Propolypeptide Of Von Willebrand Factor Research Articles

Bladder Regeneration by Collagen Scaffolds With Collagen Binding Human Basic Fibroblast Growth Factor

Studies show that basic fibroblast growth factor can promote bladder regeneration. However, the lack of targeting delivery approaches limits its clinical application. We investigated a collagen based targeting system for bladder regeneration. A collagen binding domain was added to the native basic fibroblast growth factor N-terminal to allow it to bind to collagen. Sprague-Dawley rats underwent partial cystectomy. Collagen scaffolds loaded with collagen binding domain basic fibroblast growth factor, native basic fibroblast growth factor or phosphate buffered saline were grafted to the remaining host bladders, respectively. At days 30 and 90 reconstructed bladders were evaluated by histological analysis and urodynamics. This targeting basic fibroblast growth factor delivery system induced satisfying bladder histological structures. It promoted more vascularization and smooth muscle cell ingrowth. Urodynamics revealed well accommodated bladder tissue with volume capacity and compliance. Results show that the targeting delivery system consisting of collagen binding domain basic fibroblast growth factor and collagen membranes induced better bladder regeneration at the injury site. Thus, this targeting delivery system may be an effective strategy for bladder regeneration with potential clinical applications.

Identification of Factor-XIIIa-Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100-kDa propeptide, called the propolypeptide of von Willebrand factor (pp-vWF), is generated during biosynthesis. We found that pp-vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311–123161. As such, it could form cross-linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp-vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861–867]. In this work, we identified the Gln residues in pp-vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme-reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti-dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp-vWF, only four could be modified in the factor-XIIIa-catalyzed reaction.

Activation of demineralized bone matrix by genetically engineered human bone morphogenetic protein‐2 with a collagen binding domain derived from von Willebrand factor propolypeptide

There is a large demand for new bone regeneration to restore the function during bone injuries. Bone filling materials are important in bone tissue restoration. In this study, the demineralized bone matrix (DBM) was activated with the engineering human bone morphogenetic protein-2 (BMP-2). To enhance the binding of BMP-2 to the DBM scaffolds, a collagen-binding peptide was fused to the N-terminal of BMP-2. The in vitro results showed that the engineered collagen-targeted BMP-2 (rhBMP2-v) bound to DBM scaffolds specifically and the rhBMP2-v had increased alkaline phosphatase activity in C2C12 cells. In vivo, the DBM scaffolds impregnated with rhBMP2-v showed greater effect on ectopic bone formation. Our data suggested that the collagen-based BMP-2 targeting bone repair system had greater bone inducing ability than DBM loaded with regular BMP-2.

Differential Kinetics of Cell Surface Loss of von Willebrand Factor and Its Propolypeptide after Secretion from Weibel-Palade Bodies in Living Human Endothelial Cells

The time course for cell surface loss of von Willebrand factor (VWF) and the propolypeptide of VWF (proregion) following exocytosis of individual Weibel-Palade bodies (WPBs) from single human endothelial cells was analyzed. Chimeras of enhanced green fluorescent protein (EGFP) and full-length pre-pro-VWF (VWF-EGFP) or the VWF propolypeptide (proregion-EGFP) were made and expressed in human umbilical vein endothelial cells. Expression of VWF-EGFP or proregion-EGFP resulted in fluorescent rod-shaped organelles that recruited the WPB membrane markers P-selectin and CD63. The WPB secretagogue histamine evoked exocytosis of these fluorescent WPBs and extracellular release of VWF-EGFP or proregion-EGFP. Secreted VWF-EGFP formed distinctive extracellular patches of fluorescence that were labeled with an extracellular antibody to VWF. The half-time for dispersal of VWF-EGFP from extracellular patches was 323.5 +/- 146.2 s (+/-S.D., n = 20 WPBs). In contrast, secreted proregion-EGFP did not form extracellular patches but dispersed rapidly from its site of release. The half-time for dispersal of proregion-EGFP following WPB exocytosis was 2.98 +/- 1.88 s (+/-S.D., n = 32 WPBs). The slow rate of loss of VWF-EGFP is consistent with the adhesive nature of this protein for the endothelial membrane. The much faster rate of loss of proregion-EGFP indicates that this protein does not interact strongly with extracellular VWF or the endothelial membrane and consequently may not play an adhesive role at the endothelial cell surface.

N-terminal Cleavage of the Salivary MUC5B Mucin: ANALOGY WITH THE VON WILLEBRAND PROPOLYPEPTIDE?

