Journal articles on the topic 'Clot structure'
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1
Smith, Stephanie A., and James H. Morrissey. "Polyphosphate Enhances Fibrin Clot Structure." Blood 110, no. 11 (November 16, 2007): 403. http://dx.doi.org/10.1182/blood.v110.11.403.403.
Abstract:
Abstract Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V. Our previous studies found that polyP did not affect the time to clot formation when plasma was clotted with thrombin, however, suggesting that polyP exerts its procoagulant actions upstream of thrombin. We now report that polyP enhances fibrin clot structure. Methods: Purified fibrinogen and polyP were preincubated for 15 min in multiwell plates in buffer containing CaCl2, after which clotting was initiated by adding 0.1 to 8 nM thrombin and fibrin clot formation was evaluated by quantifying the change in turbidity (A405). Mass-length ratios were calculated from scans of A400 to A800. The effect of polyP on fibrinolysis was examined by adding 8 nM plasmin to the reaction mixtures immediately prior to thrombin. Scanning electron microscopy (SEM) was employed to visualize clot structure, and time courses of covalent fibrin cross-linking were assessed by SDS-PAGE. Results: PolyP had no effect on time to clot formation, but clots formed in the presence of polyP had markedly (up to threefold) higher turbidity than clots formed in the absence of polyP (see figure), irrespective of thrombin concentration. The increased turbidity in the presence of polyP was calcium-dependent and was enhanced when fibrinogen, CaCl2, and polyP were preincubated for up to 15 min prior to initiation of clotting with thrombin. PolyP increased the mass-length ratio of fibrin, and SEM confirmed that fibers formed with polyP were thicker than those formed without polyP. The ability of polyP to enhance fibrin clot turbidity was independent of factor XIIIa activity, and polyP did not alter the rate or extent of covalent fibrin cross-linking by factor XIIIa. When plasmin was included in clotting reactions containing polyP, mean times to 50% clot lysis were 28.5 ± 0.8 min for clots without polyP but 120.4 ± 5.6 min for clots with polyP. Conclusions: PolyP alters polymerization of fibrin, resulting in fibers of higher mass-length ratio that are lysed more slowly. This effect is calcium-dependent and is enhanced by preincubation of fibrinogen with calcium and polyP. Release of polyP from activated platelets or infectious microorganisms may therefore enhance fibrin clot structure. Figure Figure
2
Mihalko, Emily, and Ashley C. Brown. "Clot Structure and Implications for Bleeding and Thrombosis." Seminars in Thrombosis and Hemostasis 46, no. 01 (October 15, 2019): 096–104. http://dx.doi.org/10.1055/s-0039-1696944.
Abstract:
AbstractThe formation of a fibrin clot matrix plays a critical role in promoting hemostasis and wound healing. Fibrin dynamics can become disadvantageous in the formation of aberrant thrombus development. Structural characteristics of clots, such as fiber diameter, clot density, stiffness, or permeability, can determine overall clot integrity and functional characteristics that have implications on coagulation and fibrinolysis. This review examines known factors that contribute to changes in clot structure and the presence of structural clot changes in various disease states. These insights provide valuable information in forming therapeutic strategies for disease states where alterations in clot structure are observed. Additionally, the implications of structural changes in clot networks on bleeding and thrombus development in terms of disease states and clinical outcomes are also examined in this review.
3
Wolberg, Alisa S., Dougald M. Monroe, Harold R. Roberts, and Maureane Hoffman. "Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk." Blood 101, no. 8 (April 15, 2003): 3008–13. http://dx.doi.org/10.1182/blood-2002-08-2527.
Abstract:
AbstractIndividuals with elevated prothrombin levels are at increased risk of venous thrombosis. To understand the mechanism behind this observation, we studied the effect of prothrombin concentration on thrombin generation and fibrin clot structure. The pattern of thrombin generation was directly related to the prothrombin level at all concentrations tested. From 0% to 300% of normal plasma levels of prothrombin, increasing the prothrombin concentration increased the initial rate, peak, and total amount of thrombin generated. Importantly, fibrin clot structure was also affected by the prothrombin concentration. Fibrin clots made from prothrombin concentrations less than 10% of plasma levels were weak and poorly formed. Fibrin clots made at 10% to 100% of plasma levels of prothrombin had similar fiber structures (mass-to-length ratio; μ). However, the fiber mass-to-length ratio decreased with increasing prothrombin levels more than 100% of plasma levels, in a dose-dependent manner. These results suggest that increased levels of prothrombin alter thrombin generation and clot structure. Specifically, elevated prothrombin levels produce clots with reduced fibrin mass-to-length ratios compared with normal clots. We hypothesize that this alteration in fibrin clot structure is an important determinant of the risk of thrombosis.
4
Smith, Stephanie A., and James H. Morrissey. "Polyphosphate enhances fibrin clot structure." Blood 112, no. 7 (October 1, 2008): 2810–16. http://dx.doi.org/10.1182/blood-2008-03-145755.
Abstract:
Abstract Polyphosphate, a linear polymer of inorganic phosphate, is present in platelet dense granules and is secreted on platelet activation. We recently reported that polyphosphate is a potent hemostatic regulator, serving to activate the contact pathway of blood clotting and accelerate factor V activation. Because polyphosphate did not alter thrombin clotting times, it appeared to exert all its procoagulant actions upstream of thrombin. We now report that polyphosphate enhances fibrin clot structure in a calcium-dependent manner. Fibrin clots formed in the presence of polyphosphate had up to 3-fold higher turbidity, had higher mass-length ratios, and exhibited thicker fibers in scanning electron micrographs. The ability of polyphosphate to enhance fibrin clot turbidity was independent of factor XIIIa activity. When plasmin or a combination of plasminogen and tissue plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of polyphosphate exhibited prolonged clot lysis times. Release of polyphosphate from activated platelets or infectious microorganisms may play an important role in modulating fibrin clot structure and increasing its resistance to fibrinolysis. Polyphosphate may also be useful in enhancing the structure of surgical fibrin sealants.
5
Celińska-Löwenhoff, Magdalena, Teresa Iwaniec, Agnieszka Padjas, Jacek Musiał, and Anetta Undas. "Altered fibrin clot structure/function in patients with antiphospholipid syndrome: association with thrombotic manifestation." Thrombosis and Haemostasis 112, no. 08 (2014): 287–96. http://dx.doi.org/10.1160/th13-11-0980.
Abstract:
SummaryWe tested the hypothesis that plasma fibrin clot structure/function is unfavourably altered in patients with antiphospholipid syndrome (APS). Ex vivo plasma clot permeability, turbidity and susceptibility to lysis were determined in 126 consecutive patients with APS enrolled five months or more since thrombotic event vs 105 controls. Patients with both primary and secondary APS were characterised by 11% lower clot permeability (p<0.001), 4.8% shorter lag phase (p<0.001), 10% longer clot lysis time (p<0.001), and 4.7% higher maximum level of D-dimer released from clots (p=0.02) as compared to the controls. Scanning electron microscopy images confirmed denser fibrin networks composed of thinner fibres in APS. Clots from patients with “triple-antibody positivity” were formed after shorter lag phase (p=0.019) and were lysed at a slower rate (p=0.004) than in the remainder. Clots from APS patients who experienced stroke and/or myocardial infarction were 8% less permeable (p=0.01) and susceptible to lysis (10.4% longer clot lysis time [p=0.006] and 4.5% slower release of D-dimer from clots [p=0.01]) compared with those following venous thromboembolism alone. Multivariate analysis adjusted for potential confounders showed that in APS patients, lupus anticoagulant and “triple-positivity” were the independent predictors of clot permeability, while “triple-positivity” predicted lysis time. We conclude that APS is associated with prothrombotic plasma fibrin clot phenotype, with more pronounced abnormalities in arterial thrombosis. Molecular background for this novel prothrombotic mechanism in APS remains to be established.
6
Carr, M. E., and S. L. Zekert. "Abnormal clot retraction, altered fibrin structure, and normal platelet function in multiple myeloma." American Journal of Physiology-Heart and Circulatory Physiology 266, no. 3 (March 1, 1994): H1195—H1201. http://dx.doi.org/10.1152/ajpheart.1994.266.3.h1195.
Abstract:
Clot retraction, measured by serum expression, is absent in some cases of multiple myeloma. Decreased clot retraction has been attributed to platelet dysfunction. A new instrument allows simultaneous measurement of platelet-mediated force development during clot retraction and of clot elastic modulus. We report 10 patients with immunoglobulin (Ig) G myeloma in whom the abnormalities of fibrin structure were quantitatively defined and platelet-fibrin interactions were assessed. Fiber mass-to-length ratios were calculated from gel turbidity. Platelet force development and clot elastic modula were measured in platelet-rich plasma gels. Fiber mass-to-length ratios for IgG myeloma patients were smaller (means +/- SE) (0.98 +/- 0.19 x 10(13) Da/cm) than for normal controls (1.36 +/- 0.06 x 10(13) Da/cm), indicating thinner fiber formation. Elastic modula of myeloma clots (51,013 +/- 14,660 dyn/cm2) were strikingly larger than modula for normal controls (23,355 +/- 1,887 dyn/cm2), indicating that such clots are mechanically less flexible. Platelet force development 1,200 s after thrombin addition was not diminished in myeloma patients (8,315 +/- 1,155 dyn) vs. controls (6,906 +/- 606 dyn). Abnormal clot retraction in myeloma appears to be primarily due to altered clot structure rather than platelet dysfunction.
7
Gersh, Kathryn, Chandrasekaran Nagaswami, and John Weisel. "Fibrin network structure and clot mechanical properties are altered by incorporation of erythrocytes." Thrombosis and Haemostasis 102, no. 12 (2009): 1169–75. http://dx.doi.org/10.1160/th09-03-0199.
Abstract:
SummaryAlthough many in vitro fibrin studies are performed with plasma, in vivo clots and thrombi contain erythrocytes, or red blood cells (RBCs).To determine the effects of RBCs on fibrin clot structure and mechanical properties, we compared plasma clots without RBCs to those prepared with low (2 vol%), intermediate (5-10 vol%), or high (≥20 vol%) numbers of RBCs. By confocal microscopy, we found that low RBC concentrations had little effect on clot structure. Intermediate RBC concentrations caused heterogeneity in the fiber network with pockets of densely packed fibers alongside regions with few fibers. With high levels of RBCs, fibers arranged more uniformly but loosely around the cells. Scanning electron micrographs demonstrated an uneven distribution of RBCs throughout the clot and a significant increase in fiber diameter upon RBC incorporation. While permeability was not affected by RBC addition, at 20% or higher RBCs, the ratio of viscous modulus (G′′) to elastic modulus (G′) increased significantly over that of a clot without any RBCs. RBCs triggered variability in the fibrin network structure, individual fiber characteristics, and overall clot viscoelasticity compared to the absence of cells. These results are important for understanding in vivo clots and thrombi.
8
Henderson, Sara J., Jing Xia, Huayin Wu, Alan R. Stafford, Beverly A. Leslie, James C. Fredenburgh, David A. Weitz, and Jeffrey I. Weitz. "Zinc promotes clot stability by accelerating clot formation and modifying fibrin structure." Thrombosis and Haemostasis 115, no. 03 (2016): 533–42. http://dx.doi.org/10.1160/th15-06-0462.
