Structure and function of murine alphaIIbbeta3 (GPIIb/IIIa): studies using monoclonal antibodies and beta3-null mice,.

The alphaIIbeta3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet alphaIIbeta3, utilizing both beta3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine alphaIIbbeta3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of alphaIIbbeta3 (60-80,000 copies/platelet). Like their human counterparts, murine alphaIIb and beta3 exhibit different electrophoretic motilities under nonreducing (aIIb 135k Da; beta3 92k Da) and reducing (aIIb 120k Da; beta3 108k Da) conditions, and the alphaIIbbeta3 complex is dissociated by EDTA at pH 8 and 37 degrees C. Murine beta3 is less susceptible to proteolysis by plasmin than is human beta3. In addition to defective platelet aggregation, mouse platelets lacking alphaIIbbeta3 and alphaVbeta3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, beta3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, beta3-null platelets have altered tyrosine phosphorylation patterns following thrombin- and collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.

Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (alphaIIbbeta3) and other proteins from hamster-mouse interspecies hybridomas.

To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.

Activation of platelets in platelet-rich plasma by rotablation is speed-dependent and can be inhibited by abciximab (c7E3 Fab; ReoPro).

BACKGROUND: Rotational atherectomy with the Rotablator catheter has improved percutaneous treatment of certain coronary atherosclerotic lesions, but the "no-reflow" phenomenon remains a serious complication. Because platelet activation by rotablation may contribute to the no-reflow phenomenon, we developed an in vitro system to test the effect of rotablation on platelets in the absence or presence of platelet GP IIb/IIIa receptor blockade with abciximab. METHODS AND RESULTS: Platelet-rich plasma (PRP) was prepared from 28 healthy human volunteers. PRP was divided into 4 samples: (1) no treatment, (2) 6D1 (anti-GP Ib), (3) c7E3 Fab (anti-GP IIb/IIIa+alpha(v)beta3), and (4) c7E3 Fab+6D1. Samples were pumped through a flow chamber containing a 2.5-mm burr rotating at various speeds and then placed in an aggregometer. PRP samples tested in the absence of antibody underwent more rapid and extensive aggregation when rotablated at 150000 and 180000 rpm compared with 0 rpm (P<0.001 at both speeds). Preincubation of platelets with c7E3 Fab decreased the slope of aggregation at each rotablation speed, with 98%, 79%, and 71% reductions at 70000, 150000, and 180000 rpm, respectively (P=0.09 for 70000 and P<0.001 for both 150000 and 180000 rpm). Preincubation of platelets with 6D1 did not decrease the slope of aggregation at any rotablation speed (P>0.5, P=0.99, and P=0.091 for 70000, 150000, and 180000 rpm). Platelet ATP release, a marker of granule release and cell damage, was markedly increased at 180000 rpm (P=0.002 compared with 0 rpm in the control group). Electron microscopy revealed extensive rotablation-induced platelet damage at 150000 and 180000 rpm, and leakage of LDH confirmed platelet lysis at these speeds (P=0.002 and P<0.001 compared with 0 rpm). CONCLUSIONS: High-speed rotablation induces platelet activation of PRP, leading to aggregation; pretreating PRP with abciximab decreases the aggregation. These data suggest that pretreatment of patients with abciximab may decrease rotablation-induced platelet aggregation during rotational atherectomy.

In vitro effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab on the activated clotting time.

BACKGROUND: In the evaluation of 7E3 for the Prevention of Ischemic Complications study (EPIC), the activated coagulation (clotting) times (ACTs) were longer in heparinized patients treated with c7E3 Fab than in those treated with placebo. The present study was designed to further investigate this observation by assessing whether the in vitro addition of c7E3 Fab to blood would affect the ACT. METHODS AND RESULTS: Native or heparinized blood obtained from normal volunteers was preincubated with antibodies c7E3 Fab (anti-GPIIb/IIIa and anti-alpha v beta 3), 10E5 (anti-GPIIb/IIIa), or LM609 (anti-alpha v beta 3). The ACTs of the heparinized, but not native samples were significantly prolonged by the addition of c7E3 Fab and 10E5 but not LM609, indicating that the prolongation was due to GPIIb/IIIa blockade. The addition of c7E3 Fab also significantly prolonged the ACTs of blood anticoagulated with the direct thrombin inhibitors hirudin and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone, indicating that the effect of c7E3 Fab was not exclusively related to decreased release of PF4, a heparin-neutralizing factor, from platelets. CONCLUSIONS: These data support the conclusion that the prolongation of the ACT in patients in EPIC was due to c7E3 Fab blockade of GPIIb/IIIa receptors. This raises the possibility that in vivo c7E3 Fab functions not only as an antiplatelet agent but also as an anticoagulant; direct in vivo data will, however, be required for assessment of this possibility.

