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Transfusion and Apheresis Science :... Feb 2008Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during... (Review)
Review
Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro- and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots that are associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo.
Topics: Blood Coagulation; Fibrin; Fibrinolysis; Hemostasis; Humans; Thrombin
PubMed: 18282807
DOI: 10.1016/j.transci.2007.12.005 -
PloS One 2023Hemostasis is the cessation of bleeding due to the formation of a blood clot. After the completion of wound healing, the blood clot is typically dissolved through the...
Hemostasis is the cessation of bleeding due to the formation of a blood clot. After the completion of wound healing, the blood clot is typically dissolved through the natural process of fibrinolysis, the enzymatic digestion by plasmin of the fibrin fibers that make up its structural scaffold. In vitro studies of fibrinolysis reveal mechanisms regulating these processes and often employ fluorescent microscopy to observe protein colocalization and fibrin digestion. In this study, we investigate the effects of labeling a fibrin network with 20 nm diameter fluorescent beads (fluorospheres) for the purpose of studying fibrinolysis. We observed fibers and 2-D fibrin networks labeled with fluorospheres during fibrinolysis. We found that the labeling of fibrin with fluorospheres can alter fibrinolytic mechanisms. In previous work, we showed that, during lysis, fibrin fibers are cleaved into two segments at a single location. Herein we demonstrate that fibrinolysis can be altered by the concentration of fluorospheres used to label the fibers, with high concentrations of fluorospheres leading to very minimal cleaving. Furthermore, fibers that are left uncleaved after the addition of plasmin often elongate, losing their inherent tension throughout the imaging process. Elongation was especially prominent among fibers that had bundled together due to other cleavage events and was dependent on the concentration of fluorophores used to label fibers. Of the fibers that do cleave, the site at which they cleave also shows a predictable trend dependent on fluorosphere concentration; low concentrations heavily favor cleavage locations at either end of fibrin fiber and high concentrations show no disparity between the fiber ends and other locations along the fiber. After the initial cleavage event bead concentration also affects further digestion, as higher bead concentrations exhibited a larger population of fibers that did not digest further. The results described in this paper indicate that fluorescent labeling strategies can impact fibrinolysis results.
Topics: Humans; Fibrinolysin; Microspheres; Fibrinolysis; Fibrin; Thrombosis
PubMed: 37027378
DOI: 10.1371/journal.pone.0284163 -
Microcirculation (New York, N.Y. : 1994) Oct 2022Vesicular trafficking dictates protein localization, functional activity, and half-life, providing a critically important regulatory step in tissue development; however,... (Review)
Review
OBJECTIVES
Vesicular trafficking dictates protein localization, functional activity, and half-life, providing a critically important regulatory step in tissue development; however, there is little information detailing endothelial-specific trafficking signatures. This is due, in part, to limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3-dimensional (3D) in vitro sprouting model to image endothelial membrane trafficking events.
METHODS
Endothelial cells were challenged to grow sprouts in a fibrin bead assay. Thereafter, spouts were transfected with fluorescent proteins and stained for various cell markers. Sprouts were then imaged for trafficking events using live and fixed-cell microscopy.
RESULTS
Our results demonstrate that fibrin bead sprouts have a strong apicobasal polarity marked by apical localization of proteins moesin and podocalyxin. Comparison of trafficking mediators Rab27a and Rab35 between 3D sprouts and 2D culture showed that vesicular carriers can be imaged at high resolution, exhibiting proper membrane polarity solely in 3D sprouts. Lastly, we imaged exocytic events of von Willebrand Factor and demonstrated a distinct imaging advantage for monitoring secretion events in 3D sprouts as compared with 2D culture.
CONCLUSIONS
Our results establish that the fibrin bead sprouting assay is well-suited for imaging of trafficking events during angiogenic growth.
Topics: Endothelial Cells; Morphogenesis; von Willebrand Factor; Fibrin
PubMed: 34415654
DOI: 10.1111/micc.12726 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... May 2022Plasma D-dimer, a special cross-linked fibrin derivative, is produced when fibrin is degraded by plasminase. During pregnancy, D-dimer increases along with the increase... (Review)
Review
Plasma D-dimer, a special cross-linked fibrin derivative, is produced when fibrin is degraded by plasminase. During pregnancy, D-dimer increases along with the increase of gestational age, and the reference value of plasma D-dimer (≤0.5 mg/L) traditionally used for the screening of venous thrombosis in the normal population is not applicable to the pregnant population. Due to the lack of uniform D-dimer detection methods or measurement units, there is currently no unified D-dimer reference values for pregnancy or puerperium. Each region or laboratory should establish its own pregnancy D-dimer reference value for different gestational weeks through blood coagulation function testing of large numbers of samples of different gestational periods. More and more studies have been conducted to investigate the association between D-dimer and venous thromboembolism (VTE) during pregnancy, gestational hypertensive disorders (GHD) and pregnancy outcome. We reviewed, herein, the generation and measurement of D-dimer, the reference values of D-dimer during normal pregnancy, and the association between D-dimer and some pathological pregnancies, intending to help clinicians develop a more thorough understanding of D-dimer during pregnancy.
Topics: Female; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Pregnancy; Reference Values; Venous Thromboembolism
PubMed: 35642169
DOI: 10.12182/20220560302 -
Biochimica Et Biophysica Acta. General... Feb 2021Thrombin activates fibrinogen and binds the fibrin E-domain (K ~ 2.8 μM) and the splice variant γ'-domain (K ~ 0.1 μM). We investigated if the loading of...
BACKGROUND
Thrombin activates fibrinogen and binds the fibrin E-domain (K ~ 2.8 μM) and the splice variant γ'-domain (K ~ 0.1 μM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability.
METHODS
A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (T) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin.