Abstract:
SummaryZinc released from activated platelets binds fibrin(ogen) and attenuates fibrinolysis. Although zinc also affects clot formation, the mechanism and consequences are poorly understood. To address these gaps, the effect of zinc on clot formation and structure was examined in the absence or presence of factor (F) XIII. Zinc accelerated a) plasma clotting by 1.4-fold, b) fibrinogen clotting by 3.5- and 2.3-fold in the absence or presence of FXIII, respectively, c) fragment X clotting by 1.3-fold, and d) polymerisation of fibrin monomers generated with thrombin or batroxobin by 2.5– and 1.8-fold, respectively. Whereas absorbance increased up to 3.3-fold when fibrinogen was clotted in the presence of zinc, absorbance of fragment X clots was unaffected by zinc, consistent with reports that zinc binds to the αC-domain of fibrin(ogen). Scanning electron microscopic analysis revealed a twofold increase in fibre diameter in the presence of zinc and in permeability studies, zinc increased clot porosity by 30-fold with or without FXIII. Whereas FXIII increased clot stiffness from 128 ± 19 Pa to 415 ± 27 Pa in rheological analyses, zinc reduced clot stiffness by 10– and 8.5-fold in the absence and presence of FXIII, respectively. Clots formed in the presence of zinc were more stable and resisted rupture with or without FXIII. Therefore, zinc accelerates clotting and reduces fibrin clot stiffness in a FXIII-independent manner, suggesting that zinc may work in concert with FXIII to modulate clot strength and stability.
9
Martinez, Marissa R., Adam Cuker, Angela M. Mills, Amanda Crichlow, Richard T. Lightfoot, Irina N. Chernysh, Chandrasekaran Nagaswami, John W. Weisel, and Harry Ischiropoulos. "Enhanced lysis and accelerated establishment of viscoelastic properties of fibrin clots are associated with pulmonary embolism." American Journal of Physiology-Lung Cellular and Molecular Physiology 306, no. 5 (March 1, 2014): L397—L404. http://dx.doi.org/10.1152/ajplung.00265.2013.
Abstract:
The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.
10
Janmey, PA, JA Lamb, RM Ezzell, S. Hvidt, and SE Lind. "Effects of actin filaments on fibrin clot structure and lysis." Blood 80, no. 4 (August 15, 1992): 928–36. http://dx.doi.org/10.1182/blood.v80.4.928.928.
Abstract:
Abstract The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament- severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.
11
Janmey, PA, JA Lamb, RM Ezzell, S. Hvidt, and SE Lind. "Effects of actin filaments on fibrin clot structure and lysis." Blood 80, no. 4 (August 15, 1992): 928–36. http://dx.doi.org/10.1182/blood.v80.4.928.bloodjournal804928.
Abstract:
The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament- severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.
12
Varin, Remi, Shasultan Mirshahi, Pehzman Mirshahi, Li Lu-Hong, Gerald Kierzek, Jean-Francois Vigneau, Jean-Pierre Marie, et al. "Improvement of Thrombolysis by Rivaroxaban, An Anti Xa Inhibitor. Potential Therapeutic Importance in Patients with Thrombosis." Blood 112, no. 11 (November 16, 2008): 3031. http://dx.doi.org/10.1182/blood.v112.11.3031.3031.
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Abstract Background and objective Rivaroxaban – an oral, direct Factor Xa inhibitor – inhibits thrombus formation and growth in animal models. We have investigated the effects of rivaroxaban on thrombolysis because impaired fibrinolysis is a risk factor for venous thrombosis and it occurs more often in patients who had a myocardial infarction. As the propensity of a clot to be degraded depends on its structure, we tested the effects of rivaroxaban on clot structure and degradability by tissue plasminogen activator (t-PA). This was done in the absence and presence of thrombomodulin because the thrombin - thrombomodulin complex is the activator of TAFI (thrombin-activatable fibrinolysis inhibitor), a potent inhibitor of fibrinolysis. Methods Clots were formed in a microchamber in the presence or absence of rivaroxaban at pharmacological concentrations (0.15 and 0.25 μg/ml). Clot structure was analyzed by confocal microscopy, and permeability calculated by measuring flow rates. Degradation was evaluated by the amount of D-dimers in the eluate of clot perfused with t-PA, in the presence or absence of thrombomodulin. Results Microscopy showed that clots formed in the presence of rivaroxaban had thicker fibers and a looser fibrin structure with larger pores than controls, leading to increased permeation rate (Darcy constant 2.16-fold and 2.45-fold higher than controls with rivaroxaban at 0.15 μg/ml and 0.25 μg/ml, respectively). This clot structure modification renders the clots more susceptible to fibrinolytic enzymes. The degradation of clots containing 0.15 μg/ml of rivaroxaban was 3.6-fold higher than that of control clots, after 90 minutes perfusion with t-PA. In addition, when clots are formed in the presence of thrombomodulin, the degradability is decreased in control, while In the presence of rivaroxaban, fibrin degradation remains enhanced. Conclusion Rivaroxaban increased thrombolysis by t-PA. This was due to a decrease in thrombin generation. Two mechanisms are involved: modification of clot structure, which makes it more accessible to thrombolytic enzymes; and decrease in TAFI activation by the thrombomodulin–thrombin complex. This property of rivaroxaban may contribute to its antithrombotic effect.
13
Faes, Camille, Anton Ilich, Erica M. Sparkenbaugh, Alisa S. Wolberg, Brian C. Cooley, Kenneth I. Ataga, Nigel S. Key, and Rafal Pawlinski. "Alteration of the Structure and Dynamics of Venous Clot Formation in Human and Murine Sickle Cell Disease." Blood 128, no. 22 (December 2, 2016): 2478. http://dx.doi.org/10.1182/blood.v128.22.2478.2478.
Abstract:
Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Traditionally, it is believed that during venous thrombosis, red blood cells (RBC) are simply trapped within fibrin-rich thrombi and do not actively affect thrombosis. However, a study from our group showed that factor XIII (FXIII) activity is critical for the retention of RBC within clots and directly affects thrombus size. Others reported that during clot contraction polyhedral shaped RBC formed a densely packed core and that SCD alters the formation of polyhedrocytes which may affect clot stability (Strauss et al, 2015, ASH abstract). We further investigated if SCD affects the structure and the dynamics of clot formation. Ex vivo clot retraction was performed using blood from sickle cell patients and Townes mice (a model of SCD). Citrated blood was added to siliconized wells of 96-well plates containing CaCl2 (10mM) and tissue factor (1pM) and incubated for 2 hours at 37°C. The number of RBC extruded from the clot was counted in serum by Hemavet™ and expressed as a percentage of initial RBC number in the anticoagulated blood. Morphology of the clots was evaluated using scanning and transmission electron microscopy (EM). Thrombosis in Townes SS (sickle) and AA (control) mice was studied using femoral vein thrombosis induced by electrolytic injury and inferior vena cava stenosis models. The number of mouse SS RBC in the serum extruded during clot contraction was dramatically reduced compared to the number of AA RBC (0.8±0.8% vs. 19.4±0.8%, n=3, p<0.0001, Fig. A). A similar result was observed for human RBC. Since SS mice and sickle patients have a lower hematocrit (HCT), we investigated if the number of RBC affects the extrusion of these cells during clot retraction. Indeed, lowering HCT in AA mouse blood reduced RBC extrusion from the clots. However, increasing HCT in SS mouse blood to that of AA blood did not increase the number of SS RBC extruded from the clot. Furthermore, inhibition of FXIIIa activity with T101 (10µM) increased the release of AA (by 64%, n=6, p<0.05) but not SS RBC from mouse clots. These data indicate that the entrapment of SS RBCs within the clot is not simply caused by lower RBC number, and is FXIII-independent. Mixing the platelet poor plasma (PPP) and cellular fraction of AA and SS mouse blood revealed that the entrapment of SS RBC is not mediated by PPP (AA blood recombined = 6.9±3.6%; SS blood recombined = 0.4±0.4%; AA cells/SS PPP = 6.7±6.4%; SS cells/AA PPP = 0.1±0.2%, n=3 per group). Clots formed ex vivo from AA blood had a gel-like, soft structure, whereas SS clots were more firm and stiff. EM demonstrated that RBC within AA clots had polyhedral shapes and were tightly packed in the central part of the clot. In contrast, most of the mouse SS RBC did not have polyhedral shapes, underwent sickling and were not compacted within the clot. They also formed long "spicule-like" processes that intertwined with fibrin fibers (Fig. C). Similar results were observed in blood of sickle patients; however, the sickled RBC phenotype was less prominent. Importantly, sickling of RBC was observed in clots formed in the inferior vena cava of SS mice, 2 hours after vessel stenosis (Fig. D). We also performed a tPA challenge assay on clots formed ex vivo from human blood and showed that SS clots challenged with low tPA concentration (0.6nM) were more resistant to fibrinolysis compared to AA clots (clot lysis time, 714±6 vs. 388.3±120.7 minutes, n=6, p=0.08). The electrolytic injury model of venous thrombosis was used to investigate the dynamics of clot formation in SS mice in vivo. Mice were infused with fluorescently labeled antibodies for fibrin (green) and platelets (red). Electrolytic injury was applied to the femoral vein; a relative intensity of fibrin and platelet accumulation was assessed by fluorescence microscopy for one hour at 10-minute intervals. SS mice have increased platelet and fibrin accumulation compared to AA mice (~ 2 fold, n=5-7, p<0.05 for 40, 50 and 60 minute time points). Interestingly, in the AA clots, platelets were mostly localized on the surface, in contrast to their widespread distribution throughout the clot in SS mice (Fig. E, yellow color). Our data demonstrated that SCD alters the structure and dynamics of venous clot formation. Experiments investigating the consequence of these observations in mouse models of stroke and pulmonary embolism are currently ongoing. Figure Figure. Disclosures No relevant conflicts of interest to declare.
14
Brown, Ashley C., Riley H. Hannan, Lucas H. Timmins, Janet D. Fernandez, Thomas H. Barker, and Nina A. Guzzetta. "Fibrin Network Changes in Neonates after Cardiopulmonary Bypass." Anesthesiology 124, no. 5 (May 1, 2016): 1021–31. http://dx.doi.org/10.1097/aln.0000000000001058.
Abstract:
Abstract Background Quantitative and qualitative differences in the hemostatic systems exist between neonates and adults, including the presence of “fetal” fibrinogen, a qualitatively dysfunctional form of fibrinogen that exists until 1 yr of age. The consequences of “fetal” fibrinogen on clot structure in neonates, particularly in the context of surgery-associated bleeding, have not been well characterized. Here, the authors examine the sequential changes in clotting components and resultant clot structure in a small sample of neonates undergoing cardiac surgery and cardiopulmonary bypass (CPB). Methods Blood samples were collected from neonates (n = 10) before surgery, immediately after CPB, and after the transfusion of cryoprecipitate (i.e., adult fibrinogen component). Clots were formed from patient samples or purified neonatal and adult fibrinogen. Clot structure was analyzed using confocal microscopy. Results Clots formed from plasma obtained after CPB and after transfusion were more porous than baseline clots. Analysis of clots formed from purified neonatal and adult fibrinogen demonstrated that at equivalent fibrinogen concentrations, neonatal clots lack three-dimensional structure, whereas adult clots were denser with significant three-dimensional structure. Clots formed from a combination of purified neonatal and adult fibrinogen were less homogenous than those formed from either purified adult or neonatal fibrinogen. Conclusions The results of this study confirm that significant differences exist in clot structure between neonates and adults and that neonatal and adult fibrinogen may not integrate well. These findings suggest that differential treatment strategies for neonates should be pursued to reduce the demonstrated morbidity of blood product transfusion.
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Gao, Chunli, Bin Bao, Chunling Bao, and Wenhui Wu. "Fungi Fibrinolytic Compound 1 Plays a Core Role in Modulating Fibrinolysis, Altering Plasma Clot Structure, and Promoting Susceptibility to Lysis." Pharmaceutics 15, no. 9 (September 14, 2023): 2320. http://dx.doi.org/10.3390/pharmaceutics15092320.
