An Experimental Model to Study the in Vivo Survival of von Willebrand Factor

Abstract

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t½α = 13 min) and a slow secondary phase (t½β = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D′-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D′-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D′-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D′-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide. To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t½α = 13 min) and a slow secondary phase (t½β = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D′-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D′-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D′-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D′-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide. von Willebrand factor (VWF) 1The abbreviations used are: VWF, von Willebrand factor; FVIII, Factor VIII; GpIbα, glycoprotein Ibα; pd-VWF, plasma-derived VWF; SPR, surface plasmon resonance; VWD, von Willebrand disease; wt-VWF, wild-type VWF; MRT, mean residence time. is a multimeric plasma protein that participates in the hemostatic process. The absence of functional VWF is associated with an abnormal bleeding tendency known as von Willebrand Disease (VWD) (1Budde U. Schneppenheim R. Rev. Clin. Exp. Hematol. 2001; 5: 335-368Crossref PubMed Scopus (56) Google Scholar, 2Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1138) Google Scholar). VWF contributes to hemostasis in a dual manner. First, it promotes the adhesion of platelets at sites of vascular injury by acting as a molecular bridge between the sub-endothelial collagen matrix and the platelet-surface receptor complex glycoprotein (Gp) Ibα/IX/V (3Ruggeri Z.M. J. Thromb. Haemost. 2003; 1: 1335-1342Crossref PubMed Scopus (361) Google Scholar). In addition, VWF serves as a carrier protein for coagulation factor VIII (FVIII) in the circulation. This chaperone function results in stabilization of the FVIII heterodimeric structure (4Weiss H.J. Sussman I.I. Hoyer L.W. J. Clin. Investig. 1977; 60: 390-404Crossref PubMed Scopus (330) Google Scholar) and protection of FVIII from premature clearance by the low density lipoprotein receptor-related protein (5Lenting P.J. Neels J.G. van den Berg B.M. Clijsters P.P. Meijerman D.W. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.J. J. Biol. Chem. 1999; 274: 23734-23739Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 6Schwarz H.P. Lenting P.J. Binder B. Mihaly J. Denis C. Dorner F. Turecek P.L. Blood. 2000; 95: 1703-1708Crossref PubMed Google Scholar). VWF is produced and stored in endothelial cells and megakaryocytes. It is synthesized as pre-pro-VWF, a single chain polypeptide with the domain structure D1-D2-D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (7Verweij C.L. Diergaarde P.J. Hart M. Pannekoek H. EMBO J. 1986; 5: 1839-1847Crossref PubMed Scopus (187) Google Scholar). The pro-VWF molecule is subject to extensive posttranslational modifications, including carboxyl-terminal dimerization (8Vischer U.M. Wagner D.D. Blood. 1994; 83: 3536-3544Crossref PubMed Google Scholar). Because of proteolytic processing, pro-VWF is further separated into two polypeptides, the VWF propeptide (also known as VWF AgII) containing the D1-D2 region, and mature VWF, which comprises the remaining domains. The main function of the propeptide is to facilitate amino-terminal multimerization of mature VWF within the post-Golgi compartments and its targeting to Weibel-Palade storage bodies in endothelial cells (9Wagner D.D. Annu. Rev. Cell Biol. 1990; 6: 217-246Crossref PubMed Google Scholar, 10Haberichter S.L. Fahs S.A. Montgomery R.R. Blood. 2000; 96: 1808-1815Crossref PubMed Google Scholar). Thus, the propeptide and mature VWF appear to interact within cellular organelles, where conditions are slightly acidic (pH < 6.5). The extent of multimerization may differ between VWF molecules and is defined by the number of dimeric subunits included (between 2 and >20). As a result, VWF circulates within plasma as a heterogeneously sized protein with its molecular mass ranging from 0.5 × 106 to over 5 × 106 Da. The average plasma level of VWF is ∼10 μg/ml (35 nm based on monomer concentrations), although a broad range is observed between individuals (11Sadler J.E. Blood. 