Sequence similarities between the oligomeric mucins (MUC2, MUC5AC, MUC5B) and the von Willebrand factor suggest that they may be assembled in a similar way. After oligomerization, a fragment corresponding to the D1 and D2 domains is released from the von Willebrand factor. This cleavage does not appear to occur in pig submaxillary mucin, the only mammalian mucin in which this cleavage has been examined thus far, but whether other oligomeric mucins undergo N terminus proteolysis is not known. Antibodies recognizing the D1, D2, D3, and the first Cys domains in MUC5B were established and used to investigate to what extent proteolytic cleavage occurs within the N-terminal part of salivary MUC5B. The antibodies against the D1 and D2 domains identified a polypeptide corresponding in size to a MUC5B fragment generated by cleavage within the D' domain analogously with the von Willebrand factor propolypeptide. The antibodies did not recognize the main mucin population, suggesting that the major part of salivary MUC5B is subjected to this cleavage. An antibody recognizing the D3 domain was used to reveal a second cleavage site in the "soluble" but not in the "insoluble" MUC5B fraction: the first structural difference observed between soluble and insoluble salivary MUC5B. The identification of these cleavage events shows that the N-terminal sites for MUC5B oligomerization are present in the D3 domain and/or in domains located C-terminal to this part of the molecule.

Tissue Transglutaminase, Coagulation Factor XIII, and the Pro-polypeptide of von Willebrand Factor Are All Ligands for the Integrins α9β1 and α4β1

We previously reported that MOLT-3 human lymphocyte-like leukemia cells adhere to tissue-type transglutaminase (tTG) through the integrin alpha(4)beta(1). We now report that G-361 human melanoma cells also adhere to tTG, although they do not express alpha(4)beta(1). G-361 cells utilize two additional integrins, alpha(9)beta(1) and alpha(5)beta(1) to adhere to tTG. Furthermore, blood coagulation factor XIII (FXIII), another member of the transglutaminase family that is highly homologous to tTG, and propolypeptide of von Willebrand factor (pp-vWF) also promoted cell adhesion through alpha(9)beta(1) or alpha(4)beta(1) in G-361 or MOLT-3 cells, respectively. In the case of pp-vWF, alpha(9)beta(1) and alpha(4)beta(1) both bind to the same site, comprised of 15 amino acid residues and designated T2-15. Moreover, SW480 human colon cancer cells stably transfected to express alpha(9)beta(1), but not mock transfectants, adhered to tTG, FXIII, pp-vWF, and T2-15/bovine serum albumin conjugate. These data identify tTG, FXIII, and pp-vWF as shared ligands for the integrins alpha(9)beta(1) and alpha(4)beta(1). This report is the first to unambiguously show that these two integrins share the same cell adhesion site within one protein and provides strong support for classifying alpha(9)beta(1-) and alpha(4)-integrins as functionally related members of an integrin subfamily.

Expression of the Molecule Detectable by Anti-Propolypeptide of von Willebrand Factor Antibody in Rat Mesangial Cells in Anti-Thy 1.1 mAb 1-22-3 Induced Glomerulonephritis: A Marker of Injured Mesangial Cells

We have previously reported that propolypeptide of von Willebrand factor (pp-vWF) binds to collagen with an affinity comparable to that of mature vWF, inhibits collagen-induced platelet aggregation, is cross-linked with laminin, and also serves as a ligand for very-late antigen 4 integrin. These observations from in vitro experiments suggest that pp-vWF is incorporated in the extracellular matrix and affects the cell-matrix interaction and that pp-vWF functions in leukocyte recruitment to inflammatory and vascular injury sites. We, therefore, hypothesize that pp-vWF might be involved in the induction and/or progression of mesangial proliferative glomerulonephritis. To test this hypothesis, we examined the kinetics of the immunostaining of the molecule detectable by an affinity-purified anti-pp-vWF antibody in rat glomeruli in monoclonal antibody 1-22-3 induced glomerulonephritis. Immunostaining by pp-vWF antibody was observed in the nuclear rim of mesangial cells in monoclonal antibody 1-22-3 induced glomerulonephritis. Positive staining first appeared on day 10 after monoclonal antibody injection, when mesangial cell proliferation and mesangial matrix expansion had already begun. Staining was still detected on day 56, when morphologic alterations observed by light microscopy had been normalized. The pp-vWF antibody recognized molecule appeared later than α-smooth muscle actin or collagen type I. Positive staining was not detected in cultured mesangial cells. It should be noted that the positive staining by pp-vWF antibody in mesangial cells was still detected in previously injured glomeruli that have almost recovered normal morphology. These observations indicate that positive staining by pp-vWF antibody could be a very useful marker for identifying a past episode of injury in mesangial cells.

