Purification of glycocalicin from human plasma.

Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.

Reconstitution of the platelet glycoprotein Ib-IX complex in phospholipid bilayer Nanodiscs.

The glycoprotein Ib-IX (GPIb-IX) complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis via the initiation of adhesion of platelets to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge of the GPIb-IX complex was obtained from studies on platelets and transfected mammalian cells expressing the GPIb-IX complex, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that the GPIb-IX complex purified from human platelets has been reconstituted into the Nanodisc. The Nanodisc-reconstituted GPIb-IX complex was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent K(d) of 0.73 ± 0.07 nM. The binding to VWF was inhibited by anti-GPIbα antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIbß monoclonal antibody RAM.1. Finally, the Nanodisc-reconstituted GPIb-IX complex exhibited ligand binding activity similar to that of the isolated extracellular domain of GPIbα. In conclusion, the GPIb-IX complex in Nanodiscs adopts a native-like conformation and possesses the ability to bind its natural ligands, thus making a Nanodisc a suitable in vitro platform for further investigation of this hemostatically important receptor complex.

[Development, identification and function assay of monoclonal antibody against platelet membrane glycoprotein Ib].

AIM: To prepare and identify a monoclonal antibody (mAb) against human platelet glycoprotein Ib and make its application. METHODS: BALB/c mice were immunized with human platelets washed, and the spleen cells of them were fused with myloma cells. A hybridoma cell was screened by indirect ELISA and cloned, and the mAb were purified from the ascites of mice. Ig subclass was analysed by double immunodiffusion. The antigen recognized by monoclonal antibody was identified by flow cytometry and radioimmunoassy, respectively. The inhibition of mAb on plasma von Willebrand factor ristocetin cofactor activity (vWF:Rcof) was investigated by enzyme linked immunosorbent assay (ELISA). RESULTS: A murine mAb against human platelet membrane glycoprotein (GP) Ib was developed and denominated as SZ-151. SZ-151 belonged to IgG1 subclass and its titer in ascites was 1:20 000. Flow cytometry and radioimmunoassy showed that the antigen recognized by monoclonal antibody SZ-151 was platelet membrane GPIb. ELISA showed that SZ-151 did not inhibit plasma von Willebrand factor ristocetin cofactor activity. CONCLUSION: A mAb, SZ-151 against platelet glycoprotein Ib was developed, which could be useful in assays of plasma von Willebrand factor ristocetin cofactor activity(vWF:Rcof) and can be used for diagnose patients with vWD.

ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor.

Thrombotic thrombocytopenic purpura is caused by congenital or acquired deficiency of ADAMTS-13, a metalloprotease that cleaves the endothelium-derived ultra-large multimers of von Willebrand factor (ULVWF). The proteolysis converts hyper-reactive and thrombogenic ULVWF into smaller and less adhesive plasma forms. Activity of ADAMTS-13 is usually measured in a static system under non-physiological conditions that require protein denaturation and prolonged incubation. We have demonstrated previously that ULVWF multimers, upon release from endothelial cells, form platelet-decorated string-like structures that are rapidly cleaved by ADAMTS-13. Here we report the direct interaction between ADAMTS-13 and VWF under both static and flowing conditions. ADAMTS-13-coated beads adhered to both immobilized VWF and ULVWF strings presented by stimulated endothelial cells. These beads adhered to VWF under both venous (2.5 dynes/cm2) and arterial (30 dynes/cm2) shear stresses. We then demonstrated that ADAMTS-13 beads adhered to immobilized recombinant VWF-A1 and -A3 domains, but soluble metalloprotease bound preferentially to the A3 domain, suggesting that the VWF A3 domain may be the primary docking site for the metalloprotease. We suggest that tensile stresses imposed by fluid shear stretch endothelial bound ULVWF multimers to expose binding sites within the A domains for circulating ADAMTS-13. The bound enzyme then cleaves within the A2 domain that lies in close proximity and releases smaller VWF multimers into the plasma. Once released, these cleaved VWF fragments become inaccessible for the metalloprotease to prevent further cleavage.

Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib.

