Platelet Membrane Proteins Research Articles

Detection of platelet activation with monoclonal antibodies and flow cytometry. Changes during platelet storage

To investigate whether changes in platelet condition during platelet storage correlate with an altered expression of platelet membrane proteins, the binding of monoclonal antibodies (MoAbs) to fresh platelets was compared with MoAbs' binding to thrombin-activated platelets and to platelets stored as platelet concentrates. The MoAbs included antibodies against the platelet glycoprotein (GP) IIb/IIIa complex and against two activation-dependent antigens, one of which was a component of the internal platelet alpha-granule membrane (GMP 140) and the other of which was a 53-kD protein derived from platelet lysosomes. The binding of MoAbs to platelets fixed with 1 percent paraformaldehyde was measured by flow cytometry. In thrombin-activated platelets, a threefold increase was found in the expression of GP IIb/IIIa over that in fresh platelets. The binding of the activation-dependent MoAbs increased from 2 to 3 percent to 70 to 80 percent of the platelets. Storage of platelet concentrates for 5 days resulted in a 60 percent increase in GP IIb/IIIa expression compared to Day 0 and increased binding of the MoAbs directed against GMP-140 from 3 to 16 percent and against the 53-kD protein from 2 to 8 percent of the platelets, respectively. These changes correlated with modifications in platelet morphology (decrease in swirling), leakage of lactate dehydrogenase, and release of beta-thromboglobulin. These data indicate that platelets become activated and are damaged during the storage of platelet concentrates.(ABSTRACT TRUNCATED AT 250 WORDS)

An integrin receptor on normal and thrombasthenic platelets that binds thrombospondin [see comments

The members of the integrin family of membrane glycoprotein heterodimer complexes function as cell surface receptors for adhesive proteins. We report here on the identification of two integrins on the surface of human platelets that bind to thrombospondin. When platelet membrane proteins are radiolabeled with 125I-lactoperoxidase, solubilized in n-octylglucoside, (Boehringer Mannheim Biochemicals, Indianapolis, IN), and applied to a column of thrombospondin-Sepharose, both complexes are bound to the column and specifically eluted with the peptide GRGDSP. One of these integrins, glycoprotein (GP) IIb-IIIa, appears to bind relatively weakly. The second integrin shares the same beta subunit (beta 3 or GPIIIa), but has a distinct alpha subunit that comigrates with the alpha subunit (alpha v) of the vitronectin receptor (VnR) on endothelial cells and reacts with a monoclonal antibody, LM142, which was raised against an integrin from M21 melanoma cells. The alpha v beta 3 integrin is present on a variety of cell types and appears to act as a receptor for thrombospondin on endothelial and smooth muscle cells. On endothelial and M21 melanoma cells this receptor is also involved in adhesion to fibrinogen, vitronectin, and von Willebrand factor (vWF). The alpha v beta 3 integrin is present at approximately equal levels on normal and thrombasthenic platelets, whereas levels of GPIIb-IIIa are greatly reduced on thrombasthenic platelets. The alpha v beta 3 integrin on thrombasthenic platelets also binds to thrombospondin-Sepharose and can be eluted with the peptide GRGDSP. These data indicate that the alpha v beta 3 integrin on platelets, endothelial cells, and smooth muscle cells functions as an Arg-Gly-Asp (RGD)-dependent receptor for thrombospondin.

An Integrin Receptor on Normal and Thrombasthenic Platelets That Binds Thrombospondin

