A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.     

The changed ultrastructure of fibrin networks during use of oral contraception and hormone replacement

Contraceptives and hormone replacement have been extensively used since the late 1950s. However, adverse effects are common and include an increased risk of cardiovascular diseases, including thrombo-embolic diseases. Previous research has shown that ultrastructure of fibrin networks may provide great insight regarding the thrombotic potential of patients. The current study investigates the scanning electron microscopy (SEM) ultrastructure of fibrin networks of individuals using oral contraceptive therapy as well as individuals using hormone replacement. We compare micrographs of these two groups with micrographs of young, healthy individuals not using oral contraception. Platelet rich plasma and thrombin was used to prepare the fibrin clots. Here we show that during contraceptive and hormone replacement use, a netted fibrin layer forms. We suggest that oestradiol use causes fibrin network changes and these changes can be seen using SEM technology. These changes may provide further evidence regarding the increased occurrence of thrombotic events during contraceptive and hormone replacement therapy.     

Scanning electron microscopy analysis of erythrocytes in thromboembolic ischemic stroke

Introduction: Erythrocytes play an important role in hemostasis and disease conditions. During ischemic stroke, erythrocytes undergo oxidative and proteolytic changes resulting in a changed cellular rheology. Methods: Blood samples were obtained from controls and thromboembolic ischemic stroke patients (within 48 h of stroke). The ultrastructure of erythrocytes was compared, using a scanning electron microscope (SEM). Abnormal morphology included codocytes, knizocytes, stomatocytes, and echinocytes. Percentage of abnormal cells was calculated, and the analyses were performed using the statistical program NCSS with the level of significance set at 0.05. A t‐test was carried out to compare the data from the erythrocyte counts of stroke patients with that of the control subjects. Results: Ultrastructural SEM results showed that there are a large percentage of erythrocytes in healthy individuals that do not have a typical discoid shape, when studying the cells using a high magnification electron microscope. Furthermore, analysis showed that variation in shape is so subtle that it is not clearly visible using a typical light microscopy blood smear analysis. Thromboembolic ischemic stroke patients presented with a significant amount of erythrocytes with abnormal morphology. Conclusion: We suggest that in healthy individuals, a typical smear would contain several nondiscoid‐shaped erythrocytes, only clearly visible at high magnification. However, thromboembolic ischemic stroke does significantly impact erythorcyte shape, and this change in morphology may result in an impaired microcirculation, as well as impaired oxygen carrying capacity. This changed morphology may further complicate the restoring of homeostasis caused by acute thromboembolic stroke.     

Cancer‐associated ischemic stroke is associated with elevated d‐dimer and fibrin degradation product levels in acute ischemic stroke with advanced cancer

Aim: Although several studies have reported various causes of ischemic stroke in patients with cancer, only a few have evaluated the clinical relevance of ischemic stroke pathogenesis to cancer. The aim of the present study was to elucidate the clinical characteristics of cancer‐associated ischemic stroke. Methods: We evaluated 154 ischemic stroke patients without cancer and 57 ischemic stroke patients with cancer who had either received continuous treatment for cancer within 5 years before to the onset of ischemic stroke, or who had been diagnosed with cancer within 1 year after the onset of ischemic stroke. Cancer patients were grouped into “cancer‐associated ischemic stroke,” the “conventional ischemic stroke,” or “other.” Results: A total of 15 patients (26%) were classified into the cancer‐associated ischemic stroke in cancer patients. In univariate analysis of the cancer‐associated ischemic stroke and the others, there were significant differences in the prevalence of hypertension, hyperlipidemia and advanced cancer (clinical stage IV), and the levels of d ‐dimer, fibrin degradation product and hemoglobin. With multivariate regression analysis of those factors, the prevalence of hypertension, hyperlipidemia and advanced cancer (clinical stage IV), and the levels of d ‐dimer and fibrin degradation product remained as statistically independent factors, which were associated with cancer‐associated ischemic stroke (n = 111, χ² = 67.21, P < 0.0001). Conclusion: In acute ischemic stroke, the cancer‐associated ischemic stroke is associated with elevated d ‐dimer and fibrin degradation products, even after controlling hypertension, hyperlipidemia and advanced cancer (clinical stage IV). Geriatr Gerontol Int 2012; 12: 468–474.     

Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases

The formation of fibrin clots that are relatively resistant to lysis represents the final step in blood coagulation. We discuss the genetic and environmental regulators of fibrin structure in relation to thrombotic disease. In addition, we discuss the implications of fibrin structure for treatment of thrombosis. Fibrin clots composed of compact, highly branched networks with thin fibers are resistant to lysis. Altered fibrin structure has consistently been reported in patients with several diseases complicated by thromboembolic events, including patients with acute or prior myocardial infarction, ischemic stroke, and venous thromboembolism. Relatives of patients with myocardial infarction or venous thromboembolism display similar fibrin abnormalities. Low-dose aspirin, statins, lowering of homocysteine, better diabetes control, smoking cessation, and suppression of inflammatory response increase clot permeability and susceptibility to lysis. Growing evidence indicates that abnormal fibrin properties represent a novel risk factor for arterial and venous thrombotic events, particularly of unknown etiology in young and middle-aged patients.     

Scanning electron microscopy of fibrin networks in rheumatoid arthritis: a qualitative analysis

Rheumatoid arthritis is a chronic inflammatory condition that affects mainly synovial joints and has an impact on approximately 1% of the Western population. The coagulation process is altered in this condition, and this is frequently complicated by thrombocytosis. Changes in fibrin morphology have been linked with inflammation, and this, in turn, plays an important role in thrombosis. Changes in the fibrin fiber formation cause the alterations observed in thrombus morphology. In the current study, the ultrastructure of platelets and fibrin networks was investigated to determine whether any morphological changes are present in these structures in patients suffering from rheumatoid arthritis. Six patients diagnosed with rheumatoid arthritis took part in this study, and their clot morphology was compared to that of control subjects. Citrated blood with and without the addition of thrombin was used. Results indicated that the fibrin networks in the arthritis patients formed thick, matted layers. This matted appearance is due to a changed ultrastructure of the minor, thin fibers. Also, in these patients, spontaneous networks were created without the addition of thrombin, which indicates an abnormal hemostatic protein functioning, and the latter is expressed as visible changes in ultrastructure.     

Plasma adrenomedullin and carotid atherosclerosis in atherothrombotic ischemic stroke

OBJECTIVE: Adrenomedullin is known to exert anti-atherosclerotic actions by inhibiting proliferation and migration of smooth muscle cells in vitro. Here we examine the relationship between the plasma concentration of adrenomedullin and ultrasonographic characteristics of carotid arteries both in ischemic stroke and in the absence of cerebrovascular disease. METHODS: We studied 61 patients with atherothrombotic ischemic stroke in the chronic phase and 50 patients without any cerebrovascular disease. Intima-media thickness and vascular lumen diameters were evaluated by carotid ultrasonography. Plasma mature-adrenomedullin was determined by radioimmunoassay. RESULTS: Plasma mature-adrenomedullin in the patients with atherothrombotic ischemic stroke in the chronic phase (2.01 +/- 0.58 fmol/ml) was significantly higher than that in the patients without any cerebrovascular disease (1.24 +/- 0.18 fmol/ml, P < 0.001). With multiple regression analysis, plasma mature-adrenomedullin was found to be predicted by: stroke status (atherothrombotic ischemic stroke versus no cerebrovascular disease), diabetes status (yes/no), left ventricular ejection fraction, internal carotid artery intima-media thickness, and common carotid artery pressure strain elastic modulus (R = 0.79; F5,105 = 85.39, P < 0.0001). CONCLUSION: Plasma mature-adrenomedullin showed significantly positive associations with carotid atherosclerosis and atherothrombotic ischemic stroke, independent of systolic blood pressure.     

Age-related changes in fibrin networks and platelets of individuals over 75: a scanning electron microscopy study showing “thrombotic preparedness”

During aging, changes in vasculature, haemostasis and endothelium, including alterations of platelets, coagulation and fibrinolytic factors, occur. Research has also reported that healthy, aged individuals have heightened coagulation enzyme activity, accompanied by signs of enhanced formation of fibrin and secondary hyperfibrinolysis. It is now believed that the impaired fibrinolytic potential in old age results in a condition that has previously been described as a systemic state of ‘‘thrombotic preparedness’’. This state is far out of proportion to the physiological needs of the person. In the current research we investigate whether this apparent changed thrombotic profile in healthy aged individuals (over the age of 75), is evident in their platelet and fibrin network ultrastructure, when compared to healthy individuals under 25 years. The main differences among young and older individuals were found in the fibrin network ultrastructure. It is concluded that with age, major fibers seem to become thinner and more sparsely arranged and that minor, thin fibers dominate it the coagulum, forming a fine netlike structure. At irregular intervals in the coagulum, thicker, fibrin fiber lattices are present; this is not found in healthy individuals. This might be due to the previously suggested enhanced fibrin formation and heightened coagulation enzyme activity. Here we therefore provide ultrastructural evidence for the thrombotic preparedness previously suggested after studying biochemistry of fibrinolysis and coagulation factors in the elderly.     

