Pathophysiology of atherothrombosis: Mechanisms of thrombus formation on disrupted atherosclerotic plaques

Atherothrombosis is a leading cause of cardiovascular mortality and morbidity worldwide. The underlying mechanisms of atherothrombosis comprise plaque disruption and subsequent thrombus formation. Arterial thrombi are thought to mainly comprise aggregated platelets as a result of high blood velocity. However, thrombi that develop on disrupted plaques comprise not only aggregated platelets, but also large amounts of fibrin, because plaques contain large amount of tissue factor that activate the coagulation cascade. Since not all thrombi grow large enough to occlude the vascular lumen, the propagation of thrombi is also critical in the onset of adverse vascular events. Various factors such as vascular wall thrombogenicity, local hemorheology, systemic thrombogenicity and fibrinolytic activity modulate thrombus formation and propagation. Although the activation mechanisms of platelets and the coagulation cascade have been intensively investigated, the underlying mechanisms of occlusive thrombus formation on disrupted plaques remain obscure. Pathological findings derived from humans and animal models of human atherothrombosis have uncovered pathophysiological processes during thrombus formation and propagation after plaque disruption, and novel factors have been identified that modulate the activation of platelets and the coagulation cascade. These findings have also provided insights into the development of novel drugs for atherothrombosis.     

Procoagulant microparticles: 'criminal partners' in atherothrombosis and deleterious cellular exchanges

Procoagulant microparticles (MP) constitute valuable hallmarks of vascular cell damage at the crossroad of atherothrombosis processes. Detectable at low concentrations in the blood flow of healthy individuals, elevated levels of procoagulant microparticles are characteristic features of most cardiovascular risk factors. Circulating MP support cellular cross-talk leading to vascular inflammation, endothelial dysfunction, leukocyte adhesion and recruitment possibly contributing to plaque growth with consecutive development of local thrombosis and altered vasomotion. Within the plaque, MP shed by apoptotic monocytes and smooth muscle cells are major determinant of plaque thrombogenicity mainly through the presence of tissue factor (TF) activity. Besides this procoagulant potential, trapped MP could contribute to plaque vulnerability through multiple pathways including angiogenesis, extracellular matrix proteolysis, recruitment of inflammatory cells, smooth muscle cell and endothelial apoptosis. Having long been considered sufficient to initiate coagulation following plaque disruption, the role assigned to plaque-bound TF does not appear physically realistic at a macroscopic scale, the swift growth of the thrombus probably involving blood-borne TF conveyed by circulating MP. As participants in crucial steps of atherosclerotic disease, MP can now be viewed as "partners in crime" in acute ischemic syndromes.     

血管血栓性疾病的发病机制和防治

血管血栓性疾病是多发病、常见病,它严重危害人类健康。其发病机制是血管中血栓形成,引起血栓形成的因素很多,危险因素有基因、环境和生活行为(如吸烟)。动脉血栓形成的基础病变是动脉粥样硬化,血小板黏附聚集在动脉血栓形成中起“扳机”作用,静脉血栓形成的主要因素是淤血和高凝。预防动脉血栓形成主要是降血脂和抗血小板黏附、聚集,而预防静脉血栓形成则抗凝和采取措施减轻淤血。动脉血栓的治疗方案主要是溶血栓和/或经皮冠脉介入术(PC I),静脉血栓的治疗方案主要是抗凝。此外,本文还介绍了微血管血栓的形成机制和防治。     

Blood coagulation in immunothrombosis—At the frontline of intravascular immunity

While hemostasis is the physiological process that prevents blood loss after vessel injury, thrombosis is often portrayed as a pathologic event involving blood coagulation and platelet aggregation eventually leading to vascular occlusion and tissue damage. However, recent work suggests that thrombosis can also be a physiological process, termed immunothrombosis, initiated by the innate immune system providing a first line of defense to locally control infection. Fibrin forms the structural basis of immunothrombotic clots and its assembly involves the concerted action of coagulation factors, platelets and leukocytes. Here, we summarize the cellular and molecular events that initiate fibrin formation during the innate immune response and discuss how aberrant activation of these pathways fosters pathologies associated with thrombosis, including disseminated intravascular coagulation and atherothrombosis.     

