Platelet Membrane Glycoproteins IIIa Research Articles

Glycoproteins IIb and IIIa and Platelet Thrombospondin in a Liposome Model of Platelet Aggregation

Platelet membrane glycoproteins IIb and IIIa and platelet thrombospondin were incorporated onto phosphatidylcholine liposomes, by freeze thawing and sonication. Protein orientation on the liposomes was confirmed by susceptibility to neuraminidase cleavage and binding to lentil lectin-Sepharose (GPIIb-IIIa liposomes) and to heparin-Sepharose (thrombospondin liposomes). Glycoproteins IIb-IIIa bound 125I-fibrinogen with Kd of 7.5 X 10(-7)M. Binding was reversible and calcium-dependent. IIb-IIIa liposomes underwent fibrinogen-dependent aggregation in the presence of 10 mM CaCl2. Maximal aggregate formation was observed with a combination of IIb-IIIa liposomes and thrombospondin liposomes. This aggregation was partially inhibited by preincubation with monoclonal antibodies to the IIb-IIIa complex. Addition of EDTA caused complete reversal of aggregates. Thrombospondin liposomes also underwent fibrinogen and calcium dependent aggregation, however, this aggregation was less than that observed with the GPIIb-IIIa liposomes. Maximal aggregate formation was observed with a mixture of IIb-IIIa liposomes and thrombospondin liposomes. These studies demonstrate that GPIIb-IIIa and thrombospondin can be incorporated into phospholipid vesicles with preservation of function. Direct evidence is provided to demonstrate that glycoprotein IIb and IIIa and fibrinogen are sufficient for platelet aggregation and to demonstrate that thrombospondin may also contribute to platelet aggregation.

A method for purifying the platelet membrane glycoprotein IIb-IIIa complex

A method has been developed for the rapid isolation of platelet membrane glycoproteins (GP) IIb and IIIa. This method produces an excellent yield and does not require the prior isolation of platelet membranes. Outdated platelets were washed and solubilized in Triton X-100. Concanavalin A affinity chromatography was used to purify a platelet glycoprotein fraction. The concanavalin A-retained glycoproteins were eluted and adsorbed with a heparin-Sepharose column to remove a major contaminant, thrombospondin. Sephacryl S-300 gel filtration was used as the final purification step to remove most fibrinogen and low-molecular-weight contaminants. Wheat germ agglutinin affinity chromatography was used to completely remove trace amounts of fibrinogen. The purified GP IIb and GP IIIa were analyzed by sucrose gradient sedimentation and found to consist of heterodimer complexes.

Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IIIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by greater than 85%. T10, an antibody specific for the GPIIb-IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex.

Platelet membrane glycoproteins (GP) IIb and IIIa form a Ca2+-dependent, heterodimer complex (GP IIb-IIIa) in detergent solution. To determine whether these glycoproteins were complexed or dissociated in intact human platelets, 125I-labeled whole platelets were lysed with an EDTA/Triton X-100 buffer, which stabilized both the complexed and dissociated forms of GP IIb and GP IIIa. The percentage of the heterodimer complex present after lysis was determined by quantitating the sedimentation of GP IIIa in sucrose gradients. The distribution of GP IIIa in sucrose gradient fractions from unlabeled platelets was detected by incubation of 125I-concanavalin A with Western blots. It was determined that GP IIb and GP IIIa existed predominantly as the GP IIb-IIIa complex in either unstimulated (92% complexed) or thrombin-stimulated (90% complexed) platelets. The GP IIb-IIIa complex in unstimulated platelets could be reversibly dissociated by brief (up to 10-min) incubations at 37 degrees C with EDTA, while longer (30-min) incubations with EDTA resulted in irreversible polymerization of GP IIb and GP IIIa. The dissociation of the GP IIb-IIIa complex by EDTA in intact cells was temperature-dependent, and the critical extracellular Ca2+ concentration for dissociation was 10(-5) to 10(-6) M. These results demonstrate that GP IIb and GP IIIa exist as a Ca2+-dependent, heterodimer complex in intact platelets and that treatment of platelets with EDTA can have multiple effects on this complex.

Effect of calcium on the stability of the platelet membrane glycoprotein IIb-IIIa complex.