Abstract:
Fibrin clot structure and function are major determinants of venous and arterial thromboembolic diseases, as well as the key determinants of the efficiency of clot lysis. Studies have revealed that fungi fibrinolytic compound 1 (FGFC1) is a novel marine pyranisoindolone natural product with fibrinolytic activity. Here, we explore the impacts of FGFC1 on clot structure, lysis, and plasminogen activation in vitro using turbidimetric, enzyme-linked immunosorbent assay, confocal and electron microscopy, urokinase, or plasmin chromogenic substrate. Clots formed in the presence of FGFC1 expressed reduced fibrin polymerization rate and maximum turbidity; however, they did not influence the lag phase of fibrin polymerization. In the absence of scu-PA (single-chain urokinase plasminogen activator), microscopy revealed that FGFC1 increased the number of protofibrils within fibrin fiber and the pore diameter between protofibrils, inducing clots to form a region of thinner and looser networks separated by large pores. The effects of FGFC1 on scu-PA-mediated plasma clot structure were similar to those in the absence of scu-PA. In addition, FGFC1 promoted the lysis of clots and increased the D-dimer concentration in lysate. FGFC1 increased the generation rate of p-nitroaniline in plasma. These results show that FGFC1 has fibrinolytic activity in plasma, leading to interference with the release of fibrinopeptide B to affect lateral aggregation of protofibrils and increase clot susceptibility to fibrinolysis by altering its structure.
16
Hantgan, Roy R., W. Gray Jerome, and Marcie J. Hursting. "No Effect of Clot Age or Thrombolysis on Argatroban’s Inhibition of Thrombin." Blood 92, no. 6 (September 15, 1998): 2064–74. http://dx.doi.org/10.1182/blood.v92.6.2064.
Abstract:
Abstract The purpose of this study was to establish the effects of clot age and thrombolysis, with either streptokinase or tissue-type plasminogen activator (tPA), on argatroban’s ability to inhibit thrombin. The antithrombotic activity of argatroban has been quantified in fibrin clot permeation and fibrin clot perfusion systems as a function of clot age and composition. Analysis of the argatroban dose-response data with a competitive inhibition model has yielded IC50 values in the low micromolar range. Results obtained in a plasma clot permeation system have also shown that argatroban is a potent inhibitor of clot-bound thrombin, independent of either clot age or the presence of hemostatically active platelets. Treatment of aged plasma clots with either streptokinase or alteplase, at therapeutic levels, increased the available thrombin activity, yet argatroban still inhibited this clot-associated thrombin with IC50 values in the low micromolar range. Scanning electron microscopy/morphometric analyses demonstrated that permeation with argatroban had no significant effects on clot structure. We conclude that argatroban is an effective inhibitor of thrombin bound to aged fibrin clots, in purified systems and in plasma clots, as well as in clots that have been treated with the thrombolytic agents streptokinase and alteplase. © 1998 by The American Society of Hematology.
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Hantgan, Roy R., W. Gray Jerome, and Marcie J. Hursting. "No Effect of Clot Age or Thrombolysis on Argatroban’s Inhibition of Thrombin." Blood 92, no. 6 (September 15, 1998): 2064–74. http://dx.doi.org/10.1182/blood.v92.6.2064.418k07_2064_2074.
Abstract:
The purpose of this study was to establish the effects of clot age and thrombolysis, with either streptokinase or tissue-type plasminogen activator (tPA), on argatroban’s ability to inhibit thrombin. The antithrombotic activity of argatroban has been quantified in fibrin clot permeation and fibrin clot perfusion systems as a function of clot age and composition. Analysis of the argatroban dose-response data with a competitive inhibition model has yielded IC50 values in the low micromolar range. Results obtained in a plasma clot permeation system have also shown that argatroban is a potent inhibitor of clot-bound thrombin, independent of either clot age or the presence of hemostatically active platelets. Treatment of aged plasma clots with either streptokinase or alteplase, at therapeutic levels, increased the available thrombin activity, yet argatroban still inhibited this clot-associated thrombin with IC50 values in the low micromolar range. Scanning electron microscopy/morphometric analyses demonstrated that permeation with argatroban had no significant effects on clot structure. We conclude that argatroban is an effective inhibitor of thrombin bound to aged fibrin clots, in purified systems and in plasma clots, as well as in clots that have been treated with the thrombolytic agents streptokinase and alteplase.© 1998 by The American Society of Hematology.
18
Baradet, T., J. W. Weisel, and L. D. Peachey. "A study of fibrin clot structure under various ionic conditions using stereoscopic IVEM images." Proceedings, annual meeting, Electron Microscopy Society of America 49 (August 1991): 194–95. http://dx.doi.org/10.1017/s0424820100085277.
Abstract:
Fibrin clots are formed by the conversion of fibrinogen into fibrin monomers which assemble to produce two-stranded protofibrils that aggregate to form fibers. These fibers may also aggregate laterally with other fibers to form larger fiber bundles. Investigations of fibrin clot structures under various ionic conditions using SEM showed dramatic differences in fiber morphology and clot structure. Fibrin clots formed under various ionic conditions were investigated by the examination of stereoscopic IVEM images. This technique provides greater depth discrimination and higher resolution images of clot ultrastructure. Details of fiber association and branching are of particular interest.Purified human fibrinogen was prepared at a concentration of 0.5 mg/ml in 0.4M, 0.2M, and 0.05M buffers (0.05M Tris-HCI, pH 7.4, with 2mM CaCl2). Fibrinogen solutions were mixed with thrombin to a final concentration of 0.3U/ml and aiiquots placed onto Formvar and carbon coated grids. After clotting for 1 hr. at room temperature, clots were fixed with 3% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.2, for 5 min. Grids were kept constantly moist to avoid collapse or syneresis.
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Flood, Veronica H., Chandrasekaran Nagaswami, Irina N. Chernysh, Hamid A. Al-Mondhiry, John W. Weisel, and David H. Farrell. "Effects of Impaired Fibrinopeptide A Cleavage on Fibrin Clot Structure: Studies with an Aα R16C Dysfibrinogen." Blood 108, no. 11 (November 16, 2006): 1617. http://dx.doi.org/10.1182/blood.v108.11.1617.1617.
Abstract:
Abstract Cleavage of fibrinopeptide A is the first step in fibrin clot formation, and mutations at the fibrinopeptide A cleavage site are the most common cause of dysfibrinogenemia. We describe here the effect on clot structure of a mutant Aα R16C fibrinogen with defective fibrinopeptide A cleavage (designated fibrinogen Hershey III). The propositus, a young child with mild bleeding symptoms, was found to be heterozygous for the Aα R16C mutation. Fibrinogen was purified from Hershey III and control plasma via glycine precipitation. Hershey III fibrinogen was only 63 ± 10% clottable with thrombin (mean ± SEM), as compared to 96 ± 0.4% for normal fibrinogen. Since the propositus was heterozygous for the mutation, the unclottable portion likely consisted of mutant homodimers, but it was still possible that normal/mutant heterodimers existed. Because the cysteine in the mutant fibrinogen prevents thrombin-mediated fibrinopeptide A cleavage, we hypothesized that incorporation of uncleaved fibrinopeptide A, if present, would affect clot structure. Western blotting was used to evaluate the presence of fibrinopeptide A in clottable and unclottable fibrinogen. For fibrinogen Hershey III, both forms showed a substantial amount of fibrinopeptide A, suggesting that mutant fibrinogen was incorporated into the final clot. No fibrinopeptide A was seen in either the clottable or unclottable fibrinogen from the normal control. Next, fibrin clots were made with thrombin, critical-point dried, and visualized via scanning electron microscopy. Visco-elastic measurements were obtained with a torsion pendulum and clot permeability was compared to that of clots formed with normal fibrinogen. The relative proportions of normal vs. mutant fibrinogen in the clottable and unclottable fibrinogen were assessed by protein sequencing. Scanning electron microscopy showed that the Hershey III clots displayed abnormal architecture with many short fibrin fibrils, consistent with premature fibril termination. Hershey III clots also had thicker fibers, with an average fiber diameter of 182 nm compared to 151 nm for the normal control. A significant difference in clot stiffness (G′), energy dissipated by viscous processes (G″), and permeability (Ks) was seen when fibrinogen Hershey III was compared to a normal control (see table). Protein sequencing of the unclottable Hershey III fibrinogen showed only the homozygous mutant form, while the fibrin clot showed approximately 50% each of the wild-type and mutant fibrinogen chains. These results support the presence of both homodimers and heterodimers in fibrinogen Hershey III, and suggest that incorporation of Aα R16C heterodimers into the fibrin clot leads to defects in fiber formation and clot structure. Mechanical Properties of Hershey III and Control Clots Hershey III Control P G′ (dyne/cm2) 10.8 37.9 0.03 G″ (dyne/cm2) 0.83 2.77 0.04 Tan δ (G″/G′) 0.077 0.076 0.79 Ks (10−7 cm2) 1.86 2.44 0.01
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Jerome, W. Gray, Stefan Handt, and Roy R. Hantgan. "Endothelial Cells Organize Fibrin Clots into Structures That Are More Resistant to Lysis." Microscopy and Microanalysis 11, no. 3 (May 12, 2005): 268–77. http://dx.doi.org/10.1017/s143192760505052x.
Abstract:
Acute myocardial infarction is a major cause of death and disability in the United States. Introducing thrombolytic agents into the clot to dissolve occlusive coronary artery thrombi is one method of treatment. However, despite advances in our knowledge of thrombosis and thrombolysis, survival rates following thrombolytic therapy have not improved substantially. This failure highlights the need for further study of the factors mediating clot stabilization. Using laser scanning confocal microscopy of clots formed from fluorescein-labeled fibrinogen, we investigated what effect binding of fibrin to the endothelial surface has on clot structure and resistance to lysis. Fluorescent fibrin clots were produced over human umbilical vein endothelial cells (HUVEC) and the clot structure analyzed. In the presence of HUVEC, fibrin near the endothelial surface was more organized and occurred in tighter bundles compared to fibrin just 50 μm above. The HUVEC influence on fibrin architecture was blocked by inhibitory concentrations of antibodies to αVor β3integrin subunits. The regions of the clots associated with endothelial cells were more resistant to lysis than the more homogenous regions distal to endothelium. Thus, our data show that binding of fibrin to integrins on endothelial surfaces produces clots that are more resistant to lysis.
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Chernysh, Irina N., Prashant K. Purohit, Chandrasekaran Nagaswami, and John W. Weisel. "Remodeling of Clots without Proteolytic Digestion." Blood 118, no. 21 (November 18, 2011): 1184. http://dx.doi.org/10.1182/blood.v118.21.1184.1184.
Abstract:
Abstract Abstract 1184 Fibrin polymerization is a necessary part of hemostasis but thrombi can obstruct blood vessels and cause heart attacks and strokes, so the possibility of reversing clot formation has significant clinical implications. It is generally assumed that fibrin polymerization is an irreversible process and that clots and thrombi are stable structures until proteolytic digestion, but this supposition has not been critically tested. Using the technique of fluorescence recovery after photobleaching (FRAP) of individual fluorescently labeled fibrin fibers in a clot, we demonstrate here for the first time that there is turnover of fibrin in an uncrosslinked clot. These conditions occur in the vasculature during early stages of clotting or thrombosis before extensive Factor XIIIa-induced crosslinking. Increase of fluorescence in the bleached area was observed as soon as 1 second after photobleaching and the intensity increased rapidly. The association/dissociation of fibrin was characterized quantitatively, with the mobile fraction representing an average of 14.1% ± 5.1% of the total fibrin. Analyses of recovery curves showed that there was a linear relationship between both the percentage of fluorescence recovery and the rate of recovery and the surface to volume ratio of the fibers. Thus, it appears that fibrin monomers or oligomers located on or near the surface of the fibers are more likely to dissociate and re-associate. The mechanism of fibrin turnover was further investigated by using the peptide GPRP, mimicking the knobs ‘A’ involved in polymerization. Surprisingly, only 0.1 mM of GPRP was necessary to dissolve completely a preformed fibrin clot, and perfusion of 0.01 mM of GPRP through a clot decreased the mobile fraction of fibrin in FRAP experiments to 6.5% ± 1.7%. The kinetic parameters of fibrin turnover characterized from FRAP recovery curves revealed that the off rate was not affected by the GPRP concentration, but the equilibrium concentration of the bound complex decreased with increasing peptide concentration, likely because the free GPRP competes with fibrin knobs ‘A’ for holes ‘a.’ As a result of the enhanced dissociation induced by this peptide, striking rearrangements in clot structure were visualized by confocal light microscopy and scanning electron microscopy. Transmission electron microscopy of structures released from the clot revealed both monomers and larger aggregates. The implications of this research are that clots and thrombi appear to be much more dynamic structures than has been previously believed. The observed modulation of clot structure in vitro suggests that in vivo clots and thrombi may be dissolved or remodeled as a result of changing the environmental conditions surrounding them. These findings may also be relevant to embolization, which could in part be a consequence of the reversibility of polymerization. Furthermore, such modulation of clot structure could be a target for potential therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.
