FAB FRAGMENTS OF MONOCLONAL ANTIBODIES SPECIFIC FOR PORCINE PLATELET MEMBRANE GLYCOPROTEINS GP IB ANDGP IIB/IIIA

1987 ◽  
Author(s):  
H Takami ◽  
W L Nichols ◽  
S E Kaese ◽  
R S Miller ◽  
J A Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments ◽  
Igg1 Subclass ◽  
Von Willebrand

For further study of the porcine hemostatic mechanism, we have prepared murine monoclonal antibodies, and F(ab')2 and Fab fragments, specific for porcine platelet membrane glycoproteins GP lb and GP Ilb/IIIa. To avoid production of antibodies to von Willebrand factor (vWF), mice were immunized with platelets obtained from pigs with severe von Willebrand,s disease. One monoclonal antibody (PP3-4C), of IgG1 subclass, caused 85% inhibition of Ristocetin-induced platelet binding of 125I-vWF (porcine) at ≥12 µg IgG/ml. PP3-4C did not affect ADP or collagen-induced platelet aggregation nor inhibit 125I-fibrinogen (porcine) binding. Pepsin and papain digestion, respectively, were used to prepare PP3-4C F(ab')2 and Fab fragments. PP3-4C F(ab')2 at concentrations ≥12 µg/ml caused 80% inhibition of washed platelet agglutination in the presence of vWF and Ristocetin, whereas Fab fragments at concentrations ≥10 µg/ml caused 60% inhibition. Another monoclonal antibody (PP3-3A), of IgG1 subclass, completely inhibited ADP or collagen-induced platelet aggregation at an IgG concentration of 6 µg/ml. At 10 µg IgG/ml PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated porcine platelets. PP3-3A did not affect vWF-dependent Ristocetin-induced platelet agglutination, nor 125I-vWF binding to platelets in the presence of Ristocetin. PP3-3A did not bind to platelets which were treated with 10 mM EDTA at 37°C for 60 min. F(ab')2 and Fab fragments were isolated from PP3-3A pepsin or papain digests. Both types of PP3-3A fragments caused 100% inhibition of ADP-induced platelet aggregation, at concentrations ≥6 yg/ml. Immunoprecipitation of surface-radiolabeled porcine platelets and subsequent SDS-PAGE demonstrated that PP3-4C recognized a glycoprotein with molecular weight of 140,000 (under reducing conditions), and 165,000 (non-reduced). PP3-3A recognized glycoproteins with molecular weights of 115,000 and 100,000 (reduced), and 130,000 and 80,000 (non-reduced). Neither monoclonal antibody bound to human platelets. These monoclonal antibodies to porcine platelet membrane glycoproteins which are analogues of human GP lb and GP Ilb/IIIa will be useful for in vitro and in vivo studies to further understanding of mammalian hemostatic mechanisms.

scholarly journals Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1740-1747 ◽  
Author(s):  
H Takami ◽  
WL Nichols ◽  
SE Kaese ◽  
RS Miller ◽  
JA Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments

Abstract We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

scholarly journals Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1740-1747
Author(s):  
H Takami ◽  
WL Nichols ◽  
SE Kaese ◽  
RS Miller ◽  
JA Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments

We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

scholarly journals Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 927-937
Author(s):  
FM LaDuca ◽  
RE Bettigole ◽  
WR Bell ◽  
EB Robson
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

scholarly journals Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 927-937 ◽  
Author(s):  
FM LaDuca ◽  
RE Bettigole ◽  
WR Bell ◽  
EB Robson
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

Antibodies Against Platelet Membrane Glycoproteins: Effect On Ristocetin-Induced Platelet Aggregation

1981 ◽  
Author(s):  
E F Ali-Briggs ◽  
C S P Jenkins ◽  
K J Clemetson
Keyword(s):  
Platelet Aggregation ◽  
Affinity Chromatography ◽  
Platelet Membrane ◽  
Receptor Activity ◽  
Free Medium ◽  
Membrane Glycoproteins ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity ◽  
Fab Fragments ◽  
Induced Aggregation

Some membrane glycoproteins (GPs) have been isolated by lectin-affinity chromatography and antibodies towards them have been raised. Platelets that have lost glycocalicin no longer respond to ristocetin-human VIII:WF, bovine VIIIR:WF, or to anti-glycocalicin or anti-GPs la and lb antibodies but are still agglutinated by anti-GPs lib and Ilia antibodies. Anti-GPs la and lb and anti-glycocalicin antibodies, IgG and Fab' fragments inhibited ristocetin- human VIIIR:WF- and bovine VIIIR:WF-induced aggregation of fixed, washed platelets and of platelets in plasma while anti-GPs Hb and Ilia antibodies were without effect.Crossed immunoelectrophorectic studies showed that glycocalicin was present on whole platelets in only trace amounts; anti-glycocalicin antibodies, however, recognized a slower migrating component. Platelets incubated in an EDTA-free medium no longer respond to ristocetin-human VIIIRrWF. Membranes isolated from such platelets contained glycocalicin which cross-reacted with a remnant of the slower migrating component. Anti-GPs la and lb antibodies gave more complex patterns but it was possible to identify the slower moving component recognized by the anti-glycocalicin antibodies.These results show that glycocalicin is not normally found as such on whole platelets but is present as a precursor which is most likely GP lb. On degradation of this precursor, glycocalicin is released from the membrane and VIIIRrWF-receptor activity is lost.

