Generation Of Thrombi Research Articles

A two-stage exacerbated hypoxia nanoengineering strategy induced amplifying activation of tirapazamine for microwave hyperthermia-chemotherapy of breast cancer

Microwave hyperthermia (MH) is an emerging treatment for solid tumors, such as breast cancer, due to its advantages of minimally invasive and deep tissue penetration. However, MH induced tumor hypoxia is still an obstacle to breast tumor treatment failure. Therefore, an original nanoengineering strategy was proposed to exacerbate hypoxia in two stages, thereby amplifying the efficiency of activating tirapazamine (TPZ). And a novel microwave-sensitized nanomaterial (GdEuMOF@TPZ, GEMT) is designed. GdEuMOF (GEM) nanoparticles are certified excellent microwave (MW) sensitization performance, thus improving tumor selectivity to achieve MH. Meanwhile MW can aggravate the generation of thrombus and caused local circulatory disturbance of tumor, resulting in the Stage I exacerbated hypoxia environment passively. Due to tumor heterogeneity and uneven hypoxia, GEMT nanoparticles under microwave could actively deplete residual oxygen through the chemical reaction, exacerbating hypoxia level more evenly, thus forming the Stage II of exacerbated hypoxia environment. Consequently, a two-stage exacerbated hypoxia GEMT nanoparticles realize amplifying activation of TPZ, significantly enhance the efficacy of microwave hyperthermia and chemotherapy, and effectively inhibit breast cancer. This research provides insights into the development of progressive nanoengineering strategies for effective breast tumor therapy.

Changes in Cardiovascular Structure and Physiology Following Polystyrene Nanoparticle Exposure

Background 
 Microplastic (MP) pollution has been a growing concern in recent decades due to the proliferation and ease of manufacturing regarding plastics products. Polystyrene (PS), being one the most abundant plastic polymers, is the subject of frequent studies due to its ubiquitous nature. Trophic transfer, inhalation, and dermal exposure are all routes by which humans may be exposed to MPs every day. There are undoubtedly more physiological consequences than we are currently aware of; however, many of these concerns are beginning to be better understood. Their distribution throughout the body dictates the potential threats to human health. The cardiovascular system is potentially the most susceptible, as it is the first medium the MPs are exposed to after entering the body. Consequently, the cardiovascular system and thrombus generation is one such area where much recent attention has been allocated. 
 Project Methods 
 Data was gathered from a variety of well-established research articles to review the relationship between PS exposure and thrombus formation in vivo. Additionally, this review looks at documented effects of MPs on platelet aggregation and discusses mechanisms through which these pathologies take place. 
 Results 
 PS plastics have varying impact on thrombus formation contingent upon both surface modifications and particle size. Aminated PS appears to be the most potent regarding the generation of thrombi in vivo. Literature on platelet aggregation is more consistent, with most studies revealing that carboxylated and aminated forms enhance aggregation while unmodified PS had insignificant effects. 
 Conclusion/Potential Impact 
 There is substantial ambiguity surrounding this field and more studies need to be conducted to reveal the full extent of pathologies caused by PS microplastics. Most studies have been done with marine life, which may not accurately reflect MPs effects in humans. Further research will allow us to begin developing viable solutions for those most susceptible to cardiovascular diseases.

Ovarian cancer circulating extracelluar vesicles promote coagulation and have a potential in diagnosis: an iTRAQ based proteomic analysis

BackgroundCirculating extracelluar vesicles (EVs) in epithelial ovarian cancer (EOC) patients emanate from multiple cells. These EVs are emerging as a new type of biomarker as they can be obtained by non-invasive approaches. The aim of this study was to investigate circulating EVs from EOC patients and healthy women to evaluate their biological function and potential as diagnostic biomarkers.MethodsA quantitative proteomic analysis (iTRAQ) was applied and performed on 10 EOC patients with advanced stage (stage III–IV) and 10 controls. Twenty EOC patients and 20 controls were applied for validation. The candidate proteins were further validated in another 40-paired cohort to investigate their biomarker potential. Coagulation cascades activation was accessed by determining Factor X activity.ResultsCompared with controls, 200 proteins were upregulated and 208 proteins were downregulated in the EOC group. The most significantly involved pathway is complement and coagulation cascades. ApoE multiplexed with EpCAM, plg, serpinC1 and C1q provide optimal diagnostic information for EOC with AUC = 0.913 (95% confidence interval (CI) =0.848–0.957, p < 0.0001). Level of activated Factor X was significantly higher in EOC group than control (5.35 ± 0.14 vs. 3.69 ± 0.29, p < 0.0001).ConclusionsOur study supports the concept of circulating EVs as a tool for non-invasive diagnosis of ovarian cancer. EVs also play pivotal roles in coagulation process, implying the inherent mechanism of generation of thrombus which often occurred in ovarian cancer patients at late stages.