Involvement of neutrophils in thrombus formation in living mice

Thrombosis is one of the major causes of human death worldwide. Identification of the cellular and molecular mechanisms leading to thrombus formation is thus crucial for the understanding of the thrombotic process. To examine thrombus formation in a living mouse, new technologies have been developed. Digital intravital microscopy allows to visualize the development of thrombosis and generation of fibrin in real-time within living animal in a physiological context. This specific system allowed the identification of new cellular partners involved in platelet adhesion and activation. Furthermore, it improved, especially, the knowledge of the early phase of thrombus formation and fibrin generation in vivo. Until now, platelets used to be considered the sole central player in thrombus generation. However, recently, it has been demonstrated that leukocytes, particularly neutrophils, play a crucial role in the activation of the blood coagulation cascade leading to thrombosis. In this review, we summarized the mechanisms leading to thrombus formation in the microcirculation according to the method of injury in mice with a special focus on the new identified roles of neutrophils in this process.     

The gut microbiota: An emerging risk factor for cardiovascular and cerebrovascular disease

Commensal gut microbiota have recently been implicated in cardiovascular disease (CVD) and cerebrovascular disease. Atherosclerotic plaque formation depends on the colonization status of the host. In addition to host nutrition and the related microbiota‐dependent metabolic changes, activation of innate immune pathways triggers the development of atherosclerosis and supports arterial thrombosis. Gnotobiotic mouse models have uncovered that activation of Toll‐like receptor‐2 by gut microbial ligands supports von Willebrand factor‐integrin mediated platelet deposition to the site of vascular injury. Depending on nutritional factors, the microbiota‐derived choline‐metabolite trimethylamine N‐oxide (TMAO) increases atherosclerotic plaque size, triggers prothrombotic platelet function and promotes arterial thrombus growth. Hence, the composition of the commensal microbiota is an emerging risk factor for CVD. Here, we provide an overview on microbiota‐dependent pathomechanisms that drive the development of CVD and arterial thrombosis.     

Proximal atherosclerotic lesion as a cause of very late stent thrombosis

Very late stent thrombosis is defined as in-stent thrombosis occurring after 1 year of an intra-coronary artery stent placement. Drug eluting stents have lately been criticized for increased reports of very late stent thrombosis. The exact cause of these very late stent thromboses is not clearly understood. Virchow's triad describes the three main factors of thrombus formation to be stasis of blood flow, endothelial injury and hypercoagulability. Based on Virchow's triad, we propose the cause of very late stent thrombosis to be formation of a de novo atherosclerotic lesion in the proximal segment of a stented artery. The de novo atherosclerotic lesion narrows the vessel lumen and causes stasis of blood flow in the distal stent. The de novo lesion can also cause myocardial ischemia creating a prothrombotic environment in the stented region. Stasis of blood flow and prothrombotic environment in the stented region can lead to the formation of very late stent thrombosis. Since atherosclerosis is a dynamic aging process in humans, we propose de novo proximal lesions in the coronary arteries can predispose to very late stent thrombosis.     

