Monoclonal antibodies against human platelet glycoprotein IIIa

Two murine monoclonal antibodies specific for human platelets were prepared and characterized by immunofluorescence, immunoprecipitation and by studying their effect on platelet function. Immunoprecipitation with lysates of normal platelets and platelets from a patient with Glanzmann's thrombasthenia revealed that the monoclonal antibodies were directed against glycoprotein GP IIIa. One of these anti-GP-IIIa antibodies (C17) inhibited both ADP- and collagen-induced platelet aggregation as well as ADP-induced fibrinogen binding to platelets. The other anti-GP-IIIa antibody (C15) also caused a complete inhibition of aggregation with collagen. However, a small, and fully reversible, 'primary wave' was observed if the platelets were stimulated with ADP when platelet-rich plasma was used. The ability to bind fibrinogen was unimpaired for platelets incubated with C15. These findings show that C15 and C17 probably recognize different determinants on GP IIIa. Neither of the monoclonal anti-GP-IIIa antibodies blocked the binding to Zwa-positive platelets of human polyclonal anti-Zwa antibodies. This implies that Zwa is different from the epitopes recognized by C15 and C17.     

Monoclonal antibodies that bind to the core of fusion-active glycoprotein 41

The heptad repeat regions HR1 and HR2 of HIV-1 gp41 can associate to form heterooligomers through helical coil-coil interactions that are believed to play a key role in virus-induced membrane fusion. The HR1/HR2 complex was proposed to be the core structure of the fusion-active conformation of gp41. Here, we show that two human monoclonal antibodies, Fab-d and 50-69, specifically recognize the putative fusion-active conformation of gp41. Fab-d binding requires the interaction between the HR1 and HR2 regions of gp41. The reactivity of human monoclonal antibody 50-69 to the C terminus of the HR1 sequence is dependent on the helical structure of HR1. It appears that HR2 is able to interact with HR1 and, subsequently, induce an epitope in HR1 that is required for 50-69 binding. Mutations that disrupt the helical structure of HR1 significantly compromise Fab-d and 50-69 binding. Although the epitopes are not identical, the ability of Fab-d to partially compete with 50-69 binding suggests a close proximity of the two epitopes. Antibodies that are able to interact with the core of the putative fusion-active gp41 may be useful in further unveiling the mechanism of HIV-induced membrane fusion.     

Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3-4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3-4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3-4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3-3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3-3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3-3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3-4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3-3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3-4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3-3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.     

Oral platelet glycoprotein IIb/IIIa inhibition

Platelet aggregation plays a central role in the pathogenesis of thrombosis and the acute coronary syndromes. When given intravenously, potent selective antagonists of fibrinogen binding to the glycoprotein (GP) IIb/IIIa receptor, the final common pathway for platelet aggregation, have been effective in the treatment of acute coronary syndromes. Their benefit ceases, however, with the end of the infusion. Aspirin reduces the incidence of secondary vascular events by 25% to 30% after an acute coronary syndrome, and clopidogrel provides modest improvement over aspirin. However, both are relatively weak antiplatelet agents that each block only one of many pathways to platelet activation and surface membrane expression of the competent GP IIb/IIIa receptor. With the success of the intravenous GP IIb/IIIa antagonists in the acute setting, recent interest has focused on the potential benefit of oral GP IIb/IIIa antagonists used long-term for secondary prevention. The oral agents tested in phase III studies thus far have not performed up to expectations, however. The following paper reviews these studies and the implications of their results.     

Inhibition of autoantibody binding to platelet glycoprotein IIb/IIIa by anti-idiotypic antibodies in intravenous gammaglobulin