The activation of human platelets by alpha-thrombin is mediated in part by cleavage of the protease-activated receptor (PAR) 1 and 4 and by the glycoprotein (Ib)alpha, (Gp(Ib)alpha), which binds with high affinity to alpha-thrombin. Recent studies have shown that the thrombin domain referred to as heparin binding site (HBS) is involved in the interaction with the platelet Gp(Ib)alpha. The HBS is rich in basic amino acids. To identify the key amino acid residues involved in the binding to Gp(Ib)alpha, we have performed alanine scanning mutagenesis of the basic HBS R93, R97, R101, R233, K236, K240, R233/K236/Q239, as well as of the neutral Q239 residues, located in different regions of the domain. For comparison, mutation at R67 within the fibrinogen recognition site (FRS) of thrombin was performed as well. Solid-phase binding experiments showed that the Kd of thrombin-GpIb interaction was reduced 22-fold for R93A, 8-fold for R97A, 13-fold for R101A, 29-fold for R233A, 21-fold for K236A, 5-fold for K240A, and 31-fold for the triple mutant R233A/K236A/Q239A, while the Q239A and R67A forms did not show any significant affinity change. The platelet activating capacity of these mutants was evaluated as well. Using gel-filtered platelets, the EC50 value of thrombin-induced aggregation was from 5- to 13-fold higher in the HBS mutants than in the WT form, and was linearly and positively correlated with the corresponding Kd values pertaining to thrombin binding to GpIb. Measurements of PAR-1 hydrolysis on the platelet membrane showed that the HBS mutants R233A, R101A, R93A, K236A, and R233/K236/Q239 forms had a reduction of the apparent kcat/Km value. These results are a consequence of a defective binding to GpIb, which is known to optimize the interaction with PAR-1 in situ. A confirm of this hypothesis came from the demonstration that the kcat/Km value pertaining to the hydrolysis by the HBS-mutated thrombins of the synthetic PAR-1 38-60 peptide in solution was similar to that one obtained with the WT form. In conclusion, these experiments provide a structural and functional mapping of the thrombin HBS subregions involved in the binding to the platelet Gp(Ib)alpha and in the cell activation.

Homozygous Pro74-->Arg mutation in the platelet glycoprotein Ibbeta gene associated with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. This complex is composed of four subunits, GPIbalpha, GPIbbeta, GPIX and GPV. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patient's platelets showed greatly reduced GPIbalpha and GPIX surface expression. Immunoblot analysis disclosed absence of GPIbalpha, GPIbbeta and GPIX in the platelets. DNA sequencing analysis revealed a novel missense mutation in the GPIbbeta gene that converts Pro (CCG) to Arg (CGG) at residue 74. Homozygosity of the mutation was confirmed by allele-specific restriction analysis, chromosome 22 microsatellite analysis and quantitative Southern blotting. The mutant GPIbbeta was normally transcribed. Transient transfection studies confirmed that mutant GPIbbeta impairs surface expression of GPIb/IX, showing that the mutation is responsible for a BSS phenotype observed in the patient.

Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen.

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affinity chromatography. The domain 3-derived peptide, thrombin, HK, factor XII, as well as antibody to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above-mentioned ligands bound to this protein. Isolation of platelet membrane proteins using a monoclonal anti-glycocalicin antibody column revealed the same HK binding protein at 74 kD, which was reactive with anti-GPIb and represents a GPIb fragment. By photoaffinity labeling, HK interacted with membrane GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, (125)I-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest that the GPIb alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3.

Glycocalicin levels in the plasma of HIV+ patients: an indicator of platelet turnover.