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Greenhalgh, Katie A., Mark W. Strachan, Saad Alzahrani, Paul D. Baxter, Kristina F. Standeven, Robert F. Storey, Robert A. S. Ariens, Peter J. Grant, Jackie F. Price, and Ramzi A. Ajjan. "BβArg448Lys polymorphism is associated with altered fibrin clot structure and fibrinolysis in type 2 diabetes." Thrombosis and Haemostasis 117, no. 02 (2017): 295–302. http://dx.doi.org/10.1160/th16-07-0554.
Abstract:
SummaryBoth type 2 diabetes (T2DM) and Bß448Lys variant of fibrinogen are associated with dense fibrin clots, impaired fibrinolysis and increased cardiovascular risk. It was our objective to investigate whether BßArg448Lys adds to vascular risk by modulating fibrin network structure and/or fibrinolysis in diabetes. The primary aim was to study effects of BßArg448Lys on fibrin network characteristics in T2DM. Secondary aims investigated interactions between gender and BßArg448Lys substitution in relation to fibrin clot properties and vascular disease. Genotyping for BßArg448Lys and dynamic clot studies were carried out on 822 T2DM patients enrolled in the Edinburgh Type 2 Diabetes Study. Turbidimetric assays of individual plasma samples analysed fibrin clot characteristics with additional experiments conducted on clots made from purified fibrinogen, further examined by confocal and electron microscopy. Plasma clot lysis time in Bß448Lys was longer than Bß448Arg variant (mean ± SD; 763 ± 322 and 719 ± 351 seconds [s], respectively; p<0.05). Clots made from plasma-purified fibrinogen of individuals with Arg/Arg, Arg/Lys and Lys/Lys genotypes showed differences in fibre thickness (46.75 ± 8.07, 38.40 ± 6.04 and 25 ± 4.99 nm, respectively; p<0.001) and clot lysis time (419 ± 64, 442 ± 87 and 517 ± 65 s, respectively; p=0.02), directly implicating the polymorphism in the observed changes. Women with Bß448Lys genotype had increased risk of cerebrovascular events and were younger compared with Bß448Arg variant (67.2 ± 4.0 and 68.2 ± 4.4 years, respectively; p=0.035). In conclusion, fibrinogen Bβ448Lys variant is associated with thrombotic fibrin clots in diabetes independently of traditional risk factors. Prospective studies are warranted to fully understand the role of BβArg448Lys in predisposition to vascular ischaemia in T2DM with the potential to develop individualised antithrombotic management strategies.
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Konings, Joke, José W. P. Govers-Riemslag, Helen Philippou, Nicola J. Mutch, Julian I. Borissoff, Peter Allan, Sumitra Mohan, Guido Tans, Hugo ten Cate, and Robert A. S. Ariëns. "Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin." Blood 118, no. 14 (October 6, 2011): 3942–51. http://dx.doi.org/10.1182/blood-2011-03-339572.
Abstract:
Abstract Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.
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de Vries, Judith J., Tamara Hoppenbrouwers, Cristina Martinez-Torres, Rezin Majied, Behiye Özcan, Mandy van Hoek, Frank W. G. Leebeek, Dingeman C. Rijken, Gijsje H. Koenderink, and Moniek P. M. de Maat. "Effects of Diabetes Mellitus on Fibrin Clot Structure and Mechanics in a Model of Acute Neutrophil Extracellular Traps (NETs) Formation." International Journal of Molecular Sciences 21, no. 19 (September 26, 2020): 7107. http://dx.doi.org/10.3390/ijms21197107.
Abstract:
Subjects with diabetes mellitus (DM) have an increased risk of arterial thrombosis, to which changes in clot structure and mechanics may contribute. Another contributing factor might be an increased formation of neutrophil extracellular traps (NETs) in DM. NETs are mainly formed during the acute phase of disease and form a network within the fibrin matrix, thereby influencing clot properties. Previous research has shown separate effects of NETs and DM on clot properties, therefore our aim was to study how DM affects clot properties in a model resembling an acute phase of disease with NETs formation. Clots were prepared from citrated plasma from subjects with and without DM with the addition of NETs, induced in neutrophils by S. aureus bacteria or phorbol myristate acetate (PMA). Structural parameters were measured using scanning electron microscopy, mechanical properties using rheology, and sensitivity to lysis using a fluorescence-based fibrinolysis assay. Plasma clots from subjects with DM had significantly thicker fibers and fewer pores and branch points than clots from subjects without DM. In addition, fibrinolysis was significantly slower, while mechanical properties were similar between both groups. In conclusion, in a model of acute NETs formation, DM plasma shows prothrombotic effects on fibrin clots.
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De Vries, Judith Juliana, Chantal Visser, Lotte Geers, Johan A. Slotman, Henrik Endeman, Marieke J. H. A. Kruip, Moniek P. M. de Maat, and The Dutch COVID & Thrombosis Coalition. "Does Fibrin Structure Contribute to the Increased Risk of Thrombosis in COVID-19 ICU Patients?" Blood 138, Supplement 1 (November 5, 2021): 3208. http://dx.doi.org/10.1182/blood-2021-145307.
Abstract:
Abstract Introduction: SARS-CoV-2 is responsible for a global pandemic, with almost 200 million confirmed cases. SARS-CoV-2 infection can lead to various disease states, from only mild symptoms in the majority of cases to severe disease, which is associated with an increased incidence of venous thromboembolism (VTE). We hypothesized that an altered fibrin network structure contributes to VTE in COVID-19 patients by affecting thrombus stability and fibrinolysis sensitivity. By studying the fibrin network of COVID-19 patients, we aimed to unravel the mechanisms that contribute to the increased risk of VTE in COVID-19 patients. Methods: Between April 2020 and December 2020, we collected plasma samples from patients with COVID-19 admitted to the intensive care unit (ICU) of the Erasmus Medical Center. We included patients with confirmed VTE diagnosed on CT-angiography, and COVID-19 patients without confirmed VTE during ICU admission. Samples were collected on admission to the ICU and after confirmed VTE or at similar time points in ICU patients without confirmed VTE. In addition, we collected plasma from COVID-19 patients at admission to general wards without confirmed VTE and from healthy controls. Clots were formed by mixing citrated plasma with thrombin (final concentration 1 U/ml) and calcium (17 mM). We imaged the clots using stimulated emission depletion (STED) microscopy, a super-resolution technique in which a depletion laser is used to selectively switch off fluorophores surrounding the focal point, thereby increasing the resolution. In these images, fibrin fiber diameters were measured using the Local Thickness plugin of ImageJ. Fiber density was quantified as percentage of area in Z-stacks of confocal microscopy images. Finally, a clot lysis assay based on turbidity was used to determine sensitivity to fibrinolysis (clot lysis time) and clot density (difference between maximum and baseline absorbance). Differences in fibrin network properties between groups were tested using One-Way ANOVA with Bonferroni post-hoc tests and linear regression with and without adjustment for fibrinogen levels. Results: We included 21 COVID-19 ICU patients with confirmed VTE, 20 COVID-19 ICU patients without confirmed VTE, 10 COVID-19 ward patients and 7 healthy controls. Mean age was comparable between the groups, while BMI was higher in COVID-19 patients than in healthy controls (Table 1). Levels of fibrinogen, D-dimer and anti-Xa were significantly higher in COVID-19 ICU patients than in COVID-19 ward patients and healthy controls. FVIII levels were significantly higher in COVID-19 ICU patients than in healthy controls, while FXIII levels were significantly lower. On admission to the ICU, clot density was significantly higher in COVID-19 ICU patients with and without confirmed VTE than in healthy controls (Figure 1 and Table 2). However, after adjustment for fibrinogen levels, this difference disappears. Clot lysis time was significantly longer in clots from COVID-19 ICU patients than in clots from healthy controls, regardless of fibrinogen levels (Table 2). COVID-19 ICU patients with confirmed VTE also showed a significant longer clot lysis time than COVID-19 ward patients. Interestingly, in the clot lysis assay, fibrinolysis did not occur in 25% of COVID-19 ICU patients with VTE versus 9.5% of COVID-19 ICU patients without VTE (Figure 2). This fibrinolysis shutdown was never observed in clots from healthy controls and COVID-19 ward patients. Fibrin fiber diameters were comparable between the groups. In the clots from plasma samples collected at admission to the ICU, there were no differences between COVID-19 ICU patients with and without VTE (Figure 2). However, when comparing clots prepared from plasma collected at the second time point (after VTE or at a similar time point for patients without VTE), we observed significant longer clot lysis times in patients with confirmed VTE (97.4 [88.5-158.8] min) than in patients without confirmed VTE (80.0 [76.0-97.8] min) (p=0.03). Finally, there were no significant changes between clots from plasma before and after VTE or between the two time points in patients without VTE, except for a decreased clot lysis time over time for COVID-19 ICU patients without confirmed VTE. Conclusion: Our results suggest that SARS-CoV-2 infection increases clot density and decreases clot susceptibility to fibrinolysis, and that these changes relate to the severity of the disease. Figure 1 Figure 1. Disclosures Kruip: Daiichi Sankyo: Research Funding; Bayer: Honoraria, Research Funding.
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Xu, Shixin, Zhiliang Xu, Oleg V. Kim, Rustem I. Litvinov, John W. Weisel, and Mark Alber. "Model predictions of deformation, embolization and permeability of partially obstructive blood clots under variable shear flow." Journal of The Royal Society Interface 14, no. 136 (November 2017): 20170441. http://dx.doi.org/10.1098/rsif.2017.0441.
Abstract:
Thromboembolism, one of the leading causes of morbidity and mortality worldwide, is characterized by formation of obstructive intravascular clots (thrombi) and their mechanical breakage (embolization). A novel two-dimensional multi-phase computational model is introduced that describes active interactions between the main components of the clot, including platelets and fibrin, to study the impact of various physiologically relevant blood shear flow conditions on deformation and embolization of a partially obstructive clot with variable permeability. Simulations provide new insights into mechanisms underlying clot stability and embolization that cannot be studied experimentally at this time. In particular, model simulations, calibrated using experimental intravital imaging of an established arteriolar clot, show that flow-induced changes in size, shape and internal structure of the clot are largely determined by two shear-dependent mechanisms: reversible attachment of platelets to the exterior of the clot and removal of large clot pieces. Model simulations predict that blood clots with higher permeability are more prone to embolization with enhanced disintegration under increasing shear rate. In contrast, less permeable clots are more resistant to rupture due to shear rate-dependent clot stiffening originating from enhanced platelet adhesion and aggregation. These results can be used in future to predict risk of thromboembolism based on the data about composition, permeability and deformability of a clot under specific local haemodynamic conditions.
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Neergaard-Petersen, Søs, Anne-Mette Hvas, Steen Kristensen, Erik Grove, Sanne Larsen, Fladia Phoenix, Zeyad Kurdee, Peter Grant, and Ramzi Ajjan. "The influence of type 2 diabetes on fibrin clot properties in patients with coronary artery disease." Thrombosis and Haemostasis 112, no. 12 (2014): 1142–50. http://dx.doi.org/10.1160/th14-05-0468.
