Platelet Surface Proteins Research Articles

Characterization of a Platelet Membrane Protein of Low Molecular Weight Associated With Platelet Activation Following Binding by Monoclonal Antibody AG-1

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.

Characterization of a platelet membrane protein of low molecular weight associated with platelet activation following binding by monoclonal antibody AG-1

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.

Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen

Platelets initially adhere to collagen via a divalent cation-dependent process supported by Mg 2+, Mn 2+, Fe 2+, Cu 2+, Zn 2+, or Co 2+ more rapidly and to a greater extent than by previously studied divalent cation-independent mechanisms. Ca 2+ not only fails to support adhesion, it is inhibitory. Platelet activation and secretion are not required for adhesion by this mechanism. Monomeric and fibrillar collagens, but not denatured collagen, effectively support divalent cation-dependent adhesion. Types I, III, and IV collagen, but not type V collagen, support adhesion. A platelet surface protein of M r 160,000, possibly identical with platelet membrane glycoprotein la, that binds to collagen with the appropriate divalent cation specificity has been identified and is the likely mediator of the initial divalent cation-dependent adhesion of platelets to collagen.

The interaction of Tb 3+ with the human platelet surface

The interaction of the lanthanide Tb 3+ with washed, human platelets was examined. When bound to the platelet surface, the fluorescence of this Ca 2+ analog was increased approximately 200-fold, most likely by a Förster mechanism involving platelet surface protein aromatic residues. The binding of Tb 3+ to the unactivated platelet was specific and saturable with an apparent approximate K d of 195 μ m. Both Ca 2+ and La 3+ effectively displaced Tb 3+ from platelet surface sites, but neither cation did so completely. Plasmin treatment of the platelet surface reduced Tb 3+ fluorescence by 68% at saturation without significantly affecting the approximate apparent K d. Activating washed, aspirinated platelets with ADP induced a 78% increase in Tb 3+ fluorescence at saturation. Tb 3+ competed effectively and completely for platelet surface-bound 45Ca 2+ with an approximate IC 50 of 10 μ m. These data indicate the potential utility of this fluorescent lanthanide in characterizing Ca 2+-binding sites on the human platelet.

Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry

Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet- rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the “platelet” light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.

Analysis of Human Platelet Glycoproteins IIb-IIIa and Glanzmann’s Thrombasthenia in Whole Blood by Flow Cytometry

Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet-rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the "platelet" light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.

Identification of binding site for heparin and other polysulfated glycosaminoglycans on human thrombocytes.

Heparin and other polysulfated glycosaminoglycans (GAGPS) can evoke an immune response when complexed to the surface of platelets in vivo. We have previously reported on the detection and characteristics of such antibodies leading to thrombocytopenia. Here we report data concerning the biochemical characterization of the heparin-/GAGPS-binding site on human thrombocytes. By means of heparin affinity chromatography, GAGPS chromatography and subsequent 'Western blotting' of specifically eluted proteins we were able to detect two proteins of apparent molecular mass of 207 and 170 kilodaltons, which could be reduced to 57 and 142 kilodaltons, respectively. The 170-kilodalton glycoprotein was stained by the carbohydrate specific PAS-reagent and by surface labeling with 3H. The second molecule binding to heparin (GAGPS)-Sepharose was detected by platelet surface labeling with 125I, protein-specific staining Coomassie brilliant blue and PAS staining. Binding studies using enzyme-labeling techniques with five monoclonal antibodies (MCA) against platelet surface proteins GPI, GPIIb/IIIa complex, GPIIIa and CRI (C3b receptor) revealed specific interaction of the 170-kilodalton heparin-binding glycoprotein with an MCA to GPI, suggesting identity or immunological cross-reactivity of these two moieties. No specific reactions were observed between the 207-kilodalton glycoprotein and any of the MCA tested. For further evaluation of the antigenicity of separated platelet proteins, an ELISA in microtiter plates was developed, and a cellular sandwich ELISA for serological detection of antibodies against whole native thrombocytes or GAGPS-derivatized proteins was set up.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification, purification, and partial characterization of plasma protein kinases

Two isomeric forms of protein kinases, FI and FII, were isolated from human plasma. These two isomeric enzymes were isolated to apparent homogeneity on NaDodSO 4-PAGE by using (NH 4) 2SO 4 fractionation, DEAE-cellulose, hydroxylapatite, Affi-Gel blue, and high-pressure liquid column chromatography. Polyclonal antibodies were obtained from immunized rabbits and both enzymes cross-reacted with each other. Furthermore, immunoaffinity-purified anti-FI and anti-FII antibodies inhibited the enzyme activity of both FI and FII. These enzymes are cyclic nucleotides, Ca 2+, calmodulin and phosphatidylserine-independent enzymes which can phosphorylate exogenously added histone, casein, protamine, phosvitin, and platelet surface proteins. The phosphorylated proteins of intact platelets by these enzymes in the presence of exogenously added [γ- 32P]ATP ranged in apparent molecular weights from 13.5K to 200K, as estimated by their mobility during NaDodSO 4-PAGE. Trypsin removed the label from the platelet surface phosphoproteins without affecting the intracellularly located phosphoproteins labeled endogenously by 32PO 4-prelabeling of intact platelets. These observations raise the possibility that these enzymes could play a role in modulating the properties of platelets through phosphorylation of the platelet surface proteins.

