PubMed Health⌕ Search

Biomedical subjects

J F Dong

Publications and source records attributed to J F Dong.

At least 19 recordsLinked to original sources
PubMed2005

Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress.

Leukocyte rolling on vascular endothelium is mediated by an interaction between P-selectin expressed on endothelial cells and P-selectin glycoprotein ligand-1 on leukocytes. This interaction reduces the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra-large von Willebrand factor (ULVWF) forms extremely long string-like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P-selectin and bound PAC-1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet-decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm(2) in a P-selectin dependent manner. These results suggest that the endothelial cell-bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.

Adult↗
PubMed2003

Sulfatides inhibit platelet adhesion to von Willebrand factor in flowing blood.

Sulfatides are sulfated glycosphingolipids present on cell surfaces that bind to adhesive proteins such as von Willebrand factor (VWF), P-selectin, laminin and thrombospondin. Previous studies have localized the sulfatide-binding site of VWF to amino acid residues Gln626-Val646 in the A1 domain. The A1 domain also contains the binding site for platelet glycoprotein Ib (GP Ib), a site that has been reported to be distinct from the sulfatide-binding site. In this study, we analyzed the interaction of sulfatides with VWF and its effect on GP Ib-mediated platelet adhesion under flow conditions. Recombinant VWF A1 domain (rVWF-A1) bound specifically and saturably to sulfatides (half-maximal concentration of approximately 12.5 microg mL(-1)), binding that was blocked by dextran sulfate (IC(50) approximately equal to 100 microg mL(-1)) but not by heparin at concentrations up to 100 U mL(-1). Furthermore, sulfatides (125 microg mL(-1)) prevented the adhesion of platelets or glycocalicin-coupled polystyrene beads to a rVWF-A1-coated surface under high shear stress. In addition, plasma VWF prebound to a sulfatide-coated surface failed to support subsequent platelet adhesion. These results provide firm evidence that sulfatides bind the VWF A1 domain at a site overlapping the GP Ib-binding site.

Cloning, Molecular↗
PubMed2001

Cholesterol sulfate: a new adhesive molecule for platelets.

BACKGROUND: Cholesterol 3-sulfate is present on a variety of cells and in human LDL, and it has been found in atherosclerotic lesions of human aorta. Its precise biological role has not yet been described. METHODS AND RESULTS: In this study, we investigated the interaction of platelets with cholesterol sulfate. Platelets adhered in a concentration-dependent and saturable manner to cholesterol sulfate but did not adhere to cholesterol, cholesterol acetate, estrone sulfate, or dehydroepiandrosterone sulfate, suggesting that the specificity of this interaction is determined not only by the cholesterol moiety but also by the sulfate group. This adhesion did not increase after platelet activation, and it was not cation-dependent. Soluble cholesterol sulfate inhibited adhesion in a concentration-dependent manner. However, antibodies against glycoprotein Ib, glycoprotein IIb/IIIa, CD36, P-selectin, von Willebrand factor, or thrombospondin had no significant effect on platelet adhesion to cholesterol sulfate. Perfusion of whole blood in a parallel-plate flow chamber resulted in the rapid and progressive adhesion of platelets to cholesterol sulfate but not to cholesterol acetate or estrone sulfate. CONCLUSIONS: Cholesterol sulfate supports platelet adhesion and may be one of the factors determining the prothrombotic potential of atherosclerotic lesions.

Antibodies↗
PubMed2001

Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions.

Under conditions of high shear stress, both hemostasis and thrombosis are initiated by the interaction of the platelet membrane glycoprotein (GP) Ib-IX-V complex with its adhesive ligand, von Willebrand factor (vWF), in the subendothelial matrix or plasma. This interaction involves the A1 domain of vWF and the N-terminal extracellular region of GP Ibalpha (His-1-Glu-282), and it can also be induced under static conditions by the modulators ristocetin and botrocetin. In this study, a panel of anti-vWF and anti-GP Ibalpha antibodies-previously characterized for their effects on ristocetin- and botrocetin-dependent vWF-GP Ib-IX-V interactions-was analyzed for their capacity to inhibit either the adhesion of Chinese hamster ovary cells expressing recombinant GP Ibalpha to surface-associated vWF under hydrodynamic flow or shear-stress-induced platelet aggregation. The combined results suggest that the shear-dependent interactions between vWF and GP Ibalpha closely correlate with ristocetin- rather than botrocetin-dependent binding under static conditions and that certain anti-vWF monoclonal antibodies are able to selectively inhibit shear-dependent platelet aggregation.

