Radio-isotopic platelet labeling with monoclonal antibody against membrane glycoprotein IIb–IIIa. Measurement of platelet adhesion/aggregation to the substrate

Institute of Experimental Cardiology, Cardiologic Scientific Center, Russian Academy of Medical Sciences, Moscow. Institute of Cardiology, Ministry of Health of the Uzbekistan Republic, Tashkent. (Presented by Academician of the Russian Academy of Medical Sciences V. N. Smirnov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 11, pp. 529–532, November, 1992.     

Inhibition of platelet aggregation by monoclonal antibodies against glycoprotein IIb–IIIa complex

Monoclonal antibodies CRC64 are obtained against Ca²⁺-dependent glycoprotein IIb–IIIa complex of the platelet membrane which possess the ability to inhibit completely fibrinogen-dependent platelet aggregation. CRC64 is directed against the epitope formed by the glycoprotein IIb–IIIa complex and does not interact with proteins isolated after platelets are treated with ethylenediamine tetraacetate. Complete, reproducible blockade of platelet aggregation caused by 5 μM adenosine diphosphate is noted in an MCA concentration of 3 μg/ml, while in the case of a stronger inductor, namely 1 U/ml thrombin, platelet aggregation is inhibited in a concentration of 5 μg/ml. F(ab′)₂ fragments are also able to inhibit platelet aggregation completely and are usually effective in concentrations lower than native monoclonal antibodies. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 10, pp. 402–405, October, 1994 Presented by V. N. Smirnov, Member of the Russian Academy of Medical Sciences     

Endothelialization and in-stent restenosis on the surface of glycoprotein IIIa monoclonal antibody eluting stent

Since the percutaneous transtuminal coronary angioplasty was introduced into China in 1984, this procedure has become widely accepted as an important step in coronary revascularization. This study shows the effect of the monoclonal antibody (mAb) on the platelet glycoprotein IIIa receptor during endothelialization and in-stent restenosis by implanting the mAb-eluting stents into iliac arteries of rabbits. The hard tissue cross sections of the stent-implanted arterial segments were made by polymethylmethacrylate embedding. Arterial intima proliferation was observed and analyzed. The endothelialization of the stent surface was observed using scanning electron microscope, whereas the ultrastructure of the neointima was observed using transmission electron microscope. After one month of stent implantation, the surfaces of both groups were covered by intact endothelial layers, but the neointimal areas and the ratio of stenosis were significantly lesser in the mAb-eluting stent group (p < 0.01). After 3 months, the ratio of stenosis in the mAb-eluting stent group was 14.67 ± 0.79, whereas that of the bare stent group was 21.58 ± 1.76 (p < 0.01). Therefore, the mAb eluting from the stent surface has the potential to accelerate endothelialization, prevent thrombosis formation due to the interaction of stent with blood, and decrease the stenosis ratio by inhibiting neointima proliferation.     

Complementary clinical benefits of coronary-artery stenting and blockade of platelet glycoprotein IIb/IIIa receptors. Evaluation of Platelet IIb/IIIa Inhibition in Stenting Investigators.

BACKGROUND: Inhibition of the platelet glycoprotein IIb/IIIa receptor with the monoclonal-antibody fragment abciximab reduces the acute ischemic complications associated with percutaneous coronary revascularization, whereas coronary-stent implantation reduces restenosis. We conducted a trial to determine the efficacy of abciximab and stent implantation in improving long-term outcome. METHODS: A total of 2399 patients were randomly assigned to stent implantation and placebo, stent implantation and abciximab, or balloon angioplasty and abciximab. The patients were followed for six months. RESULTS: At six months, the incidence of the composite end point of death or myocardial infarction was 11.4 percent in the group that received a stent and placebo, as compared with 5.6 percent in the group that received a stent and abciximab (hazard ratio, 0.47; 95 percent confidence interval, 0.33 to 0.68; P<0.001) and 7.8 percent in the group assigned to balloon angioplasty and abciximab (hazard ratio, 0.67; 95 percent confidence interval, 0.49 to 0.92; P=0.01). The hazard ratio for stenting plus abciximab as compared with angioplasty plus abciximab was 0.70 (95 percent confidence interval, 0.48 to 1.04; P=0.07). The rate of repeated revascularization of the target vessel was 10.6 percent in the stent-plus-placebo group, as compared with 8.7 percent in the stent-plus-abciximab group (hazard ratio, 0.82; 95 percent confidence interval, 0.59 to 1.13; P=0.22) and 15.4 percent in the angioplasty-plus-abciximab group (hazard ratio, 1.49; 95 percent confidence interval, 1.13 to 1.97; P=0.005). The hazard ratio for stenting plus abciximab as compared with angioplasty plus abciximab was 0.55 (95 percent confidence interval, 0.41 to 0.74; P<0.001). Among patients with diabetes, the combination of abciximab and stenting was associated with a lower rate of repeated target-vessel revascularization (8.1 percent) than was stenting and placebo (16.6 percent, P=0.02) or angioplasty and abciximab (18.4 percent, P=0.008). CONCLUSIONS: For coronary revascularization, abciximab and stent implantation confer complementary long-term clinical benefits.     