Antikoagulation bei Vorhofflimmern

Atrial fibrillation is associated with a relevant risk for ischemic stroke: Observational studies suggest that one in four to five strokes is due to atrial fibrillation. Depending on the risk profile of an individual patient, the yearly risk for a stroke is between 2% and 14%. Continuous oral anticoagulation is indicated if atrial fibrillation is accompanied by at least one additional risk factor for thromboembolic complications. This recommendation is supported by several large randomized trials. Due to their low therapeutic range, vitamin K antagonists (phenprocoumon, warfarin, and others), the most commonly used oral anticoagulants, require regular anticoagulation monitoring. If well-controlled (international normalized ratio 2-3, in elderly patients preferably 2-2.5), oral anticoagulation prevents more than half of ischemic strokes related to atrial fibrillation, while bleeding complications are rare. In the follow-up of low risk patients (CHADS2-Score 0), oral anticoagulation becomes necessary when risk factors for thromboembolic complications develop. If a stroke occurs during oral anticoagulation and an INR>2 in a patient with atrial fibrillation, other causes than thromboembolic events should be considered. New anticoagulants--especially direct thrombin antagonists--are currently evaluated in clinical trials and may in the future facilitate anticoagulation in patients with atrial fibrillation.     

Effects of gliclazide on fibrin network

Fibrin network structure is altered by diabetes, peripheral vascular disease, and by some drugs. The antidiabetic drug, gliclazide, increases fibrin fiber thickness but reduces whole network permeability. The networks are, however, more lysable. These effects are further examined in this study using electron microscopy. Changes were observed in protein concentrations in fibrin fibers, in fibrin fiber alignment and in fiber porosity. These results show that gliclazide modifies fibrin monomer polymerization so that the fibrin network is rendered more susceptible to fibrinolysis. This pharmacological action of gliclazide may be useful in the treatment of thromboembolism.     

Current clinical experience with staphylokinase in arterial thrombosis

Conclusions Sak, a profibrinolytic agent produced by S. aureus that is now readily available by recombinant DNA technology, induces efficient and rapid recanalization in patients with occlusive arterial thrombosis. Its fibrin specificity at clinically effective doses by far exceeds that of any commercially available plasminogen activator. Likewise, the speed and rate of clot lysis compare favorably with established agents, but definition of the relative benefits, especially in terms of reduction of mortality and morbidity, awaits larger comparative trials. The optimal dose and rate of infusion in patients with coronary and peripheral arterial thrombosis, as well as the value of Sak in other thromboembolic disorders (comprising deep venous thrombosis, pulmonary embolism, and ischemic stroke), remain to be established. Notwithstanding its short circulatory half-life, bolus thrombolysis with Sak appears to be feasible. The antigenicity of wild-type Sak argues against repeated administration, but limited experience with selected recombinant mutants indicates that the immunoreactivity and antigenicity of this bacterial protein can at least be attenuated while preserving fibrinolytic activity and fibrin specificity.     

D-dimer testing in ischemic stroke and cerebral sinus and venous thrombosis

D-dimer measurement is commonly included in the diagnostic workup of patients with suspected acute symptomatic deep venous thrombosis and pulmonary embolism. As a haemostatic marker, it could be theoretically useful in other thromboembolic disorders, such as acute cerebrovascular events. In this review we summarize published literature on D-dimer testing in acute ischemic stroke and cerebral sinus and venous thrombosis (CSVT), discussing possible clinical diagnostic and therapeutical applications. In ischemic stroke, mounting evidence suggests a possible role of D-dimer in the acute diagnosis of ischemic stroke subtypes, especially in identifying tromboembolic and lacunar stroke. Its prognostic role still remains unclear, due to conflicting data. D-dimer could be also an useful screening test for excluding CSVT in patients presenting with acute headache, making the presence of cerebral thrombosis unlikely with low plasma levels. In this clinical setting sensitivity and negative predictive value are comparable to that reported in the diagnosis of acute thromboembolic disease. However, more studies are needed to confirm these recent findings as well as management studies to correctly introduce D-dimer measurement in clinical daily practice of ischemic stroke and CSVT.     

