体外动态血栓形成试验在评价医用高分子材料血液相容性方面的应用研究

本文采用兔富血小板血浆进行体外动态血栓形成试验,以评价医用高分子材料的血液相容性。在观察不同医用高分子材料形成血栓过程中.发现整个过程可分为少量颗粒出现、大量颗粒涌现(即产生雪暴现象)、颗粒发生凝集、颗粒凝集回缩成块及形成圆柱形血小板血栓。本文对材料与血浆接触时间进行了研究,认为当材料与富血小板血浆接触60分钟后,可充分反映材料对富血小板血浆的作用。最后用本文建立的方法对五种高分子材料进行了测试。试验结果表明:特异血栓形成时间可作为评价医用高分子材料血液相容性的一个指标。     

活体微血栓形成过程的定量分析

在应用激发光照射荧光物质注射后的微血管来形成活体微血栓模型的基础上,开发了一种计算机图像处理方法,对微血栓形成的动态过程进行了定量的分析。利用这一方法不仅对形成过程中不同时刻微血栓形成的面积大小进行了测定,而且,进一步应用了计算机图像减影技术,将2幅不同时刻的形成过程中的图像相减,求出形成过程中血栓生长的程度和形状的变化。该方法不仅有利于血栓生长机制研究也可用作抗血栓药物的药效评价。     

大鼠软脑膜微血栓模型的建立及抗血栓药物的实验观察

以建立一个筛选抗血栓药物的体内微血栓模型为主要目的,本实验采用显微操作仪和微量注射技术,将二磷酸腺苷(ADP)溶液(1×10~(-2)M,5×10~(-2)M)微量注射到大鼠软脑膜微血管表面,在微血管中定位、定量诱发出白色的血小板性附壁血栓。通过对血栓形成过程的定量分析,观察几种体外血小板聚集抑制剂(阿斯匹林、654-2、川芎嗪)对血栓形成的影响。结果表明:在不损伤血管壁的条件下,单纯ADP(2μl/次)对微静脉的血栓诱发率达85%,病理切片结果发现血栓体主要由血小板构成。三种血小板聚集抑制剂均表现有不同程度的抗血栓作用。以上结果提示该微血栓模型在研究抗血栓药物及血栓形成机制方面可能有一定的实用价值。     

生物材料表面粘附的血小板形态变化及分类研究

本文通过狗的股动静脉短路半体内循环试验,使12种高分子材料与血液接触,然后对材料表面粘附的血小板形态结构进行大量扫描电镜观察,发现材料表面血小板粘附是一个动态过程,而且粘附的血小板并不一定都发生变性(即被激活);在早期粘附的血小板中大多未变性,在血流作用下,其中许多又重新返回血流,并不导致血栓形成。因此目前流行的把粘附血小板数目作为评价血液相容性的指标有一定局限性,会出现假阳性,而应该把变性的血小板数目作为评价指标。作者把血小板形态变化分成五型和十二个亚型,提出了半定量评价变性血小板的方法。     

夏至草醇提物对急性微循环障碍大鼠血小板功能的影响

目的观察夏至草醇提物（EEMI）对高分子右旋糖苷（Dextran500）致急性微循环障碍（AMD）大鼠血小板功能的影响。方法Wistar雄性大鼠分为夏至草组、模型组和对照组。前二组静注10% Dextran 500（10ml/kg）复制AMD模型（对照组以等量生理盐水代替）。6min后,夏至草组自颈静脉缓慢推注EEMI（1g/ml,6g/kg）,其它两组以等量生理盐水代替。40min后,观察血小板粘附与聚集功能、脑血流量和体外血栓形成。结果①模型组血小板聚集率（5min）显著低于对照组,而血小板聚集时间、解聚率及粘附率均显著高于对照组;夏至草组在不同时间点及最大的血小板聚集率均显著低于对照组和模型组,血小板聚集时间、解聚率及粘附率均显著高于对照组,但血小板粘附率显著低于模型组。②模型组及夏至草组的脑血流量、血栓长度、血栓湿重、血栓干重、血栓形成率均显著低于对照组,但夏至草组的血栓干重显著低于模型组。结论EEMI能明显降低Dextran 500致AMD大鼠的血小板聚集与粘附功能、减少血栓形成。     

东菱克栓酶对大鼠急性肠系膜微动脉血栓溶栓作用的实验研究

目的 探讨东菱克栓酶（ＤＦ－５２１）对在鼠肠系膜微动脉血栓的溶栓时间和机制。方法 采用光化学诱导法和静脉注射荧光素钠制作大鼠肠系膜微动脉血栓模型,经显微电视摄像系统和多媒体计算机图像处理技术相结合,定量分析不同剂量组ＤＦ－５２１的溶栓时间以及血管再通程度。     

