Coexpression of the vWF propeptide increases binding affinity of ΔPro-vWF for factor VIII. COS-1 cells were transfected with ΔPro-vWF in the presence or absence of the vWF propeptide expression vector pMT₂-KRX. The resulting protein was evaluated by agarose gel electrophoresis under nonreducing conditions and transferred to nitrocellulose for detection as described in the Materials and Methods and shown in (A). Lane 1, plasma-derived vWF; lane 2, ΔPro-vWF coexpressed with the propeptide; lane 3, ΔPro-vWF without the propeptide. The bracket on the left indicates the distribution of vWF multimers in plasma. The arrow indicates the migration of the dimeric form of vWF. (B) Factor VIII binding as the fluorescence per Superose microsphere monitored by flow cytometry, measured as described for Fig 4. The solid fitted curve corresponds to a model with two classes of binding sites corresponding to lower affinity sites on non–disulfide-linked N-termini and higher affinity sites on disulfide-linked N-termini. The corresponding K_Ds were 0.77 and 6.5 nmol/L. The dashed curve corresponds to the same maximum fluorescence/microsphere but a single class of binding sites with intermediate affinity, K_D of 3 nmol/L.