Characterization of Factor VIII Related Protein Synthesized by Human Endothelial Cell: A Study of Structure and Function

Abstract

SummaryCrossed immunoelectrophoresis showed that factor VIII related protein (VIII R) synthesized in the human endothelial cell (EC-VIII R) had faster electrophoretic mobility than that secreted into the culture medium (MED-VIII R). Sodium dodecyl sulphate agarose gel electrophoresis followed by radioimmunofixation showed that EC-VIII R consisted of molecules varying from 0.26 × 106 to 3.76 × 106 daltons while MED-VIII R had molecules ranging from 0.93 × 106 to greater than 10 × 106 daltons, similar to that present in plasma. The smallest VIII R molecule present in normal plasma or spent culture medium (0.93 × 106 daltons) corresponded to a tetramer of subunits of 0.22-0.24 × 106 daltons. Only molecular forms greater than 3.76 × 106 daltons possessed ristocetin cofactor activity. Sonication (15 μ. amplitued for 30 secs) effectively broke the non-covalent bonds of the VIII R multimers resulting in smaller molecules. Thus endothelial cells in culture synthesized VIII R subunits and assembled them into the higher multimeric forms on secretion. Different types of von Willebrand disease could result from defects of either of the two processes.

Structural basis of von Willebrand factor binding to platelet glycoprotein Ib and collagen. Effects of disulfide reduction and limited proteolysis of polymeric von Willebrand factor.

“…Thus, heparin-vWF interactions may be important in polymerization of vWF from dimer into oligomers at the endothelial cell surface. Since the species and amount of extracellular matrix glycosaminoglycans can be influenced by cell culture conditions, the interaction ofvWF with certain glycosaminoglycans may explain the discrepancy in vWF polymer size in the medium ofcultured endothelial cells described by several laboratories (48)(49)(50). Lastly, the affinity of vWF for sulfated glycosaminoglycans like heparin may have no role in vWF-mediated platelet adhesion in vivo.…”

Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex.

“…Intracellularly, vWf appears to be present as a series of multimers with molecular weights equivalent to those of normal plasma, reaching at least 10 X 106 mol wt (16). Most of the protein, however, appears to be present in the dimeric (14) or tetrameric (16) form. vWf has demonstrated in Weibel-Palade bodies (17), although the significance of this subcellular localization is not well understood.…”

“…Studies of vWf synthesized in cultured human umbilical vein endothelium indicate processing that includes not only glycosylation but also cleavage of a 260,000-mol-wt precursor molecule into the 220,000-mol-wt vWf subunit (14). Intracellularly, vWf appears to be present as a series of multimers with molecular weights equivalent to those of normal plasma, reaching at least 10 X 106 mol wt (16). Most of the protein, however, appears to be present in the dimeric (14) or tetrameric (16) form.…”

“…vWf has demonstrated in Weibel-Palade bodies (17), although the significance of this subcellular localization is not well understood. vWf secreted into the culture fluid of endothelial cells possesses the multimeric conformations typical of plasma vWf (14,16).…”

Rickettsial interactions with human endothelial cells in vitro: adherence and entry

“…This is important because EC differ markedly from fibroblasts. EC have been reported to (i) be rapidly pinocytic (7); (ii) express a number of unusual cell surface antigens such as factor VIII components (8,12,13), ABO antigens (15), and antithrombin III (4); and (iii) produce hemostatic effectors such as prostaglandin Iv, (3,9,19,20), factor VIIIR:Ag (5,12,14), and factor VIIIvwF (13). Thus, studies on how rickettsiae initeract with EC are necessary not only to properly ascertain the specificity and mode of rickettsial entry into their primary target cell, but also to determine the molecular pathogenesis of the hemostatic and thrombotic perturbations which characterize typhus and spotted fevers.…”