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.     

Exposure of platelet binding sites in von Willebrand factor by adsorption onto polystyrene latex particles

Von Willebrand factor molecules are flexible linear polymers composed of repeating protomeric polypeptide subunits. In the process of primary hemostasis, von Willebrand factor promotes platelet adhesion and platelet plug formation at the site of vascular injury. This biologic activity is apparently related to the multimeric size of von Willebrand factor. We simulated von Willebrand factor binding to the subendothelial surface by adsorbing purified human von Willebrand factor onto polystyrene latex particles of two different diameters, i.e., 0.312 μm and 2.02 μm. The rate and extent of ¹²⁵I-labeled von Willebrand factor binding to polystyrene was similar with both size classes of latex particles. The von Willebrand factor-coated latex beads of 2.02 μm diameter, in contrast to the smaller size, induced rapid agglutination of formalin-fixed human platelets in the absence of any other aggregating agent. Von Willebrand factor was also adsorbed from human plasma onto latex particles coated with anti-von Willebrand factor antibodies. Again, only the large beads, carrying the von Willebrand factor-antibody complex, induced agglutination of fixed platelets. Shear stress promoted the rate of von Willebrand factor adsorption to latex particles. Our results suggest that adsorption to surface exposes binding sites in human von Willebrand factor for platelets.     

Proteolytic studies on the structure of bovine von Willebrand factor

Bovine von Willebrand factor (vWF) was digested with protease I (P-I), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with P-I yielded a terminal digest consisting of an equimolar mixture of two major fragments (apparent Mr 250K and 200K). The 250-kilodalton (kDa) fragment consists of a 125-kDa chain from one subunit and a 45- and 78-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment consists of a 97-kDa chain from one subunit and a 35- and 61-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment binds to heparin, and the heparin binding domain is located on the 97-kDa polypeptide chain. This fragment also competes with labeled, native vWF for binding to formalin-fixed human platelets, with an IC50 of 12.5 micrograms/mL (65 nM). However, native vWF has an IC50 of 2.5 micrograms/mL, indicating that the affinity of the 200-kDa fragment for platelets is approximately one-fifth that of vWF. The 200-kDa fragment agglutinates platelets, but its agglutinating ability is only 5% that of the native molecule. Only the 200-kDa fragment is recognized by monoclonal antibodies 2 and H-9, which are directed against vWF and inhibit vWF binding to platelet glycoprotein Ib (GPIb). Immunological studies, using nine monoclonal antibodies directed against vWF, and the demonstration that the heparin and GPIb binding domains are located on only one fragment suggest that the two fragments are composed of different regions of the vWF subunit. Analysis of the P-I cleavage pattern suggests that all vWF subunits are not cleaved in the same fashion. The first cleavage on half of the subunits generates the 45-kDa terminal and 175-kDa intermediate digest products. The 175-kDa chain is again cleaved, producing the 97- and 78-kDa terminal polypeptide chains. However, the first cleavage of the other subunits generates the 35-kDa terminal and the 186-kDa intermediate digest product, which upon cleavage produces the 125- and 61-kDa terminal polypeptide chains. Immunological data support the asymmetric cleavage pattern. An epitope for a monoclonal antibody is present on both the 186- and 175-kDa intermediate digest products but is only found on one terminal digest fragment, the 78-kDa polypeptide chain, suggesting that the 186- and 175-kDa polypeptides are cleaved at different sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.     

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.     

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.     

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.     

Effects of diamide and dibucaine on platelet glycoprotein Ib, actin-binding protein and cytoskeleton

During extraction of platelets by 1% Triton X-100, the actin-binding protein (platelet filamin) and a 230 kDa protein are degraded by a calcium-activated thiol protease. Occurrence of degradation products of Mr 190 000 (HF-1) and 90 000 (HF-2) is a sensitive indicator of this proteolysis, and can be used to decide whether reduced amounts of the actin-binding protein in extracts are due to proteolysis or to incorporation in the Triton-insoluble (cytoskeletal) fraction. Diamide, which is a sulfhydryl-oxidizing protein cross-linker, inhibits the calcium-activated protease, polymerizes the actin-binding protein and the 230 kDa protein, increases the incorporation of glycoprotein Ib into the cytoskeletal fraction, and inhibits platelet agglutination induced by bovine von Willebrand factor. Inhibition of platelet agglutination by pretreatment with diamide is partly reversed by dibucaine which activates the calcium-activated protease. These observations are in accordance with a working hypothesis that interactions of glycoprotein Ib with cytoskeleton affect, and possibly regulate, its receptor function in the intact platelet.     

Alboaggregin-B: a new platelet agonist that binds to platelet membrane glycoprotein Ib.