2003; 101: 2089-2093Crossref PubMed Scopus (258) Google Scholar). A major part of this variation is determined by ABO blood type; the average VWF levels in persons with blood group O are ∼25% lower than those in non-O individuals (12Gill J.C. Endres-Brooks J. Bauer P.J. Marks Jr., W.J. Montgomery R.R. Blood. 1987; 69: 1691-1695Crossref PubMed Google Scholar). However, other genetic (13Orstavik K.H. Folia Haematol. (Leipz.). 1990; 117: 527-531Google Scholar, 14Carlos S.J. Almasy L. Manuel S.J. Buil A. Stone W. Lathrop M. Blangero J. Fontcuberta J. Thromb. Haemostasis. 2003; 89: 468-474Crossref PubMed Google Scholar) and non-genetic factors, such as age and physical activity (15Wannamethee S.G. Lowe G.D. Whincup P.H. Rumley A. Walker M. Lennon L. Circulation. 2002; 105: 1785-1790Crossref PubMed Scopus (423) Google Scholar, 16Yarnell J.W. Sweetnam P.M. Rumley A. Lowe G.D. Blood Coagul. Fibrinolysis. 2001; 12: 721-728Crossref PubMed Scopus (49) Google Scholar), may also contribute to this variation. These factors may influence VWF levels by modulating the balance between its biosynthesis and clearance. In the last two decades, biosynthesis of VWF has been well studied (for recent reviews see Refs. 17Denis C.V. Int. J. Hematol. 2002; 75: 3-8Crossref PubMed Scopus (79) Google Scholar and 18de Wit T.R. van Mourik J.A. Best Pract. Res. Clin. Haematol. 2001; 14: 241-255Crossref PubMed Scopus (56) Google Scholar). In contrast, little is known about the mechanism by which VWF is cleared from the circulation. However, this issue is becoming increasingly important, as illustrated by several recent reports suggesting that modified clearance plays a role in the pathogenesis of VWD type 1 (19Casonato A. Pontara E. Sartorello F. Cattini M.G. Sartori M.T. Padrini R. Girolami A. Blood. 2002; 99: 180-184Crossref PubMed Scopus (129) Google Scholar, 20Brown S.A. Eldridge A. Collins P.W. Bowen D.J. J. Thromb. Haemost. 2003; 1: 1714-1717Crossref PubMed Scopus (54) Google Scholar). One of the very few parameters that has been reported to date to influence VWF clearance is the VWF glycosylation profile (12Gill J.C. Endres-Brooks J. Bauer P.J. Marks Jr., W.J. Montgomery R.R. Blood. 1987; 69: 1691-1695Crossref PubMed Google Scholar, 21O'Donnell J. Laffan M.A. Transfus. Med. 2001; 11: 343-351Crossref PubMed Scopus (231) Google Scholar, 22Stoddart Jr., J.H. Andersen J. Lynch D.C. Blood. 1996; 88: 1692-1699Crossref PubMed Google Scholar, 23Mohlke K.L. Purkayastha A.A. Westrick R.J. Smith P.L. Petryniak B. Lowe J.B. Ginsburg D. Cell. 1999; 96: 111-120Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, D. M. Ginsburg D. A. U. A. 2002; 99: PubMed Scopus Google Scholar). by an model it has been that, in the absence of the the of VWF is reduced 2-fold D. M. Ginsburg D. A. U. A. 2002; 99: PubMed Scopus Google Scholar). in a group to the for or bleeding reduced was observed to associated with reduced VWF plasma levels D. M. Ginsburg D. A. U. A. 2002; 99: PubMed Scopus Google Scholar). These that a role in the clearance of VWF. In to the polypeptide are also to contribute to the interaction with clearance However, such region has been within the VWF Thus, the molecular basis of VWF clearance is an issue in of the as well as in of the within VWF that to To into the molecular of VWF clearance, an experimental model was employing mice for VWF. this model, the in survival of truncated as well as of VWF was with that of full-length VWF. These that the D′-D3 region of VWF in the residue Arg-1205 contribute to the clearance of VWF. VWF-deficient mice C. H. M. Wagner D.D. U. A. 1998; 95: PubMed Scopus Google Scholar) and wild-type mice used in this on a and used between and of and as by and the experimental of the a plasma-derived VWF of C. was with the this resulted in containing × VWF. in was as determined by with VWF in high or low molecular weight by an VWF by The factor VIII chain was by K. Mertens The was and as 2002; PubMed Scopus Google Scholar). VWF propeptide was a of H. C. The collagen type number and from VWF and VWF from and been C.L. Pannekoek H. J. Biochem. PubMed Scopus Google Scholar, H. Blood. PubMed Google Scholar). was by the mutation in a VWF fragment the and as is this fragment was into the containing full-length VWF was by a fragment with the containing the at in and the containing a The fragment was into the fragment was into the In this modified an that a His and a is into the VWF was by a fragment with the containing the at in and the containing an The fragment was into the fragment was into the VWF domain region containing an amino-terminal His and was by The was by a fragment from into of VWF in cells that also for of the VWF propeptide H. van M. M. Thromb. Haemost. 