A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.
By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking 0-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in 0-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein lb on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for 0-linked carbohydrates in the vWf-glycoprotein lb interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetindependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease. (J. Clin. Invest. 1992Invest. . 90:2258Invest. -2267
The adhesion molecule L-selectin (CD62L) is rapidly shed from the plasma membrane during leukocyte activation as a result of proteolytic cleavage between Lys321 and Ser322 within the extracellular domain. L-selectin is also down-modulated from the surface in response to cross-linking, possibly through a similar mechanism. To further characterize the mechanism of down-modulation, several L-selectin mutants were generated and transfected into COS cells. Wild-type L-selectin as well as mutants with one or two amino acid substitutions at the cleavage site were nearly quantitatively shed into the culture supernatant. However, mutants in which a nine-amino acid stretch that included the protease-sensitive site was either deleted or replaced with a polyglycine spacer or a comparable region of E-selectin were retained on the cell surface and not detected in the supernatant. These results are consistent with other reports describing protease resistant L-selectin mutants. We also demonstrate that when expressed in L1-2 pre-B cells, the L-selectin nine-amino acid deletion mutant (321del.9), but not wild-type L-selectin, is resistant to down-regulation induced by PMA. However, both wild-type and mutant 321del.9 are completely lost from the cell surface in response to cross-linking with an L-selectin Ab. PMA-induced- but not L-selectin cross-linking-induced down-modulation was inhibited by staurosporine. These data are consistent with the idea that the L-selectin protease(s) can tolerate minor structural alterations at the cleavage site, and that L-selectin down-modulation can be induced by more than one mechanism, at least one of which (cross-linking) is protein kinase C independent.
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