scholarly journals Normal synthesis and expression of endothelial IIb/IIIa in Glanzmann's thrombasthenia

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 809-812
Author(s):  
JC Giltay ◽  
OC Leeksma ◽  
C Breederveld ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Protein Complex ◽  
Autosomal Recessive ◽  
Umbilical Vein ◽  
Platelet Glycoprotein ◽  
Membrane Protein Complex ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Umbilical Vein Endothelial Cells

Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) IIb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesized a membrane protein complex similar to the platelet GP IIb/IIIa complex. In this article, we demonstrate that endothelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP IIb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an “endotheliopathy.”

scholarly journals Normal synthesis and expression of endothelial IIb/IIIa in Glanzmann's thrombasthenia

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 809-812 ◽  
Author(s):  
JC Giltay ◽  
OC Leeksma ◽  
C Breederveld ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Protein Complex ◽  
Autosomal Recessive ◽  
Umbilical Vein ◽  
Platelet Glycoprotein ◽  
Membrane Protein Complex ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Umbilical Vein Endothelial Cells

Abstract Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) IIb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesized a membrane protein complex similar to the platelet GP IIb/IIIa complex. In this article, we demonstrate that endothelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP IIb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an “endotheliopathy.”

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180 ◽  
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

Abstract We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

NORMAL SYNTHESIS AND EXPRESSION OF ENDOTHELIAL GP IIb/IIIa IN GLANZMANN'S THROMBASTENIA

1987 ◽  
Author(s):  
J C Giltay ◽  
O C Leeksma ◽  
C Breederveld ◽  
J A van Mourik
Keyword(s):  
Endothelial Cells ◽  
Autosomal Recessive ◽  
Umbilical Vein ◽  
Membrane Glycoprotein ◽  
Hemorrhagic Disease ◽  
Membrane Composition ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis ◽  
Umbilical Vein Endothelial Cells ◽  
Immunochemical Techniques

In Glanzmann’s thrombastenia (GT), an autosomal recessive inherited hemorrhagic disease, the platelet membrane glycoprotein (GP) IIb/IIIa complex is absent or reduced. Recently we and others demonstrated that cultured human umbilical vein endothelial cells synthesize a membrane protein complex that is structurally closely related to GP IIb/IIIa. Endothelial cells of thrombasthénie patients could, therefore be deficient in GP IIb/IIIa. We had the opportunity to culture endothelial cells isolated from the umbilical cord of a newborn with GT and to examine the plasma membrane composition of these cells. Employing a variety of immunochemical techniques, including immunoprecipitation, immunofluorescence staining and crossed immunoelectrophoresis, we demonstrated that the endothelial cells of the patient were indistinguishable from normal endothelial cells in their ability to synthesize and express GP IIb/IIIa. Our results indicate that GT is not accompanied by and "endotheliopathy".Zwa (P1A1) is an alloantigen, located on platelet GP IIIa, and, consequently, Zwa is absent on GT platelet. Data will he presented which show that not only normal endothelial IIIa carries the Zwa antigen, hut also GT endothelial cells normally express Zwa. This finding supports our view that GT endothelial GP IIb/IIIa is indistinguishable from normal endothelial GP IIb/IIIa. Moreover, this finding directly shows that the thrombastenic glycoprotein abnormality and the inheritance of Zwa antigen are controlled by different genes.

scholarly journals Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 234-237 ◽  
Author(s):  
JD Sprandio ◽  
SS Shapiro ◽  
P Thiagarajan ◽  
S McCord
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Single Class ◽  
Human Umbilical Vein ◽  
Sds Page ◽  
Umbilical Vein Endothelial Cells ◽  
Triton X

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

scholarly journals Human Endothelial Cells in Culture and In Vivo Express on Their Surface All Four Components of the Glycoprotein Ib/IX/V Complex

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2660-2669 ◽  
Author(s):  
Guoxin Wu ◽  
David W. Essex ◽  
Frank J. Meloni ◽  
Toshiro Takafuta ◽  
Kingo Fujimura ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Molecular Weights ◽  
Hel Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand

The platelet glycoprotein Ib (GpIb) complex is composed of four polypeptides: the disulfide-linked GpIbα and GpIbβ and the noncovalently associated GpIX and GpV. GpIbα contains binding sites for von Willebrand factor and for thrombin and mediates platelet adhesion to the subendothelium under conditions of high shear stress. We have previously shown the presence of GpIbα and GpIbβ mRNA and protein in cultured human umbilical vein endothelial cells (HUVECs) as well as the presence of GpIbα mRNA and protein in tonsillar endothelium. We, therefore, probed ECs for the presence of the other components of the GpIb/IX/V complex. We have identified the presence of GpIX and GpV mRNA in cultured HUVEC monolayers. The sequence of HUVEC GpIX cDNA was identical to the previously published human erythroleukemia (HEL) cell GpIX cDNA sequence. Two species of GpV mRNA, one of 3 kb and one of 4.4 kb, were found in HUVECs, whereas HEL cells displayed only the 4.4-kb species and the megakaryocytic cell line CHRF-288 contained only the 3-kb species. We previously showed that EC GpIbα protein is identical in molecular weight to platelet GpIbα. HUVEC GpIbβ, in contrast to its platelet counterpart, has a molecular weight of 50 kD and forms a correspondingly larger disulfide-bonded complex with EC GpIbα. The molecular weights of GpIX and GpV were 22 and 88 kD, respectively, identical to the corresponding platelet polypeptides. Furthermore, we have identified all four components of the complex in tonsillar vessels. Using flow cytometry, we have established that all four polypeptides of the GpIb/IX/V complex are expressed on the surface membranes of cultured HUVECs and adult aortic ECs. Furthermore, using two-color fluorescence, we have shown that all ECs expressing GpIbα also express GpIX and GpV on their surface. The ratio of GpIbα:GpIX:GpV is 1:1:0.5, which is identical to the ratio present in platelets. None of the polypeptides of the GpIb complex could be identified on the surface of human smooth muscle cells or lymphocytes. The presence of all members of the GpIb complex in the EC membrane suggests that this complex may play a role in endothelial function in vivo.

Staphylococcal Superantigen-Like 5 Activates Platelets, and Supports Platelet Adhesion Under Flow Conditions, Which Is Mediated by GPIb- Alpha and Alpha-IIb-Beta-3

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3922-3922
Author(s):  
Cees Weeterings ◽  
Carla JC de Haas ◽  
Mignon M Vughs ◽  
Philip G de Groot ◽  
Jos van Strijp ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Real Time ◽  
Video Analysis ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Direct Interaction ◽  
Platelet Glycoprotein ◽  
Set Up ◽  
Umbilical Vein Endothelial Cells ◽  
Aggregation Of Platelets

Abstract Introduction - Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus Aureus and has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Since PSGL-1 shows close homology to platelet glycoprotein Ib-alpha (GPIb), and as platelets have been shown to play an important role in neutrophil chemotaxis, we investigated whether SSL5 also interacts with platelets. Results - When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, full and irreversible aggregation was observed even in the absence of exogenous fibrinogen. Proteolysis of the extracellular part of GPIb-alpha or the addition of dRGDW abrogated platelet aggregation. SSL5 was shown to interact with glycocalicin, a soluble GPIb-alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3zeta to the actin cytoskeleton. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s−1, large stable platelet aggregates were observed. Platelet adhesion was substantially reduced by proteolysis of GPIb-alpha or addition of dRGDW. In addition, real-time video analysis showed that initial tethering of the platelets was abrogated by proteolysis of GPIb-alpha. Furthermore, when whole citrated blood was perfused over immobilized SSL5, real-time video analysis revealed that platelets activated with the PAR-1 activating peptide SFLLRN made transient contacts with the immobilized SSL5, although no permanent contacts were observed. Finally, SSL5 was shown to interact with endothelial cell matrix (ECM) from cultured human umbilical vein endothelial cells. The interaction of SSL5 with ECM enhanced aggregation of platelets from whole blood to this ECM. Discussion - Here, we show that SSL5 is capable of activation, adhesion and aggregation of platelets, in which both GPIb-alpha and alpha-IIb-beta-3 play a role. We hypothesize that platelet activation by SSL5 could be important in colonization of the vascular bed and evasion of the immune system.

scholarly journals Characterization of multiple quinine-dependent antibodies in a patient with episodic hemolytic uremic syndrome and immune agranulocytosis

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 241-248 ◽  
Author(s):  
DF Stroncek ◽  
GM Vercellotti ◽  
DE Hammerschmidt ◽  
DJ Christie ◽  
RA Shankar ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Hemolytic Uremic Syndrome ◽  
Umbilical Vein ◽  
Protein A ◽  
Platelet Glycoprotein ◽  
Staphylococcal Protein A ◽  
Capture Assay ◽  
Uremic Syndrome ◽  
Neutrophil Aggregation ◽  
Umbilical Vein Endothelial Cells