1996; 75: PubMed Scopus Google Scholar). wt-VWF, and from from by employing the as H. C. M. M. Thromb. Haemost. 1999; PubMed Scopus Google Scholar). The truncated and from by as by the and further by and (pH and stored in at by that to The multimeric structure of VWF was by employing B. A. D. Blood. 1999; PubMed Google Scholar, E. B. Lenting P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Biodistribution of or wild-type mice with radiolabeled to × 106 in containing time and and and mice with of and by the mice and and which for the of mice used time of VWF in mice with or in containing time and and and mice with of blood was by and plasma was as C.V. K. Wagner D.D. Blood. 2001; PubMed Scopus Google Scholar). to mice used for time and was only VWF levels in a E. B. Lenting P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). plasma from was used as a that 1 of plasma of VWF (35 based on a molecular mass of To of and directed the D′-D3 region used as In addition, a of was used as a plasmon employing a and as 2002; PubMed Scopus Google Scholar, E. B. Lenting P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). FVIII chain and VWF propeptide wt-VWF, and on a the as by the was used as a as it the sites for the FVIII chain and propeptide. to and was for to the to this was of to both other of the FVIII chain to VWF was in at with a rate of of the surface was by of the containing 1 The interaction between the propeptide and VWF was employing such as (pH or or (pH or at with a rate of of the surface was by of (pH two used for to and at and the of propeptide to VWF was also examined at to the of the VWF The was time the was and of and was the for from analysis used for the of the affinity as at from protein The to the = × to to the = to and to the of VWF in plasma to the These parameters used to mean residence time (MRT) employing the = Furthermore, and used to of the initial and employing = and = by the the for VWF to the into the clearance of VWF, the of this protein by radiolabeled × 106 in VWF-deficient injection or and or and for the of the initial time minor amounts of (between and of the in and These amounts that to to in the between 1 and 2 In contrast, within 3 injection of the to in the This increased to at by a to between 0.5 and h. A similar was observed in wild-type mice These that VWF is primarily to the of the of VWF from VWF-deficient mice with time mice and plasma was for VWF The recovery to in 3 injection of the revealed that the protein from plasma in a characterized by a rapid initial phase and a slow secondary phase analysis allowed the of the which and for the rapid and slow Furthermore, was to 2.8 parameters the clearance of VWF and its from of = = = = = = = = = = = in a and VWF in containing VWF are as the number of included subunits may between molecules 2 to >20). To the clearance of VWF on its extent of the survival of in high and or low and molecular weight VWF-deficient These revealed that both VWF cleared from plasma in a similar similar parameters from the experimental the clearance of both high and low molecular weight was similar to that of The was that the clearance of was similar to that of multimers, into from circulation. However, analysis of the plasma that the multimeric not into from the plasma not Thus, the rate of VWF clearance is independent of its multimeric in our experimental of and on the of of the complex protein structure of VWF, it was of further to the of the of the VWF molecule to its clearance. To this a number of VWF with carboxyl-terminal to VWF-deficient mice by to a fragment consisting of the domains a clearance similar to was observed As in similar parameters for this and In contrast, a with an amino-terminal an initial recovery that was significantly lower than that observed for = for and wt-VWF, In addition, was cleared from plasma in the initial phase than both and significantly reduced for the truncated protein with the with a deletion of the carboxyl-terminal part of the and the initial recovery was from that of However, in the rapid phase of the clearance, both from plasma than 13 in plasma 1 whereas almost 2-fold for p = and p = of the parameters revealed that both and for the truncated the may in the interaction with that the of VWF from the and within the VWF molecule may a role in its One the D′-D3 region, which seems to VWF from premature clearance. of Arg-1205 by His in an that the D′-D3 domain to in the clearance of VWF may of with to VWF with in this region as in a number of from type 1 mutation of the residue Arg-1205 to His has been to the survival of VWF in (19Casonato A. Pontara E. Sartorello F. Cattini M.G. Sartori M.T. Padrini R. Girolami A. Blood. 2002; 99: 180-184Crossref PubMed Scopus (129) Google Scholar). To the of Arg-1205 to the clearance of VWF, was in of the revealed a of multimerization for this mutant However, the protein was in VWF-deficient from the One 2 was in with 13 for < analysis that and as well as the significantly reduced with the for In contrast, the in survival of VWF a mutation in the it that this mutant was cleared from plasma to the extent as These are with the that replacement of Arg-1205 by His results in an clearance of VWF. of Arg-1205 by His to and that replacement of Arg-1205 by His the in survival of VWF us to this mutant in by a number of functional First, the of and to collagen type and to a fragment of in the of was examined by the at used to affinity which are in The revealed that and similar affinity for = and nm for and the and for collagen = and 3 nm for and the replacement of Arg-1205 by His complex its and domains for the between VWF and its type are are are are are are in a pH-dependent of to D′-D3 region, which residue comprises sites for the VWF propeptide as well as the chain of factor Therefore, of to both was analysis that and are in of affinity for the FVIII chain = and nm for and to the VWF an was to its interaction with VWF. VWF was and with VWF propeptide The interaction was examined at (between and the of the propeptide has been reported to pH-dependent (8Vischer U.M. Wagner D.D. Blood. 1994; 83: 3536-3544Crossref PubMed Google Scholar). at was of the propeptide to was markedly at lower was to a rate that was reduced of at lower of revealed that propeptide to with low affinity at of the the affinity by = and at and As for wt-VWF, a pH-dependent interaction with propeptide was also observed for similar and for the mutant and at and As in both similar in propeptide in of affinity conditions In contrast, a in of propeptide to was observed was further to and the affinity 2-fold for the mutant with (pH = = for and replacement of Arg-1205 by His is associated with of propeptide at low plasma of hemostatic are associated with an increased whereas reduced levels may in a bleeding This that plasma levels of to levels a balance between and to VWF, its has been well studied in the last two C.V. Int. J. Hematol. 2002; 75: 3-8Crossref PubMed Scopus (79) Google Scholar, 18de Wit T.R. van Mourik J.A. Best Pract. Res. Clin. Haematol. 2001; 14: 241-255Crossref PubMed Scopus (56) Google Scholar), whereas little is known its clearance. In the used VWF-deficient mice as a model to into the molecular basis of VWF is cleared from the circulation. employing radiolabeled VWF revealed that this protein was to the whereas little of other to the of VWF was In of was observed in the of in the has been for other as including and van C. K. van den B. H. van Med. 2003; Google Scholar, E. D.J. 1996; 6: Google Scholar). In of the this that are from the liver and by the the time by which the in liver and it seems that a similar is for VWF. VWF is in the liver in a is from the However, in of its and rapid in the it seems that VWF is from the by an process. the that the clearance of VWF is on the of VWF in the or However, a similar was observed in and VWF-deficient mice in of and time VWF is cleared from plasma in a characterized by a rapid initial phase and a slow phase The was to 2.8 h. In this VWF is cleared from the to the extent as reported for VWF in Jr., J.H. Andersen J. Lynch D.C. Blood. 