Blood-Contacting Biomaterials: In Vitro Evaluation of the Hemocompatibility.

Hemocompatibility of blood-contacting biomaterials is one of the most important criteria for their successful in vivo applicability. Thus, extensive in vitro analyses according to ISO 10993-4 are required prior to clinical applications. In this review, we summarize essential aspects regarding the evaluation of the hemocompatibility of biomaterials and the required in vitro analyses for determining the blood compatibility. Static, agitated, or shear flow models are used to perform hemocompatibility studies. Before and after the incubation of the test material with fresh human blood, hemolysis, cell counts, and the activation of platelets, leukocytes, coagulation and complement system are analyzed. Furthermore, the surface of biomaterials are evaluated concerning attachment of blood cells, adsorption of proteins, and generation of thrombus and fibrin networks.

Abstract B24: De-clotting tumor to improve the perfusion, distribution and efficacy of chemotherapy and nanotherapeutics

Abstract Tumors exhibit several physical and physiological barriers that limit the distribution of molecules as small as oxygen; tumor barriers are compressed vessels due to solid stress, immature vasculature with geometric resistances, high interstitial fluid pressure, and the dense extracellular matrix. Tumors are also thrombotic in nature; pancreatic, colon, gastric, lung, ovarian cancers, melanoma, glioblastoma, lymphomas, etc. are associated with a high rate of thrombosis. Solid tumors obstruct venous return which cause static blood flow, endothelial cell injury, and coagulation activation. We hypothesize that coagulation/thrombosis act as a drug transport barrier inside the tumors and the use of anticoagulants or anti-thrombotics overcome this barrier. We posit that perfusion and penetration of drugs trap in thrombotic tumor vessels, whereas the small intra-thrombotic spacing within the vessel wall and interstitial space of tumor retards the movement of particles. In this work, we show that heparin or heparin-based anticoagulants prevent clot formation in tumors, enhances blood perfusion and nanoparticle distribution, and potentiates both chemo- and nano-therapeutic efficiency in tumor treatment. We compared the vessel perfusion and their thrombi fractions in tumors of P-selectin-/- vs wild-type (WT) mice. B16f10 melanoma cells were grown subcutaneously in P-selectin-/- and WT mice and Cy7 labeled dextran (Dex-Cy7; as a marker of perfused vessels) and Cy5.5 labeled fibrinoigen (Fib-Cy5.5; a marker of blood clots) were injected simultaneously through the tail vein. B16f10 tumors in WT mice grew with high level of thrombus, as Fib-Cy5.5 concentrated inside their vessels. However, B16f10 tumors, when grown in P-selectin-/- mice, had lower accumulation of Fib-Cy5.5 and higher fractions of perfused vessels; Dex-Cy7 concentrated and overlapped with tumor vessels. Studies with several subcutaneously developed tumors—squamous (SCC7), melanoma, pancreatic (AsPC-1), glioblastoma (U87)—and an orthotopically developed pancreatic (AsPC-1-Luc) cancer suggest that tumor vessel thrombosis fractions were inversely associated with perfused vessel fractions. SCC7 was the only tumor with high Dex-Cy7 perfusion and low abundance of Fib-Cy5.5. Intravenous administration of chitosan-based nanoparticles (CNPs) showed high perfusion and distribution in SCC7 tumors. Interestingly, thermal-heating to SCC7 tumors disrupted blood vessels, activated coagulation cascades, and produced enormous thrombus in their vessels, which blocked the intratumoral distribution of CNPs. Here we address that the use of anticoagulant, preferably with low molecular weight heparin (LMWH) or its orally available LMWH-based conjugate (LHe-tetraD) as an adjuvant, leads to easier passage of chemotherapy or nanoparticles into the deep recesses of a tumor. Subcutaneous LMWH or oral LHe-tetraD pretreatment for 7 days improved the CNPs accumulation and distribution in B16f10 tumors. Doppler ultrasound analysis revealed that LHe-tetraD treatment increased B16f10 tumor blood flow and perfusion. Orthotopic B16f10 melanoma (n = 5) and AsPC-1-Luc pancreatic tumors (n = 8~10) were treated with placebo, LHe-tetraD once daily, 3 cycles of oxaliplatin/gemcitabine once per three days, and combination after 14 days of tumor inoculation. LHe-tetraD alone did not regress tumor growth; however, LHe-tetraD/oxaliplatin/gemcitabine combination markedly reduced tumor volume than oxaliplatin/gemcitabine treated group. In another set of experiment, B16f10 tumors were treated with 4 cycles of doxorubicin loaded CNPs (CNP-Dox; 3 mg/kg per three days) with or without LHe-tetraD. Co-treatment with LHe-tetraD also enhanced the tumor-inhibition efficacy of CNP-Dox than CNP-Dox only. Our results demonstrate that anticoagulant prevents the generation of tumor thrombus and represents a new way to improve the penetration and drug efficacy into solid tumors. Citation Format: Taslim A. Al-Hilal, Jeong Uk Choi, In-San Kim, Youngro Byun, Fakhrul Ahsan. De-clotting tumor to improve the perfusion, distribution and efficacy of chemotherapy and nanotherapeutics. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B24.