The Euglobulin Clot Lysis Time,a Rapid and Sensitive Method for the Assay of Fibrinolytic Activity after Venous Stasis

For the estimation of fibrinolytic activity in euglobulin precipitates after venous stasis, the euglobulin clot lysis time (ECLT) proved to be as reproducible and probably even more sensitive than the fibrin plate method (FP). Furthermore, when euglobulin precipitates from 55 healthy individuals and 36 patients with thromboembolic disease were examined, a good correlation between the two methods was observed. The present observations indicate that the ECLT is suitable for routine screening of fibrinolytic activity after venous stasis.     

Antiplatelet therapy in the treatment of cerebrovascular disease

Aspirin and the new agent ticlopidine have been the most thoroughly evaluated of the platelet-antiaggregating drugs used for the prevention of stroke and other vascular events. Numerous trials have shown aspirin to be effective in reducing the risk of myocardial infarction (MI), recurrent transient ischemic attacks, stroke, and vascular death in men at high risk for these events. Primary prevention trials have shown that aspirin reduces the risk of MI in healthy men over 50 years of age but does not reduce the risk of stroke. Two large, multicenter trials have shown that ticlopidine is effective in reducing the risk of fatal and nonfatal stroke in both men and women. Ticlopidine may also be effective in reducing the risk of recurrent stroke in patients who have had a completed thromboembolic stroke.     

Carotid atherosclerosis and ischemic stroke in young patients.

BACKGROUND: Epidemiological studies indicate a high prevalence of carotid atherosclerosis in elderly patients with ischemic stroke. The aim of this study was to investigate the presence of early carotid atherosclerotic lesions in young subjects with ischemic stroke, in the absence of the common atherosclerotic risk factors. METHODS: We studied 98 young patients with first ischemic stroke (54 males and 44 females; mean age 41.2 years; range 32-50) and 96 healthy controls. All subjects underwent ultrasonographic scanning of the carotid arteries according to a standardized protocol. RESULTS: The carotid intima-media thickness was significantly increased in the patient group (p<0.001) compared with controls. In addition, the prevalence of carotid atherosclerotic plaques was greater in the patients than in the controls (p<0.001). In particular, we detected 18 non-occlusive carotid plaques and 16 thrombotic occlusions. In 8 patients, the lesions were bilateral. The echographic pattern of the plaques was hard in 8 cases, soft in 5 cases, and mixed in the remaining 5 cases. CONCLUSIONS: We detected an increased wall thickness of the carotid arteries and an increased prevalence of carotid atherosclerotic lesions and carotid thrombotic occlusions in young patients with ischemic stroke, with a relative low incidence of cardiovascular risk factors. This finding suggests that arterial intima-media thickness per se is an important determinant of vascular disease in young patients. The data also provide indirect support for the potential role of genetic factors in the genesis of atherosclerosis in young patients.     

Hemorheological disturbances and characteristic parameters in patients with cerebrovascular disease

Whole blood and plasma viscosity were measured of 90 patients with chronic cerebrovascular disease (CVD) and 56 healthy individuals by Couette rotational viscometer Contraves Low Shear 30 at a steady flow. Plasma viscosity was measured with capillary viscometer of Ubbelohde type. Two subgroups of patients were investigated: 23 patients with transient ischemic attacks (TIAs) and 67 patients with chronic cerebral infarctions (CCI). They were compared with two control groups: 56 healthy individuals without risk factors for stroke and 37 randomly selected subjects with risk factors for stroke. It is established significant elevation of plasma viscosity in the patients with cerebral ischemia. The elevation of blood viscosity was most pronounced at shear rate of 94.5 s(-1). This comparison is confirmed by the criterion, using blood rigidity number h defined by the formula of Whittington and Harkness. Conclusion is drawn from our study that chronic ischemic cerebrovascular disorders are characterized with chronic hyperviscosity.     

Etiology and pathogenesis of thromboembolism.

