Construction of drug screening cell model and application to new compounds inhibiting FITC-fibrinogen binding to CHO cells expressing human alphaIIbbeta3.

To identify potential candidates for antiplatelet drugs, human alphaIIbbeta3 (GPIIb/IIIa) was expressed in Chinese hamster ovary (CHO) cells, which was validated by tetrapeptide RGDS (Arg-Gly-Asp-Ser) with IC(50) of 0.057 mM, supported by Basani's results [Basani, R. B., French, D. L., Vilaire, G., Brown, D. L., Chen, F., Coller, B. S., Derrick, J. M., Gartner, T. K., Bennett, J. S., Poncz, M., 2000. A naturally occurring mutation near the amino terminus of alpha IIb defines a new region involved in ligand binding to alpha IIbbeta 3. Blood 95, 180-188]. The ability of 2-(4-substituted-piperazin-1-ylacetyl)-1,2,3,4-tetrahydroisoquinoline derivatives to inhibit fibrinogen binding to alphaIIbbeta3 based on the CHO cell model was measured by flow cytometry using GPIIb/IIIa assay, and the IC(50) values of compounds 1-6 were 0.166, 0.037, 0.311, 0.025, 0.034, and 0.184 mM, respectively. Our research results indicated that the compounds with phenylsulfonyl (compounds 1 and 2) and benzoyl groups (compounds 4 and 5) at position 4 of piperazine showed higher IC(50) values of inhibiting ADP-induced human platelet aggregation. Particularly compound 4 possessed IC(50) value of approximately 6.84 nM. Additionally, a complex model of alphaIIbbeta3 with compound 4 revealed that the pharmacophore of compound 4, including m-nitro group of 4-benzene-piperazine, the nitrogen atom in the piperazine group, and 2-nitrogen of 1,2,3,4-tetrahydroisoquinoline nucleus, interacted with the hydroxyl groups of Thr125 of beta3 and Tyr166 of alpha2b by hydrogen bonds and the carboxyl group at side chain of Asp179 of alpha2b in the fashion of electrostatic interaction. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays showed that compounds 4 and 5 possess potential anti-cancer activities, suggesting a potential role of integrin-guided signal pathway in cancer therapy. Further evaluation is under investigation.

Relative efficacy of intravenous immunoglobulin G in ameliorating thrombocytopenia induced by antiplatelet GPIIbIIIa versus GPIbalpha antibodies.

Intravenous immunoglobulin G (IVIG) is used to treat idiopathic thrombocytopenic purpura (ITP). Although many patients benefit from IVIG, some are refractory to this therapy. ITP is characterized by platelet clearance mediated primarily by antiplatelet antibodies against GPIIbIIIa and/or the GPIbalpha complex. These 2 groups of antibodies may induce ITP through different mechanisms. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by anti-GPIIbIIIa versus anti-GPIbalpha antibodies in mice. Thrombocytopenia was induced in BALB/c mice using monoclonal antibodies against either mouse GPIIbIIIa (JON1, JON2, and JON3) or GPIbalpha (p0p3, p0p4, p0p5, p0p9, and p0p11). Pretreatment with IVIG significantly ameliorated ITP in all anti-GPIIbIIIa-injected animals. Conversely, IVIG failed to prevent ITP in all anti-GPIbalpha-treated mice, except for p0p4. These results were repeated in C57BL/6 mice, and with different IVIG preparations. These data in mice suggest that patients with ITP mediated by anti-GPIbalpha antibodies may be less responsive to IVIG treatment.

Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.

Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies.

Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.

Use of enzyme treatment to enhance reactivity of HLA and platelet-specific antibodies in solid-phase RBC adherence assays.

BACKGROUND: Solid-phase RBC adherence (SPRCA) assays (Immucor) detect HLA and/or platelet-specific antibodies. Pretreatment of reagent platelets with enzymes was investigated to determine whether the sensitivity of the assay could be increased. STUDY DESIGN AND METHODS: SPRCA testing, following the manufacturer's directions, was performed on 51 sera from patients with either a positive SPRCA antibody screen, suboptimal clinical responses to platelet transfusions, and/or suspected immune thrombocytopenic purpura; testing was also performed following pretreatment of the reagent platelets with bromelin, papain, or ficin. Sera from 23 patients having negative routine SPRCA antibody screens and good clinical responses to transfusion were tested as controls. Lymphocytotoxic antibody testing was also performed on selected samples. The effectiveness of enzyme treatment was judged by the increase in the proportion of reagent platelets reacting with the sample and the observed reaction strengths. RESULTS: Pretreatments of reagent platelets with all three enzymes increased the reactivity of known antibodies and detected some HLA and platelet-specific antibodies that had not reacted in routine testing. The clinical significance of the antibody specificities detected only after enzyme pretreatment was verified by a correlation with results from transfusing antigen-negative units. Only occasional false-positive results after enzyme pretreatment were observed. CONCLUSIONS: The use of enzyme pretreatment of SPRCA screening strips can provide information that is useful in selecting appropriate units for transfusion.