Antibodies, Monoclonal↗
PubMed2000

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region.

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.

Amino Acid Sequence↗
PubMed2000

Requirement of leucine-rich repeats of glycoprotein (GP) Ibalpha for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib-IX-V complex.

The platelet glycoprotein (GP) Ib-IX-V complex mediates adhesion to von Willebrand factor (vWf) in (patho)physiologic thrombus formation. The vWf-binding site on GP Ib-IX-V is within the N-terminal 282 residues of GP Ibalpha, which consist of an N-terminal flanking sequence (His-1-Ile-35), 7 leucine-rich repeats (Leu-36-Ala-200), a C-terminal flank (Phe-201-Gly-268), and a sulfated tyrosine sequence (Asp-269-Glu-282). We have used mammalian cell expression of canine-human chimeras of GP Ibalpha, corresponding to precise structural boundaries, to demonstrate the first specific requirement for individual leucine-rich repeats for binding of vWf either induced by a modulator, ristocetin, or under hydrodynamic flow. Implicit in this approach was that the GP Ibalpha chimeras retained a functional conformation, a supposition confirmed by analyzing restoration of function to reversed human-canine chimeras and demonstrating that all chimeras bound vWf activated by botrocetin, a modulator that is indiscriminate between species. Leucine-rich repeats 2, 3, and 4 of GP Ibalpha were identified as being critical for vWf adhesion to GP Ib-IX-V.

Animals↗
PubMed1999

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Animals↗
PubMed1999

[Detection of TT virus DNA in peripheral blood mononuclear cells in patients with chronic hepatitis B and hepatocellular carcinoma using PCR and in situ hybridization].

OBJECTIVE: To determine whether the TTV infection occurs in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B (of medium degree) and in HBsAg positive hepatocellular carcinoma (HCC). METHODS: TTV DNA in PBMCs was detected by nested-polymerase chain reaction(PCR) and in situ hybridization (ISH). RESULTS: 7 of 26 cases of chronic hepatitis B were positive for TTV DNA by nested-PCR with a positive rate of 26.9% that was significantly higher than that of normal control(chi 2 = 14.3, P < 0.001); 4 of 21 HBsAg positive cases were positive for TTV DNA by nested-PCR with a positive rate of 19.0%, also significantly higher than that of normal control(chi 2 = 4.86, P < 0.05). 4 of 7 TTV positive patients with chronic hepatitis B tested by nested-PCR were positive for TTV DNA by ISH in PBMC cytoplasm, with a positive rate of 57.1%. CONCLUSION: (1) A higher incidence of TTV DNA in PBMCs of patients with chronic hepatitis B and in HBsAg positive HCC was identified by nested-PCR and ISH; (2) TTV DNA was in the cytoplasm of PBMCs in patients with chronic hepatitis B.

Adult↗
PubMed1998

Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex.

The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibalpha, GP Ibbeta, GP IX, and GP V. Glycoprotein Ibalpha contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibbeta and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibalpha expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibalpha, however, was degraded when it was expressed in partial complexes with only GP Ibbeta or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibalpha expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibbeta and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibalpha to the plasma membrane. Absence of either GP Ibbeta or GP IX increased the rate of GP Ibalpha degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibalpha expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex.

Biological Transport↗
PubMed1998

Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex.

Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.

Animals↗
PubMed1997

The cytoplasmic domain of glycoprotein (GP) Ibalpha constrains the lateral diffusion of the GP Ib-IX complex and modulates von Willebrand factor binding.

To study the role of the glycoprotein (GP) Ibalpha cytoplasmic domain in the mobility of the GP Ib-IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines expressing GP Ib-IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibalpha or one of a series of GP Ibalpha truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibalpha monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibalpha was negligible. However, when only six amino acids were removed from the GP Ibalpha carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibalpha), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55-67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibalpha cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibalpha fixes the position of the GP Ib-IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.

Binding Sites↗
PubMed1997

Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex.

We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 micromol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti-GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.

Animals↗
PubMed1997

Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site.

The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ib alpha, GP Ib beta, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ib alpha, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ib alpha was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.

Allosteric Regulation↗
PubMed1997

Structure and function of the glycoprotein Ib-IX-V complex.