scholarly journals The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1031-1037 ◽  
Author(s):  
SM Silver ◽  
MM McDonough ◽  
G Vilaire ◽  
JS Bennett
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Platelet Membrane ◽  
Human Leukemia ◽  
Membrane Glycoproteins ◽  
In Vitro Synthesis ◽  
Calcium Dependent ◽  
Hel Cells ◽  
Von Willebrand

Abstract The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium- dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 164-172 ◽  
Author(s):  
Mariagrazia De Luca ◽  
David A. Facey ◽  
Emmanuel J. Favaloro ◽  
Mark S. Hertzberg ◽  
James C. Whisstock ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Platelet Membrane Glycoprotein ◽  
And Function ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481–Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf–dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700–Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf–induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494–Leu-512; CR2, Phe-536–Ala-554; CR3, Arg-578–Glu-596; CR11 and CR15, Ala-564–Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700–Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

A Novel Platelet Small-Molecule Agonist Targeting Glycoprotein Ibalpha.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4010-4010
Author(s):  
Jianfeng Yang ◽  
Zhi Chen ◽  
Weiliang Zhu ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Small Molecule ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Structural Basis ◽  
Terminal Fragment ◽  
Von Willebrand

Abstract Abstract 4010 Poster Board III-946 Introduction Interaction of glycoprotein (GP) Ibα with Von Willebrand factor (VWF) plays a critical role in platelet adhesion and signal transduction for αIIbβ3 activation under condition of high shear stress. Methods Based on the crystal structure of platelet GPIbα (PDB:1P9A), virtual screening was employed to identify active compounds. Compounds in SPECS database were docked to VWF binding site on the surface of GPIbα. The screening was carried out with the DOCK4.0 program. The 150 highest-scoring compounds were obtained for further bioassay and those with inhibitory activity of VWF binding to GPIbα were investigated the effect on platelet activation and aggregation. Results We found one compound, designated it as YC148, blocked ristocetin-induced plasma VWF binding to recombinant N-terminal fragment GPIbα (H1-V289) by ELISA method. More interestingly, YC148 did not inhibit ristocetin-induced platelet aggregation, on the contrary, it induced platelet aggregation itself in the absence of exogenous modulators such as ristocetin and botrocetin. A VWF A1 blocking antibody could not block platelet aggregation induced by YC148 despite it completely inhibited ristocetin-induced platelet agglutination. And YC148 also stimulated washed platelet aggregation where VWF was absent in the resuspension buffer. These indicated that the aggregation stimulated by YC148 could not the result from VWF binding. Flow cytomety also showed that YC148 increased P-selectin expression on platelet membrane and promoted monoclonal antibody PAC-1 binding to platelet. The platelet aggregation stimulated by YC148 was inhibited by anti-GPIbα monoclonal antibody AN51 and 6D1. Conclusion A novel exogenous small-molecule agonist was found to activate platelet through binding to GPIbα. It provides us a new tool for investigating platelet GPIb outside-in signaling pathway in platelet adhesion and aggregation. Furthermore, the structure of YC148 may provide a structural basis for developing new hemostatic drugs based on the inhibition of VWF-GPIb interaction. The effect of YC148 on platelet from Bernard-Soulier syndrome or GPIbα N-terminal fragment deficient platelet after in vitro cleavage will be further investigated. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1031-1037 ◽  
Author(s):  
SM Silver ◽  
MM McDonough ◽  
G Vilaire ◽  
JS Bennett
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Platelet Membrane ◽  
Human Leukemia ◽  
Membrane Glycoproteins ◽  
In Vitro Synthesis ◽  
Calcium Dependent ◽  
Hel Cells ◽  
Von Willebrand

The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium- dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

Aggregation to Thrombin and Collagen of Platelets from a Glanzmann Thrombasthenic Patient Lacking Glycoproteins IIb and IIIa

1989 ◽  
Vol 62 (03) ◽  
pp. 962-967 ◽  
Author(s):  
Lilian McGregor ◽  
Michel Hanss ◽  
Amal Sayegh ◽  
Juan J Calvette ◽  
Marie-Christine Trzeciak ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Membrane Glycoproteins ◽  
Albumin Solution ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Antigen Present ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 μg/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 μM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 μg/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained twodimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on Ilia), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

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