GMP-140 RIA及其临床初步观察

活化血小板是血栓的主要组份之一，通过对体内活化血小板表面或血浆内的α颗粒膜蛋白(GMP-140)的检测，对临床有关血栓性疾病的早期诊断和预后将有很大帮助。也可作为代谢性疾病、自身免疫性疾病等体内血小板活化程度的特异性指标。本文对69例健康人和142例不同病人进行了全血中血小板表面GMP-140测定，观察其临床意义。     

应用高放大率显微镜分析纤维蛋白溶解系统功能低下患者的活血细胞行为特征

目的为血栓前状态的血标本检测提供细微、动态的细胞学行为指标。方法本研究采用“清华同方”高放大率显微系统（ＴＨＭＩ—ＵＰ型,可放大１６０００倍）对ＥＬＴ（优球蛋白溶解时间）延迟、组织纤溶酶原激活物（ｔ－ＰＡ）含量减少、纤溶酶原激活物抑制物（ＰＡＩ）及ａ２纤溶抑制物活性增高的标本进行检测。结果可见明显的红细胞聚集、血小板聚集和循环内皮细胞（ＣＥＣ）,这些因子对血栓的最终形成具有重要意义。结论纤溶蛋白溶解系统各项指标的检测在临床上应用广泛,常用来反映机体血栓前状态,其特异性一直是关注焦点,本实验利用高放大率显微系统从分析活血细胞的行为出发,为血栓前状态的血标本检测提供了更微细的细胞学行为特征。     

新型溶栓药—葡激酶对实验性脑微血栓溶栓效果的观察

目的 :探讨国产葡激酶对脑微血管富血小板血栓的溶栓效果。方法 :在成功建立了金黄地鼠软脑膜微动脉富血小板血栓模型的基础上 ,通过显微—录像图像监视系统动态观察了双龙葡激酶三个剂量组的溶栓效果 ,并与生理盐水及尿激酶作对比 ,记录分析溶栓、再栓及出血等情况。结果 :四个用药组均比生理盐水对照组有显著溶栓效果 ;双龙葡激酶三个剂量组均比尿激酶对照组有显著溶栓效果。结论 :对于富血小板血栓 ,双龙葡激酶具有较好的溶解效果 ,该效果优于尿激酶。     

微血栓模型的建立及定量分析

在血栓形成的大量因素中,血小板的行为起了很重要的作用,它们的粘附、聚集等功能直接影响着血栓形成的时间和体积的大小。但是,在活体中要评价血小板功能是很难定量的。本研究分别利用了电刺激方法和荧光染料体内注射结合滤过光照射后在金黄地鼠颊囊和大白鼠肠系膜的微血管中产生血小板凝集,进而产生血栓     

何首乌水提物对光化学反应诱导的大鼠肠系膜细静脉血栓形成的抑制作用

目的:探讨何首乌水提物对光化学反应诱导的大鼠肠系膜细静脉血栓形成的抑制作用。方法:将24只雄性Wistar大鼠随机分为正常组、模型组、何首乌组和阿司匹林组,每组6只。用光化学反应法诱导肠系膜细静脉血栓形成,观察各组血栓出现时间、血栓占血管面积一半所需时间、诱导停止后5min、10min、20min和30min时,各组血栓/血管面积比;然后在各组大鼠肠系膜表面滴加甲苯胺蓝,观察细静脉周围肥大细胞脱颗粒,并计算脱颗粒的肥大细胞比率。结果:何首乌水提物可以明显延迟血栓出现时间,明显缩短血栓占血管面积一半所需时间,显著降低血栓/血管面积比,显著减少血管周围肥大细胞脱颗粒。结论:何首乌水提物能明显抑制光化学反应诱导的大鼠肠系膜细静脉血栓形成。该作用可能与其抗氧化、抑制肥大细胞脱颗粒等密切相关。     

Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo

The exact mechanism of blood vessel thrombus formation remains to be defined. Here, we introduce a new approach to probe thrombus formation in blood vessels of living animals using intravital microscopy in green fluorescent protein (GFP)-transgenic mice to simultaneously monitor platelet aggregation and procoagulant activity. To this end, GFP-expressing platelets and annexin A5 labeled with a fluorescent dye were employed to visualize and analyze platelet aggregation and markers of procoagulant activity (platelet surface phosphatidylserine (PS)). Laser-induced thrombi increased and then decreased in size with time in vessels of living animals, whereas platelet surface PS initiated at the site of injury and then penetrated into the thrombus. PS-positive platelets were predominantly localized in the center of the thrombus, as was fibrin generation. The experimental system proposed here is a valuable tool not only for investigating mechanisms of thrombus formation but also to assess the efficacy of antithrombotic drugs within the vasculature.     