Thrombus formation in the left atrium and left ventricle is primarily due to stasis of blood which causes activation of the coagulation system. Migration of thrombotic material into the circulation depends on the dynamic forces of the circulation. Atrial fibrillation is the commonest underlying cardiac disorder predisposing to thromboembolism. Rheumatic mitral stenosis, left atrial enlargement, prior myocardial infarction, hypertension, and echocardiographic left ventricular hypertrophy are risk factors for thromboembolic stroke in elderly patients with chronic atrial fibrillation. Non-valvular atrial fibrillation accounts for 45% of cardiac sources of thromboembolic stroke and includes patients with ischemic heart disease, hypertension, thyrotoxic heart disease, hypertrophic cardiomyopathy, chronic sinoatrial disorder, and idiopathic atrial fibrillation. 15% of cardiac sources of thromboembolic stroke are associated with acute myocardial infarction, 10% with left ventricular aneurysm and mural thrombi remote from an acute myocardial infarction, 10% with rheumatic valvular heart disease, and 10% with prosthetic cardiac valves. Mitral valve prolapse, mitral annular calcium, nonischemic cardiomyopathies, infective endocarditis, nonbacterial thrombotic endocarditis, left atrial myxoma, paradoxical embolism associated with congenital heart disease, calcific aortic stenosis, and complex atherosclerotic plaque within the proximal aorta also contribute to thromboembolism.     

Thromboembolic phenomena in patients with hereditary factor XI deficiency

Factor XI deficiency is an hereditary coagulopathy that is usually associated with milder tendency to bleeding with comparison to hemophilia A. While the failure of stable fibrin clot formation may lead to bleeding, it is speculated that the same process may provide a protection against thrombosis of injured arteries due to atherosclerotic plaque rupture. Whereas 2 studies indicate that hemophiliacs have decreased mortality rate from cardiovascular diseases, there is no similar data regarding factor XI deficiency patients. In here we report about 3 patients with severe factor XI deficiency who have a long-standing history of thromboembolic phenomena: 2 patients with myocardial infarctions, and one patient with transient ischemic attacks. We discuss the possible role of factor XI in thrombosis, and whether its deficiency may protect patients from thromboembolic phenomena.     

Instability of anticoagulation intensity contributes to occurrence of ischemic stroke in patients with non-rheumatic atrial fibrillation

The efficacy of anticoagulation in reducing the risk of cardiogenic embolism has been demonstrated in patients with atrial fibrillation (AF), but there are few prospective studies assessing the influence of anticoagulation stability on ischemic stroke in such patients. Accordingly, the present study investigated prospectively whether an instability of the anticoagulation intensity would affect the efficacy of the therapy in a total of 156 patients with non-rheumatic AF (NRAF) who received oral anticoagulation with warfarin. During a 2-year follow-up period, the annual event rate of ischemic stroke was 2.1%. In patients without a history of prior stroke, no ischemic stroke occurred at a higher international normalized ratio (INR> or =2.0). In contrast, patients who had had a prior stroke had no INR-dependent reduction of incidence. The coefficient of variation (CV) of measured INRs was significantly greater in patients with ischemic stroke than in those without. By multivariate analysis, only greater CV (> or =0.3) of INRs was an independent risk for ischemic stroke, although New York Heart Association functional class > or =II and treatment with diuretics were of borderline significance by univariate analysis. The present results suggest that stability of anticoagulation intensity is important to protect thromboembolic events in patients with NRAF.     

807C/T polymorphism in the platelet glycoprotein Ia gene in young patients with ischemic stroke of undetermined etiology.

Platelet membrane glycoprotein receptors mediate key reactions in arterial thrombosis. The relationship between glycoprotein Ia polymorphisms and the risk of ischemic stroke, however, remains controversial. A matched case-control study was conducted to evaluate this question in young patients. Seventy patients with ischemic stroke of undetermined etiology, with ages ranging from 15 to 50 years, and 70 healthy control individuals, matched by age, gender and ethnicity, were tested for the 807C/T genotypes. Patients were excluded if they had systemic diseases known to predispose to thrombosis or any defined etiology of ischemic stroke. The frequencies of the 807T glycoprotein Ia variant and of conventional risk factors for arterial thrombosis (hypertension, smoking, diabetes mellitus, use of oral contraceptives, levels of serum cholesterol and body mass index) were compared in stroke patients and control individuals. The 807T allele was found in 61% of patients and 53% of control individuals (matched-pair odds ratio, 1.38; 95% confidence interval, 0.69-2.74; P = 0.42). Arterial hypertension and smoking were more frequent in patients than control individuals (matched-pair odds ratio, 2.83; 95% confidence interval, 1.05-8.02; P = 0.04; and odds ratio, 3.20; 95% confidence interval, 1.10-9.97, P = 0.03, respectively). In conclusion, our results do not support an independent association between the 807C/T polymorphism and stroke of undetermined etiology. The interplay of this polymorphism with arterial hypertension in the causation of ischemic stroke requires further evaluation.