Abstract The members of the integrin family of membrane glycoprotein heterodimer complexes function as cell surface receptors for adhesive proteins. We report here on the identification of two integrins on the surface of human platelets that bind to thrombospondin. When platelet membrane proteins are radiolabeled with 125I-lactoperoxidase, solubilized in n- octylglucoside, (Boehringer Mannheim Biochemicals, Indianapolis, IN), and applied to a column of thrombospondin-Sepharose, both complexes are bound to the column and specifically eluted with the peptide GRGDSP. One of these integrins, glycoprotein (GP) IIb-IIIa, appears to bind relatively weakly. The second integrin shares the same beta subunit (beta 3 or GPIIIa), but has a distinct alpha subunit that comigrates with the alpha subunit (alpha v) of the vitronectin receptor (VnR) on endothelial cells and reacts with a monoclonal antibody, LM142, which was raised against an integrin from M21 melanoma cells. The alpha v beta 3 integrin is present on a variety of cell types and appears to act as a receptor for thrombospondin on endothelial and smooth muscle cells. On endothelial and M21 melanoma cells this receptor is also involved in adhesion to fibrinogen, vitronectin, and von Willebrand factor (vWF). The alpha v beta 3 integrin is present at approximately equal levels on normal and thrombasthenic platelets, whereas levels of GPIIb-IIIa are greatly reduced on thrombasthenic platelets. The alpha v beta 3 integrin on thrombasthenic platelets also binds to thrombospondin-Sepharose and can be eluted with the peptide GRGDSP. These data indicate that the alpha v beta 3 integrin on platelets, endothelial cells, and smooth muscle cells functions as an Arg-Gly-Asp (RGD)-dependent receptor for thrombospondin.

Regulatory control of complement on blood platelets: Modulation of platelet procoagulant responses by a membrane inhibitor of the C5b-9 complex

Antibody against a membrane inhibitor of the C5b-9 complex has been used to investigate regulatory control of the terminal complement proteins on blood platelets. Monospecific rabbit antibody (alpha-P18) was raised against the purified 18-kDa erythrocyte membrane inhibitor of C5b-9 (Sugita, Y., Nakano, Y., and Tomita, M. (1988) J. Biochem. (Tokyo) 104, 633-637). In addition to its interaction with erythrocytes, this antibody (and its Fab) bound specifically to platelet membranes. In immunoblots of cell membrane proteins prepared under non-reducing conditions, alpha-P18 bound specifically to an 18-kDa erythrocyte membrane protein and to a 37-kDa platelet membrane protein. Absorption of this antibody by platelet membranes competed its binding to the purified 18-kDa erythrocyte protein, suggesting that epitopes expressed by the erythrocyte 18-kDa C5b-9 inhibitor are common to the platelet. When bound to the platelet surface, the Fab of alpha-P18 increased C9 activation by membrane C5b-8, monitored by exposure of a complex-dependent C9 neo-epitope. Although alpha-P18 caused little increase in the cytolysis of platelets treated with C5b-9 (total release of lactate dehydrogenase less than 5%), it markedly increased the cell stimulatory responses induced by these complement proteins, including, secretion from platelet alpha- and dense granules, conformational activation of cell surface GP IIb-IIIa, release of membrane microparticles from the platelet surface, and exposure of new membrane binding sites for components of the prothrombinase enzyme complex. Prior incubation of C5b67 platelets with 100 micrograms/ml alpha-P18 (Fab) lowered by approximately 10-fold the half-maximal concentration of C8 required to elicit each of these responses (in the presence of excess C9). Incubation with alpha-P18 (Fab) alone did not activate platelets, nor did incubation with this antibody potentiate the stimulatory responses of platelets exposed to other agonists. These data indicate that a membrane inhibitor of the C5b-9 complex normally serves to attenuate the procoagulant responses of blood platelets exposed to activated complement proteins, and suggest the mechanism by which a deletion or inactivation of this cell surface component would increase the risk of vascular thrombosis.

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the .alpha.-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.

