Ursula Rauch

Thrombus formation on atherosclerotic plaques: pathogenesis and clinical consequences.

Ischemic heart disease is the most common cause of death worldwide (1). Thrombi that form on atherosclerotic lesions in coronaries are responsible for myocardial ischemia and progression of atherosclerosis (2-5). Recent pathologic, experimental, and clinical findings have led to a better understanding of the cellular and molecular mechanisms underlying thrombus formation on atherosclerotic plaques. In this review, we describe pathophysiologic mechanisms that lead to arterial thrombosis, the clinical impact of thrombosis on arterial lesions, and new therapeutic developments. With regard to the impact of arterial thrombosis on human mortality, we focus on the thrombotic process in atherosclerotic disease. Recent reviews have summarized the pathophysiologic and clinical aspects of the vulnerable plaque in the development of atherosclerotic lesions (6-9). Methods We searched MEDLINE for English-language reports on thrombosis and atherosclerosis published from 1966 to the present. Experimental, clinical, and epidemiologic studies related to the pathogenesis and pathophysiology of thrombosis on atherosclerotic lesions were reviewed, and references from identified articles were also selected. Abstracts on new aspects of therapeutic options that were presented at recent international meetings of the International Society of Thrombosis and Haemostasis and the American Heart Association were selected. Therapeutic approaches were derived from experimental studies and large clinical investigations. Pathophysiology and Clinical Impact of Thrombus Formation on Atherosclerotic Lesions The Virchow Triad As described by Virchow more than 100 years ago, occurrence of arterial thrombosis depends on the arterial vessel wall substrates, the local rheologic characteristics of blood flow, and systemic factors in the circulating blood (Table 1) (2, 3, 8, 10, 11). Table 1. The Virchow Triad Local Substrates for Thrombosis: Atherosclerotic Plaques Vulnerable atherosclerotic plaques may cause most acute coronary syndromes (12, 13). Atherosclerotic lesions are found in most major arteries, including the aorta, carotid, iliofemoral, and medium-sized arteries (such as the coronaries) (13). Focal intimal lesions may first develop in the human fetus before birth (14). Autopsy studies in persons without clinical cardiovascular disease showed that intimal alterations occur in the different vascular beds within the first 15 to 20 years of life (15-17). Using histologic characteristics, the American Heart Association has developed a standardized classification of distinct plaque types (Figure 1). Advanced atheromatous lesions are the substrates for arterial thrombosis. In advanced atheromatous lesions, the lipid core of the plaque contains pultaceous debris, apoptotic cells (such as dead macrophages and smooth-muscle cells), mesenchymal cells, and abundant free cholesterol crystals (fatty gruel) (18). The lipid core of these type IV and V lesions is rich in tissue factor, which, upon plaque rupture and exposure to the circulating blood, initiates the coagulation cascade and thrombin generation (Figure 2) (19). Smoking increases tissue factor expression in atherosclerotic plaques. Tissue factor is associated with and probably generated by activated macrophages within the plaque. The degree of plaque disruption (erosion, fissure, or ulceration) and the amount of stenosis caused by the disrupted plaque and the overlying mural thrombus are key factors for determining thrombogenicity at the local arterial site. When deep ulceration occurs, tissue factor from the atherosclerotic lipid core is exposed to flowing blood and released into the lumen (20, 21). Tissue factor interacts with factor VII and subsequently activates factor X, which leads to conversion of prothrombin to thrombin in the prothrombinase complex (Figure 2) (22, 23). The high tissue factor activity contributes to the procoagulant activity of disrupted atherosclerotic lesions and the superimposed mural thrombi (20). Disruption of advanced plaques with exposure of the highly thrombotic lipid core to the flowing blood triggers the formation of thrombi up to 6 times larger than thrombi generated by exposure of other components of the arterial wall (18). Mural thrombus formation may contribute to arterial stenosis, release vasoconstrictors from platelets, and cause ischemic symptoms (6, 8, 24, 25). The vulnerability of a plaque includes the pathoanatomic features that are related to size of the lipid pool, thickness of the fibrous cap, content and metabolic activity of lipids (26, 27), activity and density of macrophages (28, 29), and matrix metalloproteinases (30, 31). The external physical forces that expose the vessel wall to blood flow at different shear rates also influence the occurrence and progression of plaque disruption, thrombosis, and arteriosclerosis (32-35). Figure 1. Relation of lesion morphologic characteristics and phases