Purification of the Porcine Platelet GP IIb-IIIa Complex and the Propolypeptide of von Willebrand Factor

Platelet membrane glycoproteins (GP) are involved in platelet adhesion and aggregation. The glycoprotein IIb-IIa complex (GP IIb-IIIa) is a Ca2+-dependent heterodimer that binds fibrinogen and other adhesive proteins, thereby mediating platelet aggregation and adhesion. We have purified two major glycoproteins from pig platelets by Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl S-300 HR chromatography (Fitzgerald et al. Anal Biochem, 1985): i) the GP IIb-IIIa complex, GP IIb Mr = 140,000 and GP IIIa a single chain of Mr = 95,000-100,000; and ii) a predominant glycoprotein of high molecular weight, the propolypeptide of von Willebrand factor (Mr = 80,000-100,000). Western-blot analysis of the purified GP IIb-IIIa showed that only certain monoclonal antibodies against the human receptor specifically recognize the porcine complex. Differences between the porcine and human GP IIb-IIIa glycoproteins could partially explain the decreased inhibitory effects of GP IIb/IIIa-antagonists (against the human receptor) in porcine platelets.

Propolypeptide of von Willebrand Factor Is a Novel Ligand for Very Late Antigen-4 Integrin

We have previously reported that propolypeptide of von Willebrand factor (pp-vWF) promotes melanoma cell adhesion in a beta1 integrin-dependent manner. In this report, we identified the alpha subunit of the cell adhesion receptor for pp-vWF as alpha4. Human leukemia cell lines that express alpha4beta1 integrin (very late antigen-4, VLA-4), but not cell lines which lack VLA-4, attached well to pp-vWF substrate and these adhesions were completely inhibited by anti-alpha4 integrin monoclonal antibody HP2/1. Adhesion of mouse melanoma expressing alpha4 integrin was also inhibited by anti-mouse alpha4 mAb PS/2. Furthermore, transfection of human alpha4 cDNA into alpha4(-) Chinese hamster ovary cells resulted in an acquisition of adhesive activity to pp-vWF, indicating that pp-vWF is a ligand for VLA-4 integrin. Using a recombinant fragment of pp-vWF, the cell attachment site was shown to be located within amino acid residues 376-455 of pp-vWF. A series of synthetic peptides covering this region were tested for the ability to promote cell attachment and a 15-residue peptide designated T2-15 (DCQDHSFSIVIETVQ, residues numbered 395-409) promoted VLA-4 dependent cell adhesion. The peptide was also capable of inhibiting cell adhesion to pp-vWF, suggesting that this sequence represents the cell attachment site. By affinity chromatography using peptide T2-15-Sepharose, it was found that alpha4beta1 integrin complex from extracts of surface iodinated B16 cells specifically bound to the peptide. These results strongly suggest that pp-vWF is a novel physiological ligand for VLA-4.

Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100-kDa propeptide, called the propolypeptide of von Willebrand factor (pp-vWF), is generated during biosynthesis. We found that pp-vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311-12316]. As such, it could form cross-linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp-vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861-867]. In this work, we identified the Gln residues in pp-vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme-reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti-dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp-vWF, only four could be modified in the factor-XIIIa-catalyzed reaction.

Beta 1-integrin-mediated adhesion of melanoma cells to the propolypeptide of von Willebrand factor.