Antiphospholipid Antibody-Mediated Thrombotic Mechanisms in Antiphospholipid Syndrome: Towards Pathophysiology-Based Treatment.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by a persistently high titer of antiphospholipid antibodies (aPLs). In addition to pregnancy morbidity, arterial and/or venous thrombosis is another clinical feature of APS. Regardless of the type of APS, the thrombi formed by the induction of aPLs can lead to deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke and gangrene. Although the concept of APS was introduced approximately 32 years ago, its thrombogenic pathophysiology is still unclear. Therefore, patients are treated with anticoagulant and/or antiplatelet regimens just as in other thrombotic disorders even though the thrombotic pathophysiology is mainly aPLs-mediated. In this review, we provided an update of the cellular, auto-immune and genetic factors known to play important roles in the generation of thrombi. Current successful regimens are also outlined along with potential emerging treatment strategies that may lead to the optimum management of thrombotic APS patients.

Evaluating antithrombotic activity of HY023016 on rat hypercoagulable model

The generation of thrombus is not considered as an isolated progression without other pathologic processes, which may also enhance procoagulant state. The purpose of this study was to assess whether HY023016, a novel dabigatran prodrug and an oral direct thrombin inhibitor, or dabigatran etexilate, another thrombin inhibitor can improve the state of whole blood hypercoagulability in vitro/vivo. By using whole blood flow cytometry we explored the effects of HY023016 and dabigatran etexilate on thrombin and ADP-induced human platelet-leukocyte aggregation generated in vitro. With the method of continuous infusion of thrombin intravenous, we successfully established a rat hypercoagulable model and evaluated the effect of HY023016 or dabigatran etexilate in vivo. HY023016 was able to inhibit thrombin- or ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates in dose-dependent manner. Dabigatran etexilate was unable to affect ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates. Based on rat hypercoagulable model, dabigatran etexilate could reverse thrombin-induced circulatory system hypercoagulable state in a concentration-dependent manner. Dabigatran etexilate also inhibited electrical stimulation induced formation of arterial thrombus in rat under hypercoagulable state, and extracorporal circulation-induced formation of thrombus in dose-dependent manner. Compared with dabigatran etexilate, HY023016 showed nearly equal or even better antithrombotic activity, regardless of reversing the cycle of rat hypercoagulable state or inhibiting platelet-leukocyte aggregation. In surrmary, HY023016 could effectively improve hypercoagulable state of circulatory system.

The role of platelets in coagulation dysfunction in xenotransplantation, and therapeutic options

Xenotransplantation could resolve the increasing discrepancy between the availability of deceased human donor organs and the demand for transplantation. Most advances in this field have resulted from the introduction of genetically engineered pigs, e.g., α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for one or more human complement-regulatory proteins (e.g., CD55, CD46, CD59). Failure of these grafts has not been associated with the classical features of acute humoral xenograft rejection, but with the development of thrombotic microangiopathy in the graft and/or consumptive coagulopathy in the recipient. Although the precise mechanisms of coagulation dysregulation remain unclear, molecular incompatibilities between primate coagulation factors and pig natural anticoagulants exacerbate the thrombotic state within the xenograft vasculature. Platelets play a crucial role in thrombosis and contribute to the coagulation disorder in xenotransplantation. They are therefore important targets if this barrier is to be overcome. Further genetic manipulation of the organ-source pigs, such as pigs that express one or more coagulation-regulatory genes (e.g., thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39), is anticipated to inhibit platelet activation and the generation of thrombus. In addition, adjunctive pharmacologic anti-platelet therapy may be required. The genetic manipulations that are currently being tested are reviewed, as are the potential pharmacologic agents that may prove beneficial.

