Biochemistry of the platelet membrane.
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Blood Platelet Disorders↗
Biomedical subjects
K J Clemetson
Publications and source records attributed to K J Clemetson.
Thrombin receptors on human platelets.
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Humans↗
Biochemistry of platelet membrane glycoproteins.
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Chemical Phenomena↗
Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder.
Platelets from a patient with a severe lifelong bleeding tendency, which later spontaneously disappeared, lacked intact thrombospondin and glycoprotein (GP) Ia. Before disappearance of the bleeding disorder, results of coagulation studies and platelet aggregation in response to adenosine diphosphate (ADP), arachidonic acid, thrombin, A23187, epinephrine, and ristocetin were normal. In contrast, aggregation only occurred in the presence of collagen or wheat germ agglutinin at unusually high doses of these agonists. The platelets adhered normally to purified bovine and human type I collagen, and they did not spread in the presence of methylated type I collagen. No collagen-induced clot retraction was observed. Two-dimensional gel electrophoretic analyses of platelet proteins and immunologic studies showed that intact thrombospondin and GP Ia were absent. Aggregation in response to collagen could be restored by adding thrombospondin. Disappearance of the bleeding tendency occurred at the onset of menopause; subsequent analyses revealed that thrombospondin and GP Ia were present in platelets and that collagen-induced platelet aggregation was normal. These results suggest that both thrombospondin and GP Ia are essential in collagen-induced platelet aggregation. The spontaneous disappearance of the bleeding tendency may have been related to hormonal influences.
Bleeding Time↗
Residual amounts of glycoprotein Ib concomitant with near-absence of glycoprotein IX in platelets of Bernard-Soulier patients.
A study of the Bernard-Soulier syndrome in two unrelated families using different polyclonal antibodies in a sensitive immunoblot assay showed residual amounts of platelet membrane glycoprotein (GP) lb in the eight homozygotes, as well as the near-absence of GPlb beta and GPIX. The eight heterozygotes studied showed a double band pattern for GPlb and about half the normal level of GPlb beta and GPIX. Therefore, we conclude that the Bernard-Soulier syndrome is heterogeneous and is probably not due to gene deletions.
Bernard-Soulier Syndrome↗
Selective staining of platelet glycoproteins using two-dimensional O'Farrell gel electrophoresis and avidin-biotin-conjugated lectins.
A time-saving and sensitive high resolution method for the analysis and identification of platelet glycoproteins using nonradioactive compounds has been developed. Platelet proteins (50 micrograms) of normal subjects were separated by isoelectric focusing and SDS-PAGE. Proteins were either stained with silver or electroblotted onto nitrocellulose. Nitrocellulose blots were treated with the following biotinylated lectins: Abrus precatorius lectin, concanavalin A, Lens culinaris lectin, or wheat germ agglutinin. Staining was achieved by avidin-biotin-coupled peroxidase using 4-chloro-1-naphthol as the substrate. A rapid overview of platelet glycoproteins may be obtained by applying all the lectins to a single blot.
Avidin↗
[Bernard-Soulier syndrome in a Swiss family].
We report the case of a 38-year-old woman with hemorrhagic diathesis. Since early childhood she has suffered from epistaxis, gingival bleeding, ecchymoses and, since puberty, very frequent menorrhagia. Prolongation of bleeding time and thrombocytopenia (100,000 platelets/microliter) with giant platelets were observed. Since idiopathic thrombopenic purpura was suspected, splenectomy was performed. Neither this nor steroid treatment caused any improvement. The patient was able to give birth to an unaffected child, but delivery was followed by a major hemorrhage. In 1986, hysterectomy was considered because of frequent menorrhagia and, therefore, the patient's hemostasis was reinvestigated. The aforementioned abnormalities were confirmed and complementary examinations by electron microscope revealed giant platelets without granular abnormalities. While von Willebrand factor was normal, ristocetin agglutination was defective and analysis of platelet membrane glycoproteins showed absence of glycoprotein Ib. - These observations prompted the diagnosis of Bernard-Soulier syndrome. The study of the family demonstrated that 6 family members were heterozygote. This is believed to be the first large Bernard-Soulier family reported in Switzerland.
Adult↗
The glycoprotein Ib complex of human blood platelets.
Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIb alpha, which is very rich in O-linked oligosaccharides, sugar analysis revealed that GPIb beta and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal alpha-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one- or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIb beta by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPIb alpha. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.
Blood Platelets↗
Expression of platelet glycoprotein Ib alpha in HEL cells.
We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.
Asialoglycoproteins↗
Identification of two distinct regions within the binding sites for fibrinogen and fibronectin on the IIb-IIIa human platelet membrane glycoprotein complex by monoclonal antibodies P2 and P4.
The effect of two monoclonal antibodies P2 (LyP 2) or P4 (LyP 4), specific for the platelet membrane glycoprotein IIb/IIIa complex, on binding of 125I-labelled fibrinogen or 125I-labelled fibronectin to thrombin-stimulated platelets was studied. These monoclonal antibodies are directed against different determinants on the IIb-IIIa complex and react only with the complex and not with the individual glycoproteins. Fibrinogen binding to thrombin-stimulated platelets was significantly inhibited by P2 but not by P4. Fibronectin binding to thrombin-stimulated platelets was significantly inhibited by P4 but only poorly by P2. These results indicate the presence of specific regions on the glycoprotein IIb-IIIa complex which act as binding sites for fibrinogen or fibronectin. Other authors [Haverstick et al. (1985) Blood 66, 946-952; Ginsberg et al. (1985) J. Biol. Chem. 260, 4133-4138] have shown that a tetrapeptide, Arg-Gly-Asp-Ser, inhibited the binding of fibrinogen, fibronectin, and von Willebrand factor (vWf) to stimulated platelets and that fibrinogen competes with vWf and fibronectin for binding. These findings, together with previous studies, therefore indicate the presence of specific regions as well as a common region in the binding sites for fibrinogen and fibronectin on the IIb-IIIa complex.
Adenosine Diphosphate↗
Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.
Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.
Binding Sites↗
Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin.
Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is thought to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 mM calcium was immediately treated with EDTA, labelled with 125I and incubated with monoclonal antibodies P10 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with 125I-labelled platelet releasates treated for 48 h at 4 degrees C with 10mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 125I-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas 125I-labelled P12 only recognized a 400-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trypsinized, formaldehyde-fixed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calcium-treated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies P10 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.
Antibodies, Monoclonal↗
Acquired thrombasthenia due to GPIIb/IIIa-specific platelet autoantibodies.
An otherwise healthy woman developed a hemorrhagic diathesis with fluctuating clinical symptoms and laboratory findings, but without thrombocytopenia, over 8 years. In periods of bad clinical condition, a platelet defect, characteristic of thrombasthenia, was found. In contrast to classic thrombasthenia, electrophoresis of the patient's platelet membranes revealed normal amounts of glycoproteins IIb alpha, IIb beta, and IIIa in the normal positions. Monoclonal antibodies, specific for GPIIIa and GPIIb/IIIa, respectively, bound normally to the P1A1-positive platelets from the patient. Although no antibody and no platelet function inhibitor were evident in the autologous plasma, an IgG1 antibody that was bound to the patient's platelets and was directed against GPIIb/IIIa could be demonstrated. After elution from the patient's platelets, this antibody immunoprecipitated GPIIb (both subunits), IIIa, and a 200-kilodalton (kd) band (probably undissociated GPIIb/IIIa complex) from solubilized normal platelets, but did not react with thrombasthenic platelets. Adding the eluate from the patient's platelets to normal platelet-rich plasma immediately caused concentration-dependent inhibition of adenosine diphosphate (ADP)-induced and collagen-induced aggregation and also strong inhibition of ADP-stimulated fibrinogen binding. Because it was very unlikely from the patient's medical history that the antibody was caused by alloimmunization, the hemorrhagic diathesis must be interpreted as acquired thrombasthenia due to an anti-GPIIb/IIIa autoantibody.
Adult↗
Glycoprotein V is not the thrombin activation receptor on human blood platelets.
Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin-induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin-binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.
Blood Platelets↗
Structure and function of platelet membrane glycoproteins Ib and V. Effects of leukocyte elastase and other proteases on platelets response to von Willebrand factor and thrombin.