1996; 88: 1692-1699Crossref PubMed Google Scholar). The of VWF in are with the of VWF in which is A. U. 1998; PubMed Scopus Google Scholar, F. L. M. C. Blood Coagul. Fibrinolysis. PubMed Scopus Google Scholar). in the clearance rate between been reported for other including coagulation factor its in is A. M. J. K. Hematol. 1998; Google Scholar), it is reduced in mice J. Clin. Investig. PubMed Scopus Google Scholar). this the which is increased in One to the similar survival of high and low multimeric VWF which that the clearance of VWF is independent of its multimeric in our experimental This is to the that VWF is cleared than its low multimeric in as well as in Jr., J.H. Andersen J. Lynch D.C. Blood. 1996; 88: 1692-1699Crossref PubMed Google Scholar, P.L. H. L. W. B. A. W. U. Dorner F. H.J. van Mourik J.A. H.P. Blood. PubMed Google Scholar). It that high molecular weight of VWF are into in and not observed in our proteolytic of VWF in plasma not This is in with the that VWF is not by the high molecular weight VWF may to the receptor in and but not in it has been reported that VWF is not by J. Z.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This survival of VWF in our experimental model the that to compare the clearance of VWF to that of and dimeric of VWF been with to in the clearance of VWF. It that the clearance is complex in that it of the VWF that from our are the In of the similar clearance for full-length and the fragment it seems that a is in the region. to the D4-CK region, that its absence is associated with a clearance suggesting that deletion of domains to the of a However, the D4-CK domains to the region deletion of the D′-D3 this protein was cleared than the region This to a role of the D4-CK region by clearance. not us to between both which are not the clearance rate of the it seems that a is in the D′-D3 region as Furthermore, the fragment deletion of the D4-CK is cleared than the which with the D′-D3 region a role by the clearance The of the D′-D3 region in VWF clearance is by a recent by and (19Casonato A. Pontara E. Sartorello F. Cattini M.G. Sartori M.T. Padrini R. Girolami A. Blood. 2002; 99: 180-184Crossref PubMed Scopus (129) Google Scholar). observed that an Arg-1205 to His mutation within the D3 domain an increased of VWF from that it the survival is the of the an VWF mutation part of the VWF was or a in a receptor in the cellular of VWF. that in our experimental model this mutant is from the to in a reduced This that the mutation is associated with an increased clearance rate and levels of VWF are reduced in this is the recent of a mutation in the domain of VWF, which seems to a similar effect on VWF clearance J.C. M. S.L. Montgomery R.R. Blood. 2002; Scholar). It is to that VWF abnormal clearance an effect has been to date of the of this function in with the of an model it to of which for a mechanism of The mechanism by which the Arg-1205 to His mutation the clearance of VWF to One is that the mutation may an by the affinity for that receptor the clearance It that our not between the mutation part of a or a in an manner. In vitro analysis of to GpIbα, and the FVIII chain by the and D′-D3 that and the mutant are in complex with These that the Arg-1205 to His mutation not the structure and function of VWF. However, with to propeptide that the Arg-1205 mutation resulted in an impaired pH-dependent interaction and As the Arg-1205 residue is not part of the propeptide to S.L. Montgomery R.R. Blood. 2003; 101: PubMed Scopus Google Scholar), it is that the Arg-1205 to His mutation It is to functional of the reduced affinity for propeptide by in of the role of the VWF propeptide multimerization of VWF in the post-Golgi compartments and with multimeric VWF in storage in endothelial cells S.L. Fahs S.A. Montgomery R.R. Blood. 2000; 96: 1808-1815Crossref PubMed Google Scholar). A reduced affinity for propeptide may the of storage and in a premature of the VWF molecules into the circulation. This contribute to the increased of VWF in as is in not with the mutation P.M. R. J.A. A. F. Blood. PubMed Google Scholar, R. U. E. P.M. F. B. R. Thromb. Haemost. 2000; 83: PubMed Scopus Google Scholar, J. Thromb. Haemost. 2003; 1 Scholar, C. A. J. Thromb. Haemost. 2003; 1 Scholar). K. Mertens for the FVIII for the H. for the VWF M. for and J. for