Prothrombotic markers in patients with acute myocardial infarction and left ventricular thrombus formation treated with pci and dual antiplatelet therapy

BackgroundThe aim of the present study was to compare circulating levels of selected prothrombotic markers in patients suffering acute myocardial infarction (AMI) with and without left ventricular (LV) thrombus.MethodsOne hundred patients with AMI treated with PCI on the LAD and dual antiplatelet therapy were included. LV thrombus formation was detected by echocardiography and/or MRI in 15 patients. Fasting blood samples were drawn 4–5 days (baseline), 6–7 days, 8–9 days, 2–3 weeks and 3 months after the AMI for determination of haemostatic markers.ResultsWe found higher levels of soluble tissue factor (TF) and D-dimer in the LV thrombus group 4–5 days, 8–9 days and 3 months (only TF) after the AMI compared to the patients without thrombus formation (p<0.05). Patients with TF in the upper quartile at baseline had significantly higher risk for LV thrombus (OR 4.2; 95% CI 1.2 -14.5; p=0.02, adjusted for infarct size).The levels of prothrombin fragment 1+2 (F1+2) and endogenous thrombin potential (ETP) were significantly lower in the thrombus group after 8–9 days (only ETP), 2–3 weeks and 3 months. The levels of plasminogen activator inhibitor 1 activity and tissue plasminogen activator antigen did not differ between the groups.ConclusionIn the acute phase of AMI, we found higher levels of TF and D-dimer in the LV thrombus group, indicating hypercoagulability of possible importance for the generation of mural thrombus. Lower levels of F1+2, ETP and D-dimer in the thrombus group late during follow-up are probably induced by the initiated anticoagulation therapy.

New and Emerging Anticoagulant Therapy for Atrial Fibrillation and Acute Coronary Syndrome

Abstract Thrombosis is an underlying cause of many cardiovascular disorders, and generation of thrombi in the arterial circulation can lead to unstable angina, myocardial infarction, or ischemic stroke. Antithrombotic therapy is widely used, with proven benefit to prevent ischemic stroke and thromboembolic events in patients with atrial fibrillation (AF) or to prevent further ischemic complications in patients with acute coronary syndrome (ACS). Traditional anticoagulants (including unfractionated heparin, low-molecular-weight heparin, and warfarin) and antiplatelet agents (including aspirin, clopidogrel, and prasugrel) are typically used for these indications. Limitations to their use include variable pharmacokinetic and pharmacodynamic profiles, inability to inhibit fibrin-bound thrombin, risk of heparin-induced thrombocytopenia, delayed onset of action, numerous drug interactions, need for substantial laboratory monitoring and dosage titrations, hyporesponsiveness or resistance, hypersensitivity, adverse events, and bleeding. To overcome some of the limitations of traditional agents, new antithrombotic agents under development are highly selective for specific coagulation factors blocking the synthesis of thrombin. Clinicians must have an understanding of the new anticoagulants to aid in the selection of appropriate therapies for patients. We describe the most relevant phases II and III clinical trials that evaluated several recent emerging anticoagulant drugs for use in patients with AF or ACS. The advantages of many new agents include predictable pharmaco-dynamic response and pharmacokinetic parameters, allowing for fixed oral dosing with no need for laboratory monitoring. For patients with AF, dabigatran is already approved for the prevention of stroke and systemic embolism, rivaroxaban appears to be an effective alternative to warfarin in high-risk patients, and apixaban may also be an effective alternative to aspirin in patients unable to take warfarin. Otamixaban shows promise as an intravenous alternative for patients with ACS in the acute care setting. Likewise, rivaroxaban, dabigatran, and darexaban with or without dual antiplatelet therapy may be beneficial for secondary prevention of ischemic events in patients with ACS.