Treatment of platelets with human leukocyte elastase causes a rapid loss in response to von Willebrand factor and a biphasic loss in response to thrombin, first rapid then more slowly. The rapid phase corresponds to cleavage of a 45-kDa glycopeptide from the extracellular end of membrane glycoprotein GPIb. Longer treatment removes 80-kDa and 90-kDa glycopeptides and a glycopeptide corresponding to the major part of GPV. The 45-kDa and 90-kDa species could be obtained by elastase treatment of glycocalicin, the major proteolytic cleavage product of GPIb. Polyclonal rabbit antibodies against the purified 45-kDa glycopeptide had the same effect on the action of von Willebrand factor and thrombin on platelets as cleavage of GPIb by elastase. These results indicate that both the von Willebrand factor and thrombin binding sites on GPIb are located in the same region on the outside of the molecule. Thrombin activation of platelets involves at least two receptors, one on the 45-kDa glycopeptide, which when occupied causes an increase in the speed of response of the platelets to the cleavage of the second. GPV, a candidate for the second receptor, is a hydrophobic glycoprotein that is cleaved from the platelet membrane by several proteases. Proteases that do not activate platelets but degrade the second receptor remove larger fragments from GPV than do proteases such as thrombin or trypsin which activate platelets.
Blood Platelets↗
Monoclonal antibodies against human platelet membrane glycoproteins IIb-IIIa. II. Different effects on platelet function.
Preincubation of human platelets with two pairs of immunologically identical monoclonal antibodies (mab) directed against epitopes on the membrane glycoprotein IIb-IIIa complex - two of them (Gi3 and Gi5) precipitating glycoproteins IIb-IIIa, and two (Gi4 and Gi6) recognizing glycoprotein IIIa - induced slightly different inhibitory effects on platelet function. Gi3 and Gi5 blocked platelet aggregation and 14C-serotonin release from platelets induced by ADP, collagen, thrombin, arachidonic acid, ionophore A 23187, platelet activating factor and ristocetin (in the presence of divalent ions). Ristocetin-induced platelet agglutination (in the presence of EDTA) was not inhibited by these mab. Gi4 had no effect on platelet aggregation and release. Gi6 had the same effect on platelet function as Gi3 and Gi5, but only when incubated with Zw(a) (PlAl)-positive platelets with the exception that collagen-induced aggregation was not affected. We conclude that the similar, though not identical, effects on platelet function induced by mab against different epitopes on the glycoprotein complex IIb-IIIa indicate an "unspecific" interference with platelet membrane function possibly due to steric hindrance by mab of the fibrinogen receptor or by inhibition of conformational changes in the GP IIb-IIIa complex necessary for exposure of the fibrinogen binding site.
Antibodies, Monoclonal↗
Absence of platelet membrane glycoproteins IIb/IIIa from monocytes.
Two-dimensional gel electrophoresis, immunoprecipitation, and crossed immunoelectrophoresis were used in the investigation of glycoproteins IIb/IIIa in platelets, monocytes, and monocyte-derived macrophages from human blood. All techniques detected the glycoproteins in platelets but not in the mononuclear phagocytes. Similar results were obtained by immunochemistry using a monoclonal antibody against the platelet glycoproteins IIb/IIIa (revealed by a gold-labeled second antibody) which bound heavily to the platelet but not to the monocyte surface. The biochemical techniques used for the analysis of mononuclear phagocytes would have reliably detected the level of glycoproteins IIb/IIIa contributed by a 5% contamination with platelets, calculated on a per cell basis. We conclude that human monocytes and monocyte-derived macrophages lack glycoproteins IIb/IIIa. Our results further indicate that centrifugal elutriation yields monocyte preparations with minimal contamination by platelets. It seems likely that the positive results obtained by other authors were due to the presence of platelets or fragments on the monocytes.
Antibodies, Monoclonal↗
Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment.
Human platelets were surface-labeled by the periodate/NaB3H4 method or by lactoperoxidase-catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions and two-dimensional non-reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography. Chymotrypsin-treatment of surface-labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP120(4.9-5.4), GPIc and GPV. The membrane-bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s). The fact that remnants of both GPIIb and IIIa are left on chymotrypsin-treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen-binding site on platelets.
Adult↗