Virchow's triad revisited: abnormal flow

Virchow rightly recognised that blood flow plays an important role in thrombosis. The roles of blood flow in haemostasis, and in arterial, intra-cardiac, and venous thrombosis are reviewed.In streamline (laminar) flow, shear stresses are maximal at the vessel wall, and affect endothelial cell morphology and function (e.g. secretion of NO, prostacyclin,t-PA and vWF). Platelets are also concentrated at the vessel wall (due to axial concentration of red cells)where they can be activated by high shear stresses and are well-placed to interact with vWF and subendothelium,resulting in platelet adhesion and the initial stages of haemostasis. On the other hand, increasing wall shear forces increase removal of thrombin and fibrin monomer, hence stasis (induced by internal or external pressure) is required to allow fibrin formation and secondary haemostasis. Atherogenesis occurs in areas of arterial flow separation,which promotes platelet, leucocyte, LDL and fibrinogen adhesion and wall infiltration. Rheological variables (e.g. wall shear stress, viscosity, haematocrit,fibrinogen, LDL) have been correlated with the extent of ultrasonic carotid intima-media thickening. Arterial thrombosis usually follows rupture of atherosclerotic plaques and intra-plaque haemorrhage: high intra-stenotic shear stresses may activate platelets,promoting the initial platelet-rich "white-head" of arterial thrombi, while low post-stenotic shear stresses may promote the subsequent, fibrin--and red cell-rich "red tail". Blood viscosity, platelet microemboli, and activated leucocytes may each reduce post-stenotic microcirculatory blood flow, promoting infarction. Such mechanisms may explain the associations of increased levels of blood and plasma viscosity, haematocrit, white cell count, fibrinogen and vWF with risk and outcome of myocardial, cerebral and limb infarction. Areas of recirculating blood flow under low shear stresses predispose to intracardiac thromboembolism(e.g. atrial fibrillation, in which elevated fibrin D-dimer levels are normalised after cardioversion) and venous thromboembolism (fibrin D-dimer levels are associated with most risk factors). There is good evidence that reduction of venous stasis in the legs reduces the risk of venous thromboembolism.There is increasing evidence that regular exercise and avoidance of immobility reduces the risk of both arterial and venous thrombosis and also has systemic antithrombotic and anti-inflammatory effects. So: "Go with the flow!"     

D-二聚体动态监测对预测神经脊柱修复术后下肢深静脉血栓形成的意义

BACKGROUND: Deep vein thrombosis is a common postoperative complication after spinal surgery in clinical department of neurosurgery and department of orthopedics. Deep vein thrombosis is mostly related to vein intima injury, stasis and activation of blood coagulation factor. Early effective prediction can effectively avoid the adverse effects on the prognosis of patients with deep vein thrombosis. D-dimer used in the prediction of deep venous thrombosis has high sensitivity and specificity, and can be used as a sensitive predictor for deep vein thrombosis.     

Avascular necrosis of the femoral head: vascular hypotheses.

Vascular hypotheses provide compelling pathogenic mechanisms for the etiology of avascular necrosis of the femoral head (ANFH). A decrease in local blood flow of the femoral head has been postulated to be the cause of the disease. Several studies in human and animal models of ANFH have shown microvascular thrombosis. Endothelial cell damage could be followed by abnormal blood coagulation and thrombus formation with any resulting degeneration distal to the site of vascular occlusion. Other studies suggest that thrombophilia, particularly impaired fibrinolysis, plays a potential role in thrombus formation in ANFH. Reduction in shear stress due to decreased blood flow could lead to apoptosis of endothelial cells, which can ultimately contribute to plaque erosion and thrombus formation. Dysregulation of endothelial cell activating factors and stimulators of angiogenesis or repair processes could also affect the progression and outcome of ANFH. Likewise, regional endothelium dysfunction (RED), referred to as a potential defect in endothelial cells located in the feeding vessels of the femoral head itself, may also have a crucial role in the pathogenesis of ANFH. Molecular gene analysis of regional endothelial cells could also help to determine potential pathways important in the pathogenesis of ANFH.     

