Ultrastructure Of Fibrin Networks Research Articles

Clinical relevance of hypercoagulability and possible hypofibrinolysis associated with estrone and estriol.

Estrone (E1 ) and Estriol (E3 ) are endogenous female hormones, present in increased concentrations during female specific physiological processes (menopause and pregnancy respectively) that are associated with increased venous thrombotic risk. These hormones are also used as hormone therapies that are also associated with increased thromboembolism risk. Viscoelastic analysis revealed no significant difference to clot formation after hormone addition, however morphological analysis showed that the addition of both E1 and E3 result in fibrin clots composed of thinner fibrin fibers arranged in dense matted networks. These changes to the fibrin network ultrastructure are indicative of a prothrombotic state but may also indicate hypofibrinolysis. We therefore conclude that the increased risk of venous thrombosis during pregnancy and menopause may originate from a combination of hypercoagulation and a possible hypofibrinolytic mechanism of these hormones. Therefore females with a hypercoagulable tendency that fall pregnant or enter menopause need to be monitored to prevent venous thrombotic events. The decision to use hormone therapies during and after menopause should not be taken lightly and the risk-reward scale should be closely examined to ensure it does not tip towards thrombosis and subsequent thrombotic events that ultimately could have been prevented.

Clinical relevance of fibrin fiber diameter during different phases of pregnancy.

Pregnancy-related deep vein thrombosis (DVT) is most common during the late phase of pregnancy and the first 6-weeks postpartum. Pregnancy-related DVT can have long-term complications, specifically post-thrombotic syndrome (PTS). Fibrin network ultrastructure is altered during pregnancy and post-partum. It is therefore essential to evaluate fibrin fiber diameter during and after pregnancy as this may provide insight into pregnancy-related DVT and subsequent PTS. The fibrin network ultrastructure of females during different phases of pregnancy was compared to that of non-pregnant females to assess possible changes to the fibrin network morphology and fibrin fiber diameter using scanning electron microscopy micrographs. The fibrin network arrangement was more densely packed during different phases of pregnancy, corresponding to earlier findings. Fibrin diameter decreased significantly during pregnancy, with the greatest decrease occurring during the late phase of pregnancy. The fractal dimensions of fibrin micrographs increased significantly during pregnancy compared to nonpregnant females. These changes are indicative of a simultaneous hypercoagulable and hypofibrinolytic state and correspond to the increased risk of DVT and subsequent development of PTS. It is critical to identify "vulnerable" females with an inflammatory predisposition to prevent possible DVT and subsequent PTS. Modifiable risk factors like obesity and smoking should be addressed to alleviate the burden on the coagulation system. Morphological and viscoelastic techniques are crucial in assessing the coagulatory health of females during pregnancy.

Viscoelasticity and Ultrastructure in Coagulation and Inflammation: Two Diverse Techniques, One Conclusion.

The process of blood clotting has been studied for centuries. A synopsis of current knowledge pertaining to haemostasis and the blood components, including platelets and fibrin networks which are closely involved in coagulation, are discussed. Special emphasis is placed on tissue factor (TF), calcium and thrombin since these components have been implicated in both the coagulation process and inflammation. Analysis of platelets and fibrin morphology indicate that calcium, tissue factor and thrombin at concentrations used during viscoelastic analysis (with thromboelastography or TEG) bring about alterations in platelet and fibrin network ultrastructure, which is similar to that seen in inflammation. Scanning electron microscopy indicated that, when investigating platelet structure in disease, addition of TF, calcium or thrombin will mask disease-induced alterations associated with platelet activation. Therefore, washed platelets without any additives is preferred for morphological analysis. Furthermore, morphological and viscoelastic analysis confirmed that thrombin activation is the preferred method of fibrin activation when investigating fibrin network ultrastructure.

Part 2: ultrastructural changes of fibrin networks during three phases of pregnancy: a qualitative investigation.

Normal pregnancy is characterized by significant alterations in the haemostatic system accompanied by an augmented risk of thrombosis. The fibrin network ultrastructure of different phases of pregnancy, namely early pregnancy (week 8-14), late pregnancy (week 36-40) as well as post-partum (week 6-8 after birth) were compared with nonpregnant fibrin networks as well as each other to establish whether differences in fibrin network morphology exist during pregnancy. Scanning electron microscopy was employed to analyse fibrin network morphology. The fibrin networks from all phases of pregnancy appeared similar to each other, exhibiting prominent coagulant formation, an increase in the formation of minor, thin fibers, and the presence of granular globules. All three phases, however, differ from the typical fibrin network ultrastructure exhibited by the fibrin networks from nonpregnant individuals. The increase in estrogen associated with pregnancy may cause the increase in coagulation factors and ultimately the prothrombotic state characteristic of pregnancy. Since no differences were apparent between the different phases of pregnancy it suggests that activation of the coagulation system commences with pregnancy and this pro-thrombotic state continues till at least 8 weeks after birth. These results may shed light on possible pathological mechanisms employed in the development of abnormal or ailing pregnancy.