Abstract:
SummaryType 2 diabetes mellitus (T2DM) increases the risk of coronary thrombosis and both conditions are associated with altered fibrin clot properties. However, the influence of T2DM on fibrin clot properties in patients with coronary artery disease (CAD) remains unclear. We aimed to investigate the influence of T2DM on fibrin clot properties in patients with CAD. Fibrin clot structure and fibrinolysis were investigated in 581 CAD patients (148 with T2DM) using turbidimetric assays, confocal and scanning electron microscopy. Clots made from plasma and plasma-purified fibrinogen were studied, and plasma levels of inflammatory markers were analysed. T2DM patients had increased clot maximum absorbance compared with non-diabetic patients (0.36 ± 0.1 vs 0.33 ± 0.1 au; p=0.01), displayed longer lysis time (804 [618;1002] vs 750 [624;906] seconds; p=0.03) and showed more compact fibrin structure assessed by confocal and electron microscopy. Fibrinogen levels were elevated in T2DM (p< 0.001), but clots made from purified fibrinogen showed no differences in fibrin properties in the two populations. Adjusting for fibrinogen levels, T2DM was associated with C-reactive protein and complement C3 plasma levels, with the former correlating with clot maximum absorbance (r=0.24, p< 0.0001) and the latter with lysis time (r=0.30, p< 0.0001). Independent of fibrinogen levels, females had more compact clots with prolonged lysis time compared with males (all p-values< 0.001). In conclusion, T2DM is associated with prothrombotic changes in fibrin clot properties in patients with CAD. This is related to quantitative rather than qualitative changes in fibrinogen with a possible role for inflammatory proteins.
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Jen, C. J., and L. V. McIntire. "Platelet Microtubules in Clot Structure Formation and Contractile Force Generation: Investigation of a Controversy." Thrombosis and Haemostasis 56, no. 01 (1986): 023–27. http://dx.doi.org/10.1055/s-0038-1661596.
Abstract:
SummaryWhether platelet microtubules are involved in clot retraction/ contraction has been controversial. To address this question we have simultaneously measured two clotting parameters, clot structural rigidity and isometric contractile force, using a rheological technique. For recalcified PRP clots these two parameters began rising together at about 15 min after CaCl2 addition. In the concentration range affecting microtubule organization in platelets, colchicine, vinca alkaloids and taxol demonstrated insignificant effects on both clotting parameters of a recalcified PRP clot. For PRP clots induced by adding small amounts of exogenous thrombin, the kinetic curves of clot rigidity were biphasic and without a lag time. The first phase corresponded to a platelet-independent network forming process, while the second phase corresponded to a platelet-dependent process. These PRP clots began generating contractile force at the onset of the second phase. For both rigidity and force parameters, only the second phase of clotting kinetics was retarded by microtubule affecting reagents. When PRP samples were clotted by adding a mixture of CaCl2 and thrombin, the second phase clotting was accelerated and became superimposed on the first phase. The inhibitory effects of micro tubule affecting reagents became less pronounced. Thrombin clotting of a two-component system (washed platelets/ purified fibrinogen) was also biphasic, with the second phase being microtubule-dependent. In conclusion, platelet microtubules are important in PRP clotted with low concentrations of thrombin, during which fibrin network formation precedes platelet-fibrin interactions. On the other hand they are unimportant if a PRP clot is induced by recalcification, during which the fibrin network is constructed in the presence of platelet-fibrin interactions. The latter is likely to be more analogous to physiological processes in vivo.
29
Leong, Lilley, Irina N. Chernysh, Yifan Xu, Cornell Mallari, Billy Wong, Derek Sim, Adam Cuker, et al. "Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII." Blood 128, no. 22 (December 2, 2016): 3798. http://dx.doi.org/10.1182/blood.v128.22.3798.3798.
Abstract:
Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.
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Tutwiler, Valerie, Alina D. Peshkova, Giang Le Minh, Sergei Zaitsev, Rustem I. Litvinov, Douglas B. Cines, and John W. Weisel. "Fibrinolysis of Contracted Blood Clots Depends on Whether Plasminogen Activator Acts from inside or Outside." Blood 132, Supplement 1 (November 29, 2018): 3773. http://dx.doi.org/10.1182/blood-2018-99-119659.
Abstract:
Abstract Fibrinolysis involves the dissolution of polymeric fibrin networks that is required to restore blood flow through vessels obstructed by clots and thrombi. The efficiency of lysis depends on the susceptibility of fibrin to enzymatic digestion, which is governed by the structure and spatial organization of fibrin fibers as well as porosity and composition of the clot. Platelet-driven clot contraction results in compaction of the erythrocytes into the core of the clot, effectively reducing the permeability of the clot, and influences fibrin network structure. We have shown that clot contraction is reduced in blood from patients with thrombotic conditions such ischemic stroke and deep vein thrombosis, which points to the clinical importance of understanding the influence of clot contraction on efficacy of fibrinolysis. Here, we examined the effects of clot contraction on the rate of internal fibrinolysis emanating from within the clot to simulate (patho)physiological conditions, and external fibrinolysis initiated from the clot exterior to simulate therapeutic thrombolysis. Fibrinolysis was induced and the kinetics of lysis was measured in parallel in contracted versus uncontracted clots from the same citrated human blood samples. Clot formation and platelet activation were initiated with 1 U/ml thrombin and 2 mM CaCl2. Clot contraction was either unaffected or impaired by inhibiting platelet non-muscle myosin IIa (blebbistatin), actin polymerization (latrunculin A), and platelet-fibin(ogen) binding (abciximab). To examine internal fibrinolysis, 75 ng/ml of human recombinant tissue plasminogen activator (tPA) was added prior to initiation of clotting, allowing for tPA to be uniformly distributed through the clot volume and for fibrinolysis occur after the clot has formed. We used optical tracking to follow clot size in a time dependent manner. Contracted clots were completely lysed at a rate that was at least 2 times faster than clots with impaired contraction. Specifically, the average time to complete lysis was 33±4 minutes for contracted clots versus 59±3, 84±4, 75±3 minutes when contraction was impaired by blebbistatin, latrunculin A, and abciximab, respectively (p<0.001). To examine external fibrinolysis, blood spiked with purified human 125I-fibrinogen was allowed to clot and contract (unless contraction was inhibited) prior to the addition of 75 ng/ml tPA. Clots with impaired contraction released 2-4-fold more radiolabeled soluble degradation products during the first 30 minutes and continued to lyse at a rate 4-fold faster than contracted clots over the initial 4 hours following addition of tPA. This reduction of the fibrinolysis rate in contracted clots was not due to the expulsion of serum-soluble anti-fibrinolytic compounds during the contraction process because serum replacement with a buffer did not affect the lysis rate. This difference in the susceptibility of contracted and uncontracted clots to internal versus external lysis suggests that the lysis rate is dominated by the interplay of clot permeability to fibrinolytic enzymes and the spatial proximity of the fibrin fibers themselves. Despite limitations of in vitro experimental models, numerous studies on fibrinolysis have demonstrated the relevance of experimental findings to pathophysiological fibrinolysis and therapeutic thrombolysis. Enhancement of fibrinolysis in contracted blood clots is consistent with the need to dissolve mature clots once they have performed their hemostatic function in a vessel on in a wound. The reduced rates of dissolution of contracted clots in our model of externally applied tPA could account for the inefficacy of therapeutic thrombolysis of old thrombi that likely underwent more compaction compared to newer thrombi. Our studies point to the clinical importance of understanding how mechanical remodeling of clots and thrombi may influence their fibrinolytic resolution and could inform the development of improved thrombolytic therapies. This work, in part, was supported by the Program for Competitive Growth at KFU. Disclosures No relevant conflicts of interest to declare.
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Whyte, Claire S., Akriti Rastogi, Ellis Ferguson, Michela Donnarumma, and Nicola J. Mutch. "The Efficacy of Fibrinogen Concentrates in Relation to Cryoprecipitate in Restoring Clot Integrity and Stability against Lysis." International Journal of Molecular Sciences 23, no. 6 (March 9, 2022): 2944. http://dx.doi.org/10.3390/ijms23062944.
Abstract:
Loss of fibrinogen is a feature of trauma-induced coagulopathy (TIC), and restoring this clotting factor is protective against hemorrhages. We compared the efficacy of cryoprecipitate, and of the fibrinogen concentrates RiaSTAP® and FibCLOT® in restoring the clot integrity in models of TIC. Cryoprecipitate and FibCLOT® produced clots with higher maximal absorbance and enhanced resistance to lysis relative to RiaSTAP®. The fibrin structure of clots, comprising cryoprecipitate and FibCLOT®, mirrored those of normal plasma, whereas those with RiaSTAP® showed stunted fibers and reduced porosity. The hemodilution of whole blood reduced the maximum clot firmness (MCF) as assessed by thromboelastography. MCF could be restored with the inclusion of 1 mg/mL of fibrinogen, but only FibCLOT® was effective at stabilizing against lysis. The overall clot strength, measured using the Quantra® hemostasis analyzer, was restored with both fibrinogen concentrates but not cryoprecipitate. α2antiplasmin and plasminogen activator inhibitor-1 (PAI-1) were constituents of cryoprecipitate but were negligible in RiaSTAP® and FibCLOT®. Interestingly, cryoprecipitate and FibCLOT® contained significantly higher factor XIII (FXIII) levels, approximately three-fold higher than RiaSTAP®. Our data show that 1 mg/mL fibrinogen, a clinically achievable concentration, can restore adequate clot integrity. However, FibCLOT®, which contained more FXIII, was superior in normalizing the clot structure and in stabilizing hemodiluted clots against mechanical and fibrinolytic degradation.
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Undas, Anetta, Krystyna Zawilska, Mariola Ciesla-Dul, Agata Lehmann-Kopydłowska, Agnieszka Skubiszak, Katarzyna Ciepłuch, and Wiesława Tracz. "Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives." Blood 114, no. 19 (November 5, 2009): 4272–78. http://dx.doi.org/10.1182/blood-2009-05-222380.
Abstract:
Abstract We tested the hypothesis that fibrin structure/function is unfavorably altered in patients after idiopathic venous thromboembolism (VTE) and their relatives. Ex vivo plasma fibrin clot permeability, turbidimetry, and efficiency of fibrinolysis were investigated in 100 patients with first-ever VTE, including 34 with pulmonary embolism (PE), 100 first-degree relatives, and 100 asymptomatic controls with no history of thrombotic events. Known thrombophilia, cancer, trauma, and surgery were exclusion criteria. VTE patients and their relatives were characterized by lower clot permeability (P < .001), lower compaction (P < .001), higher maximum clot absorbancy (P < .001), and prolonged clot lysis time (P < .001) than controls, with more pronounced abnormalities, except maximum clot absorbance, in the patients versus relatives (all P < .01). Fibrin clots obtained for PE patients were more permeable, less compact, and were lysed more efficiently compared with deep-vein thrombosis patients (all P < .05) with no differences in their relatives. Being VTE relative, fibrinogen, and C-reactive protein were independent predictors of clot permeability and fibrinolysis time in combined analysis of controls and relatives. We conclude that altered fibrin clot features are associated with idiopathic VTE with a different profile of fibrin variables in PE. Similar features can be detected in VTE relatives. Fibrin properties might represent novel risk factors for thrombosis.
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Sidelmann, Johannes, Mikkel Brabrand, Robert Pedersen, Jørgen Pedersen, Kim Esbensen, Kristina Standeven, Robert Ariëns, Jørgen Gram, and Jonas Sjøland. "Fibrin clot structure in patients with end-stage renal disease." Thrombosis and Haemostasis 98, no. 08 (2007): 339–45. http://dx.doi.org/10.1160/th06-12-0715.