肝癌肝移植患者围手术期凝血功能变化的癌栓分组研究

目的在局部晚期肝癌肝移植患者中，观察癌栓患者和无癌栓患者围手术期凝血功能的变化。方法回顾性分析中山大学肿瘤防治中心2003年9月至2007年1月行肝移植的局部晚期肝癌患者31例。根据有无癌栓分为两组，比较两组患者移植前、术中无肝期、术后3d内的血小板（platelet，PLT）计数、血浆凝血酶原时间（prothrombin time，PT）、活化部分凝血酶原时间（activated paaial thromboplastin time，APTT）、血浆纤维蛋白原（fibrinogen，Fib）和血浆凝血酶时间（thrombin time，Tr）等凝血指标的测定结果，分析凝血功能的变化。结果与术前相比，两组患者PT、APTT和TT升高以及Fib和PLT降低以无肝期最为显著（P〈0．01），术后患者延长的PT、APTT、TT和升高INR逐渐回落。降低的Fib和PLT恢复较慢。组间比较，术前癌栓组患者Fib和PLT低于无癌栓组（P〈0．05）。术中无肝期及术后第1天，癌栓组患者PT、APTT和TT升高以及Fib和PLT降低程度都高于无癌栓组患者，围手术期癌栓组凝血物质的使用量多于无癌栓组。结论癌栓患者和无癌栓患者肝移植围手术期凝血功能变化趋势大致相同：癌栓患者围手术期低凝的因素较多，低凝状态在术中及术后1～2d表现更为突出，此类患者对凝血物质的需求较大，要注意补给。     

Changes in platelet function in the course of formation of an intravascular venous thrombus

Experiments on rabbits showed that the period of organization of a venous thrombus and the first 24 h after thrombosis is characterized by a decrease in the number of platelets and in their coagulation, adhesive, and electrophoretic activity and by an increase in their aggregation and sedimentation rate. The changes in platelet function became less marked 2–5 days after thrombosis and were actually characteristic of hemorrhage.     

Procoagulant Properties of Flow Fields in Stenotic and Expansive Orifices

In the United States, over 125,000 mechanical heart valves (MHVs) are implanted each year. Flow through the MHV hinge can cause thromboemboli formation. The purpose of this study was to examine various orifice geometries representing the MHV hinge region and how these geometries may contribute to platelet activation and thrombin generation. We also characterized these flow fields with digital particle image velocimetry (DPIV). Citrated human blood at room temperature was forced through the orifices (400 and 800 μm ID) with a centrifugal bypass pump, continuously infusing calcium chloride to partially reverse the citrate anticoagulant. Blood samples were tested for the presence of thrombin–antithrombin complex (TAT) and platelet factor 4 (PF4). Velocity and shear stress were measured with DPIV using a blood analog fluid seeded with fluorescent microbeads. The results indicate that small changes in geometry, although they do not affect the bulk flow, change the coagulation propensity as blood flows through the orifices. A more abrupt geometry allows more stagnation to occur resulting in more thrombin generation. PF4 measurements indicated similar levels of platelet activation for all orifices. DPIV showed differences in the jets with respect to entrainment of stagnant fluid. These results help to pinpoint the important parameters that lead to flow stasis and subsequent thrombus formation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A computational model based on fibrin accumulation for the prediction of stasis thrombosis following flow-diverting treatment in cerebral aneurysms

Flow diverters, the specially designed low porosity stents, have been used to redirect blood flow from entering aneurysm, which induces flow stasis in aneurysm and promote thrombosis for repairing aneurysm. However, it is not clear how thrombus develops following flow-diversion treatment. Our objective was to develop a computation model for the prediction of stasis-induced thrombosis following flow-diversion treatment in cerebral aneurysms. We proposed a hypothesis to initiate coagulation following flow-diversion treatment. An experimental model was used by ligating rat’s right common carotid artery (RCCA) to create flow-stasis environment. Thrombus formed in RCCA as a result of flow stasis. The fibrin distributions in different sections along the axial length of RCCA were measured. The fibrin distribution predicted by our computational model displayed a trend of increase from the proximal neck to the distal tip, consistent with the experimental results on rats. The model was applied on a saccular aneurysm treated with flow diverter to investigate thrombus development following flow diversion. Thrombus was predicted to form inside the sac, and the aneurysm was occluded with only a small remnant neck remained. Our model can serve as a tool to evaluate flow-diversion treatment outcome and optimize the design of flow diverters.     