Platelet membrane glycoproteins IIb and IIIa form a Ca2+-dependent heterodimer complex that contains binding sites for fibrinogen, von Willebrand factor, and fibronectin following platelet stimulation. We have studied the effect of Ca2+ on the stability of the IIb-IIIa complex using a IIb-IIIa complex-specific monoclonal antibody A2A9 to detect the presence of the complexes. Soluble IIb and IIIa interacted with A2A9-Sepharose only in the presence of Ca2+ with 50% IIb-IIIa binding requiring 0.4 microM Ca2+. In contrast, at 25 degrees C 125I-A2A9 binding to intact unstimulated platelets suspended in buffers containing EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was independent of the presence of Ca2+. However, the effect of Ca2+ chelators on 125I-A2A9 binding varied with temperature. At 37 degrees C, 125I-A2A9 binding to intact platelets became Ca2+-dependent with 50% binding requiring 0.4 microM Ca2+. This effect of temperature was not due to a change in platelet membrane fluidity because enrichment or depletion of platelet membrane cholesterol did not influence antibody binding. But, 125I-A2A9 binding to intact platelets at 25 degrees C did become Ca2+-dependent when the pH was increased above 7.4. Thus, at 1 nM Ca2+ and 25 degrees C, 50% antibody binding occurred at pH 9.0. Our studies demonstrate that Ca2+-dependent IIb-IIIa complexes are present on unstimulated platelets and that the Ca2+ binding sites responsible for the stability of these complexes are located on the external platelet surface. Our experiments also suggest that changes in platelet cytosolic Ca2+ do not regulate the formation of IIb-IIIa complexes.

Residual amounts of glycoproteins IIb and IIIa may be present in the platelets of most patients with Glanzmann's thrombasthenia

Glanzmann's thrombasthenia is an inherited bleeding disorder characterized by abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Most patients, usually designated as type I, have been reported to have undetectable levels of GP IIb and GP IIIa with the assay used. We have used polyclonal rabbit antibodies against GP IIb and GP IIIa in a sensitive immunoblot procedure capable of revealing trace amounts of these glycoproteins. Platelets from nine thrombasthenic patients, including seven with type I disease, were studied. GP IIIa, although decreased, was clearly detectable in platelets of eight patients and GP IIb was identified in five. Our findings suggest that residual quantities of GP IIb and GP IIIa are present in most patients with thrombasthenia and therefore that major deletions in the gene or genes encoding these proteins are uncommon.

Residual Amounts of Glycoproteins IIb and IIIa May Be Present in the Platelets of Most Patients With Glanzmann's Thrombasthenia

Glanzmann's thrombasthenia is an inherited bleeding disorder characterized by abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Most patients, usually designated as type I, have been reported to have undetectable levels of GP IIb and GP IIIa with the assay used. We have used polyclonal rabbit antibodies against GP IIb and GP IIIa in a sensitive immunoblot procedure capable of revealing trace amounts of these glycoproteins. Platelets from nine thrombasthenic patients, including seven with type I disease, were studied. GP IIIa, although decreased, was clearly detectable in platelets of eight patients and GP IIb was identified in five. Our findings suggest that residual quantities of GP IIb and GP IIIa are present in most patients with thrombasthenia and therefore that major deletions in the gene or genes encoding these proteins are uncommon.

Novel peripheral blood-derived human cell lines with properties of megakaryocytes.

For 18 mo, we derived 18 cell lines from 11 donors with various clinical profiles ranging from normal to leukemic. Suspension cultures were initiated with 1 X 10(6) mononuclear blood cells/ml of nutrient medium containing 10% human serum and 10% lectin-stimulated human lymphocyte conditioned medium. The cultures were monitored weekly by morphological analyses of Wright-Giemsa-stained cell preparations. All successful cultures showed a significant decline in viability during the first 3-4 wk with rate "lymphoid" cells observed in mitosis. Within the next 2 wk, the proliferating cells gave rise to a rapidly expanding population of mononuclear cells. As the cultures expanded, cell morphology became heterogeneous with respect to cell size and nuclear ploidy, with an accumulation of giant multinuclear cells that were suggestive of megakarocytes. Even though the cells did not have the classical morphology of mature platelet-forming megakaryocytes, 90% of the cells within a cell line were positive by direct or indirect immunofluorescence for the platelet membrane glycoproteins IIb and IIIa; for surface markers HLA-Dr and B2-microglobulin; for intracellular platelet-derived growth factor and platelet factor IV; and for membrane affinity or binding with serum platelet-derived growth factor and platelet factor IV. These results suggest that a blood precursor cell, most likely a primitive megakaryoblast, was isolated from the peripheral blood and was provided with an optimal culture environment for sustained growth. These cells did not mature to a more differentiated stage, perhaps owing to regulatory factor deficiencies in this in vitro system. The remarkable frequency of obtaining cell lines with megakaryocyte properties from normal peripheral blood and the capacity of some normal donors to repeatedly yield these cell lines make this cell culture system indeed unique by being selective for putative megakaryocyte precursors.

Platelet membrane glycoprotein IIb-IIIa complex incorporated into phospholipid vesicles. Preparation and morphology.

Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy.

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.