Following injury to a blood vessel, platelets rapidly adhere to the site of exposed subendothelial matrix in the vessel wall, become activated, and secrete granule contents that will recruit and activate other platelets, which then aggregate at the site and seal the defect in the vessel wall. The clot that forms prevents further blood loss. This process can become deranged in diseased blood vessels, such as coronary arteries narrowed by atherosclerosis. Platelets will also adhere to these vessels when matrix components are exposed by plaque rupture, with potentially disastrous results. The platelet glycoprotein Ib-IX-V complex plays crucial roles in both of these processes by mediating platelet adhesion to sites of blood vessel injury and by participating in the thrombin induced aggregation of platelets. This adhesion receptor complex is unique in that it is the only one of its kind. In this review, we discuss recent developments in elucidating the structure, topography, and functions of this important receptor complex.

Animals↗
PubMed1996

Biosynthetic defect in platelet glycoprotein IX mutants associated with Bernard-Soulier syndrome.

To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib-IX expression resulting from GP IX mutations described in three siblings with Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45 --> Ser) and examined the associations of the mutants with the two other subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP IX, neither of the mutants was able to increase GP Ib expression on the cell surface, either when transfected into Chinese hamster ovary (CHO) alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into wild-type CHO cells. We also evaluated whether cotransfecting wild-type or mutant GP IX with GP Ib beta would result in the appearance of GP IX on the surface of the transfected cells; the wild-type protein was detected on the surface of the cells, whereas neither mutant reached the cell surface in appreciable quantities. Immunofluorescence microscopy of permeabilized cells revealed that the failure to express mutant GP IX on the cell surface did not result from failure to synthesize the polypeptide. Both mutants were detected in intracellular compartments, albeit at lower levels than the wild-type polypeptide (the fluorescence of cells expressing the GP IX Asp21 --> Gly was consistently the lowest). Direct evidence that the mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount of GP IX coprecipitated with GP Ib beta was greatly diminished in cells expressing either mutant. These findings suggest an important role for the conserved leucine-rich motif of GP IX in the association of this polypeptide with GP Ib beta and provide further evidence for the importance of GP IX in the stability of the GP-Ib-IX complex.

Amino Acid Sequence↗
PubMed1995

Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GP Ib alpha.

The glycoprotein (GP) Ib-IX complex mediates platelet aggregation in response to high shear forces by binding von Willebrand factor (vWF) in the plasma. We investigated the possibility that the complex could mediate a similar phenomenon if expressed in nonhematopoietic cells. When agitated on a tabletop shaker, CHO and L cells expressing the full complex formed large aggregates in the presence of vWF and the modulator ristocetin. When the rate of agitation was increased, aggregation occurred without added ristocetin and appeared to require only the application of a physical force. The aggregation was homophilic and temperature-dependent and required a functional ligand-binding subunit of the GP Ib-IX complex, GP Ib alpha. Posttranslational tyrosine sulfation of GP Ib alpha was required for aggregate formation and stability. Thus, aggregation of cells expressing the GP Ib-IX complex is a unique example of a ligand-receptor interaction induced by mechanical forces and demonstrates an important biological role for sulfation of tyrosine residues.

Animals↗
PubMed1995

Expression of platelet glycoprotein (GP) V in heterologous cells and evidence for its association with GP Ib alpha in forming a GP Ib-IX-V complex on the cell surface.

The glycoprotein (GP) Ib-IX-V complex comprises four polypeptides: the subunits of the GP Ib-IX complex (GP Ib alpha, GP Ib beta, GP IX) and GP V. To determine the requirements for cell-surface expression of GPV, we transiently expressed the recombinant polypeptide in wild-type Chinese hamster ovary (CHO) cells by cotransfection with plasmids for the subunits of the GP Ib-IX complex and in CHO cells that stably express different combinations of the GP Ib-IX complex subunits. Glycoprotein V expressed alone was detectable on the cell surface, and the level was not augmented by cotransfection with any one of the subunits of the GP Ib-IX complex. However, when GP V was expressed in cells that stably express combinations of GP Ib-IX complex subunits, its expression on the cell surface was greater in all the cell lines that contained GP Ib alpha than in wild-type CHO cells. That GP V associates with GP Ib alpha was also suggested by confocal microscopy studies: GP V colocalized with GP Ib alpha in CHO alpha beta IX (cells that express GP Ib alpha, GP Ib beta, and GP IX), CHO alpha beta, and CHO alpha IX cells, but did not colocalize with GP Ib beta in CHO beta IX cells. Similarly, immunoprecipitation of GP V from cells expressing GP Ib alpha led to coprecipitation of the latter polypeptide; neither GP Ib beta nor GP IX coprecipitated with GP V from CHO beta IX cells. Taken together, these data indicate that GP V associates with the GP Ib-IX complex through a direct interaction with GP Ib alpha and establish the topology of the GP Ib-IX-V subunits on the cell surface.

Animals↗