Intravenous immunoglobulin (IVIgG) causes an acute rise in the platelet count in the majority of patients with chronic immune thrombocytopenic purpura (ITP) but the mechanism(s) of action is still unknown. We evaluated the ability of three different IVIgG preparations to inhibit the in vitro binding of autoantibody to platelet glycoprotein (GP) IIb/IIIa. ITP plasma, known to contain anti-GPIIb/IIIa antibodies, was incubated overnight with either IVIgG or bovine serum albumin (BSA) followed by measurement of the autoantibody titer. Binding of autoantibody from eight ITP patients was inhibited by IVIgG in proportion to the IVIgG concentration. Using 3.2% IVIgG, compatible with therapeutic concentrations expected in vivo, mean inhibition of autoantibody binding ranged from 20.2% to 41.3%. No inhibition by IVIgG of alloantibody binding to the same or different molecules was detected (five patients with anti-GPIIb/IIIa and two with anti-HLA alloantibodies). F(ab')2 fragments of IVIgG also inhibited the binding of both plasma autoantibodies and purified anti-GPIIb/IIIA autoantibodies prepared by elution from antigen affinity columns. A portion of the anti-idiotypic antibodies could be adsorbed from IVIgG using insolubilized, purified anti-GPIIb/IIIa autoantibody. These results show that IVIgG preparations from normal donors contain anti-idiotypic antibodies directed against idiotypes located on GPIIb/IIIa autoantibodies but do not have anti-idiotypes to platelet alloantibodies against the same or different molecules. The importance of these anti-idiotypic antibodies in the therapeutic response to IVIgG remains to be established.     

A monoclonal antibody to platelet glycoprotein IIb induces aggregation of platelet rich plasma

The monoclonal antibody (MAb) GAP 5.9 directed against platelet glycoprotein IIb is described. This MAb is able to induce aggregation of platelet rich plasma in the absence of further stimuli and likely recognizes a glycoprotein epitope not closely related to calcium binding sites on the molecule.     

Monoclonal antibodies specific for platelet glycoproteins react with human monocytes

Three monoclonal antibodies, P256 , P140, and P112 , react with the 135,000 mol wt IIb component of the glycoprotein IIb/IIa complex. They also react with a 200,000-mol wt protein present at low levels in the complex. Using immunofluorescence techniques, monoclonal antibodies P140 and P112 , but not P256 , can be shown to bind to 80% of human monocytes. However, P256 was able to immunoprecipitate the IIb/IIIa complex from detergent-solubilized monocytes, suggesting that the P256 epitope is less accessible on monocytes than on platelets. The monoclonal antibodies also precipitated molecules of approximately 200,000 mol wt from the monocytes. Two other monoclonal antibodies, J15 (specific for the IIb/IIIa complex) and AN51 (which reacts with the second major platelet glycoprotein complex, I), also react with monocytes. Binding of the monoclonal antibodies to the histiocytic cell line, U937, and promyelocytic cell line, HL-60, reflected the pattern of reaction with monocytes. The presence on monocytes of these glycoproteins, instrumental to the role of platelets in clotting, raises the possibility that monocytes might have similar functions in particular circumstances.     

Oral platelet glycoprotein IIb/IIIa receptor inhibitors--Part I

With the central importance of antiplatelet therapy in patients with coronary artery disease and the numerous positive trials with glycoprotein (GP) IIb/IIa inhibitors given intravenously, it was hoped that one could extend the benefit of IIb/IIIa inhibition to long-term treatment. Although the hypothesis that prolonged oral IIb/IIIa inhibition was appealing, many issues have been identified in the initial Phase II trials that would limit the usefulness of these compounds. Variability of the level of platelet inhibition was one major culprit that distinguished the oral compounds from intravenous ones. The problems that arose were that increased bleeding has been seen when levels of platelet inhibition are high (e.g., > 90%) and that, conversely, efficacy would likely be limited when levels of platelet inhibition were low. If further development of this class of drugs is undertaken, formal dosing studies would have to establish an oral dosing strategy that achieves appropriately high (80-95% inhibition) and steady levels of inhibition.     

Oral platelet glycoprotein IIb/IIIa receptor inhibitors--part II

Although the hypothesis of benefit from prolonged oral IIb/IIIa inhibition was appealing, the large Phase III trials have uniformly shown there was no improvement in outcome. In addition, there was an increased mortality seen in patients treated with the oral IIb/IIIa inhibitor. This latter finding is not adequately explained, but is likely a multifactorial problem of this strategy of platelet inhibition. The trials found that, even with no improvement in efficacy, there was increased bleeding, meaning that for chronic therapy with IIb/IIIa inhibition there does not appear to be a therapeutic window. Accordingly, chronic oral IIb/IIIa inhibition appears to have been well tested but has not worked. Fortunately, there are several other oral antiplatelet agents available that have shown beneficial results, including clopidogrel. In addition, other newer classes of antiplatelet agents are in earlier stages of development. Thus, agents targeted more "upstream" in platelet activation pathways may offer a more tolerable and efficacious approach to long-term antiplatelet therapy.     