Estrogen causes ultrastructural changes of fibrin networks during the menstrual cycle: A qualitative investigation

Hormonal fluctuations may influence fibrin structure. During the menstrual cycle, plasma fibrinogen levels change, mainly due to the variations of estrogen. Throughout the menstrual cycle estrogen levels peak twice, first during the mid-follicular phase and then a lower second peak during the luteal phase. In order to investigate the possible changes in the fibrin network throughout the menstrual cycle, the fibrin network ultrastructure of six healthy female participants were studied at different intervals in the menstrual cycle where differences in estrogen levels are prevalent. Blood plasma smears were prepared for scanning and transmission electron microscopy analysis. The external and internal structure of the fibrin fibers showed different morphologies throughout the menstrual cycle. The fibrin fibers were smooth during days 1-5. However, during days 12-14 of the menstrual cycle the fibrin fiber morphology started to change, becoming less smooth. During the luteal phase of the cycle (days 20-25), the network appears sticky, where the minor, thin fibers are more prominent between the thick fibers when compared to the menstrual phase. The two estrogen peaks of the menstrual cycle coincide with the changes seen in the current qualitative research, where the fibrin morphology changes during the same time as the estrogen peaks occur. Purified fibrinogen confirmed that it is indeed estrogen that causes the altered fibrin network morphology. This research is the first to show ultrastructural changes in fibrin fiber morphology resulting from estrogen changes during the menstrual cycle.

Interaction of red blood cells adjacent to and within a thrombus in experimental cerebral ischaemia

Cerebral ischaemia is associated with altered platelet and fibrin network ultrastructure indicating increased coagulation activity and resistance to fibrinolysis; which may lead to the occlusion of blood vessels. Recently, it has been shown that the addition of red blood cells to plasma has a significant effect on the structural and mechanical properties of fibrin clots and is associated with lytic resistance of thrombi. Whole blood was collected from pre-ischaemic control Sprague Dawley rats and those in which experimental cerebral ischaemia was induced by hyperglycaemic two-vessel occlusion, for the ultrastructural investigation of whole blood thrombi by scanning electron microscopy. Post-ischaemic groups were terminated at 2h, 24h and 48h subsequent to reperfusion; which were time points selected for the demonstration of initial inflammation upon neural injury, maximal neural injury and onset of regeneration. Subsequent to ischaemic insult, red blood cells transformed from normal discoid shape to form projections which allowed them to interact both with each other and with fibrin fibres in various manners. Researches have in recent years shown that inclusion of red blood cells in experimental coagula results in delayed fibrinolysis and lytic resistance. This paper shows the morphological alterations at cellular level which may elucidate the structural and mechanical strength of these clots. Through the extension of projections, red blood cells become intertwined within a thrombus to stabilise and strengthen its structure. The tighter these mechanical bonds, the more resistant thrombi are to lysis, an established characteristic of thrombi in cerebral ischaemia.

Comparing techniques: the use of recalcified plasma in comparisonwith citrated plasma alone and in combination with thrombin in ultrastructuralstudies

Fibrin plays a vital role in the coagulation process and fibrin fiber morphologycan be studied using ultrastructural techniques. When studying the ultrastructureof fibrin networks, thrombin may be added to the plasma, ensuing fibrin networkformation. The question that arises is whether there are differences in morphologywhen thrombin is added to plasma, versus morphology observed when plasma fromcitrated or recalcified citrated whole blood, is studied. The current studytherefore aimed to compare ultrastructure of platelets and fibrin networksfrom these three techniques. Results indicated comparable platelet ultrastructurebetween smears formed from the plasma of citrated blood and that of the citratedrecalcified blood. This method might give us further information regardingthe ‘natural state’ fibrin assembly and association with platelets,when studying haemostasis. However, when studying the ultrastructure of fibrinnetworks, the addition of thrombin is necessary to form an expansive, fullycoagulated layer of fibrin fibers.

Qualitative scanning electron microscopy analysis of fibrin networks and platelet abnormalities in diabetes

Diabetes is a condition defined by hyperglycaemia and these patients have a high risk of thrombosis. Previous research showed that ultrastructural changes in clot formation occur in patients in whom there are changes in the coagulation system due to, for example, an inflammatory condition. In the current study, the ultrastructures of platelets and fibrin networks were investigated in 25 diabetic patients. Plasma smears, with and without the addition of thrombin, were prepared. Results indicated that the fibrin network in all diabetic patients had thickened masses of thin, minor fibres over the major fibres, a profile typical of an inflammatory condition. A changed platelet membrane ultrastructure could also be observed in the diabetic patients that revealed typical apoptotic morphology, in whom membrane blebbing could be seen. It can, therefore, be concluded that in diabetic patients, the ultrastructure of fibrin networks show a typical systemic inflammatory profile, although platelets seem to be apoptotic.