Cell-adhesion activity of the bovine propolypeptide of von Willebrand factor (pp-vWF) was assessed by means of an in vitro assay with several cell lines of both normal and tumor-cell origin. pp-vWF promoted adhesion and spreading of B16 mouse melanoma cells and G-361 human melanoma cells. However, it could not induce adhesion of any other cell lines tested including endothelial cells, normal fibroblasts, and tumor cells of sarcoma, carcinoma, neuroblastoma and leukemia origin. A monospecific polyclonal antibody against pp-vWF, but not against fibronectin, laminin, and von Willebrand factor (vWF), completely blocked the pp-vWF-mediated adhesion, indicating that the cell adhesion was due to the pp-vWF molecule and not due to possible contamination of these three well-known adhesive proteins. The cell-adhesion activity was also observed with human pp-vWF and, furthermore, the adhesion to both bovine and human pp-vWF was not affected by a peptide containing the Arg-Gly-Asp sequence while the peptide abolished the cell adhesion to vWF. The adhesion was completely dependent on Mg2+ and inhibited by Ca2+. Inhibition by an anti-(beta 1 integrin) mAb (4B4) indicates that the receptor for this protein belongs to the beta 1-integrin family. A monoclonal antibody (TC4) among several antibodies directed against bovine pp-vWF inhibited the B16 adhesion to immobilized pp-vWF. The epitope for this monoclonal antibody lies in a central 8-kDa portion of pp-vWF, suggesting that this region is important for the cell-adhesion activity. This idea was supported by the finding that purified 8-kDa fragment promoted adhesion of B16 cells in a concentration-dependent manner. As pp-vWF shows unique cell-type specificity in its adhesion activity, which is completely different from that of fibronectin, laminin, vWF and collagen, it may be a novel type of adhesive glycoprotein that utilizes a beta 1-integrin receptor.

Propolypeptide of von Willebrand factor serves as a substrate for factor XIIIa and is cross-linked to laminin

The propolypeptide of von Willebrand factor (pp-vWF) was found to serve as a substrate for Factor XIIIa (FXIIIa). FXIIIa catalyzed polymerization of pp-vWF as well as the incorporation of monodansylcadaverine and [14C]putrescine into pp-vWF in a Ca(2+)- and time-dependent manner. The amount of putrescine incorporated into pp-vWF was increased as a function of putrescine concentration and reached a maximum of 3 mol/mol of pp-vWF, indicating that there are at least 3 glutamine residues in the pp-vWF molecule responsible for the reaction. 125I-pp-vWF was incubated with FXIIIa in the presence of various adhesive glycoproteins. Laminin, among the proteins tested, specifically formed a large molecular weight complex with 125I-pp-vWF in a Ca(2+)- and time-dependent manner. It was also found that FXIIIa catalyzed polymerization of laminin as well as the incorporation of monodansylca-daverine into laminin. The high molecular weight complexes of 125I-pp-vWF (or 125I-laminin) formed in the presence of laminin (or pp-vWF) were immunoprecipitated with anti-laminin (or anti-pp-vWF) antibodies. Taking all the data together it can be concluded that both pp-vWF and laminin have glutamine and lysine residues responsive to FXIIIa and make copolymers by virtue of FXIIIa.

Activation of phospholipases in platelets by polyclonal antibodies against a surface membrane protein

In a previous paper we demonstrated using immunochemical techniques that propolypeptide of von Willebrand factor was present on the surface of resting platelets. In the present paper we show that polyclonal antibodies against propolypeptide of von Willebrand factor induce activation of phospholipase(s) in platelets and lead to platelet aggregation. The antibody-stimulation of platelets induced the synthesis of thromboxane A 2 (TXA 2). Furthermore, the aggregation was inhibited by aspirin and an antagonist of TXA 2. Aspirin inhibited not only the aggregation but also the activation of arachidonic acid liberation from phospholipids, but the effect of aspirin on arachidonic acid liberation was cancelled by the combined effect of the antibodies and a TXA 2 mimetic agonist, which itself did not activate arachidonic acid liberation. The antibody-induced activation of arachidonic acid liberation and the aggregation were blocked by cytochalasin B. All these results obtained with antibodies were quite similar to the results obtained with collagen.

A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor.

Propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen, and we have reported that a binding domain exists in a 21.5/21-kDa fragment originated from the C-terminal portion [Takagi, J., Fujisawa, T., Sekiya, F., & Saito, Y. (1991) J. Biol. Chem. 266, 5575-5579]. The collagen-binding property of the 21.5/21-kDa fragment was compared with that of the intact pp-vWF. Although pp-v WF preferentially binds to native type I collagen fibrils, the isolated fragment no longer has this specificity and binds well to collagen of other types in the native and heat-denatured states. In order to determine the critical site that mediates this collagen/gelatin binding, several peptides were synthesized based on the primary structure of the 21.5/21-kDa fragment. Among these, a 25-residue peptide strongly inhibited the binding of the 125I-labeled 21.5/21-kDa fragment to collagen. Using this inhibitory effect as an index, the binding site was defined to the sequence as follows: WREPSFCALS. Furthermore, a decapeptide of this sequence bound to collagen and gelatin, indicating that this sequence is responsible for the binding of the 21.5/21-kDa fragment to collagen/gelatin.

