Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.     

Localization and characterization of a heparin binding domain peptide of human von Willebrand factor.

Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide. In a fluid phase binding assay, the Tyr565-Ala587 peptide competed effectively with von Willebrand factor for binding heparin. Synthesis and testing of peptides overlapping Tyr565-Ala587, as well as adjacent cationic regions, showed this core sequence to be the optimal linear binding domain. Far ultraviolet circular dichroism spectrometry of the Tyr565-Ala587 peptide suggested that the peptide undergoes conformational change upon binding heparin. The Tyr565-Ala587 peptide thus encompasses part (or all) of a functionally important heparin binding domain of von Willebrand factor. Further study of this and related peptides may be useful for exploring how heparin may influence von Willebrand factor-mediated platelet hemostasis.     

Amino group modification inhibits ristocetin cofactor activity of human von Willebrand factor

Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifications such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so.     

The dualistic mechanisms of platelet adhesion to von Willebrand factor substrates

von Willebrand factor (vWf) which serves as a necessary factor for platelet adhesion to damaged vascular subendothelium can bind to the platelet surface via two distinct receptors. Ristocetin promotes the binding of vWf to platelet membrane glycoprotein lb, whereas platelet activation by thrombin supports binding to the glycoprotein IIb/IIIa complex. Platelet adhesion to vWf substrates mediated by these two mechanisms has been compared. Both mechanisms supported similar rates of adhesion to the substrates. Whereas adhesion via the ristocetin-dependent mechanism did not require divalent cations, adhesion mediated by the thrombin-dependent mechanism required the presence of divalent cations. Modification of vWf amino groups markedly impaired the ability of the protein to support ristocetin-dependent adhesion but did not alter its ability to support thrombin-enhanced adhesion. Reduction and carboxymethylation nearly abolished the ability of vWf to support adhesion via the ristocetin-dependent mechanism, but did not substantially impair its ability to support thrombin-enhanced adhesion. Short synthetic peptides containing the sequence Arg-Gly-Asp-Ser effectively inhibited thrombin-dependent platelet adhesion to vWf substrates but had no effect on ristocetin-dependent adhesion. Substrates composed of synthetic peptides containing the Arg-Gly-Asp-Ser sequence supported thrombin-dependent adhesion but did not support ristocetin-dependent adhesion. Scanning electron microscopic examination revealed that platelets adherent via the ristocetin-dependent mechanism almost uniformly adopted a flattened and fully spread appearance. In contrast, the thrombin-enhanced mechanism of adhesion supported only a limited degree of platelet spreading on the vWf substrate.     

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocentin-von Willebrand factor.

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to 3H-labelled diazobenzene. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocentin-von Willebrand factor but did not alter the receptor for aggregated IgC. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgC. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.     

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocetin-von Willebrand factor

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to ³H-labelled diazobenzen. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocetin-von Willebrand factor but did not alter the receptor for aggregated IgG. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgG. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.     

Comparison of the primary structure of the functional domains of human and porcine von Willebrand factor that mediate platelet adhesion.

Porcine von Willebrand factor (vWF) directly aggregates human platelets in vitro indicating a conformational difference between the human and porcine molecules. We amplified and directly sequenced 1242 nucleotides of porcine vWF cDNA that encodes functional domains which mediate the binding of vWF to platelets and subendothelium. The deduced amino acid sequence corresponds to residues 473-891 of the human mature vWF subunit and is 79% homologous with the human protein. Significant differences are found in two discontinuous segments thought to be involved in the binding of vWF to platelet glycoprotein Ib. Porcine vWF lacks four contiguous residues in the first segment and has two positively charged arginine residues in the second. Three point mutations associated with human type IIB von Willebrand disease in the first segment of a botrocetin binding site are at the same position as mismatches between the pig and human. The second segment of the botrocetin site is highly conserved while the third segment shows only a 60% homology.     