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.

Investigating the effect of a platelet additive solution on apheresis platelet and fibrin network ultrastructure

In thrombotic events and diseases such as cancer, HIV/AIDS, dysfibrinogenaemia, as well as acute incidents (e.g. burn wounds), ultrastructure of platelets and fibrin networks change. In the current study, we compare the ultrastructure of platelets and fibrin networks of apheresis platelets stored in citrated human plasma (CP) and in a first-generation platelet additive solution (PAS) (T-Sol), to that of fresh donor plasma (FP). Eighteen apheresis platelet donors donated platelets on Trima®-Accel™ V5.2 and V5.1 cell separators. Six collections were stored for five days in autologous citrated plasma (CP); six collections were stored in 40% citrated human plasma and 60% PAS solution (CP/PAS) controlled, for the duration of storage, at a constant temperature (22±2°C) with continuous flat-bed agitation; and six collections were stored in conditions uncontrolled for temperature and without continuous agitation. On days 1, 3 and 5, equal volumes of human thrombin were mixed with platelets collected in either CP or CP/PAS to form a coagulum (fibrin network containing platelet aggregates), followed by preparation for scanning electron microscopy. Results were compared with platelets and fibrin networks in FP. Typically, in FP, platelet aggregates with smooth membranes and pseudopodia are seen and fibrin networks arrange to form major, thick fibers and scattered, minor, thin fibers. On day 1, in CP and in all CP/PAS units, platelet ultrastructure compared well to that of FP, although the fibrin fibers were denser, with the minor fibers forming a matted layer over the major fibers. On day 3, in platelet units uncontrolled for temperature and without continuous agitation during storage, some platelet aggregates in CP/PAS showed typical apoptotic morphology, with shrinkage and membrane damage, but comparable fibrin networks were present. On day 5 however, in those units where storage conditions were uncontrolled and where the pH had decreased to below 6.4, no platelet aggregates were seen and fibrin was arranged into short, lumpy masses with no separate major or minor fibrin fibers visible. In those units stored at 22°C with continuous flat-bed agitation, where pH was maintained >7.0, ultrastructure of platelets and fibrin network in CP/PAS was typical and similar to FP and CP at the end of five days of storage. Examining platelet and fibrin network ultrastructure may be useful, in addition to conventional laboratory analysis, in assessing the viability and potential clinical efficacy of platelets for transfusion and could play a role in the evaluation of new generation platelet additive solutions.

Comparative ultrastructure of fibrin networks of a dog after thrombotic ischaemic stroke

A cerebrovascular accident or stroke is a rare condition in dogs, but previous studies suggest that it is now increasingly being recognised. Platelets and fibrin networks are involved in haemostasis,which is disrupted during a thrombotic event. In this study we investigate the ultrastructure of the fibrin networks of a dog that had suffered ischaemic stroke, following suspected thromboembolism from clots that became dislodged during catheter maintenance (flushing with heparinised saline) 2 days after carotid artery catheter insertion. Fibrin networks of blood samples that were collected immediately after the stroke, 15 min after treatment with streptokinase and 24 h after treatment, were studied. The results were compared to those of two control dogs. During a stroke, fibrin morphology changes to form a thick, matted layer.Post-treatment ultrastructure shows that the fibrin morphology returns to that comparable to controls. Our results show that during thrombotic risk, fibrin network morphology changes visibly and reduces the fibrinolytic activity of the coagulation system.

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.

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.

Ultrastructural changes of platelets and fibrin networks in human asthma: a qualitative case study

Platelets and fibrin networks play an important role in asthma and the BALB/c asthmatic mouse model has previously been successfully used to study platelet ultrastructure. In control BALB/c mice, major, thick fibers and minor thin fibers and tight, platelet aggregates with typical pseudopodia formation, are present. Minor fibers of asthmatic mice have a netlike appearance covering the major fibers, whereas the platelets seem to form loosely connected, granular aggregates. The question that now arises is whether platelets and fibrin networks of humans with asthma will have the same ultrastructure as seen in the BALB/c asthmatic model. In order to answer this question, ultrastructure of platelets and fibrin networks from two participants (controlled asthma and uncontrolled, chronic asthma) were studied and compared with that of human controls and BALB/c asthmatic mice. Peak flow measurements of the controls and patients were also assessed. Results showed that similar platelet and fibrin network ultrastructure is found in uncontrolled, human participants and BALB/c asthmatic animals. The challenge when using animal models is always whether the model adequately mimics the human disease; the current research, therefore, shows morphological support for the use of this model in the study of asthma. These morphological results may also provide additional information to plan treatment regimes for sufferers of this very debilitating disease.