Ｆｉｌｔｅｒ Ｂｌｅｅｄｉｎｇ Ｔｉｍｅ（ＦＢＴ）による血小板機能の評価

Currently we developed a new in vitro test of hemostasis, the filter bleeding time (FBT), which is based on progressive decrease of flow rate of citrated blood through a disk filter of Woven Dacron under controlled pressure as platelet aggregates occlude the filter. FBT is defined as the time when the blood drop rate has fallen to less than 1/30sec. There was a high correlation (r=-0.91) between FBT and platelet count below 105/μl in dogs made thrombocytopenia by estradiol. There was a significant correlation (r=0.65) between FBT and Ivy Bleeding Time (IBT) in 59 patients referred because of a suspected bleeding disorder. FBT was prolonged in 3 of 3 patients with Glanzmann's thrombasthenia, 5 of 6 with von Willebrand disease and normal in 6 of 6 with hemophilia. FBT was more sensitive than IBT in detecting abnormal platelet aggregation in response to collagen, epinephrine or arachidonate. A monoclonal platelet antibody (HP1-1D), which is a specific antibody for platelet membrane glycoprotein IIb and IIIa, increased FBT in a dose dependent manner. FBT was prolonged by adding 0.1μM prostaglandin I2 and an ADP scavenge system, CP/CPK/ATP but not by adding thromboxane synthetase inhibitor, UK-37, 248 or UK-38, 485. Thromboxane synthetase inhibitors had, however, a synergistic effect on FBT when added with CP/CPK/ATP. Oral ticlopidine 250mg b. i. d. for 7 days increased FBT as well as IBT and Template bleeding time. Additional increase in FBT and skin bleeding times was produced by simultenous taking of 650mg aspirin b. i. d. for 7 days.FBT was significantly shorter in smokers than in non-smokers. It is suggested from these observations that FBT is a useful test of platelet function and has certain advantages over skin bleeding time methods.

Reconstruction of platelet proteins into phospholipid vesicles. Functional proteoliposomes.

Platelet membrane glycoproteins IIb and IIIa were reconstituted into liposomes containing phosphatidylcholine. The reconstituted vesicles bound antiplatelet antibodies and showed specific binding to thrombin-activated platelets. Prostacyclin, a known inhibitor of thrombin-activated platelet aggregation, inhibited the binding of the proteoliposomes to thrombin-activated platelets. The reconstituted vesicles also specifically bound 125I-labeled fibrinogen. This binding was insensitive to ADP but dependent on calcium ions. These data indicate that platelet glycoproteins IIb and IIIa have been successfully reconstituted into phospholipid vesicles such that their behavior is similar to that in intact platelets.

The Human Platelet Membrane Glycoprotein Complex GP IIb-IIIa Expresses Antigenic Sites Not Exposed on the Dissociated Glycoproteins

AbstractA number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in nonprecipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100–soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb-IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.

The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins

A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non- precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb- IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.

Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias.

The precise pathogenic mechanism of platelet destruction in immune thrombocytopenias is not known, although many investigators have found that platelet-associated IgG is increased in these diseases. We report here the differentiation between specific binding of anti-platelet antibody, associated with platelet destruction, and the ubiquitous presence of nonspecific, platelet-associated IgG. Using an electrophoretic separation and antibody overlay technique, we have identified a specific membrane protein that bears target platelet antigens in immune thrombocytopenias. When posttransfusion purpura serum was studied, antibody binding to the PlA1 antigen on glycoprotein IIIa was readily distinguished from the nonspecific binding of immunoglobulin to a protein of 200,000 mol wt. After reduction of disulfide bonds, the PlA1 antigenicity was not observed, and IgG bound nonspecifically to a protein band with an apparent molecular weight of 45,000. We have also identified anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura and determined their antigenic specificity. Antibodies which bind to a 100,000-mol wt protein were found in nine of thirteen patients with chronic disease. The antigens in three of these cases were studied in detail by using both reduced and nonreduced control and Glanzmann's thrombasthenic platelets. Target antigens were localized to glycoprotein IIIa, but are different from PlA1. The immune thrombocytopenic purpura antigenic system is clearly distinguished from nonspecific platelet-associated IgG. Sera from eight children with acute idiopathic thrombocytopenic purpura were also studied. In all cases, the nonspecific IgG binding to the 200,000-mol wt protein was observed. However, we were unable to demonstrate antibody binding to glycoprotein IIIa, which suggested that the acute childhood form of this disease may have a different pathogenic mechanism than that of the autoimmune chronic cases.

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain.

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.

A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

A Murine Monoclonal Antibody That Blocks Fibrinogen Binding to Normal Platelets Also Inhibits Fibrinogen Interactions With Chymotrypsin-Treated Platelets

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP-treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.

Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets.

A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.