Rapid and simple platelet function assay to assess glycoprotein IIb/IIIa receptor blockade.

BACKGROUND: The platelet glycoprotein (GP) IIb/IIIa receptor antagonist c7E3 Fab (abciximab; ReoPro) has been approved as an antithrombotic agent, and other GP IIb/IIIa antagonists, including oral agents, are under development. At present, there are no rapid and simple assays to monitor GP IIb/IIIa receptor blockade. METHODS AND RESULTS: An assay was devised based on the ability of platelets in whole blood to rapidly agglutinate fibrinogen-coated beads when stimulated with a peptide, (iso-S)FLLRN, that activates a platelet thrombin receptor but resists inactivation by plasma aminopeptidase M. Preincubation of normal blood with increasing concentrations of c7E3 Fab led to increasing inhibition of the assay, correlating with increased GP IIb/IIIa receptor blockade. Assay conditions were chosen so that agglutination was inhibited at 2 minutes when > 82% of the receptors were blocked. Similar results were obtained with the use of GP IIb/IIIa antagonists based on the arginine-glycine-aspartic acid (RGD) cell-recognition sequence. CONCLUSIONS: A simple and rapid assay sensitive to GP IIb/IIIa receptor blockade has been developed that may be helpful in optimizing GP IIb/IIIa antagonist therapy.

A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (beta 3) in a Chinese patient with Glanzmann's thrombasthenia.

A 20-year-old woman from a consanguineous family in the Hunan Province of the People's Republic of China was diagnosed as having Glanzmann's thrombasthenia based on (1) nearly a lifelong history of epistaxis, gum bleeding, petechiae, and purpura; (2) severe menorrhagia resulting in anemia and need for whole-blood transfusion; (3) normal coagulation assays; (4) prolonged bleeding time; (5) absent clot retraction; (6) decreased glass bead retention; (7) absent platelet aggregation in response to adenine diphosphate, epinephrine, and collagen; and (8) normal initial slope of platelet aggregation in response to ristocetin, but with a diminished maximal extent. The patient's platelets had a decreased level of platelet fibrinogen, but the deficiency was not as severe as in other Glanzmann's thrombasthenia patients. As judged by monoclonal antibody binding studies, surface glycoprotein (GP) IIb/IIIa (alpha IIb beta 3) expression was less than 15% of normal and alpha v beta 3 vitronectin receptor expression was 15% to 19% of normal, suggesting that the defect was in GPIIIa (beta 3). Immunoblotting of platelet lysates demonstrated decreased levels of GPIIb (approximately 30% to 35% of normal) and GPIIIa (approximately 10% of normal), and the GPIIb had undergone normal maturational processing into GPIIb heavy and light chains. Sequence analysis of the patient's GPIIIa RNA identified a G to A mutation at nucleotide 1219, predicting a Cys to Tyr substitution at residue 374. The patient's parents, who are first cousins, are asymptomatic and have only minor reductions in platelet aggregation. Direct sequencing of polymerase chain reaction-amplified cDNA and GPIIIa exon VIII indicated that the patient is homozygous and her parents are heterozygous for the mutation. Transient transfection studies in Chinese hamster ovary cells indicated that the mutation results in an 85% to 90% reduction in GPIIb/IIIa surface expression, but these cells retain the ability to mediate adhesion to immobilized fibrinogen. The relative preservation of platelet fibrinogen despite the very low level of platelet surface GPIIb/IIIa expression in this patient raises some interesting questions regarding the mechanism of fibrinogen uptake and the pathophysiology of Glanzmann's thrombasthenia.

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.

Substituting isoserine for serine in the thrombin receptor activation peptide SFLLRN confers resistance to aminopeptidase M-induced cleavage and inactivation.