Thrombus organization plays no major role in late neointimal formation after angioplasty in porcine coronary arteries.

Thrombus organization has been suggested to play a major role in late neointimal formation after coronary angioplasty. We sought to describe the time sequence of lesion formation after angioplasty in porcine coronary arteries and to quantify the relation between early thrombosis and late neointimal formation. Deep vessel wall injury was induced by conventional balloon angioplasty in the circumflex (CX) and right coronary (RCA) arteries and by retraction of a chain-encircled balloon in the left anterior descendent artery (LAD). Lesions were assessed by histomorphometry at days 0, 1, 4, 7, 14, 28, and 56 after angioplasty. A response-to-injury index (lesion area/injury length) was determined for each artery. Angioplasty led to rupture/removal of media. Thrombus was present at the exposed adventitia at days 0, 1, and 4. From day 7, neointima was observed on the luminal side of the arterial wall. All thrombus had disappeared at day 28, at which only neointima was observed. Histomorphometry revealed that lesion formation after angioplasty was a gradually increasing process from day 0 to day 28 with no further growth from day 28 to day 56. Maximal thrombus size (day 4, RCA: 0.07+/-0.04 mm, CX: 0.23+/-0.16 mm, LAD: 0.15+/-0.11 mm) was significantly smaller than late neointimal formation (day 28, RCA: 0.68+/-0.18 mm, CX: 0.63+/-0.23 mm, LAD: 0.71+/-0.18 mm) in all three arteries (p < .03). Lesion formation after angioplasty is a gradually increasing process for 4 weeks. Maximal thrombus size is about four times smaller than late neointimal formation. Thus, thrombus organization plays no major role in late neointimal formation.     

Investigation of the influence of fluid dynamics on thrombus growth at the interface between a connector and tube

Thrombus formation at the interface between connectors and tubes is a potential risk factor for complications. We investigated time-dependent relationships between formation of thrombus and hemodynamic factors at the interface between connectors and tubes using optical coherence tomography (OCT) under pulsatile flow. A swept-source OCT with the center wavelength of 1330 nm was employed. The sequential process of thrombus formation at the interface of connectors and tubes in the inlet and outlet was investigated. Connectors with and without tapers were tested using identical 50-ml air-contactless circuits. Fresh human blood from healthy volunteers was circulated under pulsatile flow. Thrombus initially formed at the interface between the connector tip and the tube. Geometries of thrombus growth were different between the 2 connectors, and between the inlet and the outlet. Growth of thrombus was observed at the interface between the connectors and tubes over time in 60 min circulation, except at the outlet part of connector without tapers. At the connector without tapers outlet, thrombus propagation length from the connector edge toward the flow downstream was comparable at 10 and 60 min (0.55 ± 0.35 vs. 0.51 ± 0.32 mm, p = 0.83). Analysis using particle image velocimetry showed the presence of a flow reattachment point 1.5 mm downstream from the connector edge. These results suggest that the flow reattachment point inhibits downstream thrombus growth. We quantitatively demonstrated sequential thrombus process at the interface between the connectors and tubes under pulsatile flow of human blood using OCT.     

Underlying mechanisms of thrombus formation/growth in atherothrombosis and deep vein thrombosis

Thrombosis remains a leading cause of death worldwide despite technological advances in prevention, diagnosis, and treatment. The traditional view of arterial thrombus formation is that it is a platelet-dependent process, whereas that of venous thrombus formation is a coagulation-dependent process. Current pathological and basic studies on atherothrombosis and venous thrombosis have revealed the diverse participation of platelet and coagulation activation mechanisms in both thrombus initiation and growth processes during clinical thrombotic events. Atherosclerotic plaque cell-derived tissue factor contributes to fibrin formation and platelet aggregation. The degree of plaque disruption and a blood flow alteration promote atherothrombotic occlusion. While blood stasis/turbulent flow due to luminal stenosis itself initiates venous thrombus formation. The coagulation factor XI-driven propagation phase of blood coagulation plays a major role in venous thrombus growth, but a minor role in hemostasis. These lines of evidence indicate that atherothrombosis onset is affected by the thrombogenic potential of atherosclerotic plaques, the plaque disruption size, and an alteration in blood flow. Upon onset of venous thrombosis, enhancement of the propagation phase of blood coagulation under blood stasis and a hypercoagulable state contribute to large thrombus formation.     