Abstract A 23-year-old woman experienced six distinct episodes of severe combined neutropenia and thrombocytopenia. At least one of the episodes was accompanied by hemodialysis-requiring acute renal failure and fragmentation hemolysis (hemolytic uremic syndrome [HUS]). In retrospect, all episodes were probably associated with the ingestion of quinine. Quinine-dependent antibodies to platelets, neutrophils, T lymphocytes, and red blood cells (RBCs) were detected in the patient's serum. By a monoclonal antibody antigen capture assay, the patient's serum contained IgG antibodies, which in the presence, but not absence, of quinine reacted with platelet glycoprotein (GP) complexes Ib/IX and IIb/IIIa, but not Ia/IIa. By immunoprecipitation assay, the serum, after addition of quinine, reacted strongly with an 85-Kd neutrophil membrane protein and weakly with 130- and 60-Kd moieties. Serum adsorbed with RBCs in the presence of quinine continued to react with platelets and neutrophils, and serum that was absorbed with platelets continued to react with neutrophils and RBCs, indicating that the antigenic targets were different on platelets, neutrophils, and RBCs. Since platelets and endothelial cells share some antigens, we tested patient serum for antibodies to human umbilical vein endothelial cells (HUVEC); no quinine-dependent antibodies to HUVEC were detected. However, her quinine-dependent antibodies not only bound to platelets and neutrophils, but also activated neutrophils. Thus, the patient's serum with quinine aggregated neutrophils, but neither agent alone caused activation. Moreover, the patient's serum with quinine (but not without) augmented the adherence of neutrophils to HUVEC. Treatment of the patient's serum with staphylococcal protein A removed the quinine neutrophil aggregation cofactor, suggesting it was due to IgG. In both neutrophil aggregation and adherence assays, decomplementation of the patient's serum markedly blunted its effect. Furthermore, the patient's serum failed to aggregate formalin-inactivated neutrophils, suggesting neutrophil activation, probably by activated complement, was necessary for aggregation and adhesivity to endothelium. We conclude that our patient's neutropenia, thrombocytopenia, lymphopenia, and anemia were due to quinine-dependent antibodies, and that these antibodies recognized epitopes that were different in the three target cell populations. We further suggest that HUS was likely secondary to the activation and adhesion of neutrophils to endothelium.

scholarly journals Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 234-237 ◽  
Author(s):  
JD Sprandio ◽  
SS Shapiro ◽  
P Thiagarajan ◽  
S McCord
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Single Class ◽  
Human Umbilical Vein ◽  
Sds Page ◽  
Umbilical Vein Endothelial Cells ◽  
Triton X

Abstract Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

scholarly journals The platelet glycoprotein IIb/IIIa-like protein in human endothelial cells promotes adhesion but not initial attachment to extracellular matrix.

1987 ◽  
Vol 105 (4) ◽  
pp. 1885-1892 ◽  
Author(s):  
C S Chen ◽  
P Thiagarajan ◽  
S M Schwartz ◽  
J M Harlan ◽  
R L Heimark
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Polyclonal Antiserum ◽  
Platelet Glycoprotein ◽  
Initial Attachment ◽  
Adhesive Interactions ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

On platelets the membrane glycoprotein IIb/IIIa complex (GPIIb/IIIa) functions in adhesive interactions with fibrinogen, von Willebrand factor, and fibronectin. However, the function of GPIIb/IIIa-like proteins on endothelial cells, as well as the ligand(s) the complex binds, is unknown. Using a highly specific polyclonal antibody we have explored the function of GPIIb/IIIa-like proteins on human umbilical vein endothelial cells (HUVE). Analysis by immunoblotting shows that this antiserum recognizes the endothelial GPIIIa-like protein of the complex. The IgG fraction of the polyclonal antiserum and its Fab' fragments detach confluent and subconfluent HUVE from extracellular substrata. The effect of the anti-GPIIb/IIIa IgG is not toxic as the detached cells maintain their viability after trypsinization and replating. Anti-GPIIb/IIIa IgG does not inhibit HUVE binding to extracellular matrix or purified fibronectin in an attachment assay despite the presence of intact GPIIb/IIIa on HUVE detached from substrate by various methods. Apparently, the GPIIb/IIIa-like protein on HUVE is important in normal HUVE adhesion to the extracellular matrix, but it is not required in the initial attachment of HUVE to extracellular matrix.

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