Peptides containing sequences derived from the new NH2 terminus of the seven-transmembrane domain thrombin receptor after thrombin cleavage can activate platelets directly. We recently demonstrated that such peptides are readily cleaved and inactivated by plasma, serum, and endothelial cell-associated aminopeptidase M. The rapid degradation and inactivation of the peptides makes it difficult to assess dose-response relationships precisely or to conduct long term incubations with cells in the presence of plasma or serum. To overcome these problems, we first substituted D-serine for the NH2-terminal L-serine in an active peptide ligand (SFLLRNPNDKY). The D-serine derivative resisted degradation in plasma, but it is less than 1% as active as the L-serine peptide. Substituting a racemic mixture of the beta-amino acid isoserine for serine in a related peptide ligand (SFLLRN) produced an active peptide ((iso-S)FLLRN) that is approximately 15-20% as potent as SFLLRN as judged by platelet aggregation. To assess the stability of the peptides in plasma, SFLLRN (1 mM) was incubated with 50% plasma for various periods of time; after 15 min, 65% of the peptide remained intact, and after 2 h only 4% remained intact. Loss of aggregating activity paralleled the loss of intact peptide. In contrast, even after 2 h of incubation with plasma, 83% of the (iso-S)FLLRN remained intact and the aggregating activity was essentially unchanged. Qualitative differences in the patterns of platelet aggregation produced by the peptides were also observed. Thus, the distinct pattern of aggregation followed by rapid disaggregation observed with submaximal aggregating doses of SFLLRN was less evident with (iso-S)FLLRN, and inhibition of aminopeptidase M by amastatin enhanced aggregation in platelet-rich plasma induced by SFLLRN but not (iso-S)FLLRN. The (iso-S)FLLRN peptide should permit improved analysis of the effects of constant levels of peptide-induced thrombin receptor stimulation in the presence of plasma or serum, or when testing the effects of the peptide on cells that contain surface-associated aminopeptidase M.

Thrombin receptor activating peptides: importance of the N-terminal serine and its ionization state as judged by pH dependence, nuclear magnetic resonance spectroscopy, and cleavage by aminopeptidase M.

Peptides derived from the recently identified thrombin receptor were tested for their ability to induce platelet aggregation in platelet-rich plasma. The 14 amino acid peptide identified as the new N-terminus after thrombin cleavage (T-14) and an 11 amino acid peptide (T-11) lacking the 3 C-terminal amino acids of T-14 were studied. Both induced platelet aggregation at micromolar concentrations, with T-11 about twice as potent as T-14. Induction of platelet aggregation by these two peptides showed an unusual pH dependence, being more potent at pH 7.2 than at pH 8.1; thrombin-induced aggregation showed a reverse pH dependence. Proton NMR studies of T-11 demonstrated that the chemical shift of the C-alpha proton of the N-terminal serine had a pH dependence that mirrored the aggregation potency. Acetylating the N-terminus of T-11 resulted in loss of aggregating activity, and this peptide did not show the pH-dependence change in chemical shift. The T-14 and T-11 peptides lost aggregating activity when incubated in plasma due to cleavage of the N-terminal serine by an enzyme identified as aminopeptidase M based on its pattern of inhibition and the ability of purified aminopeptidase M (EC3.4.11.2) to cleave the T-11 peptide. Endothelial cell aminopeptidase M was also able to cleave T-11. Inhibiting aminopeptidase M with amastatin enhanced aggregation induced by T-11 but not thrombin. These studies suggest that ionization of the N-terminus of the T-11 and T-14 peptides may be important in initiating platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromboerythrocytes. In vitro studies of a potential autologous, semi-artificial alternative to platelet transfusions.

In an attempt to overcome the limitations and drawbacks of using fresh platelets for transfusion therapy of thrombocytopenic patients, we have performed in vitro experiments on an autologous, semi-artificial alternative to platelet transfusions. Based on our previous studies of the interactions of unactivated and activated platelets with beads coated with peptides of various lengths, all of which contained the arginine-glycine-aspartic acid (RGD) cell recognition sequence, the peptide Ac-CGGRGDF-NH2 was chosen for covalent coupling to erythrocytes. A heterobifunctional crosslinking reagent (N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitrobenzene-4-sulfonic acid) was used to crosslink via the peptide's free sulfhydryl group and the erythrocyte's surface amino groups. Approximately 0.5-1.5 x 10(6) peptide molecules bound per erythrocyte after 2 h of incubation, and most of the peptides appeared to crosslink to glycophorin A. The resulting cells, termed thromboerythrocytes, interacted selectively with activated platelets to form mixed aggregates. Studies with fluid phase RGD peptides and monoclonal antibodies indicated that the RGD peptides on the thromboerythrocytes interacted with the GPIIb/IIIa receptors on activated platelets. Thromboerythrocytes could also bind to platelets adherent to collagen. There was minimal erythrocyte hemolysis during the formation of thromboerythrocytes and studies of thromboerythrocyte osmotic fragility and cellular deformability showed no significant changes from control erythrocytes. Whereas there is a 20:1 ratio of erythrocytes to platelets in the circulation of normal individuals, the erythrocytes from as little as 50 ml of blood could be transformed into the equivalent of 2 U of platelets by numbers (equivalent to 18 U of platelets by mass), and reinfused into the same individual within several hours. These data encourage us to proceed to in vivo studies to assess the hemostatic efficacy of thromboerythrocytes in thrombocytopenic animals.

Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking.

To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi-Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor-mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.

Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GPIIb/IIIa receptor. Correlation with bleeding time, platelet aggregation, and blockade of GPIIb/IIIa receptors.

We studied the dose-response effects of the F(ab')2 fragments of murine monoclonal antibodies to the platelet GPIIb/IIIa receptor (7E3 and 10E5) on in vivo platelet thrombus formation in a well-characterized monkey model in which the carotid artery is stenosed and thrombus formation is provoked and augmented by intimal damage and the infusion of subaggregating doses of epinephrine. Both antibodies abolished thrombus formation with a mean dose of -0.2 mg/kg. Ex vivo platelet aggregation was not always abolished at doses that abolished thrombus formation; similarly, bleeding times were only moderately prolonged (9.1 +/- 1.4 minutes) at these doses. Increasing the dose above that required to abolish thrombus formation consistently produced abolition of ex vivo platelet aggregation, marked prolongation of the bleeding time (14.2 +/- 1.5 minutes), and nearly quantitative blockade of GPIIb/IIIa receptors. We conclude that in a significant percentage of animals, the extent of GPIIb/IIIa blockade required to prevent vasoocclusive thrombus formation in this model is less than that required for abolition of platelet aggregation, and that the preservation of only a minority of functional GPIIb/IIIa receptors might be adequate to maintain a nearly normal bleeding time.

Collagen-platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and an indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins.

Using intact human platelets as the immunogen and a functional, collagen-coated bead agglutination assay, we have produced a murine monoclonal antibody (6F1) that blocks the interaction between platelets and collagen in the presence of Mg++. 6F1 affinity-purified the platelet glycoprotein Ia/IIa complex, and approximately 800 molecules of 6F1 bound per platelet at saturation. 6F1 nearly completely inhibited collagen-induced platelet aggregation and inhibited platelet adhesion to collagen by greater than 95% when plasma proteins were absent. Antibody 10E5, which blocks the binding of adhesive glycoproteins to GPIIb/IIIa, produced only minor inhibition (approximately 25%) of adhesion under the same circumstances. In contrast, when tested in platelet-rich plasma (PRP), 6F1 had only a minor effect on collagen-induced platelet aggregation, prolonging the lag phase but not the slope or maximum aggregation. Similarly, when collagen was precoated with plasma, 6F1 caused less inhibition of platelet adhesion (53%) than without the precoating (greater than 95%). Antibody 10E5 inhibited this adhesion by 32%, and the combination of 6F1 and 10E5 was more effective than either alone, inhibiting it by 90%. Time course studies of platelet agglutination of collagen-coated beads using PRP containing physiologic concentrations of divalent cations showed early inhibition by 6F1, indicating that the GPIa/IIa receptor operates in this environment. With more prolonged incubation, however, 6F1 was less effective; this later agglutination could be partially prevented by adding 10E5 or PGE1 to the 6F1. These data support a model wherein collagen can directly interact with GPIa/IIa and can indirectly interact with GPIIb/IIIa via intermediary adhesive proteins. The physiological significance of these interactions, and potential interactions with other receptors, remains to be established.

Inhibition of human platelet function in vivo with a monoclonal antibody. With observations on the newly dead as experimental subjects.

The F(ab')2 fragment of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor (7E3) is a potent inhibitor of both in-vitro platelet aggregation and in-vivo platelet thrombus formation in animal studies. As a first step in assessing the potential of 7E3-F(ab')2 as an antithrombotic agent for use in humans, we administered 7E3-F(ab')2 intravenously at increasing doses to a person who had just died and was being maintained on a respirator (neomort). At 0.1 and at 0.2 mg/kg body weight, 74% and 92% of the glycoprotein IIb/IIIa receptors were blocked, respectively; adenosine-diphosphate-induced platelet aggregation was inhibited by 84% and 100% at these same doses. Platelet glycoprotein Ib function remained intact, even at 0.6 mg/kg. Acute hemodynamic or hemorrhagic toxicity was not noted. This antibody fragment, a potent, immediate-acting inhibitor of platelet aggregation, may be of benefit in vaso-occlusive and thromboembolic disorders.