Quantitative comparisons of antibody-binding sites of platelet glycoprotein IIb/IIIa in aplastic anemia and idiopathic thrombocytopenic purpura

The numbers of antibody-binding sites of platelet glycoprotein (GP) IIb/IIIa on circulating platelets were analyzed using 4 kinds of antibodies in 34 aplastic anemia (AA) patients, 20 idiopathic thrombocytopenic purpura (ITP) patients, and 14 normal controls. The numbers of antibody-binding sites of CD41, CD41a, CD41b, and CD61 on platelets of the AA patients were less than in the normal controls (p <0.001). In the ITP patients, the numbers of sites for CD41 and CD41a were less than in normal controls (p <0.05). There were significant positive correlations between CD41 and CD41a, CD41b, and CD61 in the 3 groups. There were significant negative correlations between CD41 and CD41b and between CD41a and CD41b in the normal controls, but not in the AA or ITP patients. In summary, the numbers of the 4 antibody-binding sites of GPIIb/IIIa on platelets of AA and ITP patients are different from those in normal controls. Measurements of the antibody-binding sites of GPIIb/IIIa are not necessary for the differential diagnosis of AA and ITP. However, the differences in correlations between the numbers of epitopes in AA and ITP patients suggest that the epitopes of GPIIb/IIIa are altered in these diseases.     

Immunolocalization of platelet glycoprotein IIb/IIIa and P-selectin, and neutrophil-platelet interaction in human coronary unstable plaques

Platelet aggregation at the site of plaque rupture or erosion is a dominant feature in the pathophysiology of plaque destabilization. To elucidate the role of glycoprotein (GP) IIb/IIIa in coronary plaque destabilization, we immunohistochemically studied the presence of GP IIb/IIIa in coronary atherectomy specimens obtained from patients with stable angina (SAP) and unstable angina pectoris (UAP). Moreover, we immunohistochemically investigated the presence of P-selectin, which is known to be a marker of platelet activation, in these specimens. All these patients underwent atherectomy at primary atherosclerotic lesions responsible for SAP (n=25) and UAP (n=23). Frozen samples were studied with antibodies against smooth muscle cells, macrophages, neutrophils, endothelial cells, GP IIb/IIIa and P-selectin. Immunoreactive positive areas for GP IIb/IIIa, P-selectin, and macrophages, respectively, were calculated using computer-aided planimetry, and numbers of neutrophils were also counted. In the culprit lesions of UAP patients, 17 of the 23 lesions (74%) contained GP IIb/IIIa positive platelet thrombi, and all these platelet thrombi were positive for P-selectin. In contrast, in the lesions of SAP patients, 3 of the 25 lesions (12%) showed staining positivity for GP IIb/IIIa and P-selectin. Quantitatively, the percentage of GP IIb/IIIa- and P-selectin-positive area was significantly higher (GP IIb/IIIa, P<0.0005; P-selectin, P<0.0001) in patients with UAP than in patients with SAP. The number of neutrophils was significantly higher (P<0.0005) in patients with UAP than in patients with SAP. Moreover, the percentage of GP IIb/IIIa-positive area showed a significant positive correlation with the number of neutrophils (r=0.66, p<0.0001). These findings strongly suggest that platelet activation and aggregation, leading to formation of platelet thrombi, and the interaction between activated platelets and neutrophils play a pivotal role in the pathogenesis of plaque destabilization in human coronary atherosclerotic lesions.     