A functional platelet fibrinogen receptor with a deletion in the cysteine-rich repeat region of the beta(3) integrin: the Oe(a) alloantigen in neonatal alloimmune thrombocytopenia.

This report describes a new low-frequency alloantigen, Oe(a), responsible for a case of neonatal alloimmune thrombocytopenia (NAIT). In a population study none of 600 unrelated blood donors was an Oe(a) carrier. By immunochemical studies the Oe(a) antigen could be assigned to platelet glycoprotein (GP) IIIa. Sequencing of GPIIIa complementary DNA from an Oe(a) (+) individual showed deletion of a lysine residue at position 611 (DeltaLys(611)). Analysis of 20 Oe(a) (-) and 3 Oe(a) (+) individuals showed that the DeltaLys(611) form of GPIIIa was related to the phenotype. Anti-Oe(a) reacted with the DeltaLys(611), but not with the wild-type isoforms on stable transfectants expressing GPIIIa, indicating that DeltaLys(611) directly induces the expression of Oe(a) epitopes. Under nonreducing conditions the Pro(33)DeltaLys(611) variant migrated with a slightly decreased molecular weight compared to the Pro(33)Lys(611) isoform suggesting that DeltaLys(611) has an influence on the disulfide bonds of GPIIIa. The Pro(33)DeltaLys(611) GPIIIa could undergo conformational changes and bind to fibrinogen in a similar manner as the Pro(33)Lys(611) isoform. No difference was found in the tyrosine phosphorylation of pp125(FAK), suggesting that DeltaLys(611) has no effect on integrin function. In contrast to all other low-frequency antigens, the DeltaLys(611) isoform was associated with the HPA-1b, but not with the high frequency HPA-1a allele. Comparison with GPIIIa DNA from nonhuman primates indicated that the HPA-1a allele represents the ancestral form of GPIIIa. It can be assumed that the Oe(a) form did arise as a result of a mutational event from an already mutated GPIIIa allele.

Monoclonal antibodies against platelet membrane glycoproteins IIb/IIIa and Ibalpha inhibit platelet dependent thrombin generation by different mechanisms.

The antithrombotic effect of antiplatelet agents is principally due to their anti-aggregatory action, but these agents may also interfere with coagulation. We have investigated the effect of monoclonal antibodies (MAb) to platelet membrane glycoproteins (GP) IIb/IIIa and Ibalpha on thrombin generation. Antibodies to platelet membrane glycoprotein IIb/IIIa (RFGP56 and c7E3) were shown to inhibit platelet-mediated thrombin generation stimulated by both intrinsic and extrinsic methods. An antibody to GP Ibalpha (RFGP37) also inhibited thrombin generation in these systems. FITC-annexin V was used to determine the effect of these antibodies on the exposure of procoagulant phospholipids on the platelet membrane, and it was found that the anti-IIb/IIIa antibodies reduced this, whereas the anti-Ibalpha antibody caused an increase. We conclude that our monoclonal antibodies against platelet membrane glycoproteins IIb/IIIa and Ibalpha inhibit platelet dependent thrombin generation by different mechanisms.

The phenotypic profile of CD34-positive peripheral blood stem cells in different mobilization regimens.

The type of regimen used might result in mobilization of phenotypically and functionally different CD34(+) cells. We compared the phenotype of CD34(+) cells in leukapheresis products of three homogeneous groups: I, healthy individuals treated with granulocyte colony-stimulating factor (G-CSF) alone (n = 13); II, patients mobilized with G-CSF following chemotherapy (n = 16); and III, patients mobilized with G-CSF after high-dose chemotherapeutic pretreatment (n = 24). Multiparameter flow cytometry was performed for CD34(+) subpopulation analysis and focused on adhesion molecules, differentiation markers and megakaryocytic markers relevant for stem cell homing, with special reference to the importance of L-selectin expression. Regimens I and II led to higher numbers of mobilized CD34(+) cells (mean 468 x 10(6) and 491 x 10(6) CD34(+) cells per leukapheresis procedure respectively) than regimen III (mean 41 x 10(6) CD34(+) cells per leukapheresis procedure). Both the expression of L-selectin and CD54 on CD34(+) cells was significantly lower in group III, as was the percentage of megakaryocytic (CD41(+)) progenitors. A higher percentage of primitive (CD38(-) and/or HLA(-)DR(-)) CD34(+) cells was found in group III, correlating with a higher clonogenicity of the CD34(+) cells. However, when comparing the CD34(+)_ subpopulations that were also positive for L-selectin, there was no significant difference between the three regimens. A similar approach for the megakaryocytic CD34+ population resulted in an even worse quality of regimen III: 5.1% of CD34(+) being CD41(+)/L-selectin(+) compared with 9.2% and 8.9% in regimens I and II respectively. We concluded that the phenotypes of the CD34(+) cells in the G-CSF (group I) and G-CSF-chemotherapy (group II) regimens are similar, whereas the phenotype of the CD34(+) cells mobilized in the high-dose regimen (group III) displayed features that might negatively influence homing of the cells. Future studies will be directed towards regimens that will lead to the mobilization of a higher amount of CD34(+) cells with a phenotypically favourable phenotype.

In vitro efficacy of platelet glycoprotein IIb/IIIa antagonist in blocking platelet function in plasma of patients with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an important complication following administration of heparin. Platelet activation and aggregation induced by heparin/platelet factor 4/immunoglobulin complexes are thought to be the underlying mechanism for this condition, so it was hypothesized that abciximab (a humanized murine monoclonal antibody directed against the glycoprotein IIb/IIIa receptor) would prevent heparin-induced platelet aggregation and activation in plasma from patients with HIT. Platelet aggregation was tested in vitro with platelet-poor plasma (obtained from 23 patients with HIT), platelet-rich plasma (from normal donors with known reactivity), heparin (0.5 U/ml), and ascending doses of abciximab (0.07-0.56 microg/ml). The ability of abciximab to prevent platelet activation was also evaluated using flow cytometry (P selectin expression, mepacrine release, microparticle formation) and platelet factor 4 immunoassay. In vitro, abciximab inhibited heparin-induced platelet aggregation in a dose-dependent fashion (IC50 0.103 microg/ml) and inhibited microparticle formation, the expression of P-selectin, release of mepacrine and platelet factor 4. These findings suggest that abciximab may be useful in treatment of patients with HIT and warrants further clinical evaluation.

Overproduction of antiplatelet antibody against glycoprotein IIb after splenectomy in a patient with Evans syndrome resulting in acquired thrombasthenia.

We treated a 33-year-old female with Evans syndrome. She received high dose gamma globulin, prednisolone, and azathioprine, and her platelet count transiently increased. After splenectomy, the platelet count markedly increased. However, the bleeding tendency worsened and the bleeding time was prolonged. A platelet defect, characteristic of thrombasthenia, was found. Antigen-captured ELISA and Western blotting revealed that the patient's serum had an IgG autoantibody against platelet membrane glycoprotein IIb and the patient's plasma inhibited normal platelet aggregation. These findings suggest that overproduction of the antiplatelet antibody is triggered by platelet recovery due to splenectomy and affects platelet function resulting in acquired thrombasthenia.

Restenosis: is there a pharmacologic fix in the pipeline?

One of the most frustrating aspects of restenosis is that it is the result of advances in medical care (there was no restenosis before the days of balloon angioplasty), yet it seems to be resistant to all that science has to offer. Still we believe there is reason to be optimistic. We are at last beginning to see some promise from clinical trials, and data being generated confirm some of the hypotheses previously generated from animal experiments. Thus the effects seen with the GP IIb/IIIa antibody 7E3 suggest that thrombosis may be as important in its long-term sequelae as it is for acute reocclusion. The jury is still out on whether antiproliferative approaches will be a therapeutic option, but local delivery paradigms using novel formulations delivered by catheter or impregnated in stents may allow the concept to be tested without the risk of systemic toxicity. Plans are also underway for gene therapy trials, although we may have to wait for better vector technology before taking these into the coronary bed. Perhaps we should move away from the "single pill" approach and accept that, like many infections, malignancies, or even heart failure, a multifaceted approach with combination therapy will provide the first glimmer of that brighter tomorrow.

In-vitro inhibition of antiplatelet autoantibodies by intravenous immunoglobulins and Rh immunoglobulins.

Autoimmune thrombocytopenia (AITP) is caused by autoantibodies to platelet glycoprotein antigens. Intravenous immunoglobulin (i.v.IgG) and Rh immunoglobulin infusions have found great significance in the treatment of AITP patients not responding to corticosteroids and other modes of therapy. In our study, it was observed that immunoglobulins (i.v.IgG & Rh), and their Fab fragments inhibited the binding of antiplatelet autoantibodies to normal platelets, from 15.8 to 90.7% and 25.6 to 90.08% respectively; whereas, their Fc portion did not show any inhibition. The presence of specific anti-idiotypic antibodies to antiplatelet autoantibodies was established by using monoclonal antibodies to Glycoprotein IIb/IIa and Glycoprotein Ib/IX, as the specific idiotype source. The i.v.IgG and Rh immunoglobulin products reacted with the monoclonal antibodies, only through their Fab and not through the Fc portions, thereby confirming its specific anti-idiotype activity.

Prolonged inhibition of acute arterial thrombosis by high dosing of a monoclonal anti-platelet glycoprotein IIb/IIIa antibody in a baboon model.

The in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dose-finding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrombotic effect was stronger 48 h after a dose of 0.3 mg/kg administration than on the day of treatment with 0.1 mg/kg, despite the fact that comparable numbers of GPIIb/IIIa receptors were occupied on resting platelets. We postulate that with the high dose of MA-16N7C2 and after an extended period, occupied GPIIb/IIIa may be internalised by the platelets. Upon platelet activation, these receptors become reexposed but are unable to participate in thrombus formation. This is in contrast to unoccupied internal GPIIb/IIIa receptors early after a low dose of MA-16N7C2.