scholarly journals Thrombin-induced changes in platelet membrane glycoproteins Ib, IX, and IIb-IIIa complex

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1673-1678
Author(s):  
AD Michelson ◽  
MR Barnard
Keyword(s):  
Marked Decrease ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Proteolytic Fragment ◽  
Platelet Surface ◽  
Platelet Release ◽  
Induced Changes ◽  
Damaged Vessel

Platelet membrane glycoprotein Ib (GPIb) and the GPIIb-IIIa complex have central roles in the interaction of platelets with the plasma coagulation system, damaged vessel walls, and other platelets. We investigated the effects of thrombin on these glycoproteins. Monoclonal antibodies were used to assess platelet surface glycoproteins by flow cytometry, total platelet glycoprotein content by immunoassay, and glycoproteins released from platelets, also by immunoassay. Five new observations were made with regard to thrombin-induced changes in platelet membrane glycoproteins: (a) The marked decrease in platelet surface binding of antibodies directed at GPIb was not confined to antibodies directed at the von Willebrand factor binding site. (b) There was a marked decrease in platelet surface binding of an antibody directed at GPIX, with maintenance of the 1:1 ratio of platelet surface binding of antibodies directed at GPIb and GPIX. (c) Changes in platelet surface binding of antibodies were not restricted to a distinct subpopulation of platelets. (d) There was no associated platelet release of glycocalicin (a proteolytic fragment of GPIb). (e) There was no associated platelet release of the GPIIb-IIIa complex. These thrombin-induced changes may be important in modulating the reactivity of platelets with the damaged vessel wall and with each other.

scholarly journals Thrombin-induced changes in platelet membrane glycoproteins Ib, IX, and IIb-IIIa complex

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1673-1678 ◽  
Author(s):  
AD Michelson ◽  
MR Barnard
Keyword(s):  
Marked Decrease ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Proteolytic Fragment ◽  
Platelet Surface ◽  
Platelet Release ◽  
Induced Changes ◽  
Damaged Vessel

Abstract Platelet membrane glycoprotein Ib (GPIb) and the GPIIb-IIIa complex have central roles in the interaction of platelets with the plasma coagulation system, damaged vessel walls, and other platelets. We investigated the effects of thrombin on these glycoproteins. Monoclonal antibodies were used to assess platelet surface glycoproteins by flow cytometry, total platelet glycoprotein content by immunoassay, and glycoproteins released from platelets, also by immunoassay. Five new observations were made with regard to thrombin-induced changes in platelet membrane glycoproteins: (a) The marked decrease in platelet surface binding of antibodies directed at GPIb was not confined to antibodies directed at the von Willebrand factor binding site. (b) There was a marked decrease in platelet surface binding of an antibody directed at GPIX, with maintenance of the 1:1 ratio of platelet surface binding of antibodies directed at GPIb and GPIX. (c) Changes in platelet surface binding of antibodies were not restricted to a distinct subpopulation of platelets. (d) There was no associated platelet release of glycocalicin (a proteolytic fragment of GPIb). (e) There was no associated platelet release of the GPIIb-IIIa complex. These thrombin-induced changes may be important in modulating the reactivity of platelets with the damaged vessel wall and with each other.

ANALYSIS OF THE FUNCTIONAL ROLE OF PLATELET MEMBRANE GLYCOPROTEINS WITH MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
H Nagata ◽  
S Nomura ◽  
K Oda ◽  
T Kokawa ◽  
K Yasunaga
Keyword(s):  
Monoclonal Antibodies ◽  
Lymphoblastic Leukemia ◽  
Atp Release ◽  
Platelet Membrane ◽  
Calcium Flux ◽  
Antibody Concentration ◽  
Membrane Glycoproteins ◽  
Induce Platelet Aggregation ◽  
Platelet Surface ◽  
Induced Aggregation

