Dense Fibrin Network Research Articles

Increased Thrombin Generation and Fibrin Formation In Premature Aging BMAL1 Deficient Mice

IntroductionThe occurrence of venous thromboembolism is strongly age-dependent and has a substantial morbidity and mortality. While the incidence is<1 per 1000 per year up to an age of 50, it thereafter increases rapidly end exponentially. The cause of this increased risk is still unclear, however immobility, decreased muscular tone, aging of the veins and the presence of other or more risk factors and diseases than young individuals all contribute to this increased risk. Whether age-induced circulatory changes and/or increased levels of blood coagulation levels are responsible for the increased thrombogenicity remains unclear. We recently developed a method in which these problems were overcome enabling the measurement of thrombin generation (TG) in a small aliquot of blood. The advantage of the use of this whole blood assay is that the small volume enables us to apply this assay to mice and the use of whole blood enables us to include the effect of blood cells on TG. ObjectivesBy modifying the classic plasma Calibrated Automated Thrombogram (CAT) assay, we were able of measuring TG in whole blood in mice. The objective was to validate this assay in mice blood and to examine the rate and extent TG in a mouse model of premature aging. MethodsTG was assayed in 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. The Bmal1-KO mice have an impaired circadian behavior and demonstrate loss of rhythmicity in the expression of their target genes. They are known to have a reduced lifespan and display symptoms of premature aging. Mice blood samples were taken from the orbital sinus using a capillary anticoagulated with citrate (3.8%). Coagulation and therefore TG was initiated with a solution containing calcium, tissue factor (TF) and a thrombin specific substrate. Varying the TF concentration revealed an optimal of 0.5 pM. At the end of the TG assay, the samples were fixated, prepared for and analysed by scanning electron microscopy (SEM). ResultsThe intra-assay variations (CV%) in mice blood of the endogenous thrombin potential (ETP), peak height, lagtime, time-to-peak and velocity index were 10% or less (n=24). The whole blood TG test showed that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437±7 nM.min; mean±SD, n=5) when compared with wild type mice (ETP=220±45 nM.min; mean±SD, n=5). The peak heights also differed significantly (p=0.027). However, differences in lagtime, TTP and velocity index were not significantly different (p-values were respectively 0.45, 0.15 and 0.74). Applying SEM we found that Bmal1 deficient mice display a more dense fibrin network with smaller pores compared to WT-mice. ConclusionsThe whole blood TG assay in mice blood revealed to be reproducible and we demonstrated that aging Bmal1-KO mice have a hypercoagulable state when compared with WT mice indicating that a higher thrombotic risk in elderly is partially due to a more procoagulation state. Disclosures:No relevant conflicts of interest to declare.

Reduced Plasma Fibrin Clot Permeability and Susceptibility to Lysis in Patients with Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is associated with an increased risk of thromboembolism. Its mechanism is still unclear. Altered fibrin clot properties have been reported in patients with thromboembolism and those with chronic inflammatory states. We investigated whether fibrin characteristics are abnormal in IBD. Ex vivo plasma fibrin clot permeability (Ks), compaction, turbidity, and efficiency of fibrinolysis were assessed in 85 consecutive patients with IBD, including 47 with ulcerative colitis (UC) and 38 with Crohn's disease (CD), all with no history of thromboembolism. Forty-eight patients matched for age and sex served as controls. Compared with controls, patients with UC and CD had 29.5% and 35.7% lower Ks associated with 13.8% and 23.1% lower compaction, respectively (all P < 0.001). Patients with UC and CD had higher maximum clot absorbance (+8.9%, P = 0.008, and +15.2%, P < 0.0001, respectively), higher maximum D-dimer released from clots (D-D(max), +27.0%, P = 0.01, and +28.7%, P < 0.0001, respectively), and prolonged clot lysis time (+19.0%, P < 0.0001, and +25.5%, P < 0.0001, respectively). Lag phase was similar in both group of patients. D-D(max) was the only parameter that differed between patients in the UC and CD groups, being higher in CD (P = 0.04). The multiple linear regression model showed that in patients with UC, but not with CD, Ks, compaction, lysis time, and D-D(max) were all independently associated with disease activity. In patients with CD, Ks and lysis time were independently predicted by fibrinogen and C-reactive protein. Both UC and CD are characterized by formation of dense fibrin networks relatively resistant to lysis. Prothrombotic clot phenotype might represent a novel mechanism increasing thrombotic risk in IBD.