Abstract:
SummaryFibrin clots with reduced permeability, increased clot stiffness and reduced fibrinolysis susceptibility may predispose to cardiovascular disease (CVD). Little is known, however, about the structure of fibrin clots in patients with end-stage renal disease (ESRD).These patients suffer from a high risk of CVD in addition to their chronic low-grade inflammation. Using permeability, compaction and turbidity studies in 22 ESRD patients and 24 healthy controls, fibrin clots made from patient plasma were found to be less permeable (p<0.001), less compactable (p<0.001), and less susceptible to fibrinolysis (p<0.001) than clots from controls.The maximum rate of turbidity increase was also higher for the patients than controls (p<0.001), and scan-ning electron microscopy revealed higher clot density of fibrin fibers in clots from patients than clots from controls (p<0.001). Patients had higher plasma concentrations of fibrinogen, C-reative protein and interleukin 6 than controls.These plasma markers of inflammation correlated significantly with most of the fibrin structure characteristics observed in the patients. In contrast, plasma markers of azothemia showed no such correlations. The results suggest that in ESRD patients fibrin clots are significantly different from healthy controls, and that the fibrin structure characteristics in the patients are associated primarily with the inflammatory plasma milieu rather than with level of azothemia.
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Davies, Nia Anne, Nicholas Kim Harrison, Roger H. Keith Morris, Simon Noble, Matthew James Lawrence, Lindsay Antonio D’Silva, Laura Broome, et al. "Fractal dimension (df ) as a new structural biomarker of clot microstructure in different stages of lung cancer." Thrombosis and Haemostasis 114, no. 12 (2015): 1251–59. http://dx.doi.org/10.1160/th15-04-0357.
Abstract:
SummaryVenous thromboembolism (VTE) is common in cancer patients, and is the second commonest cause of death associated with the disease. Patients with chronic inflammation, such as cancer, have been shown to have pathological clot structures with modulated mechanical properties. Fractal dimension (df) is a new technique which has been shown to act as a marker of the microstructure and mechanical properties of blood clots, and can be performed more readily than current methods such as scanning electron microscopy (SEM). We measured df in 87 consecutive patients with newly diagnosed lung cancer prior to treatment and 47 matched-controls. Mean group values were compared for all patients with lung cancer vs controls and for limited disease vs extensive disease. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. Significantly higher values of df were observed in lung cancer patients compared with controls and patients with extensive disease had higher values than those with limited disease (p< 0.05), whilst conventional markers failed to distinguish between these groups. The relationship between df of the incipient clot and mature clot microstructure was confirmed by SEM and computational modelling: higher df was associated with highly dense clots formed of smaller fibrin fibres in lung cancer patients compared to controls. This study demonstrates that df is a sensitive technique which quantifies the structure and mechanical properties of blood clots in patients with lung cancer. Our data suggests that df has the potential to identify patients with an abnormal clot micro-structure and greatest VTE risk.
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de Vries, Judith J., Charlotte J. M. Snoek, Dingeman C. Rijken, and Moniek P. M. de Maat. "Effects of Post-Translational Modifications of Fibrinogen on Clot Formation, Clot Structure, and Fibrinolysis." Arteriosclerosis, Thrombosis, and Vascular Biology 40, no. 3 (March 2020): 554–69. http://dx.doi.org/10.1161/atvbaha.119.313626.
Abstract:
Objective: Post-translational modifications of fibrinogen influence the occurrence and progression of thrombotic diseases. In this systematic review, we assessed the current literature on post-translational modifications of fibrinogen and their effects on fibrin formation and clot characteristics. Approach and Results: A systematic search of Medline, Embase, Cochrane Library, and Web of Science was performed to find studies reporting post-translational modifications of fibrinogen and the effects on clot formation and structure. Both in vitro studies and ex vivo studies using patient material were included. One hundred five articles were included, describing 11 different modifications of fibrinogen. For the best known and studied modifications, conclusions could be drawn about their effect on clot formation and structure. Oxidation, high levels of nitration, and glycosylation inhibit the rate of polymerization, resulting in dense clots with thinner fibers, while low levels of nitration increase the rate of polymerization. Glycation showed different results for polymerization, but fibrinolysis was found to be decreased, as a consequence of increased density and decreased permeability of clots. Acetylation also decreases the rate of polymerization but results in increased fiber diameters and susceptibility to fibrinolysis. Other modifications were studied less or contrasting results were found. Therefore, substantial gaps in the knowledge about the effect of post-translational modifications remain. Conclusions: Overall, post-translational modifications do affect clot formation and characteristics. More studies need to be performed to reveal the effects of all post-translational modifications and the effects on thrombotic diseases. Expanding the knowledge about modifications of fibrinogen can ultimately contribute to optimizing treatments for thrombotic diseases.
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Gaule, Thembaninkosi G., and Ramzi A. Ajjan. "Fibrin(ogen) as a Therapeutic Target: Opportunities and Challenges." International Journal of Molecular Sciences 22, no. 13 (June 28, 2021): 6916. http://dx.doi.org/10.3390/ijms22136916.
Abstract:
Fibrinogen is one of the key molecular players in haemostasis. Thrombin-mediated release of fibrinopeptides from fibrinogen converts this soluble protein into a network of fibrin fibres that form a building block for blood clots. Thrombin-activated factor XIII further crosslinks the fibrin fibres and incorporates antifibrinolytic proteins into the network, thus stabilising the clot. The conversion of fibrinogen to fibrin also exposes binding sites for fibrinolytic proteins to limit clot formation and avoid unwanted extension of the fibrin fibres. Altered clot structure and/or incorporation of antifibrinolytic proteins into fibrin networks disturbs the delicate equilibrium between clot formation and lysis, resulting in either unstable clots (predisposing to bleeding events) or persistent clots that are resistant to lysis (increasing risk of thrombosis). In this review, we discuss the factors responsible for alterations in fibrin(ogen) that can modulate clot stability, in turn predisposing to abnormal haemostasis. We also explore the mechanistic pathways that may allow the use of fibrinogen as a potential therapeutic target to treat vascular thrombosis or bleeding disorders. Better understanding of fibrinogen function will help to devise future effective and safe therapies to modulate thrombosis and bleeding risk, while maintaining the fine balance between clot formation and lysis.
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Weisel, John W. "Multiple Approaches to Visualizing Fibrin Clot Structure and Assembly." Microscopy and Microanalysis 3, S2 (August 1997): 329–30. http://dx.doi.org/10.1017/s1431927600008539.
Abstract:
Fibrin clot formation is necessary for maintaining the integrity of the vasculature via physiological processes of hemostasis and wound healing and is also involved in pathological processes, such as thrombosis and atherosclerosis. A variety of structural and biophysical approaches has been used to examine intermediates in the formation of clots and to visualize in vitro clots and ex vivo thrombi.Structures at all stages of polymerization have been examined to learn about molecular mechanisms of assembly. Fibrinogen is a polyfunctional, multi-domain protein that is essential for platelet aggregation and for the formation of the three-dimensional network of fibrin fibers which is the structural basis of the clot. Distinctive functions for several of fibrinogen's domains in the fibrin assembly process have been elucidated. Enzymatic removal of the fibrinopeptides exposes binding sites in the central region which then interact with complementary sites at the ends of a neighboring molecule to yield fibrin oligomers.
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Gantioqui, Jorell, Ivan Stevic, Paul Y. Kim, Keith K. Lau, Anthony K. C. Chan, and Howard H. W. Chan. "The Architecture Of Fibrin Clots Formed From Plasma With Low Platelet Levels Are Less Altered In The Presence Of Factor-Specific Anticoagulants Compared With Unfractionated Heparin." Blood 122, no. 21 (November 15, 2013): 578. http://dx.doi.org/10.1182/blood.v122.21.578.578.
Abstract:
Abstract Background Current guidelines for treating patients with thromboembolism and concomitant thrombocytopenia are based on anecdotal data and experts’ opinion. We previously described an in vitro assay using thromboelastography (TEG) to evaluate the effects of anticoagulants on plasma clot formation in the presence of autologous platelets. We showed that specific TEG clotting profiles for plasmas with predefined platelet counts in the presence of fondaparinux, rivaroxaban and dabigatran were less compromised compared with clots containing unfractionated heparin (UFH) and dalteparin. In this study, we further characterize the combined effects of anticoagulants and low platelet counts on the architecture of fibrin clots in plasma. Aim To determine the effect of various anticoagulants on the surface structure of fibrin clots in plasma when generated with autologous platelets at varying levels. Methods Scanning electron microscopy (SEM) was used to assess the surface structure of clots. Fresh human platelet-rich plasma and platelet-poor plasma (PPP) were obtained from the same donor and then the platelets were subsequently added back to PPP so that the final plasma samples contained predefined levels of platelets (PPP, 30,000, and 150,000 /mL). Each reaction mixture contained 30 µg/mL corn trypsin inhibitor, and one of the following anticoagulants: unfractionated heparin (UFH 0.3 IU/mL), dalteparin (1.0 IU/mL), fondaparinux (1.0 IU/mL), rivaroxaban (150 ng/mL) or dabigatran (180 ng/mL). For each condition, clotting was initiated with 10 mM CaCl2 and 1.2 pM tissue factor on a 0.02 µm Millipore membrane. After a 3-hr incubation, the clots were washed with phosphate buffered saline (pH 7.4), fixed with 2% v/v glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and then stained with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. The samples were dehydrated with gradient ethanol series, critical point dried, mounted onto stubs, and then gold sputter-coated for SEM examination at 20,000×. The porosity, fibril width and the number of fibrils were quantified for each clot generated. Results In the absence of anticoagulants, reducing the amount of platelets from 150,000 /mL to <10,000/mL in PPP had minimal effect on the porosity of clots, although there was a reduction in the number of fibrils by approximately 30% in the PPP sample (p < 0.05) and the fibrils were generally thicker (Fig. 1). Addition of anticoagulants to plasma containing 150,000/mL of platelets minimally changed the clot structure, except for UFH and dalteparin, which significantly increased the number of fibrils. Anticoagulants profoundly changed the clot structure as platelet levels fell below 150,000/ml. The greatest effects were observed for UFH, followed by dalteparin and then fondaparinux. The changes included an increase in porosity of clots (p < 0.05), a reduction in the number of fibrils (p < 0.05), and an increase in the width of fibril strands (p < 0.05). For the newer factor-specific anticoagulants, rivaroxaban changed the clot structure similar to fondaparinux in low platelets conditions, but dabigatran changed the clot structure less than rivaroxaban. Summary/Conclusion The present study supports our previous TEG observations that anticoagulants intensified the negative effects of low platelets during the formation of fibrin clots. This SEM study also demonstrates that anticoagulant therapy, in particular UFH or dalteparin, substantially changes the fibrin clot structure. This information provides evidence supporting the current clinical practice of withholding anticoagulants for patients with platelet count <30,000/mL. In addition, these in vitro data suggest that fondaparinux and the new factor-specific oral anticoagulants may be safer because clot structure is better preserved when compared to the traditional heparinoids. Yet, the absence of an antidote for these anticoagulants is a major hurdle in testing this hypothesis clinically because patients with severe thrombocytopenia inherit a high risk of bleeding. Disclosures: No relevant conflicts of interest to declare.
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Faes, Camille, Anton Ilich, Amandine Sotiaux, Erica M. Sparkenbaugh, Michael W. Henderson, Laura Buczek, Joan D. Beckman, et al. "Red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease." Blood 133, no. 23 (June 6, 2019): 2529–41. http://dx.doi.org/10.1182/blood.2019000424.
Abstract:
Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa–independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.
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Varin, Remi, Shahsultan Mirshahi, Pehzman Mirshahi, Jean Chidiac, Gerald Kierzek, Jean-Pierre Marie, Massoud Mirshahi, Claudine Soria, and Jeannette Soria. "Effect of Rivaroxaban, An Oral Direct Factor Xa Inhibitor, On Whole Blood Clot Permeation and Thrombolysis: Critical Role of Red Blood Cells." Blood 114, no. 22 (November 20, 2009): 1064. http://dx.doi.org/10.1182/blood.v114.22.1064.1064.