Transient effect of aspirin on collagen-induced platelet accumulation

A piece of collagen fiber (catgut) was placed into polyethylene tubing and perfused with heparinized blood. The perfusion pressure was monitored, and the pressure rise indicated platelet accumulation around collagen leading to occlusion of the tubing. Continuous slow infusion of prostacyclin into the blood or monoclonal antibody against von Willebrand factor completely prevented thrombus formation. Aspirinization of blood (0.1-2 mM) resulted in a concentration-dependent delay of the onset of thrombus growth, but even at the highest aspirin concentration, the thrombus did finally grow and at the same rate as that of the nonaspirinated control platelets. These findings question the clinical anti-thrombotic benefit of aspirin as platelet release inhibitor.     

Hypoxia-reoxygenation contributes to increased frequency of venous thromboembolism in air travellers

Commercial air travel is widespread, with close to 2 billion people traveling each year. The association between venous thromboembolism (VTE) and confined sitting has been established and a correlation between the distance traveled and its incidence had been documented. Most studies underscore stasis caused by immobility as the cause of VTE and ignore the possible contribution of additional factors including coagulation and the blood vessel wall. Recent studies indicate that hypobaric hypoxia may contribute to blood vessel wall alteration and activation of coagulation. We suggest that air travel associated hypobaric hypoxia may lead to endothelial injury and initiation of a pro-coagulatory response, effects which are augmented by the reoxygenation upon landing, thus culminating in thrombosis. Prevention of air travel associated hypoxia and reoxygenation injury may decrease the incidence of flight associated VTE.     

Blood compatibility of a newly developed trileaflet mechanical heart valve

An ideal heart valve prosthesis, which has both the flow dynamic properties and blood compatibility of a tissue valve prosthesis and the durability of a mechanical prosthesis, does not exist. The Triflo trileaflet mechanical heart valve (MHV; Triflo Medical Inc., Irvine, CA) is a newly developed MHV prosthesis with the following design goals: central flow, minimal flow disturbance and stasis around the hinge region, and durability. The current study was conducted to evaluate the blood compatibility of a 29 mm Triflo MHV in the mitral position of eight calves for 5 months without any postoperative anticoagulation. Whole blood platelet aggregometry and the Xylum Clot Signature Analyzer (Xylum Corporation, Scarsdale, NY) were used to evaluate the postoperative changes in platelet and coagulation functions. Full autopsies, histological examinations of major internal organs, and scanning electron microscopy analyses of the explants were performed. Early termination occurred in two cases; one was because of valve thrombosis on the 25th day, and the other was killed because of a nonvalvular complication on the 105th day. The valve thrombosis was attributed to prolonged ventricular fibrillation at the time of valve replacement surgery. Whole blood platelet aggregometry and clot signature analyzer parameters did not show any sign of activation of platelets or the coagulation system. No hemolysis was observed. There was no macroscopic valve thrombosis or embolism observed in the remaining seven cases. Scanning electron microscopy analyses showed clean leaflet and valve ring surfaces, with only occasional minute platelet aggregations. Excellent blood compatibility of the Triflo MHV was demonstrated in this study.     

Computational Model of Device-Induced Thrombosis and Thromboembolism

A numerical model of thrombosis/thromboembolism (T/TE) is presented that predicts the progression of thrombus growth and thromboembolization in low-shear devices (hemodialyzers, oxygenators, etc.). Coupled convection–diffusion-reaction equations were solved to predict velocities, platelet agonist (ADP, thromboxane A2, and thrombin) concentrations, agonist-induced and shear-induced platelet activation, and platelet transport and adhesion to biomaterial surfaces and adherent platelets (hence, thrombus growth). Single-platelet and thrombus embolization were predicted from shear forces and surface adhesion strengths. Values for the platelet-biomaterial reaction constant and the platelet adhesion strength were measured in specific experiments, but all other parameter values were obtained from published sources. The model generated solutions for sequential time steps, while adjusting velocity patterns to accommodate growing surface thrombi.Heparinized human blood was perfused (0.75 ml/min) through 580 μm-ID polyethylene flow cells with flow contractions (280 μm-ID). Thrombus initiation, growth, and embolization were observed with videomicroscopy, while embolization was confirmed by light scattering, and platelet adhesion was determined by scanning electron microscopy.Numerical predictions and experimental observations were similar in indicating: 1) the same three thrombotic locations in the flow cell and the relative order of thrombus development in those locations, 2) equal thrombus growth rates on polyethylene and silicon rubber (in spite of differing overall T/TE), and 3) similar effects of flow rate (1.5 ml/min versus 0.75 ml/min) on platelet adhesion and thrombosis patterns.     