IgG and IgM antibodies to platelet membrane glycoprotein antigens in acute childhood idiopathic thrombocytopenic purpura

In contrast to the situation in chronic idiopathic thrombocytopenic purpura (ITP) it has not been known whether acute childhood ITP has an autoimmune aetiology and to what extent autoantibodies directed to platelet autoantigens appear during the transient course of this disease. In the present study of 21 children with acute ITP an immunoblot technique was applied to detect serum antibodies to electrophoretically (SDS-PAGE) separated normal donor platelet membrane proteins. Platelet antigen binding antibodies detected by alkaline phosphatase conjugated protein A or anti-IgM antibody were observed in 13 out of 21 (62%) patients while controls were negative. In nine children antibodies were directed to antigens localized on the three major membrane glycoproteins. GPIb (four), GPIIb (one) and GPIIIa (five). Antibodies to a 250 kDa antigen were noted in one case and to smaller 25-52 kDa proteins in 12 patients. Four patients had IgG as well as IgM platelet antibodies while in nine only IgM was found. The reactions were eliminated after absorptions of sera with fresh platelets. In all of three tested patients the glycoprotein antigen specific antibodies could be detected in acid eluates prepared from the absorbing platelets. The presence of antibodies, predominantly IgM, to platelet surface antigens in a majority of the children with acute ITP, may suggest an autoimmune process, albeit transitory, similar to that in chronic ITP.

Antibodies to platelet membrane glycoprotein antigens in three cases of infectious mononucleosis‐induced thrombocytopenic purpura

Infectious mononucleosis may occasionally be complicated by purpura, but the mechanism of the thrombocytopenia is not known in detail. In the present study, 3 children with mononucleosis-associated purpura were found to have marked elevations of platelet-associated immunoglobulins and circulating platelet binding IgG and IgM. Employing electrophoretically (SDS-PAGE) separated normal platelet membrane proteins in an immunoblot assay, serum IgG and IgM antibodies were found to be directed to antigenic determinants situated on glycoprotein GP IIb (140 kDa) in all patients, but also on smaller proteins of molecular weights between 30 and 52 kDa. 24 control sera were negative. Positive reactions were eliminated after absorption of sera with fresh platelets. These results demonstrate autoantibodies to platelet surface membrane proteins in infectious mononucleosis-induced purpura and suggest a transient autoantibody-mediated platelet destruction as the cause of thrombocytopenia in these patients.

Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species

The small proteoglycans (PG) of bone consist of two different molecular species: one containing one chondroitin sulfate chain (PG II) and the other, two chains (PG I). These two proteoglycans are found in many connective tissues and have Mr = 45,000 core proteins with clear differences in their NH2-terminal sequences. Using antisera produced against synthetic peptides derived from the human PG I and PG II NH2 termini, we have isolated several cDNA clones from a lambda gt11 expression library made against mRNA isolated from human bone-derived cells. The clones, which reacted with antisera to the PG II peptide, were sequenced and found to be identical with the PG II class of proteoglycan from human fibroblasts known as PG-40 or decorin. The clones reacting to the PG I antisera, however, had a unique sequence. The derived protein sequence of PG I showed sufficient homology with the PG II sequence (55% of the amino acids are identical, with most others involving chemically similar amino acid substitutions) to strongly suggest that the two proteins were the result of a gene duplication. PG II (decorin) contains one attached glycosaminoglycan chain, while PG I probably contains two chains. For this reason, we suggest that PG I be called biglycan. The biglycan protein sequence contains 368 residues (Mr = 42,510 for the complete sequence and Mr = 37,983 for the secreted form) that appears to consist predominantly of a series of 12 tandem repeats of 24 residues. The repeats are recognized by their conserved leucines (and leucine-like amino acids) in positions previously reported for a diverse collection of proteins (none of which is thought to be proteoglycans) including: two morphogenic proteins (toll and chaoptin) in the fruit fly; a yeast adenylate cyclase; and two human proteins, the von Willebrand Factor-binding platelet membrane protein, GPIb, and a rare serum protein, leucine-rich glycoprotein.