Abstract:
Abstract Abstract 1064 Poster Board I-86 Introduction: Decreased fibrinolysis has been reported in venous thrombosis. Thrombus degradation depends on its structure: thicker fibrin fibers are permeable to blood flow and highly susceptible to fibrinolytic enzymes, while thinner fibers are poorly permeable to flow and are resistant to fibrinolysis. Thrombin concentration present at the time of gelation profoundly influences fibrin clot structure: decrease in thrombin generation leads to the formation of thick fibrin fibers and to a decrease in activation of thrombin-activated fibrinolysis inhibitor (TAFI). Rivaroxaban, an oral direct factor Xa inhibitor, is in late stage clinical development for the prevention and treatment of venous and arterial thrombosis. The objective of this study was to evaluate the effect of Rivaroxaban on whole blood (WB) clot structure and degradability by t-PA. Compared to plasma clots, WB clots might better represent the in vivo formed thrombi. Methods: 1- Clots were formed by adding to WB or to corresponding plasma, low concentration of tissue factor and calcium in the presence or absence of Rivaroxaban at therapeutic concentrations (0.15 and 0.25 μg/ml). 2- Clot permeability was calculated by measuring the flow rate of liquid through the clot. It was expressed as Darcy constant. 3- Clot degradability was evaluated by D dimers generation during clot perfusion with plasminogen and tissue-type plasminogen activator (t-PA). Results: 1- In the absence of Rivaroxaban, WB clots had a lower porosity than that of corresponding plasma clots: Darcy constant of WB clots was 3.1 –fold lower than that of plasma clots. This decreased porosity of WB clots leads to thrombolysis resistance by preventing access of fibrinolytic enzymes to fibrin network: D dimers generation in t-PA-perfused clots for 60 min was 38 -fold lower in WB clots compared to plasma clots. 2- Rivaroxaban increased the permeation rate of WB clots and thrombolysis by t-PA: the addition of Rivaroxaban at 0.15 μg/ml in WB (corresponding in fact to plasma concentration of 0.25 μg/ml), increased the Darcy constant by 5.5 –fold and the clot degradability in 60 min by 108 -fold. These effects of Rivaroxaban were higher in WB clots than in corresponding plasma clots, as Rivaroxaban at 0.25 μg/ml in plasma clots increased the Darcy constant by 2.5-fold and clot degradation by 9.6-fold. In the presence of Rivaroxaban, the Darcy constant and the degradability of WB clots and of plasma clots were nearly identical. 3- To explain the greater efficacy of Rivaroxaban on WB permeation constants and thrombolysis in comparison to plasma clots a) we tested the possibility for Rivaroxaban to reduce the entrapment of red blood cells (RBC) into the network of fibrin as RBC can be responsible for fibrin pore occlusion. This possibility was excluded since Rivaroxaban had no effect on clot permeation rate in clots formed by clotting purified fibrinogen with thrombin in the presence or in the absence of RBC (condition in which there is no generation of thrombin): RBC induced a 2.5 times decrease in permeation rate due to entrapment of RBC into fibrin network, regardless of presence or absence of Rivaroxaban. b) we analyzed the effect of RBC on thrombin generation and its modification by Rivaroxaban: the addition of 0.1 ml RBC diluted ½ to 0,2 ml plasma increased the thrombin generation (540 % of control without RBC). This is probably due to exposure of phosphatidyl serine at surface of RBC during thrombin generation. The increase in thrombin generation by RBC was reduced to 140 % in presence of Rivaroxaban at 0.15 μg/ml. This is explained by Rivaroxaban's inhibition of factor Xa bound to cells. Conclusion: Thrombin generation was greater in WB than in plasma, leading to a lower porosity and degradability of WB clots as compared to plasma clots. Rivaroxaban, by decreasing thrombin generation, increased clot permeability and degradability to the same level in WB clots and plasma clots. This property of Rivaroxaban may contribute to its antithrombotic effect. This study received a support from Bayer-Schering-Pharma France. Disclosures: No relevant conflicts of interest to declare.
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Undas, Anetta. "Fibrin clot properties and their modulation in thrombotic disorders." Thrombosis and Haemostasis 112, no. 07 (2014): 32–42. http://dx.doi.org/10.1160/th14-01-0032.
Abstract:
SummaryAccumulating evidence indicates that accelerated formation of fibrin clots composed of compact, highly-branched networks with thin fibres which are relatively resistant to plasmin-mediated lysis can be commonly observed in patients with venous or arterial thrombosis. This review discusses characteristics of fibrin clot structure and function in patients with various thromboembolic manifestations, in particular myocardial infarction, ischaemic stroke and venous thromboembolism, based on the publications till December 2013. Moreover, factors will be presented that in vivo unfavourably determine altered fibrin clot properties in thrombotic disorders and modalities that can improve clot phenotype.
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Dargaud, Yesim, Jean C. Bordet, Chantal Huchon, and Claude Negrier. "How can We Predict Efficacy of rFVIIa in Hemophilia Patients with Inhibitors?" Blood 114, no. 22 (November 20, 2009): 3487. http://dx.doi.org/10.1182/blood.v114.22.3487.3487.
Abstract:
Abstract Abstract 3487 Poster Board III-424 Hemophilia patients with inhibitors are treated with bypassing agents for which hemostatic efficacy is unpredictable. While both activated prothrombin complex concentrate and recombinant activated factor VII (rFVIIa) have demonstrated excellent safety profiles, neither product is a universal hemostatic agent and the variability of response to bypassing agents complicates the treatment in these patients. Moreover, the lack of a validated laboratory assay to measure the effectiveness of bypassing agents dramatically limits the optimisation of treatment strategies. As the final enzyme generated by bypassing agents is thrombin, thrombin generation assay (TGA) could theoretically be used for monitoring rFVIIa. However, TGA does not reflect the stability of the fibrin clot and its resistance to fibrinolysis which are essential parameters of hemostasis. We have therefore evaluated the use of an additional method that might provide complementary information on fibrin clot structure and stability, and would allow a better prediction of the biological efficacy of rFVIIa. In the absence of FVIII/FIX, fibrin fibres are abnormally thick and clots are overly susceptible to fibrinolysis. After treatment with rFVIIa, clots are less porous and fibrin fibres are thinner as assessed using scanning electron microscopy. Using whole blood thromboelastography (TEG) measuring viscoelastic changes of fibrin throughout clot initiation, formation and fibrinolysis, we developed an in vitro model to assess fibrin clot stability and resistance to fibrinolysis. The aim of the present study was to evaluate the correlation between the modifications of the fibrin clot structure and the stability of the fibrin clots obtained in the presence of rFVIIa. METHODS The in vitro effect of rFVIIa was tested in 6 severe hemophilia A patients at doses of 90 – 180 – 270 μg/kg. Thrombin generation (TG) was measured in platelet rich plasma using the CAT method in the presence of TF 1pM. After addition of rFVIIa, the improvement in TG capacity was compared to normal values obtained from 80 control males. Fibrin clots obtained from the TG measurements were studied by scanning electron microscopy (SEM) and fibrin diametres were measured (700 measurements on each sample). In the same samples, the stability of fibrin clots obtained before and after addition of rFVIIa was assessed using TEG-5000™. Clot resistance to fibrinolysis was recorded in the presence of TF 0.5pM and tPA 0.125μM. TEG-tPA and SEM results were compared to those obtained in 30 healthy control males. RESULTS A dose dependent increase of TG was observed in the presence of increasing doses of rFVIIa (p<0.0001; ANOVA). In the presence of rFVIIa 90μg/kg, TG capacity was significantly improved in all patients (p=0.0023; Mann Whitney), and was completely normalized in 4 patients while 2 others needed higher doses of rFVIIa to normalize their TG. The fibrin fibre diameters were thicker (217±16 nm; p<0.0001) in all hemophilia patients in comparison with controls (170±24 nm). After addition of rFVIIa 90μg/kg, the fibrin clot structure was modified and the diameter of fibrin fibres was dramatically decreased in all patients (184±11 nm; p=0.006). A further improvement of fibrin clot structure was observed with rFVIIa 180μg/kg in only one patient. TEG-tPA showed a dose-dependent improvement of fibrin clot stability in the presence of rFVIIa (p<0.0001; ANOVA). A reverse correlation was observed between fibrin fibre diametres and resistance of fibrin clots to fibrinolysis (r=-0.68, p=0.001; Spearman test). CONCLUSION This data demonstrates a statistically significant correlation between clot structure and its stability. The combined use of TGA with TEG-tPA may allow physicians to better evaluate the individual response of patients to bypassing agents. The clinical validity of the minimal individual dose of rFVIIa normalizing both TGA and TEG-tPA needs to be verified in clinical studies. Disclosures: No relevant conflicts of interest to declare.
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Weisel, John W., Tatiana Lebedeva, Chandrasekaran Nagaswami, Vincent M. Hayes, Walter Massefski, Rustem I. Litvinov, Lubica Rauova, Thomas J. Lowery, and Douglas B. Cines. "Polyhedrocytes: Compressed Polyhedral Erythrocytes In Contracted Blood Clots and Thrombi." Blood 122, no. 21 (November 15, 2013): 452. http://dx.doi.org/10.1182/blood.v122.21.452.452.
Abstract:
Background Contraction of blood clots is necessary for hemostasis, wound healing and to restore flow past obstructive thrombi. However, little has been known about the structure of contracted clots and mechanisms of contraction. Erythrocytes, biconcave cells that are highly deformable to allow their passage through the microvasculature, are abundant in venous thrombi, and to a lesser extent in arterial thrombi. Erythrocytes promote hemostasis, but their participation in clot contraction has not been reported. Here we study the mechanisms of clot contraction and the roles of erythrocytes, platelets and fibrin, and show that erythrocyte shape change into compressed polyhedrocytes allows tight packing consistent with the major function of clots to stem bleeding. Methods Whole blood was clotted by recalcification and addition of thrombin or kaolin, while following the process of clotting, including contraction, with a new technique using T2 magnetic resonance. We examined the structure and composition of contracted whole blood clots by scanning electron microscopy and confocal light microscopy. Results Contracted clots display a remarkable structure, with a close-packed, tessellated array (or mosaic tiling of space) of compressed polyhedral erythrocytes (called polyhedrocytes) on the interior and a meshwork of fibrin and platelet aggregates on the exterior. Little fibin and few platelets were found on the interior of the contracted clots. The same results were obtained with both thrombin and kaolin as activators of clotting and also with reconstituted human blood and clots prepared from mouse blood. Confocal microscopy of hydrated clots confirms the results of scanning electron microscopy. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a closely packed polyhedral array. To assess the density of packing of the polyhedral erythrocytes, we replaced the water surrounding the clots with D2O and observed by T2 magnetic resonance that hydrogen/deuterium exchange for the contracted clots was very slow, consistent with their very tightly packed, almost impermeable structure. The same polyhedrocyte structures were observed from in vivo thrombi aspirated by cardiologists from the coronary arteries of ST-elevation myocardial infarction patients. Summary/Conclusions We have observed a previously undiscovered, naturally occurring erythrocyte function and morphology, closely packed polyhedra, in contracted clots and thrombi, and an unexpected spatial redistribution of platelets and fibrin that occurs during contraction. Clot contraction is an essential part of hemostasis, since both human genetic disorders of platelet myosin IIA and megakaryocyte myosin IIA-knock out mice show a bleeding phenotype. These observations on contracted clots imply that they are stiff, rigid structures that can form an impermeable, watertight seal. On the one hand, contraction of clots within the vasculature may relieve obstruction of blood vessels and allow recanalization, especially in the venous system. On the other hand, these results account for long-standing clinical observations that fibrinolysis is greatly prolonged following clot contraction, since perfusion or diffusion of lytic enzymes into these tightly packed polyhedral erythrocytes would be nearly impossible. These results suggest a vital role for erythrocytes and clot contraction in hemostasis and wound healing. Disclosures: No relevant conflicts of interest to declare.
