Structural analysis of the von Willebrand factor gene located on chromosome 12 is complicated by the presence of a partial unprocessed pseudogene on chromosome 22q11-13. The structures of the von Willebrand factor pseudogene and corresponding segment of the gene were determined, and methods were developed for the rapid differentiation of von Willebrand factor gene and pseudogene sequences. The pseudogene is 21-29 kilobases in length and corresponds to 12 exons (exons 23-34) of the von Willebrand factor gene. Approximately 21 kilobases of the gene and pseudogene were sequenced, including the 5' boundary of the pseudogene. The 3' boundary of the pseudogene lies within an 8-kb region corresponding to intron 34 of the gene. The presence of splice site and nonsense mutations suggests that the pseudogene cannot yield functional transcripts. The pseudogene has diverged approximately 3.1% in nucleotide sequence from the gene. This suggests a recent evolutionary origin approximately 19-29 million years ago, near the time of divergence of humans and apes from monkeys. Several repetitive sequences were identified, including 4 Alu, one Line-1, and several short simple sequence repeats. Several of these simple repeats differ in length between the gene and pseudogene and provide useful markers for distinguishing these loci. Sequence differences between the gene and pseudogene were exploited to design oligonucleotide primers for use in the polymerase chain reaction to selectivity amplify sequences corresponding to exons 23-34 from either the von Willebrand factor gene or the pseudogene. This method is useful for the analysis of gene defects in patients with von Willebrand disease, without interference from homologous sequences in the pseudogene.

Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr 1605 -Met 1606 scissile bond in the VWF A2 domain depends on a Glu 1660 -Arg 1668 segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme-substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage Ϸ20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.enzyme kinetics ͉ thrombotic thrombocytopenic purpura ͉ fluid shear stress

Introductionvon Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays an essential role in tethering platelets at the site of vascular injury. VWF is synthesized in endothelial cells, where a portion is stored in granules called Weibel-Palade bodies as "unusually large" or "ultralarge" multimers (ULVWF) and secreted upon endothelial stimulation. 1,2 Secreted ULVWF multimers bind platelets with relatively high affinity and are thought to be prothrombotic. ULVWF is cleaved into smaller and less dangerous multimers by the metalloprotease ADAMTS13, a member of the A Disintegrin And Metalloprotease with ThromboSpondin type I repeat family. [3][4][5] Inherited or acquired deficiency of ADAMTS13 causes life-threatening microvascular thrombosis that is characteristic of thrombotic thrombocytopenic purpura. 4,6,7 Conversely, mutations in von Willebrand disease type 2A cause bleeding by increasing the cleavage of VWF by ADAMTS13 and impairing platelet adhesion. [8][9][10] Therefore, normal hemostasis depends on the precise regulation of VWF proteolysis.ADAMTS13 cleaves the Tyr 1605 -Met 1606 bond in the A2 domain of VWF, but this bond is buried and relatively inaccessible until the A2 domain is unfolded, presumably by tensile force in vivo. 10,11 The shear stress required to apply this force will vary depending on whether VWF is immobilized at the vessel wall or moving with the flowing blood, and whether platelets are bound to it. The rate of VWF cleavage also can be modulated by cofactors that bind to the A1 domain, including platelet GPIb and heparin. 12 Thus, ADAMTS13 is presented with VWF multimers in plasma or on endothelial cell surfaces that vary in their susceptibility to cleavage, with or without attached platelets.The relevance of each of these potential substrates to the catabolism of VWF is unknown. Several studies suggest that VWF strings on endothelial cells must be cleaved to inhibit thrombus growth, 13,14 but the role of proteolysis in the fluid phase has not been established. Therefore, the cleavage of VWF by ADAMTS13 was assessed in a cone-plate viscometer to minimize the contribution of surface interactions. The results indicate that proteolysis of fluid phase VWF-platelet complexes is likely to determine the steady state size distribution of circulating VWF multimers in vivo. Methods Recombinant ADAMTS13Full-length human ADAMTS13 with a C-terminal V5 tag was expressed in TRex 293 cells (Invitrogen) as described previously 15 and partially purified by anion exchange chromatography. In brief, conditioned medium containing recombinant ADAMTS13 was supplemented with proteinase inhibitors (0.1 mol/L D-Phe-Pro-Arg-chloromethane and 144 mol/L phenylmethylsulfonyl fluoride and applied to tandem columns of HiTrap Q Sepharose (2 ϫ 5 mL; GE Healthcare, Chalfont St Giles, United Kingdom). The columns were washed with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and developed with a linear gradient of 0 to 50 mM CaCl 2 in 20 mM Tris-HCl, pH 8.0, and 100 mM NaCl. Fractions containing ADAMTS13 were combined,...

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Deficiency of blood coagulation factor V or tissue factor causes the death of mouse embryos by 10.5 days of gestation, suggesting that part of the blood coagulation system is necessary for development. This function is proposed to require either generation of the serine protease thrombin and cell signaling through protease-activated receptors or an activity of tissue factor that is distinct from blood clotting. We find that murine deficiency of prothrombin clotting factor 2 (Cf2) was associated with the death of approximately 50% of Cf2 ؊/؊ embryos by embryonic day 10.5 (E10.5), and surviving embryos had characteristic defects in yolk sac vasculature. Most of the remaining Cf2 ؊/؊ embryos died by E15.5, but those surviving to E18.5 appeared normal. The rare Cf2 ؊/؊ neonates died of hemorrhage on the first postnatal day. These studies suggest that a part of the blood coagulation system is adapted to perform a developmental function. Other mouse models show that the absence of platelets or of fibrinogen does not cause fetal wastage. Therefore, the role of thrombin in development may be independent of its effects on blood coagulation and instead may involve signal transduction on cells other than platelets.

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys 2008 , Cys

ADAMTS13 belongs to the "a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH 2 -terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half-time of 7 h and the rate-limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro-ADAMTS13. Second, furin-deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro-ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both AD-AMTS13 and von Willebrand factor, pro-ADAMTS13 cleaved pro-von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.