新型抗血小板多肽类药物研究进展

抗血小板治疗在血栓性疾病的防治中发挥重要作用,血小板膜糖蛋白 GP IIb/IIIa 受体的活化是血小板聚集的最终共同通路。目 前的研究发现,多种新型多肽能与 GP IIb/IIIa 受体特异性结合,从而发挥抗血小板聚集的药理作用。分类综述含有或类似 RGD 序列和非 RGD 序列的新型抗血小板多肽类药物研究进展。     

New therapeutic approaches to combat arterial thrombosis: better drugs for old targets, novel targets, and future prospects

Cardiovascular disease and stroke are predominant causes of death in developed countries. Rupture of atherosclerotic plaque in an artery wall and the ensuing thrombotic events are the triggers for acute ischemic injury in these diseases. Platelet activation and aggregation play key roles in this process of atherothrombosis. Anti-platelet drugs thus provide the primary therapeutic strategy to combat these diseases. Dual therapy with aspirin and clopidogrel is the current standard of care for most patients, but it has significant limitations. This provides an impetus for developing new anti-platelet drugs. One new drug has received FDA approval recently; prasugrel targets the platelet P2Y(12) receptor, just like clopidogrel. Several other new drugs are showing great promise in clinical trials and appear to be nearing approval. Some of these drugs have traditional targets on the platelets; others, such as vorapaxar, terutroban, and sarpogrelate, generate more excitement as they are directed against novel targets.     

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.     

ABCB1、CES1 和 CYP2C19 基因多态性与 氯吡格雷抵抗的相关性研究进展

氯吡格雷是目前全球临床使用最为广泛的血小板受体抑制剂,但其抗血小板效应存在明显个体差异,部分病人服用常规剂量氯吡格 雷后存在抵抗现象,甚至发生不良临床事件。多项研究表明,ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的产生发挥重要作用。 简介氯吡格雷体内吸收与代谢机制和氯吡格雷抵抗的定义,综述 ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的影响。     

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.     

The value of using polymorphisms in anti-platelet therapy

Objectives and Backgrounds

Cardiovascular events occure as a result of various risk factors, such as uric acid (UA), inflammation, hormones and other materials that induce C- reactive protein (CRP) expression. These factors lead to complement activation, and endothelial damages. Damaged endothelial cells release heparan sulfate which inhibits tissue factor activity and von Willed brand factor (VWVF) and causes aggregation. Finally this cascade of events cause platelets aggregation and leads to heart ischemia and cardiovascular events.

Discussion

Anti-platelet therapy is an interesting premise. Anti-platelet resistance patients and bleeding as a result of using ticagrelor and prasugrel should be considered in this treatment methods. Anti-platelet drugs such as clopidogrel are prescribed in cardiovascular events. Platelets have VWF receptors and P2Y12 receptors on their surface, and thus, targeting these receptors can be useful in treatment. The active metabolites of clopidogrel bind to P2Y12R and inhibit ADP binding; thus, clopidogrel inhibits aggregation by interfering in several events as a result of the inhibition of ADP attachment to P2Y12R of the platelet. However, the polymorphisms of P2Y12 and other genes mentioned in Table 1 showed treatment resistance in anti-platelet therapy, highlighting that these SNPs can be helpful in anti-platelet therapy.

Conclusion

The knowledge of these SNPs may decrease the number of unwanted effects that endanger patients with cardiovascular diseases and avoids ineffective anti-platelet therapy in several patients. Clopidogrel, ticagrelor, prasugrel, and aspirin and CYP2C19 and their SNPs are very important subjects in anti-platelet therapy. To present the importance of using pharmacogenetics in anti-platelet therapy, we discuss here the association between these drugs and the SNPs for therapeutic resistance.

     

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.     

Platelet membrane glycoproteins: role in primary hemostasis and component antigens

The biochemical details of the platelet surface as they relate to normal platelet function have been elucidated through study of labeled membranes from both normal platelets and those with congenitially defective function. Several cytoadhesive glycoprotein complexes which are integral components of the platelet membrane have been demonstrated to act as important receptors for extracellular matrix macromolecules. Glycoproteins Ia/IIa (collagen receptor), Ic/IIa (fibronectin receptor), and IIb/IIIa (fibrinogen receptor) belong to a family of cytoadhesive complexes called the integrins, while glycoprotein Ib/IX, the major von Willebrand receptor, has different features. These same major glycoproteins comprise all of the alloantigens and most of the autoantigens that have been characterized. Glycoprotein IIb/IIIa contains the alloantigens, PlA (Zw), Bak (Lek), and Pen (Yuk), as well as the most frequent target antigenic sites for anti-platelet autoantibodies. Because a number of platelet alloantigens were discovered independently by more than one group, nomenclature is confusing at present, although a system analogous to that used for histocompatibility antigens has been proposed. Precise identification of the antigenic epitopes has not yet been accomplished for all of the platelet antigens. Current research efforts include characterization of antigenic epitopes, elucidation of mechanisms by which platelet immunization occurs, and determination of the clinical implications of the presence of various platelet antibodies.     

Plateletworks: a novel point of care platelet function screen

Platelet function is critically important in the acute-care settings of cardiopulmonary bypass surgery and percutaneous coronary intervention, which are commonly associated with the adverse vascular events of hemorrhage and thrombosis, respectively. To improve outcomes, it has been suggested that patients should be screened for platelet count and function periprocedurally, and therapeutic intervention including the possible use of thrombolytics and adequate anticoagulation or administration of antiplatelet agents, should be utilized. Antiplatelet therapy including aspirin (acetylsalicylic acid), the thienopyridines (clopidopgrel), and parenteral anti-glycoprotein (GP) IIb/IIIa agents (abciximab, tirofiban, and eptifibatide) are recognized as clinically important in patients at risk of developing thrombotic events. Recently, it has been recognized that empiric therapeutic administration of these agents may be suboptimal in clinical environments because of interpatient variability with regard to platelet count, platelet response, receptor concentration on the platelet, and other factors. Hence there is a clinical need to monitor such therapies on an individual basis. Traditional platelet tests including light transmission aggregometry (LTA) are inconvenient for acute diagnostic testing because of the complexity of the test and the requirement for specialty training. Hence, 'near-patient' test systems have recently been introduced. Plateletworks is an in vitro diagnostic, point-of-care test platform that has demonstrated utility in monitoring platelet response to all current antiplatelet agents including aspirin and clopidogrel.     

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).     

Quantitation and characterization of human platelet glycoprotein IIIa by radioimmunoassay

Glycoprotein IIIa was quantitated in human platelets by radioimmunoassay using antisera specific to platelet membranes and purified glycoprotein IIIa. Glycoprotein IIIa and glycoprotein IIb were isolated from washed platelets by Triton X-114 extraction followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioiodinated glycoprotein IIIa was further purified by affinity chromatography on Lentil lectin-Sepharose 4B. Purified glycoprotein IIb showed little crossreactivity with 125I-labeled glycoprotein IIIa using the anti-platelet membrane or anti-glycoprotein IIIa antisera on a competition inhibition radioimmunoassay. The expression of glycoprotein IIIa epitopes were the same for the purified glycoprotein IIIa and glycoprotein IIIa in Triton X-100 solubilized platelets. A 66 kDa protein derived from glycoprotein IIIa by limited proteolysis of platelet membranes also expressed the same epitopes as intact glycoprotein IIIa. Solubilized platelets contained approximately 16 micrograms of total glycoprotein IIIa antigen per 10(9) cells. The level of glycoprotein IIIa determined by radioimmunoassay in one patient with Glanzmann's thrombasthenia amounted to 6.7% of normal and it was close to the values obtained by other methods.     

血小板功能负性调控机制

在血液循环系统中,血小板在抑制因子的作用下,处于静息状态。当机体出血或外界因素刺激时,血小板活化,产生聚集、黏附和释放反应,释放出二磷酸腺苷(ADP)、花生四烯酸(AA)、血小板活化因子和5-羟色胺等物质,招募更多的血小板黏附于出血处,从而启动凝血过程,发挥止血作用。当止血反应完成后,血小板发生解聚,恢复到静息状态。然而,在病理条件下,血小板的内在解聚能力下降,形成过度活化的血小板,产生病理性血栓,导致急性缺血性心血管疾病的发生。临床使用抗血小板药物控制血小板的活化,治疗急性缺血性心血管疾病。然而,目前临床上常用的抗血小板药物发挥抗血小板活化作用的同时,影响了血小板正常的生理性止血作用,产生出血等副作用。因此,我们需要研发新型抗血小板药物,使其既能发挥抗血小板作用,又能减少出血等副作用。本文将对血小板负性调控机制进行综述,为进一步研究抗血小板药物提供思路。     

Stent thrombosis associated with first-generation drug-eluting stents: issues with antiplatelet therapy

Notice of the rare but catastrophic occurrence of stent thrombosis in association with deployment of drug-eluting stents has focused attention on the adequacy of the current dual antiplatelet regimen of aspirin and clopidogrel. Some patients will not respond to clopidogrel and a glycoprotein (GP) IIb/IIIa inhibitor may be strongly considered during stenting procedures, especially in high-risk patients or those not receiving pretreatment with clopidogrel. Insisting upon and confirming adherence to antiplatelet therapy are complicated tasks, especially because the reasons for premature discontinuation are myriad, from cost to bleeding complications to the need for minor surgery. Nevertheless, the concern about adherence to antiplatelet therapy represents a new and significant clinical reality in our stenting era, one previously less appreciated with the deployment of bare metal stents. (Neth Heart J 2007;15:148-50.)     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Familial occurrence of Glanzmann thrombasthenia]

Glanzmann's thrombasthenia, known also as Glanzmann's disease, is an autosomally inherited hemorrhagic disease with unique abnormalities of platelet functions. Authors present a large family in which Glanzmann's disease was diagnosed in the father and two sons. An analysis of platelet membranes enabled diagnosis of Glanzmann's thrombasthenia type II. A decrease in clot contractibility, fibrinogen binding to blood platelets, and decreased glycoprotein IIb and IIIa levels with marked impairment of GP IIb and IIIa complexes formation were characteristic for affected family members. One daughter died 8 days after birth with the symptoms of hemorrhagic diathesis. Mother and remaining three sons are healthy without the signs of Glanzmann's disease.     

Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action

The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 10³ per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10^?3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A₂ synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(?) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.     

Preparation and biological activity of novel tricyclic GPIIb/IIIa antagonists

Antagonists of the glycoprotein GPIIb/IIIa are a promising class of antithrombotic agents offering potential advantages over present antiplatelet agents (i.e., aspirin and ticlopidine). Novel tricyclic nonpeptidal GPIIb/IIIa antagonists have been prepared and evaluated in vitro as antagonists of fibrinogen binding to the purified GPIIb/IIIa receptor and as inhibitors of platelet aggregation. The work presented demonstrates the robustness of the benzodiazepinedione (BZDD) scaffold, which can be functionalized at the N¹---C² amide as well as at C⁷, to provide structural diversity and allow optimization of the physiochemical and pharmacological properties of the BZDD based GPIIb/IIIa antagonists. In addition, the resulting new class of tricyclic GPIIb/IIIa antagonists could be used to probe for additional binding interactions on the GPIIb/IIIa receptor and perhaps lead to BZDD based GPIIb/IIIa antagonists with increased potency. The tricyclic molecules reported herein demonstrate that a heterocyclic ring can be fused to the benzodiazepinedione scaffold with retention of anti-aggregatory potency and in the case of tetrazole 30i, increased potency relative to the bicyclic analogue 1c.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Summary Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist20 (S 1197). Compound20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure-activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the β-amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

Inhibition of platelet function by abciximab or high-dose tirofiban in patients with STEMI undergoing primary PCI: a randomised trial

BackgroundIn patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary PCI, few data exist on the magnitude of platelet activation, aggregation and dosing of glycoprotein (GP) IIb/IIIa receptor inhibitors. Methods Sixty STEMI patients were randomised to abciximab, to high-dose tirofiban or to no additional GP IIb/IIIa inhibitor treatment. Platelet activation (P-selectin expression) was measured using flow cytometry and the level of inhibition of platelet aggregation was assessed using the Plateletworks assay. Additionally, the PFA-100 with the collagen/adenosine-diphosphate cartridge (CADP) was used to compare the levels of platelet inhibition. All measurements were performed at baseline (T₀), immediately after (T₁), 30 minutes (T₂), 60 minutes (T₃) and 120 minutes (T₄) after primary PCI. Results The level of platelet activation in both GP IIb/IIIa receptor inhibitor treated groups was significantly lower compared with the control group at all time points after primary PCI (p=0.04). Also the administration of the currently recommended dose of abciximab resulted in significantly lower levels of inhibition of aggregation compared with high-dose tirofiban (p<0.0001). In addition, the CADP closure times were significantly prolonged in both GP IIb/IIIa inhibitor treated groups compared with the control group at time points T1 (p=0.006) and T4 (p<0.0001). Conclusion The administration of high-dose tirofiban resulted in a significantly higher inhibition of platelet aggregation compared with the currently recommended dose of abciximab. Large clinical trials are needed to assess whether this laboratory superiority of high-dose tirofiban translates into higher clinical efficacy. (Neth Heart J 2007;15:375-81.)