Isolation and Characterization of a Platelet Membrane Protein Related to the Vitronectin Receptor

Glycoprotein IIb-IIIa is the most prominent Arg-Gly-Asp (RGD)-binding adhesion receptor on platelets. By affinity chromatography on an immobilized RGD peptide, we have investigated the possible existence of other platelet-associated adhesion receptors that bind RGD peptides. When an octyl glucoside extract of surface-radioiodinated platelets was applied to an affinity matrix of KYGRGDS-coupled Sepharose 4B, a 160-kDa-labeled protein (P160) and GPIIb-IIIa bound and were specifically eluted by soluble GRGDSP peptide, but not by the variant GRGESP peptide. Furthermore, a dodecapeptide corresponding to fibrinogen gamma 400-411 eluted only GPIIb-IIIa but not P160 from the RGD affinity matrix. Characterization of P160 by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the O'Farrell gel electrophoresis system indicated that P160 is a component of platelet GPIc. GoH3, a monoclonal antibody recognizing the alpha subunit of the very late antigen-6, failed to immunoprecipitate P160 from the RGD eluate, indicating that it did not contain the very late antigen-6 alpha subunit. In immunoblots, P160 reacted specifically with a polyclonal anti-peptide antibody recognizing the alpha subunit of the vitronectin receptor (VnR), but not with the monoclonal anti-GPIIb antibody PMI-1, suggesting that P160 is the alpha subunit of platelet VnR. This possibility was further substantiated by the complete identity between the determined amino-terminal sequence of P160 and the known sequence of the VnR alpha subunit. Moreover, direct association of P160 with a beta subunit having an apparent molecular weight similar to that of GPIIIa was demonstrated by immunoprecipitation with LM609, an anti-VnR complex monoclonal antibody. These results indicate that the VnR complex is present on platelets and may play a functional role in platelet adhesive reactions.

Small Molecular Weight GTP-binding Proteins in Human Platelet Membranes: Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.

Platelet Aggregation Is Stimulated by Lactose-lnhibitable Snake Venom Lectins

Five lactose-specific lectins from snake venoms were tested for the ability to stimulate the aggregation of human platelets. Three of the lectins, bushmaster (Lachesis muta), cottonmouth (Ancistrodon piscivorous leukostoma) and rattlesnake (Crotalus atrox) lectins, consistently stimulated secretion and aggregation. Thrombolectin (Bothrops atrox) occasionally caused aggregation. Copperhead (Agkistrodon contortrix contortrix) lectin did not by itself cause platelet aggregation. Lactose, a specific inhibitor of hemagglutination mediated by these lectins was a potent inhibitor of lectin-induced aggregation. Antiserum specific for bushmaster lectin inhibited aggregation by bushmaster lectin. In contrast, the same antiserum and anti-cottonmouth lectin serum enhanced aggregation by low levels of the other lectins. A variety of substances were assayed in the aggregometer for the ability to inhibit aggregation in response to these lectins. Both secretion and aggregation were inhibited by PGI2 and PGE1. Furthermore, lectin-induced aggregation was completely blocked by trifluoperazine and partially blocked by indomethacin. Monoclonal antibodies specific for GP IIb/IIIa (AP2, A2A9, LJP5, LJCP8) but not monoclonals directed against other platelet membrane proteins (AP1 and AP3) inhibited lectin-induced aggregation. The peptide Arg-Gly-Asp-Ser but not Arg-Ala-Asp-Ser was a potent inhibitor of aggregation.

Application of analytical isotachophoresis in the study of platelet membrane proteins in patients with chronic myeloproliferative disorders.

The supernatants of dispase-treated platelets from normal persons and patients with myeloproliferative disorders were analysed by capillary isotachophoresis. The examined myeloproliferative disorders were three cases of chronic myelogeneous leukemia, one case of chronic neutrophilic leukemia, one case of essential thrombocythemia and one case of polycythemia vera. An additional peak was revealed in the samples from the patients with chronic myelogeneous leukemia. Platelets from the other three patients showed no additional bands. Isotachophoresis will be useful not only to analyse the superficial peptide on the cell membrane but also to define the peptide associated with malignant transformation.