Diagnosis of Glanzmann's thrombasthenia and carrier detection using monoclonal antibodies to platelet glycoprotein IIb and IIIa in immunoblotting.

Glanzmann's thrombasthenia is a rare hemorrhagic syndrome, characterized by a quantitative or functional defect of the platelet glycoprotein GPIIb-IIIa complex. The authors describe a method to diagnose thrombasthenic patients and identify carrier subjects by using monoclonal antibodies specific for GPIIb and GPIIIa in an immunoblotting technique. The immunoreaction patterns of two thrombasthenic patients lacking GPIIb or GPIIIa, respectively, are shown. The described method produces further evidence concerning the biochemical heterogeneity of Glanzmann's thrombasthenia.     

Monoclonal antibodies that recognize the native human thyrotropin receptor

Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor. The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established. An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational. In contrast, two IgGl antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting). The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both of the IgGl antibodies to be assigned to the region 125–369. None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgGl antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor.     

Monoclonal antibodies to rabbit hepatocyte myosin that cross-react with human liver myosin

We prepared polyclonal and monoclonal antibodies against myosin purified from rabbit hepatocytes. Immunoblotting analyses revealed that the polyclonal antibody and four (HM1, HM2, HM3 and HM4) of five monoclonal antibodies reacted with myosin heavy chain. Chymotryptic cleavage of myosin yielded a 130 kDa fragment comprising the tail portion of the myosin heavy chain and a 67 kDa fragment comprising the ATP-binding active site of the myosin head. All active antibodies reacted with epitopes localized in the 130 kDa fragment. Monoclonal antibodies HM3 and HM4 and the polyclonal antibody reacted strongly with myosin heavy chains from a human liver homogenate prepared from a surgically resected liver specimen. Immunocytochemical analyses showed that myosin is localized along the plasma membrane as well as around the bile canaliculi in both rabbit and human hepatocytes. Immunocytochemical analyses on liver blocks obtained from those patients who suffered various types of diseases accompanying cholestasis clearly indicated a marked increase in pericanalicular myosin. This altered myosin localization is analogous to that observed in phalloidin-treated liver. Thus, myosin localization, determined using these antibodies, can provide a valid morphological basis for diagnosing the pathological state of the patient liver.     

Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons

The antithrombotic efficacy of the monoclonal antibodies 6B4-Fab and MA-16N7C2 against platelet glycoprotein (GP) Ib and GP IIb/IIIa, respectively, on acute platelet-mediated thrombosis was evaluated in a baboon model of femoral artery stenosis, which is a modification of the original Folts model: platelet thrombi form on the injured stenosed artery, producing cyclic flow reductions (CFRs). A dose of 0.6 mg/kg 6B4-Fab significantly reduced the CFRs by 59 +/- 15%, whereas 2 mg/kg 6B4-Fab completely abolished the CFRs without prolongation of the bleeding time. MA-16N7C2 inhibited CFRs by 43 +/- 8% at a dose of 0.1 mg/kg and abolished the CFRs at a dose of 0.3 mg/kg but with a significant prolongation of the bleeding time. Finally, the combination of 0.6 mg/kg 6B4-Fab and 0.1 mg/kg MA-16N7C2 fully prevented the CFRs without prolongation of the bleeding time. The present study demonstrates that the inhibition of platelet GP Ib function by 6B4-Fab is a powerful intervention to prevent platelet thrombus formation in injured arteries without prolongation of the bleeding time; the latter is in contrast to the result after the inhibition of GP IIb/IIIa. Moreover, we demonstrate that combining a GP Ib blocker with a GP IIb/IIIa blocker can achieve a strong antithrombotic effect without increasing the bleeding time. This provides new information that will be beneficial in designing clinical therapeutic approaches.     

Detection of glycoprotein IIb and IIIa by monoclonal antibodies

Murine monoclonal antibodies were produced against human platelet membranes and screened on platelets by a ¹²⁵I protein A radioimmunoassay. Several clones produced platelet specific antibodies as they showed no reaction with peripheral blood lymphocytes, neutrophils, bone marrow (excluding megakaryocytes) or several cell lines. Two antibodies (designated anti-HuPl-mla and anti-HuPl-mlb) were of particular interest in that although platelet specific they were non-reactive with platelets from a thrombasthenic patient. In functional assays these two antibodies could specifically inhibit ADP and collagen induced aggregation of platelets and release of ATP, retard platelet aggregation and ATP release induced by epinephrine, and inhibit ADP induced platelet fibrinogen binding. These two antibodies appear to recognize glycoproteins IIb and IIIa as analysis by SDS-PAGE using radiolabelled membranes revealed a two chain structure of molecular weight 112 000 and 122 000 daltons when run after reduction and 87 000 and 140 000 daltons non-reduced.     

Monoclonal antibodies to human pancreatic carcinoma

Hybridoma cultures were produced by the fusion of SP2 mouse myeloma cells with spleen cells from mice immunized with human pancreatic carcinoma cells. After limiting dilutions, three monoclonal antibodies, YPan1, YPan2, and YPan3, which bound to immunizing cells but not to normal human skin fibroblasts, were further characterized. The three monoclonal antibodies were found to bind to all seven pancreatic carcinoma cell lines but not to other carcinoma cell lines tested except some colon carcinoma cell lines. When human tissue sections were examined using immunohistochemical techniques, the three monoclonal antibodies identified antigens in the pancreatic carcinomas and some normal pancreases, but only YPan1 showed strong positive staining. No cross-reactivity was seen in sections of other carcinomas tested except some colon carcinomas. The results suggest that these monoclonal antibodies may be usefully applied to the detection of pancreatic carcinomas.     

Monoclonal antibodies to human intrinsic factor

Mice were immunized with human intrinsic factor, and their lymph node cells were fused with a myeloma cell line by standard hybridoma techniques. Eleven of the resulting 227 hybridomas secreted immunoglobulin G capable of binding to intrinsic factor-cobalamin complex. Cloning by limiting dilution gave 6 clones secreting anti-intrinsic factor antibodies that bound human intrinsic factor-cobalamin complex with affinities of 13-116 nM; 3 antibodies also bound rabbit intrinsic factor-cobalamin complex. Five antibodies inhibited to some degree the binding of cobalamin by intrinsic factor, and 2 also prevented attachment of intrinsic factor-cobalamin complex to guinea pig ileal receptors. Anti-rabbit intrinsic factor antibodies specifically precipitated a peptide of molecular weight 53,000, corresponding to the molecular weight of rabbit intrinsic factor from homogenates of rabbit gastric mucosal explants biosynthetically labeled with [35S]methionine and from culture medium in which the explants were incubated. Indirect fluorescence immunocytochemistry with the antibodies in human and rabbit gastric mucosal sections showed intense selective staining of parietal cells. These results (a) document species differences between human and rabbit intrinsic factors not previously demonstrable with polyclonal anti-intrinsic factor sera; (b) confirm earlier evidence that cobalamin binding and receptor functions occur at separate sites in intrinsic factor; and (c) provide a useful approach to studying structure-function relations of the intrinsic function molecule.     

Thrombocytopenia after treatment with platelet glycoprotein IIb/IIIa inhibitors

Abciximab, eptifibatide, and tirofiban are the three drugs approved by the US Food and Drug Administration designed to block platelet aggregation by binding glycoprotein IIb/IIIa. Results from large therapeutic trials demonstrate that all three agents induce thrombocytopenia in approximately 1% to 5% of cardiac patients. Thrombocytopenia is typically rapid in onset and antibody mediated. In vitro evidence suggests abciximab-induced thrombocytopenia is associated with antibodies directed to murine sequences within the chimeric anti-IIb/IIIa molecule. In addition, abciximab, eptifibatide, and tirofiban also function as mixed agonist-antagonists in vivo, inducing neoepitopes within the glycoprotein IIb/IIIa receptor that may react with pre-existing or induced antibodies. Screening for the development of these antibodies may reduce the incidence of thrombocytopenia associated with these agents, but it may limit their chronic usage.