Immunological detection of propolypeptide of von Willebrand factor on platelet surface

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet α-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.

Collagen-binding domain within bovine propolypeptide of von Willebrand factor.

Two reduced/alkylated fragments of bovine propolypeptide of von Willebrand factor (pp-vWF) that inhibit pp-vWF binding to collagen were isolated. One is a tryptic fragment of molecular mass of about 30 kDa and inhibits the binding at a molar concentration about 20 times higher than the intact pp-vWF. Amino acid sequence of this fragment was determined almost completely, and it was revealed that this fragment corresponded to the carboxyl-terminal region of pp-vWF molecule beginning with Phe557. The other active fragment was obtained by lysyl endopeptidase digestion. This migrated as a 21.5/21-kDa doublet in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but deglycosylation of this doublet resulted in production of single species of 19 kDa. The difference between the doublet constituents, therefore, was of carbohydrate composition. The extent of inhibition of collagen-binding by this 21.5/21-kDa fragment was comparable to that of the 30-kDa fragment, and furthermore, location of this fragment in the molecule was established to be between Phe570 and Lys682. These were the only fragments among those obtained by proteolytic digestions that had significant competitive effect on the binding of intact pp-vWF to collagen. These results strongly suggest that at least one collagen-binding site should be present in the carboxyl-terminal region of bovine pp-vWF extending from residue 570 to 682.

Monoclonal antibodies that inhibit binding of propolypeptide of von Willebrand factor to collagen

We reported previously that bovine propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen. To determine the collagen-binding sites of pp-vWF we generated monoclonal antibodies (mAbs) against bovine pp-vWF. One mAb, designated TC8, very strongly inhibited the binding of pp-vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp-vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp-vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while TC6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N-terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp-vWF, the locations of these fragments in the primary structure were estimated to be Phe570--Lys682 for the 21-kDa fragment and Glu281--Lys375 for the 18-kDa fragment. These data suggest that pp-vWF contains at least two collagen-binding sites which lie within or close to the regions between Phe570--Lys682 and Glu281--Lys375.

The pro-polypeptide of von Willebrand factor is required for the formation of a functional factor VIII-binding site on mature von Willebrand factor

We have established that a recombinant von Willebrand Factor (vWF) mutant (vWFdelpro) that lacks the propolypeptide, in contrast with mature wild-type vWF, with which it is identical in terms of primary amino acid sequence, is not able to form a complex with Factor VIII. Wild-type vWF (flvWF) and vWFdelpro were expressed in AtT-20 cells. Under the culture conditions employed, completely processed multimerized flvWF and dimeric vWFdelpro were secreted into the medium. FlvWF and vWFdelpro were compared for their Factor VIII-binding properties in two distinct assay systems. In a direct binding assay, purified human Factor VIII was shown to bind to flvWF that had been immobilized on the surface of microtitre wells by using an anti-vWF monoclonal antibody. In contrast, Factor VIII did not bind to immobilized vWFdelpro. In a competition assay, fluid-phase flvWF appeared to inhibit efficiently the binding of Factor VIII to immobilized vWF isolated from plasma, whereas vWFdelpro did not influence Factor VIII binding. From these observations, it is argued that the pro-polypeptide serves an essential role in the post-translational processes that lead to the expression of a functional Factor VIII-binding site on the mature vWF subunit.

Induction of specific storage organelles by von Willebrand factor propolypeptide

Endothelial cells store the multimeric adhesive glycoprotein von Willebrand factor (vWf), which promotes the formation of a platelet plug at the site of vessel injury. To investigate the packaging of vWf into the granules called Weibel-Palade bodies, we expressed pro-vWf cDNA and cDNA lacking the prosequence in a variety of cell lines. Storage granules formed only in cells that contain a regulated pathway of secretion. Furthermore, packaging required the prosequence. Pro-vWf, lacking the C-terminal region involved in interchain disulfide bonding, formed granules. We conclude that the signal for storage is universal in that an adhesive glycoprotein can be stored by a hormone-secreting cell; the storage of vWf is independent of its covalent multimeric structure; the unusual rod shape of Weibel-Palade bodies is due to vWf; and the vWf propolypeptide is necessary for the formation of vWf storage granules.

A Collagen-binding Glycoprotein from Bovine Platelets Is Identical to Propolypeptide of von Willebrand Factor

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.