of progression of coronary atherosclerosis to clinical findings. Figure 2. The tissue factor pathway activation of coagulation. Blood Rheology and Thrombus Formation Acute changes in rheologic characteristics induced by vasoconstriction, plaque disruption, and thrombus formation induce changes in the shear rate of flowing blood. Thrombus formation increases with increasing shear force (32). Shear force is directly related to flow velocity and inversely related to the third power of the lumen diameter. Thus, acute platelet deposition after plaque disruption depends on arterial size and the geometric changes and degree of narrowing after disruption. Changes in geometry may increase platelet deposition, whereas a sudden protrusion of plaque contents or growth of thrombus at the injury site may create severe stenosis and thrombotic occlusion. Most platelets are deposited at the apex of a stenosis, which is the site of maximal shear force (32, 36). Mechanical forces associated with blood flow influence the vascular tone, arterial structure, and location of arterial lesions (32, 36). Thrombin causes vasoconstriction when endothelium is absent or dysfunctional. Arterial vasoconstriction increases the shear force. Systemic risk factors for atherosclerosis, such as smoking, hyperlipidemia, or diabetes mellitus, may cause endothelial dysfunction, promote vasoconstriction, and increase shear force and platelet deposition. Contribution of Systemic Risk Factors to Thrombosis Systemic factors, including changes in lipid and hormonal metabolism, hyperglycemia, hemostasis, fibrinolysis, and platelet and leukocyte function, are associated with increased blood thrombogenicity or a systemic hypercoagulable state (37). Approximately one third of patients with acute myocardial infarction and coronary thrombosis have plaque erosions that occurred on nonruptured, moderately stenotic plaques. The patients who developed coronary thrombosis on erosions had systemic risk factors associated with a hypercoagulable state (38). Lipoprotein(a), a known risk factor for coronary heart disease, has a structure similar to that of plasminogen and may reduce plasmin formation and impair thrombolysis (39). Elevated low-density lipoprotein cholesterol levels increase blood thrombogenicity (40) and growth of thrombus under defined rheologic conditions (41). Reducing low-density lipoprotein cholesterol levels using statins decreased thrombus growth by approximately 20% (41). The reduction of total vascular events, including death, coronary events, and stroke, by lipid-lowering therapy with statins was documented in several large prospective clinical trials (42-48). Reduced low-density lipoprotein cholesterol levels may also decrease vasoconstriction and the size of the lipid core (41). High plasma levels of cathecholamines potentiate platelet activation, enhance vasospasm, and increase the incidence of sudden death after emotional and physical stress in patients with acute cardiovascular events. Smoking increases catecholamine release, causes endothelial dysfunction, and is associated with increased levels of fibrinogen (49). Increased plasma fibrinogen level is an independent risk factor for complications in patients with atherosclerotic disease (50-52). Fibrinogen, factor VIIIc, and von Willebrand factor were shown to be positively related to C-reactive protein (53). C-reactive protein, like fibrinogen, is a protein of the acute-phase response and a sensitive marker of low-grade inflammation. Increased levels of C-reactive protein have been reported to predict acute coronary events (54-56). C-reactive protein seems to be a useful marker for predicting risk for thrombotic events. Whether C-reactive protein reflects only the extent of the acute-phase reaction in response to nonspecific events, such as myocardial ischemia or atherosclerosis, or whether it may directly participate in the process of thrombus formation at the site of the atherosclerotic vessel is not known (56). Diabetic patients, especially those whose diabetes is poorly controlled, have increased blood thrombogenicity, due in part to glycosylation of collagen and proteins and increased levels of plasma fibrinogen and plasminogen activator inhibitor-1 (57-62). Platelets from patients with diabetes have increased reactivity and hyperaggregability and expose a variety of activation-dependent adhesion proteins (63, 64). Abnormal platelet function is reflected by increased platelet consumption and prolonged accumulation of thrombocytes on the altered vessel wall (64-66). In addition, more leukocyteplatelet aggregates circulate in the blood of patients with diabetes and diabetic vasculopathy (64). The prothrombotic state in diabetes is also associated with increased expression of monocyte procoagulant activity in the presence of diabetic microalbuminuria (67). Increased procoagulant activity in diabetes is attributed to leukocytes (64, 65, 67), which may in part activate the tissue factor pathway (68) and contribute to the high blood thro