Ambivalent roles of carboxypeptidase B in the lytic susceptibility of fibrin

BackgroundRemoval of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, which also removes arginines that are exposed at the time of fibrinogen clotting by thrombin.ObjectiveTo evaluate the impact of alterations in fibrin structure mediated by constitutive carboxypeptidase activity on the function of fibrin as a template for tissue plasminogen activator-(tPA) induced plasminogen activation and its susceptibility to digestion by plasmin.Methods and resultsWe used the stable carboxypeptidase B (CPB), which shows the same substrate specificity as TAFIa. If 1.5 – 6 μM fibrinogen was clotted in the presence of 8 U/mL CPB, a denser fibrin network was formed with thinner fibers (the median fiber diameter decreased from 138 – 144 nm to 89 – 109 nm as established with scanning electron microscopy). If clotting was initiated in the presence of 5 – 10 μM arginine, a similar decrease in fiber diameter (82 -95 nm) was measured. The fine structure of arginine-treated fibrin enhanced plasminogen activation by tPA, but slowed down lysis monitored using fluorescent tPA and confocal laser microscopy. However, if lysis was initiated with plasmin in CPB-treated fibrin, the rate of dissolution increased to a degree corresponding to doubling of the plasmin concentration.ConclusionThe present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but conversely, CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion.

Molecular Mechanisms, Thermodynamics, and Dissociation Kinetics of Knob-Hole Interactions in Fibrin

Polymerization of fibrin, the primary structural protein of blood clots and thrombi, occurs through binding of knobs 'A' and 'B' in the central nodule of fibrin monomer to complementary holes 'a' and 'b' in the γ- and β-nodules, respectively, of another monomer. We characterized the A:a and B:b knob-hole interactions under varying solution conditions using molecular dynamics simulations of the structural models of fibrin(ogen) fragment D complexed with synthetic peptides GPRP (knob 'A' mimetic) and GHRP (knob 'B' mimetic). The strength of A:a and B:b knob-hole complexes was roughly equal, decreasing with pulling force; however, the dissociation kinetics were sensitive to variations in acidity (pH 5-7) and temperature (T = 25-37 °C). There were similar structural changes in holes 'a' and 'b' during forced dissociation of the knob-hole complexes: elongation of loop I, stretching of the interior region, and translocation of the moveable flap. The disruption of the knob-hole interactions was not an "all-or-none" transition as it occurred through distinct two-step or single step pathways with or without intermediate states. The knob-hole bonds were stronger, tighter, and more brittle at pH 7 than at pH 5. The B:b knob-hole bonds were weaker, looser, and more compliant than the A:a knob-hole bonds at pH 7 but stronger, tighter, and less compliant at pH 5. Surprisingly, the knob-hole bonds were stronger, not weaker, at elevated temperature (T = 37 °C) compared with T = 25 °C due to the helix-to-coil transition in loop I that helps stabilize the bonds. These results provide detailed qualitative and quantitative characteristics underlying the most significant non-covalent interactions involved in fibrin polymerization.

Abstract TP254: Clot Dissolution Is Better With Ultrasound Assisted Thrombolysis For Fresh Clots With Higher Cholesterol Content