Eight monoclonal antibodies were obtained which recognized platelet surface antigens of these, 5 (NNKYl-32, NNKY2-5, NNKY2-6, NNKY2-11, NNKY2-18 ) recognized GP IIb-IIIa complex, 2 (NNKY5-4, NNKY5-5 ) recognized GP lb and 1 (NNKYl-19) recognized CD 9 antigen. They were used to research the platelet membrane antigens.Monoclonal antibodies that recognize CD 9 antigen, which exists on the surface of platelets, acute lymphoblastic leukemia cells, eosinophils and other tissue, are known to act as an aggregating agent to platelets and NNKYl-19 was fond to induce platelet aggregation accompanied by ATP release. NNKY5-4 had no effect on platelet functions. NNKY5-5 inhibited aggregation induced by ristocetin but had no effect on aggregation induced by ADP, collagen, thrombin, and NNKYl-19. NNKYl-32, 2-5, 2-6, 2-11, and 2-18 inhibited aggregation induced by ADP, collagen, thrombin, and NNKYl-19, although slight release of ATP was recognized when NNKYl-19-induced aggregation was completely inhibited by NNKYl-32. Mutual inhibition of binding to platelet membranes between the 3 groups of monoclonal antibodies was not recognizedNNKYl-19-induced aggregation was associated with a lag time that was plo-longed in inverse proportion to antibody concentration. Aspirin had almost no effect on NNKYl-19-induced aggregation. A TXA2 receptor antagonist, a calci-um-channel blocking drug and EDTA inhibited NNKYl-19-induced aggregation. These results indicate that GP I b, GP IIb-IIIa complex and the cyclooxygenase pathway are not involved in NNKYl-19-induced platelet activation, that the target of NNKYl-19 on the platelet membrane is same as that of TXA2, and that the mechanism of activation by NNKYl-19 is related to calcium flux.

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

scholarly journals Plasmin-induced redistribution of platelet glycoprotein Ib

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2005-2010 ◽  
Author(s):  
AD Michelson ◽  
MR Barnard
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glycoprotein Ib ◽  
Autologous Plasma ◽  
Proteolytic Fragment ◽  
Platelet Surface ◽  
Microfilament System

Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin- induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).

scholarly journals Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1524-1527 ◽  
Author(s):  
X Du ◽  
L Beutler ◽  
C Ruan ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Proteolytic Fragment ◽  
Number Of Binding Sites ◽  
Membrane Bound ◽  
The Individual ◽  
Triton X ◽  
Murine Monoclonal Antibodies ◽  
Combined Data

Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

Anti-Glycoprotein Ib/IX and llb/llla Antibodies in Patients with Antiphospholipid Antibodies

1994 ◽  
Vol 71 (05) ◽  
pp. 571-575 ◽  
Author(s):  
Monica Galli ◽  
Maria Daldossi ◽  
Tiziano Barbui
Keyword(s):  
Antiphospholipid Antibodies ◽  
Cross Reactivity ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Platelet Destruction ◽  
Platelet Surface ◽  
Immune Mediated ◽  
Platelet Antigen ◽  
History Of ◽  
Associated Conditions