Photoaffinity labeling of platelet thrombin-binding proteins

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl- N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins; platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequences of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or α 2-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.

Labeling of platelet surface proteins with 125I by the Iodogen method

A procedure for the 125I-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of 125I. Best labeling results were obtained at low platelet concentrations (3–5 × 10 8 platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 μg of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.

Surface Labeling of Human Platelets by Reductive Methylation

A simple, reproducible method for the specific labeling of the surface proteins of human platelets with tritium is described. The labeled platelets were analyzed by two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by fluorography and the results compared with those obtained by conventional methods. Reductive methylation gave an intense labeling of membrane glycoproteins. There was no cross-linking of the labeled membrane proteins by formaldehyde nor could clear evidence be found that inner proteins were labeled by permeation of the reagents through the plasma membrane. As well as previously described platelet membrane glycoproteins, several others could be detected that were not invariable seen using labeling techniques.

The role of platelet surface proteins reacting with heterologous antibodies.

Platelets were labeled with surface-labelling agents which identified up to six platelet membrane proteins. Three of these labelled proteins were recognised by a specific anti-platelet membrane serum by immunoprecipitation. Affinity fractionation of platelets with insolubilised IgG obtained from the anti-platelet membrane serum indicated a greater reaction between the antiserum and the platelet surface. Fab fragments from the antiserum inhibited ristocetin-induced aggregation of platelets and partially inhibited collagen-induced aggregation but did not affect ADP aggregation. Crossed immunoelectrophoresis of platelets using the anti-platelet membrane serum showed that the antiserum was specific for platelet membranes.

Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Previous reports have described conflicting results concerning the glycoprotein (GP) and protein composition of Bernard-Soulier platelets. In view of this controversy we have analyzed the platelets of four Bernard-Soulier patients using improved single and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An absence of staining for carbohydrate of membrane GP Ib was characteristic for the platelets of each patient. Major periodate-Schiff staining bands corresponding to membrane GP IIb, IIIa, and IIIb were clearly detected and their presence was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis. The protein content of the Bernard-Soulier platelets was increased two- to fourfold. However, analysis of their protein composition using 7-12% acrylamide gradient gels showed normal polypeptide profiles. Lactoperoxidase-catalyzed 125I-labeling of the Bernard-Soulier platelet surface proteins was followed by SDS-polyacrylamide gel electrophoresis and autoradiography. No labeling in the Ib position was detected whereas the other major membrane GP, including Ia and IIa, were normally located. In contrast, GP Ib was clearly detected by periodate-Schiff staining and autoradiography when normal human platelets that had been exhaustively treated with neuraminidase before the lactoperoxidase-catalyzed iodination were analysed. No abnormalities were detected in the GP patterns of membranes isolated from the patients' erythrocytes. Only a severe molecular abnormality or possible deletion of GP Ib could account for this major platelet lesion in the Bernard-Soulier syndrome.

Glanzmann's thrombasthenia: studies of surface proteins of platelets and red cells with (125I)-diazotized diiodosulfanilic acid and SDS-polyacrylamide gel electrophoresis.

Glanzmann's thrombasthenia: studies of surface proteins of platelets and red cells with (125I)-diazotized diiodosulfanilic acid and SDS-polyacrylamide gel electrophoresis.

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point ( [formula omitted]) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ( pI ) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.

The effect of ambient storage on platelet membrane structure and response to thrombin.