Procoagulant Soluble Tissue Factor Is Released From Endothelial Cells in Response to Inflammatory Cytokines

Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-α and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-α (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-α and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223±0.0069 versus 0.0012±0.0006 for control, P<0.001 and 0.0022±0.0004 versus 0.0012±0.0007, P<0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-α stimulation, supporting factor Xa generation after relipidation (6.55±1.13 U versus 2.99±0.59 U in control supernatant, P<0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.

Blood thrombogenicity in type 2 diabetes mellitus patients is associated with glycemic control

OBJECTIVES This study was designed to determine whether blood thrombogenicity is related to chronic glycemic control in type 2 diabetes mellitus (T2DM). BACKGROUND Type 2 diabetes mellitus is associated with accelerated atherosclerosis and a high rate of arterial thrombotic complications. Whether increased blood thrombogenicity is associated with glycemic control has not been properly tested. METHODS Forty patients with T2DM with hemoglobin A1c (HbA1c) > or =7.5% were selected. Maintaining their current hypoglycemic therapies, patients were randomized into a conservative (diet modification plus placebo) or intensive (diet modification plus troglitazone) hypoglycemic regimen for three months. Blood thrombogenicity was measured at baseline and after three months with the Badimon ex vivo perfusion chamber and assessed as platelet-thrombus formation. The repeated measurements allowed every patient to be his/her own control. RESULTS Patients in both groups (48% and 74% of the conservative and intensive groups, respectively) improved glucose control (HbA1c reduction > or =0.5%), showing a significant decrease in blood thrombogenicity. A significant positive correlation was observed between the reduction in thrombus formation and the reduction in HbA1c (r = 0.47, p < 0.01). The reduction in HbA1c achieved by both treatments was comparable. Patients without glycemic improvement showed no change in blood thrombogenicity. Improved glycemic control was the only significant predictor of a decrease in blood thrombogenicity. CONCLUSIONS In T2DM, there is an association between improved glycemic control and blood thrombogenicity reduction. The effect of glycemic control on the thrombotic complications of T2DM patients deserves further investigation.

Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing

Summary There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).

Circulating tissue factor and thrombosis.

Tissue factor (TF) on circulating microparticles has recently received much attention as a factor in myocardial infarction. We have developed systems by which we have been able to investigate the thrombogenic potential of blood-borne TF. Thrombi develop when native human blood is passed over either collagen-coated glass slides or over pig arterial media. These thrombi immunostain for TF even when the substrate contains none. Moreover, we have demonstrated that the deposited TF is active because the thrombi contain fibrin; fibrin deposition and thrombotic mass are both inhibited by the inclusion of a potent TF-inhibitor in the perfusions. We have also shown that leukocyte-derived particles attach to platelets in a reaction mediated by adhesion proteins.

Tissue factor, the blood, and the arterial wall.

Thrombogenic tissue factor (TF) on cell-derived microparticles is present in the circulating blood of patients with acute coronary syndromes. Recently, we reported that leukocytes transfer TF-positive particles to platelet thrombi, making them capable of triggering and propagating thrombus growth. This observation changes the original dogma that vessel-wall injury and exposure of tissue factor within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF-positive leukocyte particles is dependent on the interaction of CD15 and TF with platelet thrombi. The inhibition of TF transfer and TF activity suggests a novel therapeutic approach to the prevention of thrombosis that may prove to be effective in disorders associated with increased blood TF.

Leucettines, a Class of Potent Inhibitors of cdc2-Like Kinases and Dual Specificity, Tyrosine Phosphorylation Regulated Kinases Derived from the Marine Sponge Leucettamine B: Modulation of Alternative Pre-RNA Splicing

We here report on the synthesis, optimization, and biological characterization of leucettines, a family of kinase inhibitors derived from the marine sponge leucettamine B. Stepwise synthesis of analogues starting from the natural structure, guided by activity testing on eight purified kinases, led to highly potent inhibitors of CLKs and DYRKs, two families of kinases involved in alternative pre-mRNA splicing and Alzheimers disease/Down syndrome. Leucettine L41 was cocrystallized with CLK3. It interacts with key residues located within the ATP-binding pocket of the kinase. Leucettine L41 inhibits the phosphorylation of serine/arginine-rich proteins (SRp), a family of proteins regulating pre-RNA splicing. Indeed leucettine L41 was demonstrated to modulate alternative pre-mRNA splicing, in a cell-based reporting system. Leucettines should be further explored as pharmacological tools to study and modulate pre-RNA splicing. Leucettines may also be investigated as potential therapeutic drugs in Alzheimers disease (AD) and in diseases involving abnormal pre-mRNA splicing.

PAR-1 contributes to the innate immune response during viral infection

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3-induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1(-/-) mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1(+/+) mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1(-/-) mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1(+/+) mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman® gene assays. Only two of the tested Taqman® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Genomic expression profiling of human inflammatory cardiomyopathy (DCMi) suggests novel therapeutic targets.

The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage.