Synthesis and pharmacology of modified amidine isoxazoline glycoprotein IIb/IIIa receptor antagonists

Selective antagonism of the platelet GPIIb/IIIa receptor represents an attractive mechanism for the prevention and treatment of a number of thrombotic disease states. The antiplatelet activity of the oral GPIIb/IIIa receptor antagonists DMP 754 and DMP 802 have been disclosed. In this paper, the synthesis and biological evaluation of a series of potent N-substituted benzamidine isoxazolines are explored. The effect of benzamidine substitution on the duration of antiplatelet efficacy in dog is presented.     

Platelet glycoprotein IIb/IIIa receptor antagonists derived from isoxazolidines

A series of isoxazolidines has been synthesized as mimetics of the RGD sequence and was evaluated as antagonists of the platelet glycoprotein IIb/IIIa receptor. These compounds were shown to be highly potent GPIIb/IIIa antagonists, exhibiting submicromolar potencies.     

Differences between platelet and microparticle glycoprotein IIb/IIIa.

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).     

Molecular cloning and expression of glycoprotein IIb and IIIa

Objective To amplify the cDNA genes of GPIIb, GPIIIa, then construct the eukaryotic expression carriers of GPIIb and GPIIIa respectively, finally establish CHO cell lines stably expressing GPIIb and GPIIIa. Methods Human erythroleukemia (HEL) cells were cultured for total RNA extraction. RT-PCR was accomplished using the specific GPIIb, GPIIIa primers designed according to Genbank by Primer 5, then each of cDNAs were obtained. The expressive vector pcDNA3.1(+) and PCR products were cut by NheI and HindIII, and then the fragements were directly cloned to pcDNA3.1(+) because of having the same adhesive ends. Then pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were transfected into CHO cells respectively by Lipofectamine 2000. The cell lines expressing GPIIb, GPIIIa were screened by G418. Then the Chinese hamster ovary (CHO) cell lines were examed through flow cytometry (FCM) and RT-PCR to detect the expression of GPIIb, GPIIIa in CHO cells. Results The cDNAs of GPIIb and GPIIIa were amplidied by RT-PCR, and the pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were constructed respectively. By sequencing and double digestion, pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were all correct. Expression of GPIIb and GPIIIa were detected on transfected CHO cells by FCM and RT-PCR. Conclusions (1) Succeeded in constructing pcDNA3.1(+)IIb, pcDNA3.1(+)IIIa. (2) Succeeded in getting the cell lines expressing GPIIb, GPIIIa.     

Platelet glycoprotein IIb/IIIa receptor blockade in coronary artery disease

Glycoprotein IIb/IIIa inhibitors represent a new promising class of antiplatelet medications. Their use in acute coronary syndromes and for patients undergoing percutaneous coronary intervention has been the subject of a number of large controlled trials using both the intravenous and the oral forms. In this review, we present a systematic overview of these trials.     

Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation

The platelet integrin glycoprotein (GP) IIb/IIIa, which mediates platelet aggregation, has been the target for novel antiplatelet agents, the GPIIb/IIIa antagonists. Several GPIIb/IIIa antagonists have been developed based on the peptide RGDS present in adhesion proteins, including the principle ligand fibrinogen. The apoptosis enzyme, procaspase-3, contains an RGD-recognition sequence and is activated by RGDS. We examined the effects of RGDS and several GPIIb/IIIa antagonists on cell death and procaspase-3 activation in rat neonatal cardiomyocytes. These antagonists do not recognize rat integrins, yet RGDS, orbofiban, and xemilofiban induced dose-dependent apoptosis and procaspase-3 activation in cardiomyocytes over 72 h, particularly under hypoxic conditions. Scrambled peptide, the monoclonal antibody 7E3 or integrelin (a peptide containing a KGD sequence), had little or no effect. Immunoprecipitation of procaspase-3 followed by treatment with the compounds showed that procaspase-3 was activated directly by RGDS, orbofiban, xemilofiban, and by monoclonal 7E3 antibody, the latter demonstrating that compounds must enter cells to induce apoptosis through caspase activation. Integrelin had no effect. Binding studies with (3)H-SC52012B, a GPIIb/IIIa antagonist analogue of orbofiban, showed no specific binding to cardiomyocytes, but the radioligand accumulated intracellularly over 72 h. (3)H-SC52012B also bound directly to human recombinant caspase-3 (K(d), 59 +/- 2 nm), and this was prevented by orbofiban, xemilofiban, and the monoclonal 7E3 antibody but not by integrelin. Finally confocal microscopy showed that RGDS co-localized with caspase-3 inside the cell. These data show that RGDS and its mimetics induce cardiomyocyte apoptosis by direct activation of procaspase-3.     

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

Effects of diamide and iodoacetamide on the expression of the glycoprotein IIb/IIIa complex on blood platelets

We have examined the effects of two agents that alter platelet thiol-disulphide status on platelet aggregation and on the ability of platelets to bind a monoclonal antibody (M148) that is directed toward an epitope on the glycoprotein IIb/IIIa complex. The immediate effect of both diamide and iodoacetamide is to enhance aggregation but after further incubation diamide, but not iodoacetamide, inhibits platelet aggregation. Incubation of platelets with diamide, but not iodoacetamide, is accompanied by a marked increase in the amount of M148 that binds to platelets. This is presumably a reflection of an altered distribution of glycoproteins on the platelet surface. It is known that diamide, but not iodoacetamide, leads to polymerisation of cytoskeletal proteins in platelets. Thus evidence is provided that agents that interact with the cytoskeleton inhibit platelet behaviour via an effect on surface glycoproteins.     

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

Glycoprotein IIb/IIIa receptor antagonists in acute myocardial infarction and primary coronary stenting: Not always effective

Glycoprotein IIb/IIIa antagonists have been shown to be effective in reducing thrombotic complications prior to high-risk coronary interventions. Some studies have reported improved coronary flow after abciximab in slow or no-reflow phenomenon. We report a case in which abciximab did not clear the thrombotic occlusion or restore artery flow. Further studies are needed into the refractory no-flow phenomenon.     

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.     

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex.

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Chrysoptin is a potent glycoprotein IIb/IIIa fibrinogen receptor antagonist present in salivary gland extracts of the deerfly

Salivary gland lysates of the deerfly (genus Chrysops) contain chrysoptin, an inhibitor of ADP-induced platelet aggregation, which presumably assists the fly in obtaining a blood meal. Chrysoptin has now been isolated, and its cDNA has been cloned and expressed. Chrysoptin was purified to homogeneity using anion exchange and hydrophobic interaction chromatography and found to be a protein with a molecular mass of 65 kDa as determined by gel electrophoresis. N-terminal amino acid sequencing allowed for the synthesis of degenerate oligonucleotides that led to cloning, from salivary gland specific mRNA, of the cDNA encoding this platelet inhibitor. No RGD sites are present in the predicted sequence. A search of GenBank(TM) did not reveal significant sequence homology between chrysoptin and other proteins. The molecular mass predicted from the cDNA was 59 kDa. Predicted glycosylation and phosphorylation sites may account for this difference in molecular mass, as recombinant chrysoptin expressed in Sf21 cells had a molecular mass of 65 kDa, matching that of the natural protein. Chrysoptin functions by inhibiting the binding of fibrinogen to the fibrinogen/glycoprotein IIb/IIIa receptor on platelets with an IC(50) of 95 pmol. These results reveal that insect salivary glands are a source of fibrinogen receptor antagonists.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

Summary We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20–40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10–70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Ca(2+)-dependent structural transitions of the platelet glycoprotein IIb-IIIa complex. Preparation of stable glycoprotein IIb and IIIa monomers

The platelet membrane glycoprotein (GP) IIb-IIIa complex is the receptor for adhesive proteins on activated platelets that mediates platelet aggregation. In the present study, factors affecting the structural stability of the purified GP IIb-IIIa complex and the dissociated subunits were investigated. Purified GP IIb-IIIa was incubated in various Ca2+ concentrations, and the percentage of dissociated subunits was quantitated by sucrose gradient sedimentation. Two Ca(2+)-dependent transitions were observed, one at about 60 microM Ca2+, where half of the complexes became dissociated, and the other at 0.1 microM Ca2+, where half of the dissociated subunits became incapable of reforming heterodimer complexes when higher Ca2+ concentrations were readded. This loss in ability to reform heterodimer complexes was caused primarily by a Ca(2+)-dependent transition in GP IIIa, leading to an apparent unfolding of this subunit, followed by the formation of high molecular weight aggregates. The formation of these aggregates was time- and temperature-dependent and could not be reversed by added Ca2+. Although Mg2+ prevented dissociation of GP IIb-IIIa, it failed to promote reassociation of the dissociated subunits. Based on these findings, conditions were developed for the preparation of dissociated GP IIb and GP IIIa such that 70% of the subunits remained functional in that they retained the ability to reform heterodimer complexes.