Rapid Purification and Partial Characterization of Human Platelet Glycoprotein IIIb: Interaction with thrombospondin and its role in platelet aggregation

Glycoprotein IIIb (also known as glycoprotein IV) is a major glycoprotein present on the surface of human platelets. Recent studies suggest that glycoprotein IIIb may be a receptor site for thrombospondin. Thrombospondin, a multifunctional adhesive glycoprotein released from stimulated platelets, plays an important role in the stabilization of platelet aggregates. In this study, a new method for the purification of glycoprotein IIIb is described. Glycoprotein IIIb was isolated from Triton X-114 platelet membrane extracts, under nondenaturing conditions, by tandem anion-exchange and size exclusion fast protein liquid chromatography. The purified glycoprotein had the same apparent molecular mass (88 kDa) under nonreducing or reducing conditions. The tryptic peptide map of the purified protein was identical to that of bona fide glycoprotein IIIb as isolated from two-dimensional polyacrylamide gels of platelet membrane proteins. In addition, the purified glycoprotein was recognized by an anti-GPIIIb monoclonal antibody (OKM5). The purified glycoprotein specifically bound to thrombospondin in the presence of calcium. Monospecific anti-GPIIIb antibodies interfered with the expression of endogenous thrombospondin on thrombin-activated platelets and partially inhibited collagen- and thrombin-induced platelet aggregation without a significant effect on platelet secretion. Glycoprotein IIIb, by interacting with thrombospondin on the activated platelet surface, may play an important role in the platelet aggregation process.

Effects of taxol and colchicine on platelet membrane properties

The effects of the assembly-disassembly of microtubule on the membrane lipid fluidity, the fragmental motion of sulfhydryl groups of membrane proteins, and the functions of shape change and hypotonic shock response (shrinkage ratio) in human platelets were studied. We have employed electron spin resonance (ESR) utilizing spin labeles for bilayer lipids or membrane proteins and microtubule reactive reagents to change microtubule assembly. Both, taxol, a microtubule stabilizing agent, and colchicine, a microtubule disrupting agent, did not affect the platelet membrane lipid fluidity detected by 5- or 16-doxylstearate. On the other hand, the mobility of sulfhydryl groups detected by 4-maleimide-tempo increased by taxol treatment and decreased by colchicine . Moreover, the temperature sensitivity of platelets below 20°C decreased by taxol treatment, but unchanged by colchicine treatment. This behavior was similar to the shape change ability or the shrinkage ratio of platelets pre-treated with taxol, in which microtubule disassembly was inhibited due to taxol binding to microtubule. Therefore, it is confirmed that the assembly-disassembly state of microtubule which may control the ability of platelets to change shape, influences the mobility of sulfhydryl groups in platelet membrane proteins and its temperature sensitivity.

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an αβγ-subunit structure. The α of the major IAP substrate had a molecular mass of 40 kDa and differed from that of G i 1 or G o previously purified from brain membranes. The partial amino acid sequences of the 40 kDa α completely matched with the sequences which were deduced from the nucleotide sequences of the human G i 2 α gene. On the other hand, the α of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against α of brain G i 1 (G i 1 α). These results indicate that the major IAP substrate present in human platelet membranes is a product of the G i 2 α gene.

G n-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet

The 27 kDa platelet membrane protein (G n27) that binds [α- 32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617–620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti- ras p21 antibody and 22–23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from G n27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431–439] was also resolved from G n27. At the levels normally present in cell membranes, only G n-proteins bound significant amounts of [ 32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.