Blood platelets rapidly lose hemostatic function during in vitro storage at room temperature. In view of recent studies implicating platelet surface proteins in some aspects of platelet function, we have examined the effect of platelet storage on membrane structure and function. Platelets obtained from a single donor by double plateletpheresis were stored for seven days at 22 C with constant agitation. On succeeding days, platelets were removed and analyzed for membrane surface structure determined by iodination of membrane surface proteins, membrane function determined by serotonin uptake and equilibrium binding of thrombin to surface receptors, and platelet (unction determined by serotonin release and platelet aggregation in response to thrombin. The earliest changes in membrane surface structure were observed within 48 hours and consisted of decreased iodination of membrane surface proteins, and increased iodination of membrane proteins with estimated molecular weights of 260,000, 240,000, and 84,000. No changes were observed in the overall protein composition of the platelet, but changes in membrane function were observed. Relative uptake of serotonin decreased from 89 to 37 per cent and appeared to occur in parallel with the changes in membrane surface proteins. Changes in thrombin binding, on the other hand, were progressive throughout the storage period. The number of molecules of thrombin bound with high affinity decrease from 1,040 to 520 per platelet while the number of molecules bound with low affinity decreased from 103,600 to 4,500. No persistent alterations were observed in the affinity of thrombin for its receptors. Thrombin‐induced platelet aggregation and release of serotonin decreased in parallel with the changes in thrombin binding.

A comparison of techniques used to study externally oriented proteins and glycoproteins of human blood platelets

The surface of whole washed human platelets was labelled by a variety of methods specific for proteins or for carbohydrates. The labelled platelets were solubilized and analysed by electrophoresis by means of a Laemmli slab gel system. Galactose oxidase plus tritiated sodium borohydride weakly labelled five glycoprotein bands indicating that few free terminal galactose residues are present on platelet membrane glycoproteins. Pretreatment with neuraminidase before galactose oxidase and tritiated sodium borohydride greatly increased both labelling intensity and the number of bands labelled indicating that most of the glycoprotein chains terminate with sialic acid. After treatment, glycoprotein Ib moved to a slightly higher molecular weight. Labelling with periodate and tritiated borohydride gave intensely labelled glycoprotein bands which correlated well with those observed by PAS staining. Lactoperoxidase plus 125I labelled protein bands, which could be correlated with the bands labelled by the carbohydrate specific techniques. Simultaneous analysis of platelets from single donors labelled by a variety of techniques offers a reliable approach to the investigation of the structures of the platelet surface proteins and glycoproteins.

Binding of pyridoxal phosphate to human platelets: Its effect on platelet function

Pyridoxal phosphate, the coenzyme form of vitamin B 6, exerted a concentration-dependent inhibition of epinephrine-induced platelet aggregation, in vitro . A dose-dependent inhibition of [2- 14C]-5-hydroxytryptamine release from platelets was also observed. Likewise, the coenzyme also blocked the ADP, thrombin and collagen-induced platelet aggregation and release. The fluorographs of platelets treated with pyridoxal phosphate and sodium borotritide indicated three predominently labeled platelet protein components of M r = 170,000, 150,000 and 90,000. One of these polypeptides was characterized as platelet glycoprotein I. The amount of tritium that was incorporated into these proteins showed a dependence on the concentration of the coenzyme and the label. The inhibitory effect of pyridoxal phosphate appears to be due to its specific binding to these platelet-surface proteins which may be involved in various platelet functions.

Studies on platelet plasma membranes. I. Characterization of surface proteins of human platelets labeled with diazotized (125i)-diiodosulfanilic acid.

A polar, nonpenetrating compound of high specific activity, diazotized (125I)-diiodosulfanilic acid (DD125ISA), has been developed as a label for exposed proteins of the human platelet plasma membrane, and platelet proteins and the pattern of labeling have been studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). That DD125ISA binds specifically to membrane proteins was demonstrated by: (1) the specific activity of isolated membrane protein was five to seven times that of whole platelet protein and (2) no proteins of intact platelets were labeled which were not represented in the isolated plasma membrane. That the DD125ISA-labeled membrane proteins were exposed on the cell surface was demonstrated by: (1) DD125ISA-labeled proteins were altered by trypsin treatment of intact, labeled platelets and (2) the pattern of labeling produced by reaction of isolated membranes with DD125ISA was quite different from that produced by the labeling of intact platelets. Analysis of platelet membrane proteins by SDS-PAGE demonstrated the glycoproteins previously described at 150,000 daltons (termed glycoprotein I) and 92,000 daltons (glycoprotein III) but we could discriminate two apparently distinct glycoproteins in the intermediate region (IIa: 125,000 daltons, and II: 118,000 daltons). Glycoproteins I and III were constant whereas IIa was clearly visible only in unreduced samples and II was predominant in reduced samples. Reaction of DD125ISA with intact platelets resulted in equal labeling of three of these four membrane glycoproteins (IIa, II, and III). The pattern of exposed proteins on the platelet surface labeled by DD125ISA was different from lactoperoxidase-131I, which labeled predominantly the 92,000 dalton glycoprotein, as demonstrated by simultaneous SDS-PAGE analysis. Therefore three glycoproteins of the human platelet plasma membrane are exposed to a radioisotope probe on the platelet surface and are accessible for contact interactions.