SummaryAntiphospholipid antibodies, namely lupus anticoagulant (LA), anticardiolipin (aCL) type A and type B antibodies, are frequently associated with immune-mediated thrombocytopenia. Antiphospholipid antibodies have been suggested to bind to the phospholipids of the platelet membrane, thus participating to the process of platelet destruction, which leads to thrombocytopenia. However, a clear antiphospholipid (aPL) demonstration of such a role has never been given for antibodies. Conversely, autoantibodies directed against membrane-associated glycoproteins (GP) have been shown to be pathogenet- ically linked to the development of thrombocytopenia in patients with idiopathic thrombocytopenic purpura. For this reason, we have measured anti-GPIb/IX and GPlIb/IIIa IgG in the plasma of 68 patients with aPL antibodies by ELISA. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was used.Twenty-seven out of 68 patients with antiphospholipid antibodies (40%) had increased plasma levels of anti-GP antibodies. In particular, 7 of them had elevated anti-GPIIb/IIIa levels only, 6 had anti-GPIb/IX antibodies only, whereas in the remaining 14 cases both types of autoantibodies were found elevated. The level of anti-GP antibodies in plasma did not correlate with age, sex, clinical associated conditions, history of thrombosis, IgG aCL titer or the presence of a phospholipid- dependent inhibitor of coagulation. In contrast, a statistically significant association between thrombocytopenia and high anti-GP antibody titer was observed (p = 0.0458).To establish whether there was cross-reactivity between antiphospholipid and anti-GP antibodies, adsorption experiments were performed using eardiolipin-containing liposomes or washed, normal, resting platelets. When patients’ plasma was mixed with resting washed platelets, only anti-GP antibodies were adsorbed on platelet membrane and subsequently eluted from platelets. Conversely, binding to cardio- lipin-containing liposomes but not to platelet surface was demonstrated for antiphospholipid antibodies.In conclusion, our data indicate that high levels of anti-platelet glycoprotein antibodies are more frequently found in patients with antiphospholipid antibodies and thrombocytopenia and that they might be responsible, more than LA and/or aCL antibodies, for the development of thrombocytopenia.

scholarly journals Plasmin-induced redistribution of platelet glycoprotein Ib

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2005-2010 ◽  
Author(s):  
AD Michelson ◽  
MR Barnard
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glycoprotein Ib ◽  
Autologous Plasma ◽  
Proteolytic Fragment ◽  
Platelet Surface ◽  
Microfilament System

Abstract Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin- induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).

scholarly journals Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1524-1527 ◽  
Author(s):  
X Du ◽  
L Beutler ◽  
C Ruan ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Proteolytic Fragment ◽  
Number Of Binding Sites ◽  
Membrane Bound ◽  
The Individual ◽  
Triton X ◽  
Murine Monoclonal Antibodies ◽  
Combined Data

Abstract Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.

scholarly journals Exposure of fibrinogen receptors on fresh and stored platelets by ADP and epinephrine as single agents and as a pair

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1054-1059 ◽  
Author(s):  
G Di Minno ◽  
AM Capitanio ◽  
P Thiagarajan ◽  
J Martinez ◽  
S Murphy
Keyword(s):  
Marked Decrease ◽  
Adenosine Diphosphate ◽  
Platelet Concentrates ◽  
Specific Receptor ◽  
Surface Binding ◽  
Normal Response ◽  
Platelet Surface ◽  
Aggregation Response ◽  
Fibrinogen Binding ◽  
Complex Thought

Abstract Platelet concentrates stored at 22 degrees C have a marked decrease in their aggregation response to adenosine diphosphate (ADP) or epinephrine but a normal response to these agents when used as a pair. Since platelet stimulation involves exposure of receptors for fibrinogen, we studied fibrinogen binding to platelets from fresh and stored concentrates. Following stimulation with 10 microM ADP or 20 microM epinephrine, platelet suspensions from fresh concentrates bound 125I-fibrinogen in a reaction that reached completion within 30 min. Significantly less binding occurred in suspensions from platelet concentrates that had been stored for 5 days at 22 degrees C. When stimulated by ADP and epinephrine as a pair (2 microM each), binding of fibrinogen to platelets was complete within 10–15 min and was not significantly decreased in suspensions from stored concentrates. We also investigated the effect of storage on the glycoprotein IIb-IIa complex, thought to be a specific receptor for fibrinogen on the platelet surface. Binding of a monoclonal antibody specific for this complex (B59.2) to platelet suspensions was unaffected by 5 days of storage. Furthermore, B59.2 inhibited aggregation, secretion, and fibrinogen binding of fresh and stored platelets stimulated with the pair of agents just as it did with single agents. We conclude that storage for 5 days at 22 degrees C impairs the exposure of fibrinogen receptors on platelets in response to ADP or epinephrine when used as single agents, without affecting the glycoprotein IIb-IIIa complex quantitatively. The function of the receptor is normal in response to the pair of agents.

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