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Ajjan, Ramzi, Bernard C. B. Lim, Kristina F. Standeven, Robert Harrand, Sarah Dolling, Fladia Phoenix, Richard Greaves, et al. "Common variation in the C-terminal region of the fibrinogen β-chain: effects on fibrin structure, fibrinolysis and clot rigidity." Blood 111, no. 2 (January 15, 2008): 643–50. http://dx.doi.org/10.1182/blood-2007-05-091231.
Abstract:
Fibrinogen BβArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bβ-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BβLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BβArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.
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Landi, Elia, Marco Mugnaini, Tunahan Vatansever, Ada Fort, Valerio Vignoli, Elvira Giurranna, Flavia Rita Argento, et al. "Advancing Thrombosis Research: A Novel Device for Measuring Clot Permeability." Sensors 24, no. 12 (June 9, 2024): 3764. http://dx.doi.org/10.3390/s24123764.
Abstract:
Thromboembolism, a global leading cause of mortality, needs accurate risk assessment for effective prophylaxis and treatment. Current stratification methods fall short in predicting thrombotic events, emphasizing the need for a deeper understanding of clot properties. Fibrin clot permeability, a crucial parameter in hypercoagulable states, impacts clot structure and resistance to lysis. Current clot permeability measurement limitations propel the need for standardized methods. Prior findings underscore the importance of clot permeability in various thrombotic conditions but call for improvements and more precise, repeatable, and standardized methods. Addressing these challenges, our study presents an upgraded, portable, and cost-effective system for measuring blood clot permeability, which utilizes a pressure-based approach that adheres to Darcy’s law. By enhancing precision and sensitivity in discerning clot characteristics, this innovation provides a valuable tool for assessing thrombotic risk and associated pathological conditions. In this paper, the authors present a device that is able to automatically perform the permeability measurements on plasma or fibrinogen in vitro-induced clots on specific holders (filters). The proposed device has been tailored to distinguish clot permeability, with high precision and sensitivity, between healthy subjects and high cardiovascular-risk patients. The precise measure of clot permeability represents an excellent indicator of thrombotic risk, thus allowing the clinician, also on the basis of other anamnestic and laboratory data, to attribute a risk score to the subject. The proposed instrument was characterized by performing permeability measurements in plasma and purified fibrinogen clots derived from 17 Behcet patients and 15 sex- and age-matched controls. As expected, our results clearly indicate a significant difference in plasma clot permeability in Behcet patients with respect to controls (0.0533 ± 0.0199 d vs. 0.0976 ± 0.0160 d, p < 0.001). This difference was confirmed in the patient’s vs. control fibrin clots (0.0487 ± 0.0170 d vs. 0.1167 ± 0.0487 d, p < 0.001). In conclusion, our study demonstrates the feasibility, efficacy, portability, and cost-effectiveness of a novel device for measuring clot permeability, allowing healthcare providers to better stratify thrombotic risk and tailor interventions, thereby improving patient outcomes and reducing healthcare costs, which could significantly improve the management of thromboembolic diseases.
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Ząbczyk, Michał, Joanna Natorska, and Anetta Undas. "Factor XIII and Fibrin Clot Properties in Acute Venous Thromboembolism." International Journal of Molecular Sciences 22, no. 4 (February 5, 2021): 1607. http://dx.doi.org/10.3390/ijms22041607.
Abstract:
Coagulation factor XIII (FXIII) is converted by thrombin into its active form, FXIIIa, which crosslinks fibrin fibers, rendering clots more stable and resistant to degradation. FXIII affects fibrin clot structure and function leading to a more prothrombotic phenotype with denser networks, characterizing patients at risk of venous thromboembolism (VTE). Mechanisms regulating FXIII activation and its impact on fibrin structure in patients with acute VTE encompassing pulmonary embolism (PE) or deep vein thrombosis (DVT) are poorly elucidated. Reduced circulating FXIII levels in acute PE were reported over 20 years ago. Similar observations indicating decreased FXIII plasma activity and antigen levels have been made in acute PE and DVT with their subsequent increase after several weeks since the index event. Plasma fibrin clot proteome analysis confirms that clot-bound FXIII amounts associated with plasma FXIII activity are decreased in acute VTE. Reduced FXIII activity has been associated with impaired clot permeability and hypofibrinolysis in acute PE. The current review presents available studies on the role of FXIII in the modulation of fibrin clot properties during acute PE or DVT and following these events. Better understanding of FXIII’s involvement in the pathophysiology of acute VTE might help to improve current therapeutic strategies in patients with acute VTE.
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Bowley, Sheryl R., and Susan T. Lord. "Fibrinogen variant BβD432A has normal polymerization but does not bind knob “B”." Blood 113, no. 18 (April 30, 2009): 4425–30. http://dx.doi.org/10.1182/blood-2008-09-178178.
Abstract:
AbstractFibrinogen residue Bβ432Asp is part of hole “b” that interacts with knob “B,” whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed BβD432A has normal polymerization, we hypothesized that Bβ432Asp is not critical for knob “B” binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from BβD432A. Surprisingly, the structure (rfD-BβD432A+GH) showed the peptide GHRP was not bound to hole “b.” We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking. The turbidity and the rate of γ-γ dimer formation for BβD432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BβD432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than “B:b” interactions. We conclude that hole “b” and “B:b” knob-hole binding per se have no influence on fibrin polymerization.
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Pan, Xiaoxi, Yun Gong, Ya Xu, Robert Ariens, and Michael Routledge. "Urban Particulate Matter Induces Changes in Gene Expression in Vascular Endothelial Cells that Are Associated with Altered Clot Structure In Vitro." Thrombosis and Haemostasis 118, no. 02 (2018): 266–78. http://dx.doi.org/10.1160/th17-05-0362.
Abstract:
Background Particulate matter contained in ambient air pollution has been associated with cardiovascular diseases in several epidemiological studies. Objective The aim of this study was to investigate the potential for urban particulate matter to induce changes in clot structure through interaction with vascular endothelial cells. Methods We examined the structure of clots formed on human umbilical vascular endothelial cells that had been treated with various types of particles versus those formed on untreated cells. Particles used were standard reference particulate matter from diesel engine emissions (SRM2975) and urban ambient collection (SRM2787). Results There was a dose-dependent increase in fibre density in clots formed on particle-treated endothelial cells. It was also found that exposure to the particles induced increased expression of tissue factor and reduced expression of thrombomodulin genes as measured by real-time polymerase chain reaction and increased expression of von Willebrand factor and plasminogen activation inhibitor-1 as measured by ELISA. Conclusion These changes are consistent with increased procoagulant activity of air pollution particulate matter–treated endothelial cells and suggest that particulate matter has the potential to promote clot formation through changes induced in endothelial genes controlling clot formation.
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Moiseiwitsch, Nina, Kimberly A. Nellenbach, Nina A. Guzzetta, Ashley C. Brown, and Laura Downey. "Ex Vivo and In Vivo Evaluation of Fibrinogen Concentrate to Mitigate Post-Surgical Bleeding in Neonates." Blood 138, Supplement 1 (November 5, 2021): 1034. http://dx.doi.org/10.1182/blood-2021-153823.
Abstract:
Abstract Introduction: Bleeding is a serious complication among neonates undergoing cardiopulmonary bypass (CPB) and it is linked to significant morbidity and mortality. Current standard of care treatment for bleeding after CPB focuses on the transfusion of adult blood products, including platelets and cryoprecipitate. However, prior work by Nellenbach et al. has demonstrated structural differences between neonatal and adult clotting components. Importantly, neonatal and adult fibrin do not fully integrate during clot formation which may contribute to ineffective clot formation and/or increased thrombotic risk following transfusion of adult cryoprecipitate to neonates. There has been increased interest in using human fibrinogen concentrate (HFC) in treating bleeding in the post-CPB neonate; however, HFC has not been validated in this population through evidence-based means. This study analyzed structural and degradation properties of post-CPB clots +/- the ex vivo addition of HFC and compared structural and degradation properties of post-CPB clots after the in vivo transfusion of HFC versus cryoprecipitate. Methods: Human neonatal plasma samples were collected from patients undergoing CPB at the Children's Hospital of Atlanta. For ex vivo studies, samples were taken at baseline, post-bypass, and post-transfusion of cryoprecipitate (n = 18 patients). Clots were formed for analysis from samples alone as well as post-bypass samples with the addition of 0.5 or 0.9 mg/mL HFC (RiaSTAP, CSL Behring) and structure was examined through confocal microscopy. Clot degradation was assessed through a microfluidic fibrinolysis assay. For in vivo studies, samples were taken at baseline, post-transfusion of cryoprecipitate or HFC, upon ICU arrival, and at 24 hours post-surgery (n = 36 patients). Clots were formed from samples and structure was examined through confocal microscopy. Clot degradation was assessed through a plate-based fibrinolysis assay. Results: In ex vivo studies, clot structural analysis demonstrated no significant differences in fiber density between samples collected at different time points (baseline = 0.541 ± 0.105, post-bypass = 0.431 ± 0.111, post-transfusion = 0.594 ± 0.170). The addition of 0.5 mg/mL or 0.9 mg/mL HFC to post-bypass samples led to a significant increase in fiber density (0.5 mg/mL HFC=0.654 ± 0.158, p=0.02; 0.9 mg/mL HFC= 0.797 ± 0.193, p<0.0001). Functional microfluidic analysis of clot degradation demonstrated significantly faster degradation times among post-bypass samples when compared to baseline samples (baseline degradation rate = 11.061 ± 6.087, post-bypass degradation rate = 25.906 ± 9.990 microns/hour, p=0.04). The addition of 0.5 mg/mL HFC resulted in a slower degradation rate from the original post-CPB degradation rate, but did not reach statistical significance (0.5 mg/mL HFC=14.091 ± 2.241, p=0.14). However, the addition of 0.9 mg/mL HFC resulted in a significantly slower degradation rate (0.9 mg/mL HFC=8.594 ± 6.087, p=0.01). Studies comparing in vivo transfusion of cryoprecipitate and HFC demonstrated no significant difference between treatment groups in clot density or degradation rate for any sample time point. Conclusion: We identify patterns in structural properties of clots formed after the transfusion of HFC that are consistent with successful hemostasis. However, caution is warranted regarding potentially thrombotic risks and should be carefully analyzed in future studies. Figure: Effect of Ex Vivo HFC Addition on Clot Structure and Degradation. (A) Representative confocal imaging of clots formed from different samples and HFC dosages (scale = 50 um). (B) Effect of HFC Addition on Clot Fiber Density. Addition of both 0.5 and 0.9 mg/mL HFC dosages to post-bypass sample result in statistically significant increases in fiber density compared to post-bypass samples. (C) Effect of HFC Addition on Clot Degradation Profiles. Addition of 0.9 mg/mL HFC to post-bypass sample leads to statistically significant slower fibrinolysis. Figure 1 Figure 1. Disclosures Brown: Selsym Biotech, Inc.: Other: Co-Founder and CEO. OffLabel Disclosure: RiaSTAP (human fibrinogen concentrate) is FDA approved for the treatment of congenital hypofibrinogenemia.
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Collet, Jean-Philippe, Jennifer L. Moen, Yuri I. Veklich, Oleg V. Gorkun, Susan T. Lord, Gilles Montalescot, and John W. Weisel. "The αC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis." Blood 106, no. 12 (December 1, 2005): 3824–30. http://dx.doi.org/10.1182/blood-2005-05-2150.
Abstract:
The functions of the αC domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at Aα chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of α251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of α251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the αC domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of α251 fibrin clots differed markedly from control clots; α251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the αC domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked α251 and control clots showed that γ chain cross-linking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of α251 clots, monitored with both macroscopic and microscopic methods, was faster than lysis of control clots. In conclusion, these studies provide the first definitive evidence that the αC domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis.