The effect of long-term clopidogrel use on neointimal formation after percutaneous coronary intervention

OBJECTIVE: The purpose of this study was to evaluate the long term effect of clopidogrel-based antiplatelet therapy on neointimal formation. METHODS: This study comprised 78 patients with typical stable angina pectoris or documented myocardial ischaemia, and with only one angiographic lesion in one native coronary artery undergoing successful stent implantation without predilatation with C-reactive protein levels < or =5 mg/l at 72 h after the procedure. All patients received dual antiplatelet therapy with 75 mg/day clopidogrel and 300 mg/day aspirin for four weeks. Clopidogrel was switched to isochronous placebo in half of the patients (n=39) at the end of the fourth week. This allocation was maintained for 20 weeks, and at week 24 of the study, coronary angiography and intravascular ultrasound imaging were performed again in all cases in order to evaluate the changes that had occurred in the in-stent neointimal formation; rates of restenosis were also recorded RESULTS: At the end of the follow-up period, angiographic stenosis diameter and restenosis rates were smaller in the clopidogrel group than in the placebo group (23.3% versus 35.6%, p=0.05 and 5.12% versus 10.25%; p=0.03 respectively); the intravascular ultrasonographic neointimal cross sectional area was also smaller in the clopidogrel group (3.6 +/- 2.7 mm(2) versus 5.2 +/- 2.5 mm(2), p=0.03). CONCLUSIONS: Long-term clopidogrel administration significantly reduced neointimal formation at the stent site as well as reducing major clinical events in patients who did not develop high-risk systemic inflammatory response after percutaneous coronary intervention.     

Antithrombotic effect of ticlopidine on He-Ne laser-induced thrombus formation in rat mesenteric microvessels.

The inhibitory effects of ticlopidine and acetylsalicylic acid (ASA) on thrombus formation in rat mesenteric microvessels were studied. The results were compared with the effect of the drugs on platelet aggregation in citrated whole blood. He-Ne laser-induced thrombus formation in arterioles and in venules was significantly inhibited by 100 mg/kg ticlopidine. In contrast, a higher dose of ASA (300 mg/kg) was needed to inhibit thrombus formation and the effects of ASA were observed only in arterioles and not in venules. In addition, although the inhibition by ASA in arterioles was not strong it followed a dose-dependent manner, suggesting that the differential effect of ASA on platelets and endothelium may not be evident in vivo. Ticlopidine and ASA strongly inhibited ADP-induced whole blood platelet aggregation, but not collagen- or thrombin-induced platelet aggregation.     

Experimental thrombus formation in mesenteric microvessels: evaluation of a method

Platelet thrombi in rats are produced at sites of electrically induced damage in mesenteric venules. The thrombus formation response is dependent on the temperature of the bathing fluid and varies with electrode polarity. Cathodal stimulation causes vasoconstriction and induces platelet aggregation after a slight delay, while anodal stimulation induces the rapid formation of aggregates containing some white cells and erythrocytes, without any vasoconstriction. The effects of cathodal and anodal stimulation can be simulated by topical application of isotonic saline adjusted to high or low pH, respectively.Ultrastructural studies showed that aggregated platelets remaining at the stimulation site lose some of their organelles after 10 min (platelet emboli were found in liver venules of gerbils after thrombi had been produced in the ileal veins).The thrombus formation response cannot be satisfactorily quantified by measuring either rate or duration of embolisation, but recording the number of thrombi produced by a group of “threshold” stimuli is a simple, semi-objective means of assessment, and gives reproducible results in control animals.     

Mechanisms of thrombus formation and lysis

Maintenance of a patent vasculature is critical to provide nutrient blood flow to dependent tissues. This is normally facilitated by vessels composed of actively nonthrombogenic endothelium and blood that contains both nonactivated platelets and inactive coagulation proenzymes. Following vessel injury, active hemostasis results from vasoconstriction, adherence and aggregation of activated platelets, and coagulation enzyme activation. The resulting thrombus contains a mixture of blood cells and the insoluble product of coagulation, fibrin, which further stimulates the activation of another blood enzyme system known as the fibrinolytic system. This results in the conversion by plasminogen activators of the circulating plasma proenzyme plasminogen into the active fibrinolytic enzyme plasmin, which dissolves the clot into degradation products. Vascular patency ultimately is restored and healing is thus facilitated. The hemostatic system is highly regulated by a variety of processes. Derangement of regulation can lead to disease manifesting either as thrombosis or hemorrhage. Furthermore, improved understanding of the molecular interactions involved in these processes has led to design of newer therapeutic interventions targeted toward amelioration of the sequelae of thromboembolic disease.     

In vivo thrombus formation in murine models

Platelets play a central role in hemostasis, but also in atherothrombosis, as they rapidly adhere to tissue and to one another as a response to any vascular injury. This process involves a large number of surface receptors, signaling pathways, and enzymatic cascades as well as their complex interplay. Although in vitro experiments proved successful in both identifying new receptors and pathways and developing potent and selective antithrombotic drugs, in vitro research cannot mimic the myriad hemodynamic and spatiotemporal cellular and molecular interactions that occur during the generation and propagation of thrombi in vivo. Animal models, and, with the availability of genetically modified mouse strains and of modern intravital imaging techniques, mouse models in particular, have opened new ways to identify both individual roles and the interplay of platelet proteins in complex in vivo settings. In vivo models revealed the important role of, eg, Gas6 or blood coagulation factor XII in thrombus formation, and results obtained in in vivo models raised the interesting possibility that (physiologic) hemostasis and (pathologic) thrombosis might represent 2 mechanistically different processes. This review summarizes in vivo findings that contributed significantly to our understanding of hemostatic and thrombotic processes and which may help to guide future research.     

肝素诱导的血小板减少性血栓形成1例

患者,女,60岁,因呼吸困难、不能平卧、双下肢水肿4d、晕厥2次于2005年3月5日入院。入院后根据临床表现及心脏超声、心电图、肺灌注扫描诊断为肺动脉血栓栓塞。溶栓后给予肝素5000U负荷量,以800U/h微泵泵入,持续48h后给予低分子肝素5000U,q12h,ip,同时给予华法林2·5mg/d等治疗。患者呼吸困难缓解,可平卧,双下肢水肿消退,周径对称。入院时查血常规WBC6·43×109/L,RBC4·33×1012/L,Hb139g/L,PLT222×109/L;PT12s,APTT30s,INR1·1。患者应用肝素第5天复查血常规:WBC3·67×109/L,RBC3·75×1012/L,Hb118g/L,PLT12×109/L,IN…     

Noncardiogenic pulmonary edema associated with clopidogrel: a serious but unexpected side effect of clopidogrel

Clopidogrel has a favourable side effect profile, in which the most serious complications are related to bleeding diathesis. Drug reactions represent frequently encountered clinical problems that can also be life-threatening. In the present case, serious noncardiogenic pulmonary edema was observed as an idiosyncratic reaction after clopidogrel use. Using the Naranjo adverse drug reactions probability scale, a possible likelihood of this putative interaction between clopidogrel and pulmonary edema was calculated. Such an adverse effect of clopidogrel has never been reported before. The present report shows the importance of recognizing rare adverse reactions of newer drugs, such as clopidogrel, in clinical practice.     

Targeting platelet receptor function in thrombus formation: The risk of bleeding

In this review, we presume that the process of thrombus formation, as assessed in whole blood flow studies and in experimental (murine) thrombosis studies, reflects the platelet responses in human haemostasis and thrombosis. Following this concept, we give an up-to-date overview of the main platelet receptors and signalling pathways that contribute to thrombus formation and are used as targets in (pre)clinical intervention studies to prevent cardiovascular disease. Discussed are receptors for thrombin, thromboxane, ADP, ATP, prostaglandins, von Willebrand factor, collagen, CLEC-2 ligand, fibrinogen and laminin. Sketched are the consequences of receptor deficiency or blockage for haemostasis and thrombosis in mouse and man. Recording of bleeding due to (congenital) platelet dysfunction or (acquired) antiplatelet treatment occurs according to different protocols, while common laboratory methods are used to determine platelet function.     

Clot-promoting effect of platelet-vessel wall interaction: influence of dietary fats and relation to arterial thrombus formation in rats.

A small piece of vascular tissue punched from a rat aorta is able to clot plasma. This coagulation process is promoted by blood platelets, especially after their activation. Thrombin, generated by this clotting process, plays a key role in vessel-wall induced platelet activation. Vascular prostacyclin inhibits vessel-wall-induced clotting of platelet-rich plasma, possibly by inhibiting platelet activation. Type and amount of dietary fats were shown to influence vessel-wall-induced clotting via at least four different mechanisms, namely: by modifying vascular prostacyclin formation; by affecting the clotting potency of the vascular tissue per sec; by an effect on some platelet property, probably connected with platelet activation; by influencing a plasma factor. Each of these mechanisms, as well as the nature of vessel-wall-induced coagulation, requires further investigation.     

Intracardiac thrombus formation in association with hydatid disease

Cardiac hydatid disease is rare, but life threatening. A pedunculated hydatid cyst which mimicked a left atrial myxoma was diagnosed on two-dimensional echocardiography. The degree of cardiac involvement at necropsy was much greater than indicated by either echocardiography or limited cardiac catheterization. The suggestion that only echocardiography be performed before surgery is undertaken may be inappropriate in many cases.     

Platelet PECAM-1 inhibits thrombus formation in vivo

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.     

Function of protein kinase CK2 in thrombus formation

Thrombus formation is dependent on the interaction of platelets, leukocytes and endothelial cells as well as proteins of the coagulation cascade. This interaction is tightly controlled by phospho-regulated pathways involving protein kinase CK2. A growing number of studies have demonstrated an important role of this kinase in the regulation of primary and secondary hemostasis. Inhibition of CK2 downregulates the expression of important adhesion molecules on platelets and endothelial cells, such as glycoprotein (GP)IIb/IIIa, P-selectin, von Willebrand factor and vascular cell adhesion molecule. Moreover, the reduced CK2-dependent phosphorylation of different coagulation factors prevents the conversion of fibrinogen to fibrin. Targeting these mechanisms may open the door for the development of novel anti-thrombotic therapies.     

Hirudin interruption of heparin-resistant arterial thrombus formation in baboons

To determine the role of thrombin in high blood flow, platelet-dependent thrombotic and hemostatic processes we measured the relative antithrombotic and antihemostatic effects in baboons of hirudin, a highly potent and specific antithrombin, and compared the effects of heparin, an antithrombin III-dependent inhibitor of thrombin. Thrombus formation was determined in vivo using three relevant models (homologous endarterectomized aorta, collagen-coated tubing, and Dacron vascular graft) by measuring: (1) platelet deposition, using gamma camera imaging of 111In-platelets; (2) fibrin deposition, as assessed by the incorporation of circulating 125I-fibrinogen; and (3) occlusion. The continuous intravenous infusion of 1, 5, and 20 nmol/kg per minute of recombinant hirudin (desulfatohirudin) maintained constant plasma levels of 0.16 +/- 0.03, 0.79 +/- 0.44, and 3.3 +/- 0.77 mumol/mL, respectively. Hirudin interrupted platelet and fibrin deposition in a dose-dependent manner that was profound at the highest dose for all three thrombogenic surfaces and significant at the lowest dose for thrombus formation on endarterectomized aorta. Thrombotic occlusion was prevented by all doses studied. In contrast, heparin did not inhibit either platelet or fibrin deposition when administered at a dose that maximally prolonged clotting times (100 U/kg) (P greater than .1), and only intermediate effects were produced at 10-fold that dose (1,000 U/kg). Moreover, heparin did not prevent occlusion of the test segments. Hirudin inhibited platelet hemostatic function in concert with its antithrombotic effects (bleeding times were prolonged by the intermediate and higher doses). By comparison, intravenous heparin failed to affect the bleeding time at the 100 U/kg dose (P greater than .5), and only minimally prolonged the bleeding time at the 1,000 U/kg dose (P less than .05). We conclude that platelet-dependent thrombotic and hemostatic processes are thrombin-mediated and that the biologic antithrombin hirudin produces a potent, dose-dependent inhibition of arterial thrombus formation that greatly exceeds the minimal antithrombotic effects produced by heparin.     

心脏血栓患者52例临床分析

目的:分析52例经食管超声明确诊断心脏血栓患者临床病史、左心房及左心室内径、射血分数、心脏附壁血栓情况及检测的凝血指标、高敏C反应蛋白（hsCRP）等。方法: 病例回顾分析,选择2006年10月~2011年10月首都医科大学宣武医院住院患者新诊断证实心脏有血栓形成者52例。结果: 52例心脏血栓患者中,男性34例（65%）,女性18例（35%）,男性显著高于女性（P<005）。年龄18~83（60±16）岁。心功能（NYHA）分级Ⅲ级及以上31人,占60%。心房颤动是第1并发症,其次为缺血性脑血管病、高血压病、冠心病。血栓并发心房颤动患者中,心脏瓣膜病占47%。共发现70个栓子,左心耳发生率最高。按照血栓部位分组,发现左房及左心耳血栓患者左心房内径显著增大,其他部位血栓者左心室内径显著增大,差别均有显著性（P<005）。hsCRP在左房及左心耳血栓患者中低于其他部位血栓患者,两组之间差异显著（P<005）。结论: 房颤与血栓形成关联密切,左心耳部位血栓发生风险最高,临床上对心功能不全、房颤、缺血性脑血管病者要积极行经食道超声检查以明确诊断。     

DNAse-dependent,NET-independent pathway of thrombus formation in vivo

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)–activated neutrophils play a key role in initiating the tissue factor–dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

Thrombosis is one of the major causes of human mortality. Ischemic stroke, myocardial infarction, and pulmonary embolism are the most common thrombotic complications. Platelets have been described as the main drivers of thrombosis. Activated platelets directly participate in thrombus formation by interacting with fibrinogen, secreting different platelet agonists and participating in activation of the blood coagulation cascade. Recently, neutrophils in addition to platelets were described to play an important role in thrombus formation in both the arteries and veins. We previously showed that in a laser-induced injury model, neutrophils are the first cells present at the site of injury. Neutrophils interact with the injured vessel wall are activated by adenosine triphosphate (ATP) and generate tissue factor (TF) on their surface, leading to the generation of thrombin, which is involved in the recruitment of platelets (1, 2).Activated neutrophils have also been found to play an important role in the veins in a deep vein thrombosis (DVT) model through the secretion of neutrophil elastase (NE), leading to the degradation of TF pathway inhibitor (TFPI) and sustained activity of TF at the site of injury (3, 4). Once activated, neutrophils may produce neutrophil extracellular traps (NETs). NETosis involves different reactive oxygen species (ROS)-dependent and ROS-independent intracellular pathways and leads to decondensation of DNA by different histone deaminases and secretion of DNA and various proteins, such as the histones and enzymes originally present in neutrophil granules. NETosis involves different intracellular pathways depending on the neutrophil agonists used (5). The most commonly used agonists are phorbol myristate acetate (PMA), calcium ionophore, lipopolysaccharide (LPS), and bacteria (Streptococcus agalactiae), which have been shown to induce NETs within a few hours. ROS generation, the family of peptidyl arginine deiminase (PAD) enzymes, and neutrophil serine proteases such as NE are differentially required by different stimuli (6). The intracellular pathways may be dependent on ROS generation by NADPH oxidase and/or on histone citrullination by PAD4, MPO, and NE (7). In all cases, NETs produced upon stimulation are proteolytically active, kill bacteria, and are composed mainly of chromosomal DNA and proteins originally present in neutrophil granules.In vitro studies have shown that NETs may participate in platelet aggregation and activation of the blood coagulation cascade. Indeed, NETs are involved in thrombosis in several ways. NETs form scaffolds for the adhesion of platelets, red blood cells, and platelet adhesion molecules (fibrinogen, fibronectin, and von Willebrand factor [vWF]) (8). Moreover, these scaffold components trigger platelet activation and blood coagulation. For instance, histones such as histones H3 and H4 can interact with platelets via fibrinogen (9) or Toll-like receptor (TLR) 2 and 4 (10), resulting in platelet aggregation and thrombin generation (11). Additionally, neutrophil serine proteases such as NE, MPO, and cathepsin G enhance both the extrinsic coagulation pathway via proteolysis of TFPI (12) and the intrinsic coagulation pathway by binding and activating fXII (13). Although it is difficult to distinguish NETs from activated neutrophils in vivo, the main proof of concept that NETs indeed play a role in thrombus formation has come from experiments using DNase-I in mice. In a DVT model, injection of 100 U DNase-I significantly reduces the size of the thrombus (13).Since activated neutrophils are key cells involved in thrombus formation following laser-induced injury, the aim of the present study was to identify the role played by NETs in this model. We first used wild-type (WT) mice treated (or not) with DNase-I to investigate the role of NETs in vivo in a laser-induced model of thrombosis. We found that infusion of DNase-I into mice decreased thrombus growth, fibrin generation, neutrophil recruitment, and elastase release at the site of injury. However, surprisingly, we were not able to detect NETs at the site of thrombosis by electron microscopy. In contrast, we observed that DNase-I hydrolyzed ATP into adenosine diphosphate (ADP) and ADP into adenosine. Interestingly, both ATP and ADP are agonists of neutrophils and platelets, whereas adenosine inhibits neutrophil activation.In conclusion, we propose that DNase-I hydrolyzes ATP and ADP and induces adenosine production, leading to the inhibition of thrombus formation in vivo.