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains.

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.     

Fibrinogen and glycoprotein IIb/IIIa localization during platelet adhesion. Localization to the granulomere and at sites of platelet interaction.

Platelet membrane glycoprotein IIb-IIIa plays a focal role in primary hemostasis by serving as the cell surface receptor for fibrinogen. Recent studies by several groups have suggested that GPIIb-IIIa, which is dispersed randomly in the resting cell, undergoes migration leading to receptor clustering after platelet activation. The authors have investigated this activation-dependent relocation of fibrinogen receptors on platelets adherent to a standardized artificial surface. The correlative use of immunogold electron microscopy, ligand-gold binding, and stereo (three-dimensional) electron microscopy (EM) revealed specific localization of fibrinogen and its receptor at points of platelet to platelet interaction. Fibrinogen distribution on the plasma membrane, studied through the use of fibrinogen-gold conjugates with whole-mount adherent platelets, was primarily over the granulomere and at the cell periphery corresponding to sites of platelet-platelet interaction. Compared with the general hyalomere, fibrinogen density over the granulomere and at contact regions was increased 12-fold and 22-fold, respectively, and the specificity of binding at these sites was verified by positive competition with native fibrinogen, one of its degradation products (Fragment D1), and by monoclonal antibodies (HP1-1d and AP-2) specific for GPIIb-IIIa. The distribution of receptor antigens, localized by immunogold EM using antibodies against GPIIb-IIIa, also was localized over the hyalomere, where fibrinogen did not bind. To understand this apparently nonfunctional hyalomere GPIIb-IIIa further, correlative immunocytochemistry was performed using polyclonal and monoclonal antibodies for GPIIb and GPIIIa simultaneously. Colocalization of the antigens was observed consistently over the granulomere and at regions of cell contact, whereas the hyalomere antigens tended to be nonassociated. The studies document GPIIb-IIIa as a function complex at sites of cell interaction where fibrinogen binds.     

抗血小板糖蛋白IIb/IIIa Fab抗体的原核表达及其生物活性

目的 :研制抗血小板膜糖蛋白IIb/IIIaFab抗体。方法 :通过间接免疫荧光试验和血小板聚集抑制试验 ,选取鼠源抗血小板糖蛋白IIb/IIIa单克隆抗体 (mAbP14 0 )。从分泌该mAb的杂交瘤细胞株 (P14 0 )中 ,克隆到抗体轻链基因和重链Fd段基因 ,构建原核表达重组质粒p3MH/P14 0к Fd ,并在XLI Blu菌株中进行表达。采用钴亲和层析法对P14 0Fab抗体进行纯化 ,用SDS PAGE、ELISA和Westernblot等方法 ,对P14 0Fab抗体进行检测 ,并通过血小板聚集抑制试验 ,观察P14 0Fab抗体的抗栓活性。结果 :SDS PAGE和Westernblot表明 ,纯化的P14 0Fab抗体的相对分子质量 (Mr)约为 4 70 0 0。ELISA的结果显示 ,P14 0Fab抗体可与人血小板特异性结合。在体外ADP诱导的血小板聚集试验中 ,P14 0Fab抗体对血小板聚集的抑制作用成剂量依赖性 ,IC50 的平均值为 16 .85mg/L。结论 :成功地研制出具有抗栓活性的抗血小板膜糖蛋白IIb/IIIa的Fab抗体     

Platelet kinetics in dogs treated with a glycoprotein IIb/IIIa peptide antagonist

Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists have been highly effective inhibitors of platelet aggregation in preclinical studies and in clinical trials. However, decreased platelet counts have been documented in preclinical studies and in some patients receiving GPIIb/IIIa antagonists. We evaluated changes in platelet kinetics and fate in dogs receiving the GPIIb/IIIa receptor antagonist RPR 109891 orally for 4 days. Dogs receiving RPR 109891 had a 22-52% decrease in platelet count with the nadirs at 3-5 days after initiation of treatment. Platelet survival time was reduced by 19%, and platelet half-life was reduced by 63%. Indium-111-labeled platelets were rapidly cleared from the blood within 1 hour after administration of RPR 109891 on treatment days 1 and 2. This clearing was associated with a sharp increase in radioactivity in spleen but not in liver or lung. Platelet clearance was markedly attenuated on treatment days 3 and 4. Platelet counts returned to baseline within 1 week after discontinuation of treatment. These data indicate that RPR 109891 causes rapid and selective sequestration of platelets in the spleen.     

Platelet glycoprotein IIb/IIIa inhibition with coronary stenting for acute myocardial infarction.

BACKGROUND: When administered in conjunction with primary coronary stenting for the treatment of acute myocardial infarction, a platelet glycoprotein IIb/IIIa inhibitor may provide additional clinical benefit, but data on this combination therapy are limited. METHODS: We randomly assigned 300 patients with acute myocardial infarction in a double-blind fashion either to abciximab plus stenting (149 patients) or placebo plus stenting (151 patients) before they underwent coronary angiography. Clinical outcomes were evaluated 30 days and 6 months after the procedure. The angiographic patency of the infarct-related vessel and the left ventricular ejection fraction were evaluated at 24 hours and 6 months. RESULTS: At 30 days, the primary end point--a composite of death, reinfarction, or urgent revascularization of the target vessel--had occurred in 6.0 percent of the patients in the abciximab group, as compared with 14.6 percent of those in the placebo group (P=0.01); at 6 months, the corresponding figures were 7.4 percent and 15.9 percent (P=0.02). The better clinical outcomes in the abciximab group were related to the greater frequency of grade 3 coronary flow (according to the classification of the Thrombolysis in Myocardial Infarction trial) in this group than in the placebo group before the procedure (16.8 percent vs. 5.4 percent, P=0.01), immediately afterward (95.1 percent vs. 86.7 percent, P=0.04), and six months afterward (94.3 percent vs. 82.8 percent, P=0.04). One major bleeding event occurred in the abciximab group (0.7 percent); none occurred in the placebo group. CONCLUSIONS: As compared with placebo, early administration of abciximab in patients with acute myocardial infarction improves coronary patency before stenting, the success rate of the stenting procedure, the rate of coronary patency at six months, left ventricular function, and clinical outcomes.     

抗血小板糖蛋白Ⅵ单克隆抗体的制备及其功能研究

目的：制备抗血小板糖蛋白Ⅵ（GPⅥ）单克隆抗体，观察其在体外抗血小板黏附和聚集功能。方法：采用基因重组技术体外表达血小板糖蛋白Ⅵ胞外区重组蛋白（rGPⅥ）。以rGPⅥ免疫小鼠，经细胞融合及筛选后制备抗GPⅥ单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convulxin及ADP诱导的血小板聚集的影响；利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果：正确构建了rGPⅥ表达载体pET-20b（＋）-GPⅥ，rGPⅥ在原核细胞中有效表达。rGPⅥ能够被抗Penta-His单抗和抗GPⅥ多抗识别。制备的抗GPⅥ单克隆抗体SZ118能够识别rGPⅥ，并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convulxin诱导的血小板聚集，呈抗体剂量依赖性；对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明，SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论：成功制备抗GPⅥ单克隆抗体SZ118，该抗体与血小板有良好的结合能力，显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

抗血小板糖蛋白VI单克隆抗体的制备及其功能研究

目的:制备抗血小板糖蛋白VI(GPVI)单克隆抗体,观察其在体外抗血小板黏附和聚集功能。方法:采用基因重组技术体外表达血小板糖蛋白VI胞外区重组蛋白(rGPVI)。以rGPVI免疫小鼠,经细胞融合及筛选后制备抗GPVI单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convu lxin及ADP诱导的血小板聚集的影响;利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果:正确构建了rGPVI表达载体pET-20b(+)-GPVI,rGPVI在原核细胞中有效表达。rGPVI能够被抗Penta-H is单抗和抗GPVI多抗识别。制备的抗GPVI单克隆抗体SZ118能够识别rGPVI,并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convu lxin诱导的血小板聚集,呈抗体剂量依赖性;对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明,SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论:成功制备抗GPVI单克隆抗体SZ118,该抗体与血小板有良好的结合能力,显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

Eluting characteristics of a platelet glycoprotein receptor antibody using a PLLA-coated stent

Drug-eluting stents (DES) have emerged as a recognized alternative to treat stent restenosis but many questions remain regarding the optimal type and eluting characteristics of both drug and stent. The first component of the study examines the extent of surface coating of PLLA (poly(L-lactic acid)) on a Nitinol stent. The second characterizes the adsorption and elution rates of monoclonal mouse anti-human platelet glycoprotein (GP) IIIa antibody SZ-21 from a PLLA-coated surface. The PLLA coating was examined by fluorescence staining and image analysis using the Image Processing Box of MATLAB. Stents exposed to the monoclonal mouse anti-human platelet GP IIIa antibody were tested for their adsorption characteristics by radioisotope technique with (125)I-labelled SZ-21. The elution rates were then measured in looped circuits at different velocities (10 or 20 ml/min) and durations (30 min up to 312 h). Results showed that the fluorescence staining and image analysis showed a striking difference in the extent of coating between PLLA-coated stents and SZ-21 eluting stents on the gray-scale distribution of Nitinol surfaces. The amount of SZ-21 adsorbed onto the PLLA-coated stents was dependent on the concentration and duration of immersion in the solution. The method of preparation the mAb eluting stent significantly influenced the elution characteristics for a continuous perfusion of more than 300 h. The eluting curve was biphasic with initial rapid elution for the first 24 h followed by a gradual slow elution. These results indicate that the Image Processing Box of MATLAB appears to be a useful method for semi-quantitative analysis of fluorescence images. Furthermore, SZ-21 can be passively adsorbed onto PLLA-coated stents and predictably influenced by the concentration and duration of immersion. These studies may pave the way to developing stent-based delivery of a potent anti-platelet agent.     

Eluting characteristics of a platelet glycoprotein receptor antibody using a PLLA-coated stent

Drug-eluting stents (DES) have emerged as a recognized alternative to treat stent restenosis but many questions remain regarding the optimal type and eluting characteristics of both drug and stent. The first component of the study examines the extent of surface coating of PLLA (poly(L-lactic acid)) on a Nitinol stent. The second characterizes the adsorption and elution rates of monoclonal mouse anti-human platelet glycoprotein (GP) IIIa antibody SZ-21 from a PLLA-coated surface. The PLLA coating was examined by fluorescence staining and image analysis using the Image Processing Box of MATLAB. Stents exposed to the monoclonal mouse anti-human platelet GP IIIa antibody were tested for their adsorption characteristics by radioisotope technique with ¹²⁵I-labelled SZ-21. The elution rates were then measured in looped circuits at different velocities (10 or 20 ml/min) and durations (30 min up to 312 h). Results showed that the fluorescence staining and image analysis showed a striking difference in the extent of coating between PLLA-coated stents and SZ-21 eluting stents on the gray-scale distribution of Nitinol surfaces. The amount of SZ-21 adsorbed onto the PLLA-coated stents was dependent on the concentration and duration of immersion in the solution. The method of preparation the mAb eluting stent significantly influenced the elution characteristics for a continuous perfusion of more than 300 h. The eluting curve was biphasic with initial rapid elution for the first 24 h followed by a gradual slow elution. These results indicate that the Image Processing Box of MATLAB appears to be a useful method for semi-quantitative analysis of fluorescence images. Furthermore, SZ-21 can be passively adsorbed onto PLLA-coated stents and predictably influenced by the concentration and duration of immersion. These studies may pave the way to developing stent-based delivery of a potent anti-platelet agent.     

抗血小板膜糖蛋白Ib单克隆抗体的制备、鉴定及其功能的初步研究

目的:制备抗人血小板膜糖蛋白Ib(GPIb)单克隆抗体(mAb),并进行生化鉴定与初步探讨其临床应用.方法:采用血小板免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合.经间接ELISA法筛选和克隆化培养,获得1株抗人GPIb mAb的杂交瘤细胞,纯化其腹水获得mAb;免疫双扩散鉴定其抗体亚类;流式细胞术和免疫放射法鉴定...