Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure.

Abstract This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2-type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcγRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcγRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation.

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The effect of antibodies to human platelet membrane and cytoplasmic myosins on myosin ATPase activity and platelet aggregation

Thrombosis Research ◽

10.1016/0049-3848(85)90189-6 ◽

1985 ◽

Vol 38 (5) ◽

pp. 567-576 ◽

Cited By ~ 3

Author(s):

I. Peleg ◽

A. Eldor ◽

A. Muhlrad ◽

U. Groschel-Stewart ◽

I. Kahane

Keyword(s):

Platelet Aggregation ◽

Atpase Activity ◽

Human Platelet ◽

Myosin Atpase ◽

Platelet Membrane ◽

Myosin Atpase Activity

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Cloning of the human platelet F11 receptor: a cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation

Blood ◽

10.1182/blood.v95.8.2600.008k28_2600_2609 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2600-2609

Author(s):

Malgorzata B. Sobocka ◽

Tomasz Sobocki ◽

Probal Banerjee ◽

Cipora Weiss ◽

Julie I. Rushbrook ◽

...

Keyword(s):

Cell Adhesion ◽

Platelet Aggregation ◽

Membrane Protein ◽

Adhesion Molecule ◽

Platelet Adhesion ◽

Human Platelet ◽

Physiological Role ◽

Immunoglobulin Superfamily ◽

Predicted Amino Acid Sequence ◽

Platelet Surface

This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2-type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcγRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcγRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation.

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Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

Blood ◽

10.1182/blood.v66.1.92.92 ◽

1985 ◽

Vol 66 (1) ◽

pp. 92-98 ◽

Cited By ~ 41

Author(s):

SJ Shattil ◽

LF Brass ◽

JS Bennett ◽

P Pandhi

Keyword(s):

Platelet Aggregation ◽

Surface Membrane ◽

Broad Band ◽

Adenosine Diphosphate ◽

Platelet Membrane ◽

Membrane Glycoproteins ◽

Fibrinogen Receptor ◽

Activated Platelets ◽

Discrete Band ◽

Functional Consequences

Abstract The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

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Human platelet membrane protein

Biochemistry ◽

10.1021/bi00804a002 ◽

1970 ◽

Vol 9 (2) ◽

pp. 200-205 ◽

Cited By ~ 16

Author(s):

Ralph L. Nachman ◽

Barbara Ferris

Keyword(s):

Membrane Protein ◽

Human Platelet ◽

Platelet Membrane

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Further Observations On The Possible Association Of Human Platelet Membrane Glycoproteins IIb And IIIa

10.1055/s-0038-1652278 ◽

1981 ◽

Author(s):

J-P Rosa ◽

D Pidard ◽

T Kunicki ◽

A T Nurden

Keyword(s):

Platelet Aggregation ◽

Membrane Proteins ◽

Divalent Cation ◽

Human Platelet ◽

Human Platelets ◽

Platelet Membrane ◽

Membrane Glycoproteins ◽

Normal Human ◽

Triton X

Studies are described which represent a continuation of our investigation into the role of membrane glycoproteins (GP’s) IIb and IIIa during human platelet aggregation. The surface proteins of washed platelets were labelled with 125I by the lactoperoxidase-catalysed method prior to membrane isolation by the glycerol lysis procedure. Solubilisation of the membrane proteins by triton X-100 was followed by their analysis by crossed immunoelectrophoresis (CIE) using a rabbit antibody prepared against normal human platelets. In the absence of divalent cation chelation GP IIb and Ilia were contained within a single 125I-labelled immunoprecipitate. When the isolated membranes were solubilised by triton X-100 in the presence of 5mM EDTA, GP IIb and IIIa formed distinctand separate immunoprecipitates during CIE. In order to further investigate this finding 125I-labelled membrane proteins solubilised by triton X-100 in the presence or absence of EDTA were subjected to centrifugation for 18 h at 100,000 g over a 10-40% sucrose gradient containing the nonionic detergent. The results confirmed that in the presence of divalent cations lib and Ilia were associated in a complex, and that this complex is dissociated by EDTA. The IgG..L is an alloantibody isolated from a polytransfused thrombasthenic patient that has been shown in previous studies to inhibit ADP-induced platelet aggregation and the binding of 125I-fibrinogen to normal human platelets in the presence of ADP. When the IgG..L was incorporated in an intermediate gel during CIE it was shown to precipitate the complex containing IIb/IIIa but under identical conditions it did not precipitate the individual glycoproteins dissociated by EDTA. Divalent cation-mediated changes in the orientation of lib and Ilia in the platelet membrane should be considered in assessing the role of these GP’s in platelet function.

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Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

Blood ◽

10.1182/blood.v66.1.92.bloodjournal66192 ◽

1985 ◽

Vol 66 (1) ◽

pp. 92-98

Author(s):

SJ Shattil ◽

LF Brass ◽

JS Bennett ◽

P Pandhi

Keyword(s):

Platelet Aggregation ◽

Surface Membrane ◽

Broad Band ◽

Adenosine Diphosphate ◽

Platelet Membrane ◽

Membrane Glycoproteins ◽

Fibrinogen Receptor ◽

Activated Platelets ◽

Discrete Band ◽

Functional Consequences

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

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HIP2: A NEW MONOCLONAL ANTI-HUMAN PLATELET ANTIBODY WITH STIMULANT FUNCTION

10.1055/s-0038-1643505 ◽

1987 ◽

Author(s):

W J Chen ◽

Z Chen ◽

C X Bao

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Immunofluorescence Assay ◽

Platelet Membrane ◽

Weak Reaction ◽

Platelet Surface ◽

Capillary Endothelial Cells ◽

Induced Aggregation ◽

Formalin Fixed ◽

Platelet Antibody

A monoclonal anti-human platelet antibody(MoAb), HIP2 (IgG3) which induced irrevesible aggregation of platelet in association with the release of serotonin and thromboxane B2 formation is described. Indirect immunofluorescence assay (IFS) showed that the antibody binded to platelets and megakryocytes, and gave a weak reaction with aortic, liver and capillary endothelial cells. Electrophoresis of radiolabelled antigen showed that HIP2 recognized platelet membrane glycoprotein lib (130KD). The purified HIP2 MoAb induced aggregation with normal PRP, not with thrombasthenia platelets, formalin-fixed platelets, washed platelets and EDTA-PRP. Washed platelet aggregation with HIP2 could be restored by adding normal plasma or serum into medium, but not by inactivated serum at 56°C for 30 minutes and fibrinogen, The results suggested that the aggregation induced by HIP2 needed Ca++ and complement in medium. HIP2-induced aggregation was completely inhibited by calmodulin inhibitor, compound 48/80, and partially inhibited by aspirin, apyrase, ATP, antimycin A and phentolamine. Anti-glycoprotein Ilia MoAb(SZ-21) inhibited HIP2-induced platelet aggregation, did not block HIP2 binding on platelet surface in IFS. HIP2 no affected platelet adhision on glass. HIP2 prolonged koalin cephalin clotting time in PRP and interruped whole blood retraction. Under electron microscopy, the fibrin formation of clots in the presence of HIP2 was much less than the control. Recently, we found that the platelets from patients with myelo-proligerative diseases had decreased response to HIP2 aggregation. So that, HIP2 MoAb may be a very useful tool not only for study of the platelet membrane structure associated with function of platelet and platelet physiology, but also of the pathogenesis of some platelet diseases.

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