While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature

Abstractvon Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T → A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A → T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

Abstract-The von Willebrand factor (VWF) propeptide (VWFpp) is critical for the targeting of VWF multimers to storage granules. VWFpp alone efficiently navigates the storage pathway in AtT-20 and endothelial cells and chaperones mature VWF multimers to storage granules when the two proteins are expressed in cis or in trans. To further define the role of VWFpp in granular sorting, we examined its ability to sort an unrelated protein, C3␣ into the regulated secretory pathway.

Summary.  Background: The large von Willebrand factor (VWF) propeptide (VWFpp) plays a critical role in the multimerization and regulated storage of the mature VWF protein. Although our laboratory and others have identified mutations in von Willebrand disease patients that disrupt VWF multimerization, little is known about the affect of mutations on the regulated storage of VWF. Patients/Methods: We identified a heterozygous 18 base pair, in‐frame deletion in exon 12 of the VWF gene in a patient with an unusual, dimer‐intense multimer pattern. This deletion results in loss of amino acids 436–442 of VWFpp, which include one cysteine. Results: Through expression studies, we demonstrate reduced secretion, loss of VWF multimerization, and defective regulated storage of the variant VWF. The loss of VWF storage is secondary to loss of propeptide storage resulting from an apparently defective sorting signal on VWFpp. Suprisingly, coexpressed wild‐type VWF or VWFpp functioned in trans to partially restore multimerization of VWF from the variant allele. Conclusions: The deletion of six amino acids in VWFpp results in defects in VWF processing, regulated storage, and function. Although VWFpp may usually function in a homotypic fashion, acting on its own mature VWF subunit, VWFpp may retain the ability to function in trans on VWF expressed from the variant allele.

von Willebrand disease (VWD) is the most common inherited human bleeding disorder and is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWF is a secreted glycoprotein that circulates as large multimers. While reduced VWF is associated with bleeding, elevations in overall level or multimer size are implicated in thrombosis. The zebrafish is a powerful genetic model in which the hemostatic system is well conserved with mammals. The ability of this organism to generate thousands of offspring and its optical transparency make it unique and complementary to mammalian models of hemostasis. Previously, partial clones of zebrafish vwf have been identified, and some functional conservation has been demonstrated. In this paper we clone the complete zebrafish vwf cDNA and show that there is conservation of domain structure. Recombinant zebrafish Vwf forms large multimers and pseudo-Weibel-Palade bodies (WPBs) in cell culture. Larval expression is in the pharyngeal arches, yolk sac, and intestinal epithelium. These results provide a foundation for continued study of zebrafish Vwf that may further our understanding of the mechanisms of VWD.

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T → A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A → T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

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The deficiency of Factor VIII (FVIII) leads to hemophilia A, a severe X-linked bleeding disorder. To date, it is still unknown with certainty where FVIII is synthesized and how the storage pool of desmopressin acetate (DDAVP)-releasable FVIII is established. In vitro studies performed by our laboratory have shown that FVIII will store with VWF in endothelial cells if FVIII expression is induced by transfection or transduction. This FVIII is released by agonist stimulation. Furthermore, our studies have demonstrated that FVIII is not taken up from culture media by cells in culture and transfused FVIII does not re-establish the releasable pool in vivo. In this study, we generated 2 transgenic mouse lines in which human B-domain deleted FVIII (hFVIII) expression is under control of the endothelial-specific Tie 2 promoter (Tie2F8). This transgene was then bred into FVIII-deficient mice so that FVIII expression was restricted to endothelium. Functional FVIII activity (FVIII:C) was quantitated by a chromogenic assay. FVIII antigen (FVIII:Ag) and VWF antigen (VWF:Ag) were quantitated by ELISA. RT-PCR was used to analyze transcription of the transgene. Phenotypic correction was assessed by tail clip survival test, electrically-induced femoral venous thrombus formation, and ferric chloride induced carotid thrombus formation studies. The results demonstrated that the levels of FVIII in our transgenic mice were 559 ± 161 mU/ml in a higher expression line and 186 ± 31 mU/ml in a lower expression line. Tie2F8 transgene transcription was most abundant in lung and heart tissues. All transgenic mice survived tail clipping and achieved hemostasis within 6 hours. Both arterial and venous thrombi formation models demonstrated significant improvement of hemostasis at sites of injury. Confocal microscopy demonstrated recombinant FVIII stored together with VWF in endothelial cells, with especially high expression in the outlet branches of the aorta. Furthermore, this endothelial-specific expressed FVIII can be co-released together with VWF by epinephrine. The ratios of plasma FVIII:C and VWF:Ag post-epinephrine treatment to pre-epinephrine were determined, and the mean ± SEM were 1.68 ± 0.13 and 2.08 ± 0.34, respectively, at 30 minutes post-epinephrine treatment. Our results demonstrate that targeting human FVIII expression to endothelial cells corrects the murine hemophilia A phenotype and re-establishes a releasable pool of FVIII together with VWF.