Glycocalicin (GC) is the carbohydrate-rich portion of platelet membrane glycoprotein Ib(alpha) that can be cleaved from circulating platelets by proteases. The plasma GC level is an indicator of platelet turnover. Using an ELISA for GC, we assayed the plasma of 20 normal children (age 6 to 13 years), 50 HIV+ children (ages 4 to 18 years), 32 normal adults (ages 21 to 53 years), and 50 HIV+ adults (ages 24 to 66 years). The results were adjusted for individual platelet counts to give GC indexes (GCI). The normal children and the normal adults had significantly different GCI distributions (P = .002). In both normal and HIV+ individuals the GCI decreased with increasing platelet count (-.73 < r < -.34). Twenty-eight percent of the HIV+ children and 28% of the HIV+ adults had elevated GCI values. The majority of these elevated values occurred in patients with platelet counts >100,000/microL. Neither the GCI nor the platelet count was correlated with viral load. The platelet count, however, was weakly correlated with the CD4 count in both children (r = .31) and adults (r = .30) infected with HIV. Also, the CD4 count was weakly and inversely correlated with GCI in HIV+ adults (r = -.34) and in children (r = -.24). We conclude that increased GCI and, by implication, increased platelet turnover is a relatively common feature of advanced HIV disease. Furthermore, GCI may be elevated in HIV+ patients even with a platelet count >100,000/microL, suggesting increased platelet turnover before thrombocytopenia develops.

Physical proximity and functional interplay of the glycoprotein Ib-IX-V complex and the Fc receptor FcgammaRIIA on the platelet plasma membrane.

Although the glycoprotein (GP) Ib-IX-V complex and FcgammaRIIA are distinct platelet membrane receptors, previous studies have suggested that these structures may be co-localized. To determine more directly the proximity of GP Ib-IX-V and FcgammaRIIA, we assessed the effects of anti-GP Ibalpha monoclonal antibodies on FcgammaRIIA-mediated platelet aggregation and on the direct binding of polymeric IgG to human platelets. In addition, we directly examined the proximity of FcgammaRII and GP Ib-IX-V using flow cytometric fluorescence energy transfer and immunoprecipitation studies. Preincubation of platelets with either of two monoclonal antibodies (AN51 or SZ2) directed against GP Ibalpha completely blocked platelet aggregation by polymeric IgG. Similarly, these antibodies totally inhibited platelet aggregation by two strains of viridans group streptococci known to induce aggregation via FcgammaRIIA. In addition, AN51 and SZ2 significantly reduced the binding of polymeric IgG to washed fixed platelets. When assessed by flow cytometry, significant levels of bidirectional energy transfer were detected between FcgammaRIIA and GP Ibalpha, indicating a physical proximity of less than 10 nm between these receptors. This energy transfer was not due to high receptor density, because no homoassociative energy transfer was seen. Moreover, immunoprecipitation of FcgammaRIIA from platelet lysates also co-precipitated GP Ibalpha. These results indicate that GP Ibalpha and FcgammaRIIA are co-localized on the platelet membrane and that this association is not random.

Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein IBalpha.

Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ibalpha in ABP-280. A melanoma cell line lacking ABP-280 was stably transfected with the cDNAs coding for GP Ib-IX, then transiently transfected with cDNA coding for various carboxyl-truncates of ABP-280. Immunocapture assays and co-immunoprecipitation experiments from detergent-lysed cells showed that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an ABP-280 fragment comprising repeats 17-19 bound GP Ibalpha. Deletion from either end abolished binding. Individual or multiple repeats of ABP-280 were expressed as fusion protein in bacteria and purified; structural folding was evaluated, and binding to GP Ib-IX was assessed. Binding depended on the presence of repeats 17-19. None of the individual repeats were able to bind to GP Ib-IX. These findings demonstrate that residues 1850-2136 comprising repeats 17-19 contain the binding site for GP Ib-IX.

Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha.

The zeta-form 14-3-3 protein (14-3-3zeta) regulates protein kinases and interacts with several signaling molecules. We reported previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX, was associated with a 29-kDa protein with partial sequences identical to 14-3-3zeta. In this study, the interaction between GPIb-IX and recombinant 14-3-3zeta is reconstituted. Further, we show that the 14-3-3zeta binding site in GPIb is within a 15 residue sequence at the C terminus of GPIb-alpha, as indicated by antibody inhibition and direct binding of 14-3-3zeta to synthetic GPIb-alpha cytoplasmic domain peptides. The 14-3-3zeta binds to recombinant wild type GPIb-IX but not to the GPIb-alpha mutants lacking C-terminal 5 or more residues, suggesting that the C-terminal 5 residues of GPIb-alpha are critical. Similarity between the GPIb-alpha C-terminal sequence and the serine-rich regions of Raf and Bcr kinases suggests a possible serine-rich recognition motif for the 14-3-3 protein.