A new protein, called alboaggregin-B (AL-B), has been isolated from Trimeresurus albolabris venom by ion-exchange chromatography. It agglutinated platelets without the need for Ca2+ or any other cofactor. The purified protein showed an apparent molecular mass on SDS-PAGE and gel filtration of about 23 kDa under nonreducing conditions. Ristocetin did not alter the binding of AL-B to platelets or affect AL-B-induced platelet agglutination. Agglutinating activity was not dependent on either proteolytic or lectin-like activity in AL-B. Binding analysis showed that AL-B bound to platelets with high affinity (Kd = 13.6 +/- 9.3 nM) at approximately 30,800 +/- 14,300 binding sites per platelet. AL-B inhibited the binding of labeled bovine von Willebrand factor (vWF) to platelets. Monoclonal antibodies against the 45-kDa N-terminal domain of platelet glycoprotein Ib inhibited the binding both of AL-B and of bovine vWF to platelets, and also inhibited platelet agglutination induced by AL-B and bovine vWF. Specific removal of the N-terminal domain of GPIb by treatment of the platelets with elastase or Serratia marcescens protease reduced the binding of labeled AL-B and bovine vWF to platelets and blocked platelet agglutination caused by both agonists. Monoclonal antibodies to glycoprotein IIb/IIIa, to bovine vWF, and to bovine serum albumin did not show any effect on the binding of AL-B to platelets. Our results indicate that the binding domain for AL-B on platelet GPIb is close to or identical with the one for vWF. This new protein may be a very useful tool for studying the interaction between platelets and vWF.     

Isolation and characterization of a collagen binding domain in human von Willebrand factor

von Willebrand factor binds to fibrillar type I collagen in a rapid, temperature-independent, reversible, specific, and saturable manner. Evaluation of binding isotherms by Scatchard-type analysis demonstrated that 6-18 micrograms of von Willebrand factor bind per mg of collagen, with Ka between 2 and 8 X 10(8) M-1. Five distinct tryptic fragments, purified under denaturing and reducing conditions and representing over 75% of the molecular mass of the von Willebrand factor subunit, were tested for their capacity to inhibit the von Willebrand factor-collagen interaction. Complete inhibition was obtained with a 52/48-kDa fragment at a concentration of approximately 1 microM. The location of this fragment in the subunit was established to be between Val-449 and Lys-728. Fifteen monoclonal antibodies against the 52/48-kDa fragment inhibited von Willebrand factor binding to collagen. Six antibodies against other portions of the von Willebrand factor subunit had no inhibitory effect. The tryptic fragment was a competitive inhibitor of von Willebrand factor binding to collagen and, therefore, recognizes the same interaction site as the intact molecule. These studies precisely define a domain in the von Willebrand factor subunit that interacts with type I collagen.     

The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane

Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (means +/- SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib alpha (22 000 +/- 2700, n = 3), WM 23, epitope on GP Ib alpha (21 000 +/- 3400, n = 3), FMC 25, epitope on GP IX (20 100 +/- 2700, n = 3), and FMC 25 (Fab')2 (27 100 +/- 800, n = 2).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.     

Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.     

von Willebrand factor. A protein which binds at the cell surface interface between platelets

von Willebrand factor (VWF) functions in platelet aggregation, a form of cellular interaction. In vitro analysis of platelet aggregation, as measured by the platelet aggregometer, requires addition of a promoter such as the glycopeptide antibiotic ristocetin. Native multimeric VWF (Mr = 1-20 X 10(6)) can be reduced with sulfhydryl reagents to a monomeric state (Mr = 2 X 10(5)). In this study, the binding of bovine VWF and ristocetin to bovine platelets was investigated using fluorescence anisotropy of derivatized monomer protein and ristocetin and also by radioisotope methods using 125I-labeled monomer and native protein. Ristocetin bound to bovine platelets but not to VWF. VWF binding to formaldehyde-fixed platelets was dependent on the presence of a promoter such as ristocetin. The monomer and multimer VWF bound equally well in the presence of low ristocetin concentrations. Under these conditions, plots of VWF binding versus platelet concentration were sigmoidal, indicating positive cooperativity with respect to platelets. At higher (100 micrograms/ml) ristocetin concentrations, the binding curve was no longer sigmoidal. Ristocetin promoted the formation of small platelet aggregates, an effect that was amplified by the presence of VWF. In fact, all conditions which resulted in monomer or multimer VWF binding to platelets also caused formation of platelet aggregates observed by light microscopy. These combined results were consistent with VWF binding only to the interface between proximal platelets. High affinity binding could be provided by the presence of two cell surfaces and the resulting multiple binding interactions. Polycations, such as poly(L-lysine) and Polybrene, also promoted the formation of platelet aggregates and facilitated the binding of VWF to platelets. Physiological platelet activators such as thrombin, ADP, and collagen also facilitated VWF binding to native platelets and caused platelet aggregation. It appears possible that any process which causes the surface membranes of platelets to become spatially close will allow expression of VWF activity.     

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli

We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.