Interaction between platelet receptor and iloprost isomers

Iloprost, a stable analog of prostacyclin, has been used for studying the interaction between prostacyclin and its effector cells such as platelets and vascular cells. The compound is usually prepared as a mixture of 16(S) and 16(R) stereoisomers. In this work, we compared the biological activity and platelet receptor binding characteristics between the two isomers. The 16(S) isomer was 20-times more potent than the 16(R) in inhibiting collagen-induced platelet aggregation. Equilibrium binding of iloprost isomers to platelet membrane receptors measured by rapid filtration method revealed that the specific binding data of 16(S) isomer was fit for a single binding species with K d of 13.4 nM and B max 665 fmol/mg protein. By contrast, the K d and B max of 16(R) isomer were 288 nM and 425 fmol/mg, respectively. To further assess different binding behavior of these two isomers, association rate was measured. The observed association rate of the S isomer was 0.036 s −1 and 0.001 s −1 for the R isomer at 15 nM iloprost and 2 mg/ml platelet membrane proteins. We postulate that the striking difference in the association rate with resultant difference in binding affinity and biologic activity between the two isomers was due to fitting of the molecule to the receptor channel. The 16(S) form has a more favorable orientation for fitting into the receptor. We conclude that the two iloprost isomers must be considered as two entirely different compounds when iloprost is used as the ligand for quantifying prostacyclin receptor binding.

Identification and isolation of a platelet GPIb-like protein in human umbilical vein endothelial cells and bovine aortic smooth muscle cells.

Glycoprotein Ib (GPIb) is an intrinsic platelet membrane protein that plays a major role in platelet adherence and mediates ristocetin-dependent platelet von Willebrand factor binding. Recent reports that the platelet membrane glycoprotein complex IIb/IIIa is expressed in several cell types prompted us to look for GPIb expression in other vascular cells. Immunoperoxidase staining of human stomach and skin histologic sections with polyclonal as well as monoclonal anti-GPIb antibody revealed the presence of GPIb in the endothelial cell and smooth muscle cell layers. Western blotting using monospecific polyclonal anti-GPIb antibodies confirmed the presence of immunoreactive GPIb in human umbilical vein endothelial and bovine aortic smooth muscle cell cultures. Fab fragments of a monoclonal anti-GPIb antibody were used to immunoprecipitate [3H]leucine labeled GPIb from metabolically labeled cells. The GPIb in these cells was functional as measured by ristocetin-dependent cell agglutination and by vWF binding. Endothelial cells as well as smooth muscle cells bound 125I-labeled vWF in a ristocetin-dependent manner, with a Kd of 7.9 nM.

Distinction between glycoprotein IIIa and the 100-kDa membrane protein (aggrogin) mediating ADP-induced platelet activation

Previous studies from our laboratories showed that 5′- p-fluorosulfonylbenzoyl adenosine (FSBA) inhibits ADP-induced platelet shape change, aggregation, and exposure of fibrinogen sites while covalently binding to 100-kDa platelet membrane protein (aggregin) on the intact platelet. Chymotrypsin digests aggregin to a fragment of 70 kDa, abolishing the inhibition, and also cleaves platelet glycoprotein IIIa (GPIIIa) (100 kDa) to a 70-kDa fragment containing the P1A 1 epitope. We questioned whether these platelet membrane proteins were distinct. Both 5′- p-[ 3H]sulfonylbenzoyl adenosine (SBA)-labeled aggregin and 125I-GPIIIa were precipitated by polyclonal antibodies to a 100-kDa fraction of platelet membranes, but aggregin was not precipitated by a monospecific antibody to P1A 1 which precipitates GPIIIa. Further a monospecific polyclonal antibody to immunopurified GPIIIa coupled to protein A-Sepharose adsorbed GPIIIa but not aggregin. Similarly, both aggregin and GPIIIa were precipitated by a polyclonal antibody to an isolated 70-kDa component of platelet membrane but only GPIIIa was precipitated by the monoclonal antibody to GPIIIa, (SSA6). Two patients with Glanzman's thrombasthenia whose platelet membranes contained <5% GPIIIa as assayed by monoclonal antibody binding (A 2A 6), incorporated [ 3H]SBA to the same extent as normal individuals. Furthermore, FSBA inhibited ADP-induced shape change with a similar concentration dependence for both thrombasthenic and normal platelets. Finally, mobility of GPIIIa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was decreased following reduction with dithiothreitol whereas that of [ 3H]SBA-labeled MP 100 was not altered. We conclude that GPIIIa and aggregin are distinct platelet membrane proteins.

Identification of Bakb, a new platelet-specific antigen associated with posttransfusion purpura

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, “Har,” obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.