Hemostatic evaluation in bleeding disorders from native blood. Clinical experience with the hemostatometer

Primary hemostasis (PH), i.e., hemostatic platelet plug formation, and the subsequent coagulation were recorded and quantified from the same nonanticoagulated venous blood sample with the use of the Haemostatometer. In addition, platelet thrombus formation induced by interaction of flowing native blood with a collagen fiber under low shear rates (450 s-1) was simultaneously analyzed by this device. The effect of monoclonal antibodies (MoAbs) directed against von Willebrand's factor antigen (vWF:Ag), platelet glycoprotein Ib (GPIb) and the GPIIb/IIIa complex, and fibrinogen were studied. PH was significantly inhibited by MoAbs against vWF:Ag, GPIIb/IIIa, and fibrinogen but was unaffected by antibody against GPIb. Collagen-induced thrombosis was prevented by MoAbs against vWF:Ag and GPIb, slightly inhibited by antifibrinogen, and unaffected by blockage of platelet membrane GPIIb/IIIa. The effect of a single 600-mg dose of aspirin was monitored, and abnormal PH was still detectable five days later. From the 13 hemophiliacs tested, 7 showed significantly prolonged PH. In von Willebrand's disease, a characteristic defect of PH with significant inhibition or absence of collagen-platelet interaction was observed in all the 11 patients. PH was greatly prolonged in both of the two patients with storage pool deficiency. The technique detected improvement of platelet function, i.e., PH in all of six patients with bleeding disorders after replacement therapy or DDAVP infusion. The authors conclude that the Haemostatometer technique is a sensitive test for determining platelet dysfunction and monitoring efficacy of factor-replacement or DDAVP therapy.     

Advancements in left ventricular assist devices to prevent pump thrombosis and blood coagulopathy

Mechanical circulatory support (MCS) devices, such as left ventricular assist devices (LVADs) are very useful in improving outcomes in patients with advanced-stage heart failure. Despite recent advances in LVAD development, pump thrombosis is one of the most severe adverse events caused by LVADs. The contact of blood with artificial materials of LVAD pumps and cannulas triggers the coagulation cascade. Heat spots, for example, produced by mechanical bearings are often subjected to thrombus build-up when low-flow situations impair washout and thus the necessary cooling does not happen. The formation of thrombus in an LVAD may compromise its function, causing a drop in flow and pumping power leading to failure of the LVAD, if left unattended. If a clot becomes dislodged and circulates in the bloodstream, it may disturb the flow or occlude the blood vessels in vital organs and cause internal damage that could be fatal, for example, ischemic stroke. That is why patients with LVADs are on anti-coagulant medication. However, the anti-coagulants can cause a set of issues for the patient—an example of gastrointestinal (GI) bleeding is given in illustration. On account of this, these devices are only used as a last resort in clinical practice. It is, therefore, necessary to develop devices with better mechanics of blood flow, performance and hemocompatibility. This paper discusses the development of LVADs through landmark clinical trials in detail and describes the evolution of device design to reduce the risk of pump thrombosis and achieve better hemocompatibility. Whilst driveline infection, right heart failure and arrhythmias have been recognised as LVAD-related complications, this paper focuses on complications related to pump thrombosis, especially blood coagulopathy in detail and potential strategies to mitigate this complication. Furthermore, it also discusses the LVAD implantation techniques and their anatomical challenges.