Correction of Various Abnormalities in Cultured Porcine von Willebrand Endothelial Cells by Inhibition of Plasminogen-Dependent Protease Activity

Abstract

AbstractCultured aortic endothelial cells (EC) from severe homozygous von Willebrand (vWd) pigs were compared to cultured normal pig EC at the same passage (passage 3, 5, 7, and 9). In vWd EC cultures, the quantity of immunologically identifiable (immunofluorescence staining), fibronectin-containing microfilaments was considerably decreased. The distribution and composition of the vWd subendothelial (SE) components was altered as indicated by quantitative polyacrylamide slab gel/autofluorography; total protein synthesis (incorporation of 14C-proline) and collagen synthesis (hydroxy 14C-proline-containing protein) was decreased and total protein and collagen turnover increased (two-fold). The interaction of washed pig platelets with vWd SE was decreased (1.27 × 103 ± 100 platelets/sq cm of SE for vWd versus 1.58 × 105 ± 1.32 × 104 platelets/sq cm of SE for normal) with no spread platelets associated with the vWd SE; and finally, plasminogen (Pmg) dependent protease activity was 2–3-fold higher (after 48 hr) than in normal EC cultures when assayed by growing cells directly in 12BI-labeled fibrin-coated chambers in the presence of added human Pmg. Short-term inhibition (3 passages) of this increased Pmg-dependent protease activity in vWd EC cultures with either 20% heat-deactivated Pmg-free fetal calf serum (FCS) or 20% heat-deactivated FCS plus 2 × 10–5M Trasylol resulted in the partial or complete correction of the structural and functional abnormalities in cultured vWd porcine ECs, described above. These inhibition data suggest that increased Pmg-dependent protease activity in cultured vWd porcine aortic ECs may be responsible, in part, for the expression of one or more of the SE-associated defects in porcine vWd ECs, as studied in a culture system, and may also play a role in the expression of the molecular defect(s) in porcine vWd in vivo.