Recombinant VWF D'D3, A1A2A3, A1, A2 and A3 all contained a 66His tag (NOVAGEN, San Diego, CA, USA) ( Figure 5A ). The preparation of VWF A1, A2 and A3 was described previously [26, 27, 31] . Plasmid construction, expression and purification of VWF A1A2A3 were similar to VWF A3 [27] . Two primers were used for amplifying VWF A1A2A3: 59-gga tcc GCA GGA GCC GGG AGG C-39 and 59-ctc gag TCC AGA GCA CAG TTT GTG-39 (Lowercase letters for BamHI and XhoI sites). Four recombinant VWF domains including A1A2A3, A1, A2 and A3 were all expressed in inclusion bodies. After purification, protein refolding was achieved using an 8 to 0 M urea linear gradient. Plasmids encoding A2-12, A2-23 (VWF114), A2-1, A2-2 and A2-3 (VWF73) ( Figure 5B ) were constructed similarly in the GST fusion vector pGEX-6P-1 (Amersham Biosciences, Piscataway, NJ). These recombinant plasmids were prepared by use of primers as follows: A2-12 (59-cgg gat cc GAC CTT GCC CCT GAA GCC CCT C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG ACC CTG GCT GAC CAA GAA GCT G-39), A2-23 (59-cgg gat cc GCA CAG TCC AAA GGG GAC ATC C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG CCT CTG CAG CAC CAG GTC AGG A-39), A2-1 (59-cgg gat cc GAC CTT GCC CCT GAA GCC CCT C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG CTC GCT GAA GGG GTA CTC CAC AG-39), A2-2 (59-cgg gat cc GCA CAG TCC AAA GGG GAC ATC C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG ACC CTG GCT GAC CAA GAA GCT G-39) and A2-3 (59-cgg gat cc GAC CGG GAG CAG GCG CCC AAC C-39 and 9-cgg aat tc TCA GTG ATG GTG ATG GTG ATG CCT CTG CAG CAC CAG GTC AGG A-39). Lowercase letters indicate added restriction enzyme sites (BamHI and HindIII) and the underlined sequence was the inserted C-terminal 66His-tag. The GST fusion proteins were expressed and purified by chromatography on Ni-NTA agarose (QIAGEN GmbH, Hilden, Germany) as described [21] . All five recombinant GST fusion proteins were mainly expressed in soluble fractions at low temperatures.