Experience with vascular grafts in total artificial heart replacement

In total artificial heart replacement the pumps are attached to the vascular system with the help of connectors. These consist of a woven Dacron vessel graft to which a short silastic segment is vulcanized. In 36 calves surviving total artificial heart replacement between one and seven months (average 85 days) the morphological alterations due to interfacial reactions were studied: thrombus formation and neointimal fibrous hyperplasia at the anastomoses. In 15 calves (41.7%) thrombus growth within the outflow tract led to anastomotic stenosis: pulmonary artery 14 (93.3%), aorta-anastomosis 1 (2.8%), both vessels 1 (2.8%), in combination with pannus growth in atrial location 13 (86.7%). In 73.3% the pannus consisted of infected organized thrombus imitating the course of septic vegetative endocarditis. Two calves were reoperated in order to remove the vegetative thrombi, one successfully. In five animals pulmonary stenosis was the main cause of death. The presence and location of excessive tissue growth and thrombus formation within the outflow tract are also inherent to the fluid mechanical design of the valve. Neointimal fibrous hyperplasia at the anastomoses of the grafts seems to be a reparative process started up by platelet-induced subendothelial cell proliferation in response to intimal injuries.     

Correlation of in vivo clot deposition with the flow characteristics in the 50 cc penn state artificial heart: a preliminary study

Flow stasis in an artificial heart may provide a situation where thrombus develops. Should part, or all, of the clot dislodge, a thromboembolism may lead to stroke(s), neurologic deficits, or even death. In an effort to determine if the regime of low shear or stasis exists, a two-dimensional particle image velocimetry (PIV) system was implemented to measure the velocity field within the 50 cc Penn State Artificial Heart. The velocity measurements were decomposed nearest the wall to obtain wall shear rates along the bottom of the chamber. The PIV measurements were made in three image planes across the depth of the chamber to reconstruct a surface distribution of the wall shear rates at the bottom over the entire heart cycle. The wall shear rate is shown to be spatially nonuniform, with persistently low wall shear rates. An area near the front edge of the chamber at the bottom showed wall shear rates not exceeding 250 s(-1). This was an area of clot formation seen in vivo, suggesting a link may exist between the low wall shear rate zone and thrombus formation.     

Flow vectorial analysis in an artificial implantable lung

An artificial implantable lung would be a useful device to support patients awaiting lung transplantation. A suitable device must offer low resistance and adequate gas exchange, be impermeable to plasma, and nonthrombogenic. Although plasma permeability is an intrinsic quality of the materials, the other requirements are largely a function of device geometry, particularly as it relates to fluid dynamics. Using a CAD system and the requirements of a membrane surface area of 1.5 m2 and an inlet outlet port distance of 12 cm, we designed 10 models that varied in their other dimensions. Computational fluid dynamic (CFD) software was applied to the models to determine which minimized regions of low flow velocity. A prototype built to these specifications was used in an in vivo ovine experiment to verify the CFD predictions. The prototype was placed in parallel to the native pulmonary circulation (pulmonary artery to left atrium) for 120 minutes while the activated coagulation times were kept between 110 and 120 seconds and device flow was maintained between 1.5 and 2.5 L/min. Examination of the prototype confirmed a correlation between predicted areas of low flow and thrombus formation. Although nearly identical low flow velocity conditions exist at both the inlet and outlet ports, thrombus formation occurs only near the outlet port. This finding agrees with detailed vectorial analysis, which predicts a more complex flow pattern near the outlet port. Although near the inlet port flow vectors are nearly parallel, near the outlet port flow vectors collide. This area of flow collision corresponds to the area of thrombus formation in vivo. The addition of microflow vectorial analysis to flow velocity predictions allows for improved accuracy in predicting regions at risk of thrombosis in an artificial implantable lung.     

The effect of convective flows on blood coagulation processes

Two mathematical models of clot growth in the fluid flows have been considered. The first one is the model of embolus growth in a wall-adjacent flow. The effect of hydrodynamic flows on proceeding chemical reactions and the backward effect of the growing clot on the flow are taken into account. The growing thrombus is assumed to be porous and having low permeability, that is in good agreement with experimental data. The exact solutions determining the distribution of a fluid velocity close to the embolus have been used. Numerical analysis of these solutions have demonstrated that hydrodynamic flows can essentially affect the processes of blood coagulation, and consequently on the clot structure. Their presence might lead to the destruction of chemical fronts having a cylindrical symmetry and formation of the so-called chemical spots. The second model describes the initial stage of thrombus growing in the hemorrhage into a natural internal space. It permits accounting for vessel geometry and provides studying the effects of geometric parameters on fluid flows and coagulation processes. The process of thrombus growth is shown to depend on the ratio of typical values of blood velocity in the vessel and rate of chemical reactions.