Background: Intravenous tissue plasminogen activator (IV-tPA) remains the only drug for arterial recanalization in acute ischemic stroke (AIS). Clot composition differs according to the stroke etiology- clot from heart being fresh and rich in fibrin while the clot from an ulcerated plaque is rich in cholesterol. These characteristics might influence the therapeutic response to tPA as well as ultrasound-enhanced thrombolysis. We evaluated the impact of cholesterol content and age of the clot on its dissolution with tPA and ultrasound in an in-vitro model. Methods: Clot was prepared by mixing horse blood with Calcium-chloride solution. Cholesterin was added to increase cholesterol level up to 100 mg/dL. Periodic serum replacement was used to ‘age’ the clot. The clots were exposed to continuous 2-MHZ diagnostic transcranial Doppler (TCD) ultrasound (acoustic intensity 340mW/cm2) during thromolysis with tPA (355 IU/ml). Efficiency of thrombolysis was calculated as the percentage of clot weight-loss after 60 minutes. Clot characteristics after lysis were evaluated with surface emission microscope (SEM). Results: Significant increase in the weight of clot occurred with its increasing age (from 2 to 10 hours, the clot-weight increased from 37.28±2.87mg to 51.56±5.34mg; p&lt;0.005). Exposure to ultrasound and tPA resulted in significant reduction in the clot-weight at 1 hour (from 35.50±3.23% to 24.71±2.11%; p&lt;0.005). Although, no significant differences were noted between clot-cholesterol levels and lysis with TCD or tPA alone, combination of these modalities induced significant lysis in clots with higher cholesterol levels (clot-weight reduced by 41.68±2.3%) as compared to clots with normal cholesterol (30.60±4.10%; p&lt;0.005). SEM demonstrated thicker and denser fibrin network in aged clots while higher number of activated platelets were noted on the surface of cholesterol-rich clots. Conclusion: Ultrasound-assisted thrombolysis with tPA work better for fresh thrombi with higher cholesterol levels. Our findings provide insights into the differential recanalization rates in acute ischemic stroke of various etiologies

Abstract 521: The Thrombotic Potential of the Endothelium Is Regulated by Hemodynamic Flow

BACKGROUND: Vascular wall mechanisms underlying human arterial and venous thrombosis are still evolving. TNFα and other cytokines have been shown to be potent mediators of endothelial cell-derived tissue factor in vitro, which mediates fibrin deposition in the vascular wall. Circulating levels of TNFα detected in humans with cardiovascular disease are approximately 0.05 ng/ml. METHODS: Human endothelial cells were co-cultured with or without smooth muscle cells in the presence or absence of human-derived, region-specific hemodynamics. Endothelial cells were exposed to TNFα at various concentrations and incubated in human, platelet-free-plasma supplemented by Alexa 488-labeled fibrinogen. Conversion of A488-fibrinogen to A488-fibrin and deposition on the endothelium was quantified by confocal microscopy. RESULTS: In static cultures, samples treated with 1ng/ml TNFα demonstrated minimal fibrin deposition whereas samples treated with 10ng/ml or 20ng/ml exhibited dense fibrin networks (1.17e5 vs. 2.69e7 vs. 3.61e7 mean fluorescence intensity, respectively). Fibrin deposition was tissue factor-dependent. In contrast, endothelial/smooth muscle cell co-cultures primed with inflammatory-prone hemodynamics derived from the internal carotid sinus and 0.05ng/ml TNFα deposited a dense fibrin network (1.9e7 mean fluorescence intensity). Identical experiments in which only endothelial cells were cultured yielded similar results demonstrating the result is hemodynamic-specific and not a consequence of the presence of smooth muscle cells. CONCLUSIONS: Hemodynamic priming of endothelial cells in vitro shifts the dose-dependent fibrin deposition response to TNFα into a more human physiological range, two orders of magnitude below that required to induce fibrin deposition in static endothelial cell cultures. Similar approaches can be used to investigate the differential mechanisms underlying vascular wall-mediated venous and arterial thrombosis.

Plasma and cellular contributions to fibrin network formation, structure and stability

Growing evidence suggests that fibrin network structure and stability are important determinants of haemostasis and thrombosis, with alterations in fibrin structure implicated as a causative mechanism in various haemostatic and thrombotic disorders. In haemophilia, for example, deficiency of factor VIII or IX reduces the rate and peak of thrombin generation and produces coarse fibrin clots that show increased susceptibility to fibrinolysis. More recently, studies have shown significant effects of cellular activity and integrin composition on fibrin network and stability. Platelets support the formation of a dense, stable fibrin network via interactions between the alphaIIbbeta3 integrin and the fibrin network, whereas tissue factor-bearing cells regulate fibrin structure and stability predominantly via procoagulant activity. Highly procoagulant extravascular cells (e.g. fibroblasts and smooth muscle cells) support the formation of dense fibrin networks that resist fibrinolysis, whereas unstimulated intravascular cells (e.g. endothelial cells) produce coarser networks that are susceptible to fibrinolysis. Moreover, cellular contributions produce heterogeneous clots in which fibrin network density and stability decrease with increasing distance from the cell surface. Together, these findings suggest that specific plasma and cellular mechanisms link thrombin generation, clot stability and haemostatic or thrombotic outcome. Understanding these mechanisms may provide new therapeutic targets in the management of bleeding and thrombotic disorders.

Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability

AbstractFibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cell's surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between β3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.

Hemocompatibility of diamondlike carbon–metal composite thin films

We have investigated the hemocompatibility of diamondlike carbon–silver composite and diamondlike carbon–titanium composite thin films prepared using a multicomponent target pulsed laser deposition process. These materials were examined using transmission electron microscopy, Raman spectroscopy, nanoindentation, electrochemical charge transfer testing, and platelet adhesion testing. Cross-sectional transmission electron microscopy revealed that silver self-assembles into nanoparticle arrays within the diamondlike carbon matrix in the diamondlike carbon–silver composite film. On the other hand, titanium self-assembles into alternating nanometer-thick titanium carbide layers within the diamondlike carbon matrix in the diamondlike carbon–titanium composite film. The hemocompatibility of these materials was examined using electrochemical charge transfer testing and platelet adhesion testing. A few small, widely scattered crystals were observed on the surface of the unalloyed diamondlike carbon film exposed to platelet rich plasma. On the other hand, dense fibrin networks with densely aggregated platelets were observed on the surfaces of diamondlike carbon–silver and diamondlike carbon–titanium composite thin films exposed to platelet rich plasma. Electrochemical testing revealed that the time constant for the diamondlike carbon thin film ( λ = 1) was significantly higher than those for the diamondlike carbon–silver and diamondlike carbon–titanium composite thin films. These results suggest possible uses for diamondlike carbon thin films and diamondlike carbon–metal composite thin films as coatings in next generation cardiovascular implants.

Fate of fibrin sealant in pericardial space

Background. Although fibrin sealant (Beriplast, Aventis Behring, Marburg, Germany) has been widely used as a supplementary measure for hemostasis during cardiac surgery in Europe and is becoming popular in the United States, the pharmocokinetics of fibrin sealant applied in pericardial space has not been elucidated. Methods. A small incision was made on the epicardial surface of the left ventricle of a rat, and the incision was sutured. Total 0.2 ml of fibrin sealant containing iodine 125 ( 125I)-labeled fibrinogen, aprotinin, blood coagulation factor XIII and thrombin was applied to the area around the suture line. Results. Distributions of 125I-labeled fibrinogen in the heart on postoperative days 1, 3, 7, and 14 were 48.2% ± 1.8%, 20.7% ± 2.2%, 0.15% ± 0.02%, and 0.01% ± 0.02%, respectively. The radioactivity was negligible in the blood, liver, spleen, and kidney except for the thyroid in which the radioactivity increased to 7.9% ± 0.7% and 4.3% ± 0.4%, respectively, on postoperative days 7 and 14. Iodine 125-labeled fibrinogen concentrations of the heart and other organs showed a similar change in the time course of distribution. Dense and thick fibrin network, observed on postoperative day 1, had dissipated and was thinner with collagen formation by postoperative day 7. Conclusions. Fibrin sealant applied to the pericardial cavity regresses rapidly and plays an important role in wound healing.

The Vicious Cycle of Nonhealing Neointima in Fabric Vascular Prostheses

Delayed neointimal healing of a fabric vascular prosthesis was investigated in an animal study focusing on the relationship between red thrombus, fibrinolysis, and endothelialization on the luminal surface. Fabric vascular prostheses were implanted into the descending aortas of 72 dogs. Fifty-nine grafts were explanted from 1 h to 1,705 days after implantation. One hour after implantation, the graft wall was red in color due to fresh thrombus; however, at 1 day the luminal surface became white. Red thrombus reappeared at 1 week and remained present in the long-term. Microscopically the initial red thrombus contained numerous erythrocytes. The white thrombus at 1 day was composed of a dense fibrin network without erythrocytes. At 2 days numerous lacunae appeared in the fibrin layer, and at 3-5 days cavernae and low density fibrin areas were present secondary to fibrinolysis. These areas allowed the blood components to infiltrate into the fibrin layer, and as a result red thrombus reformed within it. The thrombi on the luminal surface in the long-term was always red in color and composed of complicated, multiple stages of thrombus formation, i.e., fresh thrombus with erythrocytes, dense fibrin without erythrocytes, low fibrin density areas, lacunae and cavernae in the fibrin layer, and blood component infiltration into these spaces. Thrombus was always newly formed and present, and involuted in parallel due to fibrinolysis, suggesting that these phenomena perpetuated in a vicious cycle. However, at the anastomoses fibrinolysis was present, but blood component infiltration was prevented by the endothelial cell lining. These results suggest that endothelialization may arrest the vicious cycle of nonhealing neointima in fabric vascular prostheses.