Monoclonal antibody against the platelet glycoprotein (GP) IIb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs.

Localized thrombosis was produced in the left anterior descending (LAD) coronary artery of open chest dogs by constricting a segment so as to produce greater than 90% stenosis (reducing blood flow to 40 +/- 10% of baseline), and placing a thrombus in the segment immediately proximal to the stenosis by inducing endothelial cell injury and instilling a mixture of blood and thrombin. Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) at a rate of 15-30 micrograms/kg per min for 30 or 60 min in eight dogs induced coronary artery reperfusion within 23 +/- 7 min (mean +/- SD), but reocclusion occurred despite heparin anticoagulation in all but one of these dogs within 7 +/- 5 min. Intravenous injection of 0.8 mg/kg of the F(ab')2 fragment of a monoclonal antibody (7E3) directed against the platelet GPIIb/IIIa receptor, prevented reocclusion in 10/10 dogs during an observation period of 2 h (P less than 0.001 vs. rt-PA alone). The antibody abolished ADP-induced platelet aggregation and markedly prolonged the bleeding time. Intravenous aspirin or dipyridamole prevented reocclusion for 1 h or more in only 2/7 and 1/6 dogs, respectively. We conclude that the monoclonal antibody is very effective in preventing reocclusion after successful thrombolysis of occluded coronary arteries with rt-PA.

Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model.

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

Studies on the binding of an alloimmune and two murine monoclonal antibodies to the platelet glycoprotein IIb-IIIa complex receptor.

The platelet-binding characteristics of three different antibodies that completely block adenosine diphosphate (ADP)-induced platelet fibrinogen binding and react with glycoproteins IIb, IIIa, or both, were studied. Two of the antibodies are murine monoclonal antibodies (10E5 and 7E3), and the third is an alloimmune antibody produced by a patient with Glanzmann's thrombasthenia who has received multiple transfusions (E.S.). The two monoclonals differ in that 7E3, but not 10E5, binds to dog platelets as well as to human platelets. In mutual competition studies, 7E3 did not interfere with 10E5 binding, indicating that both antibodies could bind to the complex simultaneously. E.S. IgG produced only minor inhibition of 10E5 binding, but nearly complete inhibition of 7E3 binding, suggesting that its epitope lies closer to the 7E3 epitope than the 10E5 epitope. Ethylenediaminetetraacetic acid (EDTA) pretreatment of intact platelets at 22 degrees C and pH 7.75 did not affect 10E5 binding, but significantly inhibited 7E3 binding. Similar treatment of intact or solubilized platelets at high pH and temperature produced splitting of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex and loss of both 10E5 and 7E3 binding. The 10E5 bound equally well to unactivated and activated platelets, and even rapid fixation of whole blood produced only a modest decrease in 10E5 binding. Preincubation of platelets with either E.S. IgG or 10E5 partially prevented EDTA from splitting the complex, and both monoclonal antibodies were able to bind to such complexes despite the EDTA treatment. These studies indicate that the binding of antibodies to several different epitopes on the GPIIb-IIIa complex can result in similar functional properties in blocking fibrinogen binding and platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of dog platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor.

To assess the potential of monoclonal antibodies that inhibit platelet function in vitro as in vivo therapeutic agents, F(ab')2 fragments (0.17 to 0.81 mg/kg) of a murine monoclonal antibody (7E3) that binds to platelet glycoproteins IIb and/or IIIa and blocks platelet aggregation induced by ADP were infused into three dogs. Soon after infusion, platelets recovered from the dogs showed a decreased aggregation response to adenosine diphosphate, with the highest dose producing nearly total inhibition. These platelets also showed decreased ability to bind 125I-7E3, which was assumed to reflect occupancy of the sites by the unlabeled F(ab')2 fragments. At the highest dose, the binding decreased by 85%, reflecting the binding of approximately 44,000 molecules of 7E3 F(ab')2 per platelet. Platelet counts decreased after antibody infusion by less than 20%, and none of the dogs showed spontaneous bleeding. Both the aggregation and binding results reverted toward normal within one day. We conclude that it is possible to profoundly inhibit platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antiplatelet antibody without producing spontaneous hemorrhage or significant thrombocytopenia.