. Glycocalicin is solubilized during freezing and thawing of platelets in buffer, immunoquantitation (Laurell) of solubilized glycocalicin showed that EDTA(3,6mM), NEM (4.0mM) and IAA(4.0mM)reduced solubilization by 87%, 65% and 57%, respectively, and also inhibited calcium-dependent protease of platelet homogenatos indicating that the solubilization may be enzyme-mediated.

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