Fiberoptic study on the effects of transluminal angioplasty in experimental occlusive arterial thrombosis

Percutaneous transluminal coronary angioplasty has been proposed as definitive therapy for coronary recanalization of occluded coronary arteries in patients with acute myocardial infarction (AMI). The effects of transluminal angioplasty (TA) on experimental occlusive canine arterial thrombi that closely simulated the clinical condition was examined by a fiberoptic method. Experimental arterial thrombosis was produced by endothelial denudation and induction of luminal stenosis. Eighteen dogs that showed total occlusion of the iliac artery with thrombi were evaluated. Seven dogs (group A) with 6-hour-old thrombi received 20,000 IU/kg intravenous urokinase (UK) but did not show recanalization. TA was performed with a Gruentzig or Simpson-Robert balloon catheter and its effect was evaluated by a vascular fibroscope. Eight dogs (group B) with 6-hour-old thrombi underwent primary TA. After TA, less than 50% luminal obstruction with residual thrombi was visualized in five dogs (71%) of group A and four dogs (50%) of group B. Residual thrombi showed a doughnut-like or globular type shape and consisted of dense fibrin networks and compact platelet aggregates. All dogs in group B received 20,000 IU/kg intravenous UK after TA, but most of them showed progression of thrombus size despite UK infusion. In conclusion, the results suggest (1) that TA is effective in recanalization of an occluded artery with aged thrombus that is resistant to thrombolytic therapy and (2) that vascular fiberscope is a useful method for evaluation of the effects of TA on occlusive arterial thrombus.

Experimental canine arterial thrombus formation and thrombolysis: A fiberoptic study

We produced arterial thrombi that simulated the clinical condition in canine iliac arteries by endothelial denudation and partial occlusion. The processes of thrombus formation and thrombolysis were examined by vascular fiberscope. Laminar mural thrombi developed in all 21 preparations 10 minutes after blood reperfusion, with stenosis formation. Over 50% luminal obstruction with thrombi occurred in 10 preparations 30 minutes after inducing stenosis. Total occlusion with thrombi occurred in four preparation (31%) at 1 hour, and in all nine at 3 hours after inducing stenosis. Usually, red thrombus at the denudated region grew in size distal to the sited of stenosis. Histologically, thrombi of less than 1 hour duration consisted of a loose fibrin network and those of over 3 hours' duration consisted of a dense fibrin network. Infusion of 144,000 IU of urokinase (UK) reduced the size of thrombi less than 1 hour old during fiberoptic observation. However, UK infusion of similar dose failed to recanalize two of four preparations with 3-hour-old thrombi. In conclusion, arterial thrombi that closely simulate those observed clinically can be made by endothelial denudation and partial occlusion, and the vascular fiberscope provides a useful method for analysis of thrombosis and thrombolysis.

Surface structure of the human term placenta and of the uterine wall post partum in the screen scan electron microscope

The human term placenta after normal and toxemic pregnancies, the membranes, and the endometrial surface post partum was examined by means of the screen scan electron microscope. This method permits a stereoscopic view on tissue surfaces with magnifications up to ×20,000. Anatomical relations between maternal erythrocytes and the syncytiotrophoblastic surface of the tertiary chrionic villi can thereby be visualized. The villous surface of the mature human placenta consists of a relatively flat brush border of microvilli. In the region of infarctions the terminal chrionic villi are covered by a dense fibrin network, and maternal erythrocytes there are fixed by fibrin very similar to intravascular clot formation. Placental septa are wrapped by a trophoblastic layer where single chorionic villi insert. The paraplacental exchange surface of the membranes is stabilized by coarse collagen fibrils, and the coverture of the amnion cells resembles rudimentary microvilli. Cytotrophoblastic cells near the vessel entries in the basal plate are linked like cells in an epithelial layer. Postpartum hemostasis in utero is partially managed by a clotting process, the result of which can be demonstrated as a fibrinous “wallpaper” extended on the internal surface of the uterus.