Binding of ADAMTS13 to von Willebrand factor

ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. ADAMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was approximately 4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K(d) was 14 nm. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K(d) of 206 nm, whereas ADAMTS13 truncated after the spacer domain had a K(d) of 23 nm, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by approximately 3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by approximately 2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.     

Immunoelectron-microscopic studies of human platelet thrombospondin, Von Willebrand factor, and fibrinogen redistribution during clot formation

Summary The relative distributions of the human platelet -granule proteins fibrinogen, thrombospondin, and von Willebrand factor were mapped by immunoelectron microscopy in thin cryosections of activated platelets, platelet aggregates, and clots during the first 24 h ofin vitro clot formation. In early activated platelets, the results suggest that the canalicular system constitutes a significant component of the external platelet surface, and may act as a compartment for biochemical reactions occurring during granule relase. Further, detection of coagulation proteins by various non-morphological procedures may reflect protein contained within canalicular elements. Later in the release process, von Willebrand factor was detected as a major antigen on the platelet canalicular and plasma membranes; thrombospondin, on the other hand, showed minimal binding to platelets and only limited binding to the extensive fibrin network. Comparison of radioimmunoassays of supernatants of thrombin-stimulated platelets in plasma, clotted whole blood, and Triton X-100 platelet releasates indicated that virtually all of the platelet thrombospondin appears in serum. These data confirm the immunocytochemical results indicating that very little platelet thrombospondin binds to the platelet surface, compared with von Willebrand factor, studied here under the same conditions, which binds extensively to the platelet membrane following release and clot formation.     

Differential Involvement of ERK2 and p38 in platelet adhesion to collagen

We investigated the role of two MAP kinases, ERK2 and p38, in platelet adhesion and spreading over collagen matrix in static and blood flow conditions. P38 was involved in collagen-induced platelet adhesion and spreading in static adhesion conditions, whereas ERK2 was not. In blood flow conditions, with shear rates of 300 or 1500 s(-1), ERK2 and p38 displayed differential involvement in platelet adhesion, depending on the presence or absence of the von Willebrand factor (vWF). Low collagen coverage densities (0.04 microg/cm2) did not support vWF binding. During perfusions over this surface, platelet adhesion was not affected by the inhibition of ERK2 phosphorylation by PD 98059. However, abolishing p38 activation by SB 203580 treatment reduced platelet adhesion by 67 +/- 9% at 300 s(-1) and 56 +/- 2% at 1500 s(-1). In these conditions, the p38 activity required for platelet adhesion depends on the alpha2beta1 collagen receptor. At higher collagen coverage densities (0.8 microg/cm2) supporting vWF binding, the inhibition of ERK2 activity by PD 98059 decreased adhesion by 47 +/- 6% at 300 s(-1) and 72 +/- 3% at 1500 s(-1), whereas p38 inhibition had only a small effect. The ERK2 activity required for platelet adhesion was dependent on the interaction of vWF with GPIb. In conclusion, ERK2 and p38 have complementary effects in the control of platelet adhesion to collagen in a shear stress-dependent manner.     

Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr1605-Met1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more C-terminal TSP1 and CUB domains are dispensable in vitro.     

Characterization of the platelet agglutinating activity of thrombospondin

Thrombospondin (TSP) is a glycoprotein secreted from the alpha-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets. Platelets fixed with formaldehyde following activation by thrombin are agglutinated by exogenously added TSP. Fixed, nonactivated platelets are not agglutinated. The platelet agglutinating activity of TSP is optimally expressed in the presence of 2 mM each of Mg2+ and Ca2+. Reduction of the disulfide bonds within the TSP molecule inhibits its platelet agglutinating activity. TSP bound to the surface of fixed, activated platelets can be eluted by the addition of disodium ethylenediaminetetraacetate. This approach was exploited to identify the region of the TSP molecule containing the platelet binding site. The binding site resides within a thermolytic fragment of TSP with Mr 140 000 but is not present in the Mr 120 000 fragment derived from the polypeptide of Mr 140 000. Since both the Mr 140 000 and 120 000 fragments contain fibrinogen binding sites, this finding suggests that the binding of TSP to the platelet surface requires interaction with other platelet surface components in addition to fibrinogen. The observation that fibrinogen only partially inhibits the TSP-mediated agglutination of fixed, activated platelets is consistent with this interpretation.     

