Crossed 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.
Keywords
Human endothelial cells - Factor VIII related protein - Multimeric forms - Ristocetin cofactor