Hydraulic Analysis and Optimization in Impeller of Magnetically Levitated Centrifugal Blood Pump

This article, on the basis of the magnetically levitated pumps developed by Ibaraki University, talks about fluid dynamic analysis of pump and optimization of parameters of the impellers. The pump uses blood as medium, with a flow rate Q of 5L/min, a pressure head H of 100mmHg. By using software of ANSYS CFX, the article analyzes the effect of shape, angle, height, length and thickness of the impeller on performance chart and fluid field distribution inside the pump. To fulfill the human requirements in blood pump, an optimization analysis is conducted to improve the uniformity of fluid field, reduce power and efficiency of the pump. As result, meeting the flow and pressure head condition, single curvature impeller is found to have lower speed, better efficiency and more uniform flow field, this helps to prevent the generation of thrombus and hemolytic.

Filamentous thrombi during left-sided sheath-assisted catheter ablations

This editorial refers to ‘Left atrial Lasso catheter thrombus aspiration’ by D. Sorajja et al. , Europace 12(10):1506–1507. Sorajja et al .'s1 case report highlights the problem of thrombi developing during a complex left-sided procedure in conjunction with the use of long sheaths. The dramatic growth in the number of catheter ablations for atrial fibrillation and other left atrial arrhythmias has brought into sharp focus the incidence of thrombo-embolic complications, most frequently cerebrovascular in nature. In a recent world-wide survey, cerebrovascular accidents including transient ischemic attacks and strokes were observed in 0.94% of patients.2 Typically, these embolic events may be the result of either dislodgement of a pre-existing intra-left atrial thrombus or the occurrence of air embolism or the generation of thrombus, coagulum, or char during the procedure. The probability of encountering a pre-existing thrombus in the left atrium can be diminished significantly by ensuring effective anticoagulation for 4–6 weeks leading up to the catheter intervention and excluding a transoesophageal echo detectable thrombus within the 24 h preceding the intervention. Despite an INR within the therapeutic range, the anti-thrombotic protection afforded by oral anticoagulants is unlikely to be 100% (more likely, 60–70%) and screening by transoesophageal echocardiography is a useful and effective second level of protection. During the procedure, air emboli can be easily prevented by adhering to basic … *Corresponding author. Tel: +41 22 3727202; fax: +41 22 3727229, Email: dipen.shah{at}hcuge.ch

Combination Antithrombotic Therapies

Atherothrombosis is the major pathophysiological process responsible for the occurrence of severe ischemic events in patients with cardiovascular diseases. In the United States, atherothrombosis strongly influenced mortality in 2004: One in 2.8 deaths was due to CVD, 1 in 5 deaths to coronary heart disease, and 1 in 17 deaths to stroke.1 Because cardiovascular disease is a progressive and systemic disease, long-term antithrombotic therapies that effectively target the entire arterial vasculature and modulate the key components responsible for thrombus generation are essential to improve patient outcomes. Because platelet activation is determined by multiple receptor-mediated signaling pathways, clinical studies have evaluated the efficacy of multidrug administration in the prevention of atherothrombotic complications.2,3 The major concern with these therapies is the critical balance between anti-ischemic effect and bleeding risk. This review summarizes our understanding of the role of combination antiplatelet therapies in the treatment and prevention of atherothrombosis. Platelet activation and aggregation play a pivotal role in the generation of occlusive thrombus at the site of coronary arterial plaque rupture. In addition, platelets influence various endothelial and inflammatory responses during the initiation and progression of atherosclerosis. Under normal conditions, anucleate circulating platelets are in a quiescent state. Healthy vascular endothelium prevents adhesion and activation of platelets by producing antithrombotic factors such as CD39 (ectoADPase), prostaglandin I2, nitric oxide, heparin, matrix metalloproteinase-9, protein S, and thrombomodulin.3,4 Endothelial activation and denudation and frank atherosclerotic plaque rupture expose the subendothelial matrix and release prothrombotic factors during acute coronary syndromes (ACS) and percutaneous interventions. These processes result in localized platelet adhesion and platelet activation. After adhesion to the exposed subendothelial matrix, platelets are activated by shear and the soluble agonists thromboxane A2 (TxA2), ADP, and thrombin. TxA2 is produced from arachidonic acid, which originates from membrane phospholipids and …

Epsilon-aminocaproic acid (EACA).

Epsilon-aminocaproic acid (EACA) is a synthetic inhibitor of the plasmin-plasminogen system. It is the only potent antifibrinolytic agent which is commercially available in the United States. Effective blood levels of the drug are readily obtainable with either oral or intravenous administration, with very high levels of the drug being found in the urine since the drug is greatly concentrated during excretion. EACA has been used in numerous clinical situations to control bleeding and has been claimed to be an effective agent in subarachnoid hemorrhage, genitourinary bleeding from many causes, and in dental surgery in hemophiliacs. It may also be effective in several less well studied situations such as prophylaxis of bleeding episodes in hemophiliacs, control of menorrhagia, gastrointestinal bleeding, obstetrical bleeding and in bleeding following cardiac and thoracic surgery. Major side effects from EACA include hypotension, cardiac arrhythmias, rhabdomyolysis, and generation of thrombi. The incidence of thrombotic events secondary to the inhibition of the fibrinolytic system by EACA is unknown, but may be particularly increased in those patients who have some underlying predisposition to develop thrombosis. The potential benefit from the use of EACA must be weighed against the possible serious complications, particularly the development of widespread thrombi.

In vivo response to vascular injury in the absence of factor IX: Examination in factor IX knockout mice

Introduction Recently, in vitro models of coagulation have called into question the traditional conception of Factor IX as an intrinsic pathway protein, essential to propagation of coagulation but not central to the initiation of hemostatic plug, which has been thought instead to involve TF/FVIIa interactions with factor X and platelets. We hypothesized that the activation of factor IX, and its role in a factor IXa/FVIIa “tenase” complex leading to thrombin generation, plays a more important role than that of TF/FVIIa complex activation of factor X in the early hemostatic response to vascular injury. In vivo modeling is possible because of the generation of factor IX −/− mice. Materials and methods We used two models of arterial vascular injury, histological examination following mechanical carotid artery disruption and intravital microscopy of a mesenteric arteriole subsequent to ferric chloride arteriolar injury to examine mice having complete deficiency of factor IX (FIX −/−). Results Both injury models demonstrate that platelet rich thrombi /hemostatic plug in FIX −/− mice is dramatically reduced as compared to wild type mice under conditions of high shear; in fact, no platelet thrombi (> 20 μm) were observed in the intravital experiments. Interestingly, the platelet defect is more striking than that described in mice lacking fibrinogen and/or von Willebrand factor. Conclusions The results suggest TF/FVIIa → FX pathway is insufficient for effective platelet aggregation in the presence of high flow, requiring factor IX at the convergence of both intrinsic and extrinsic pathways. Following platelet adhesion, factor IX is required for normal platelet aggregation in vivo, as well as thrombin generation and propagation of occlusive thrombus at the site of vascular injury.

Model study of blood flow through a stent in the aorto-iliac bifurcation.

Atherosclerotic ailments play a major role in industrial societies. Atherosclerotic disease causes stenotic narrowing of the arterial vessel system. These narrow passages can be widened with balloon angioplasty and stents are introduced to keep the passage open. In this study, stents designed for the aorto-iliac bifurcation are considered. Most of these stents used in peripheral arteries are self-expanding stents made from Nitinol, a thermo-reactive alloy. The insertion is done under radiographic control. However, the delicate metallic structure of the stent usually casts only a weak radiographic shadow and thus is difficult to detect. A stent with an innovative radio opaque tantalum marker (Luminexx, C.R. BARD--Angiomed, Karlsruhe), overcomes this problem. However, the markers extend into the blood flow and the question arises whether the markers may cause the generation of thrombi. In a model study in an enlarged model of the aorto-iliac bifurcation with an inserted stent the flow was visualized. The enlarged scale permits the observation and video recording of the flow in great detail. The Reynolds similarity is kept. A subsequent analysis of the flow in the vicinity of the markers shows a short residence time. These results indicate that the additional markers do not increase the risk of thrombus generation.

Determining wall shear stress in artificial blood pumps of heart assist devices

The walls in blood pumps are made of artificial material and thus are thrombogenic to a lesser or larger degree. Also the flow plays a role: a blood flow with no flow separations and stagnation zones is required to avoid the generation of thrombi. A precondition for solving this problem is the assessment of the wall shear rate. However this parameter is difficult to assess because of the deformability of the walls and the pulsation of the flow. Two methods are proposed to estimate the wall shear stress in bloodpumps. The paint erosion method allows a characterisation of the flow near the wall. The second method is a special development of standard Particle Image Velocimetry (PIV). A vector field of the flow close to the wall results. Both methods should permit the assessment of the wall shear stress in bloodpumps.

The impact of molecular medicine upon early cardiovascular drug development.

Heart disease remains the leading cause of death and morbidity in the western world despite significant advances in cardiovascular (CVS) therapeutics. Since the 1960s clinical pharmacologists have been very much involved in bringing forward new classes of CVS drugs. This started with the introduction of β-adrenoceptor blockers in the early 1960s and has continued with calcium antagonists, ACE inhibitors and more recently endothelin antagonists. The principles for classical drug design have been based upon interaction with: receptor sites (β-adreno-ceptor blockers), enzymes (thrombolytics and anticoagulants) and ion channels (calcium antagonists, antiarrhythmics). The advent of molecular biology has added a fourth principle for CVS drug development – that of molecular therapeutics. While classical approaches will still provide new CVS drugs (e.g. platelet IIb/IIIa receptor antagonists and directly acting thrombin inhibitors), there is now anticipation that molecular therapeutics will provide a very specific means of intervention thus potentially avoiding some of the problems associated with the non specificity of conventional approaches, e.g. side-effects associated with lack of cardioselectivity with β-adrenoceptor blockers, risk of bleeding with anticoagulants, cough with ACE inhibitors, controversy following retrospective analysis of the use of calcium antagonists. In the broader sense molecular therapeutics includes both gene therapy and oligonucleotide therapy. However the advances in genetic analysis (identification of polymorphisms associated with diseases) mean that there may be a further impact upon drug development – namely the use of known gene polymorphisms to select patient populations at risk. We will review the status of each of these and prospects for the future.

Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells. Role of Ca2+ mobilization and protein kinase C activation.

Tissue factor (TF) is a low molecular weight glycoprotein that initiates the clotting cascade and is considered to be a major regulator of coagulation, hemostasis, and thrombosis. TF is not expressed in the intima or media of normal adult blood vessels. Accordingly, it has been hypothesized that the initiation of intravascular coagulation may require the "induced" expression of TF in the vessel wall. We report that TF mRNA and protein are rapidly and markedly induced in early and late passaged vascular smooth muscle cells (VSMC) by growth factors (serum, platelet-derived growth factor, epidermal growth factor), vasoactive agonists (angiotensin II), and a clotting factor (alpha-thrombin). The induction of TF mRNA by these agents is dependent upon mobilization of intracellular Ca2+ and is blocked by Ca2+ chelation. In contrast to other growth factor-responsive genes, such as KC and c-fos, downregulation of protein kinase C activity by prolonged treatment with phorbol esters fails to block agonist-mediated TF induction. This raises the possibility that protein kinase C activation may not be necessary for TF mRNA induction in VSMC. VSMC may play a role in the generation or propagation of thrombus through the induction of TF, particularly in settings, such as those associated with acute vessel injury, where the endothelium is denuded and the VSMC are exposed to circulating blood.

Suppression of plasminogen activator inhibitor 1 release by fibrin from human umbilical vein endothelial cells

The effect of fibrin stimulation on the fibrinolytic potential in cultured human umbilical vein endothelial cells (HUVEC) was investigated in the normal state and aged state. The amount of antigen of the two fibrinolytic factors, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), was determined using ELISA and the ABC method, respectively. When a fibrin clot was overlayered on the normal HUVEC, the secretion of t-PA or PAI-1 from the HUVEC was greatly changed. That is, PAI antigen was decreased 3-fold and t-PA antigen increased slightly in the conditioned medium. On the other hand, when the aged HUVEC were stimulated by a fibrin clot, PAI antigen was increased 3-fold and t-PA antigen did not change in the conditioned medium. When the level of fibrinolytic activity in the conditioned medium was expressed as the molar ratio of PAI and t-PA (PAI/t-PA), the value in the fibrin-stimulated normal HUVEC was markedly reduced (a 3.5-fold decrease) when compared with that of the non-stimulated normal HUVEC, reflecting a profibrinolytic state. On the other hand, the value in the fibrin-stimulated aged HUVEC was markedly increased (a 5-fold increase) when compared with that of the non-stimulated aged HUVEC, reflecting a antifibrinolytic state. Actinomycin D- or cycloheximide-treated HUVEC showed no response to the fibrin stimulation. We conclude that the level of HUVEC-mediated fibrinolytic activity was regulated mainly by the production and secretion of PAI from the HUVEC to protect against the generation of thrombi. In the aged HUVEC, the regulatory mechanism acts in an opposite manner and a thrombotic process may be induced.