Characterization of a fragment of bovine von Willebrand factor that binds to platelets

Bovine von Willebrand factor was digested with human plasmin in order to isolate and characterize a fragment that can bind to human platelets. A terminal plasmin digest of bovine von Willebrand factor is composed of five fragments, ranging in relative molecular weight (Mr) from 250,000 to 35,000. The major fragment has a Mr of 250,000 and consists of four disulfide-linked polypeptide chains with Mr from 69,000 to 35,000. The Mr 69,000 and 49,000 polypeptides possess carbohydrate moieties, as indicated by their reaction with periodate-Schiff reagent. Gel filtration studies suggest that, at physiological ionic strength, four of the Mr 250,000 fragments associate into a limited noncovalent oligomer. Monoclonal antibodies were prepared against native von Willebrand factor and used to characterize the distribution of epitopes on native vWF and the Mr 250,000 major fragment. Two of the monoclonal antibodies that recognize the major fragment (2 and H-9) inhibit platelet agglutination. The Mr 250,000 fragment binds to human platelets, and the binding is inhibited by monoclonal antibodies 2 and H-9. The Mr 250,000 fragment does not agglutinate platelets, consistent with a requirement for high molecular weight oligomers of von Willebrand factor for platelet agglutination. The Mr 250,000 fragment can compete with intact, bovine von Willebrand factor for binding to human platelets. However, its affinity is one-tenth that of intact von Willebrand factor.     

Gramicidin-S: Structure-activity relationship

The two side chain amino groups of the two L-ornithine residues in gramicidin-S seem to be important for the antibacterial activity of the molecule, since complete acetylation, formylation, carbamylation, deamination, trinitrophenylation, succinylation, maleylation of the antibiotic caused 90–95 % loss of the antibacterial activity of the antibiotic. However this modification leads to only 12–30% loss of the hemolytic activity. Monoacetyl- and monoformyl gramicidin-S with a free amino group retains nearly 50% of the antibacterial activity of the molecule. It seems, therefore, that the two amino groups contribute equally to the antibacterial activity of gramicidin-S Presented at 53rd Annual General Body Meeting of the Society of Biological Chemists (India), New Delhi, October, 1984     

Identification of amino acid residues essential for heparin binding by the A1 domain of human von Willebrand factor

Platelet adhesion is mediated by von Willebrand factor (VWF) that binds platelet glycoprotein Ib (GPIb). Previous observations suggested that heparin competitively inhibits the binding of VWF to GPIb and may down-regulate platelet adhesion. We performed charged-to-alanine scanning mutagenesis of domain A1 and studied dose-dependent binding to heparin-Sepharose beads. Mutations at Lys1362 and Arg1395, at which the GPIb binding was markedly decreased, showed 41% and 42% binding, respectively. Clustered mutations in the segments 1332KDRKR1336 and 1405KKKK1408, which have been proposed as heparin binding sequences, showed 72% and 52% binding, respectively. However, single alanine substitutions within these clusters showed normal binding. Our findings suggest that heparin may inhibit the binding of VWF to GPIb by interacting with GPIb binding and interpret why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.     

Human von Willebrand factor: a multivalent protein composed of identical subunits

A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.     

The domain of platelet glycoprotein I as recognized by various antibodies, both monoclonal and polyspecific

Platelet membrane glycoprotein Ib plays a major role in the binding of factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium. We have used polyspecific and monoclonal antibodies to glycoprotein Ib and have demonstrated that both antibodies were directed to glycoprotein Is, a soluble fragment of glycoprotein Ib. By showing an inhibition of the binding of factor VIII/von Willebrand factor to control platelets in presence of the antibodies, it can be concluded that glycoprotein Is is involved in these binding sites.     

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 alpha-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.     

Identification of the disulphide bonds in human platelet glycocalicin

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献