RESULTS
Large increases in T indicated that calcium led to protein stabilization (0 vs. 2 mM Ca) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the T of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔT = 9.3 °C, similar to the ΔT when fibrinogen was converted to fibrin monomer (ΔT = 8.8 °C) or to fibrin (ΔT = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) T values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction.
CONCLUSIONS
TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin.
SIGNIFICANCE
The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.
Topics: Amino Acid Chloromethyl Ketones; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinogens, Abnormal; Humans; Oligopeptides; Protein Stability; Thrombin; Transition Temperature
PubMed: 33276061
DOI: 10.1016/j.bbagen.2020.129805 -
BioMed Research International 2017Prothrombotic fibrin clot phenotype, involving faster formation of dense meshwork composed of thinner and highly branched fibers that are relatively resistant to... (Review)
Review
Prothrombotic fibrin clot phenotype, involving faster formation of dense meshwork composed of thinner and highly branched fibers that are relatively resistant to plasmin-induced lysis, has been reported in patients with not only myocardial infarction or stroke, but also venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT), and/or pulmonary embolism (PE). Prothrombotic fibrin clot phenotype, in particular prolonged clot lysis time, is considered a novel risk factor for VTE as well as venous thrombosis at unusual location, for example, cerebral sinus venous thrombosis, retinal vein obstruction, and Budd-Chiari syndrome. Growing evidence from observational studies indicates that abnormal fibrin clot properties can predict recurrent DVT and PE and they are involved in serious complications of VTE, for example, thromboembolic pulmonary hypertension and postthrombotic syndrome. The purpose of this article is to review our current understanding of the role of fibrin clot structure and function in venous thrombosis with emphasis on clinical issues ranging from prognosis to therapy.
Topics: Fibrin; Humans; Pulmonary Embolism; Recurrence; Risk Factors; Venous Thrombosis
PubMed: 28740853
DOI: 10.1155/2017/8196256 -
Acta Biomaterialia Mar 2023The mechanical stability of blood clots necessary for their functions is provided by fibrin, a fibrous gel. Rupture of clots leads to life-threatening thrombotic...
The mechanical stability of blood clots necessary for their functions is provided by fibrin, a fibrous gel. Rupture of clots leads to life-threatening thrombotic embolization, which is little understood. Here, we combine experiments and simulations to determine the toughness of plasma clots as a function of fibrin content and correlate toughness with fibrin network structure characterized by confocal and scanning electron microscopy. We develop fibrin constitutive laws that scale with fibrin concentration and capture the force-stretch response of cracked clot specimens using only a few material parameters. Toughness is calculated from the path-independent J integral that includes dissipative effects due to fluid flow and uses only the constitutive model and overall stretch at crack propagation as input. We show that internal fluid motion, which is not directly measurable, contributes significantly to clot toughness, with its effect increasing as fibrin content increases, because the reduced gel porosity at higher density results in greater expense of energy in fluid motion. Increasing fibrin content (1→10mg/mL) results in a significant increase in clot toughness (3→15 N/m) in accordance with a power law relation reminiscent of cellular solids and elastomeric gels. These results provide a basis for understanding and predicting the tendency for thrombotic embolization. STATEMENT OF SIGNIFICANCE: Fibrin, a naturally occurring biomaterial, is the major determinant of the structural and mechanical integrity of blood clots. We determined that increasing the fibrin content in clots, as in some thrombi and fibrin-based anti-bleeding sealants, results in an increase in clot toughness. Toughness corresponds to the ability to resist rupturing in the presence of a defect. We couple bulk mechanical testing, microstructural measurements, and finite element modeling to capture the force-stretch response of fibrin clots and compute toughness. We show that increased fibrin content in clots reduces porosity and limits fluid motion and that fluid motion drastically alters the clot toughness. These results provide a fundamental understanding of blood clot rupture and could help in rational design of fibrin-containing biomaterials.
Topics: Humans; Fibrin; Thrombosis; Plasma; Fibrosis
PubMed: 36642339
DOI: 10.1016/j.actbio.2022.12.028 -
Journal of Thrombosis and Haemostasis :... Jul 2007Information on the structural origins of clot stability is necessary for understanding the functions and pathology of fibrin clots and thrombi, but is also important for... (Review)
Review
Information on the structural origins of clot stability is necessary for understanding the functions and pathology of fibrin clots and thrombi, but is also important for interpreting correctly the results of a variety of clinical diagnostic systems and technologies used daily to assess the hemostatic potential in patients. Fibrin polymerizes to make clots with a great diversity of structural, biological, physical and chemical properties, depending on the conditions of formation, and correlations have been established between these clot properties and many pathophysiological conditions. Clot stability refers to both viscoelastic properties, which are important because the clot essentially fulfills mechanical functions, and fibrinolytic properties, because the clot must be efficiently dissolved in a timely manner. The structure of the fibrin clot, which can be characterized in terms of a branched network, directly affects the clot's fibrinolytic and viscoelastic properties, which are remarkable and unique among polymers. Basic mechanisms underlying both the mechanical and fibrinolytic characteristics of fibrin are described. Some of the known correlations between clot structure and mechanical and fibrinolytic properties are summarized.
Topics: Fibrin; Fibrinolysis; Humans; Microscopy, Confocal; Microscopy, Electron, Scanning; Thrombosis
PubMed: 17635717
DOI: 10.1111/j.1538-7836.2007.02504.x -
Journal of the Mechanical Behavior of... Sep 2022Blood clots form at the site of vascular injury to seal the wound and prevent bleeding. Clots are in tension as they perform their biological functions and withstand...
Blood clots form at the site of vascular injury to seal the wound and prevent bleeding. Clots are in tension as they perform their biological functions and withstand hydrodynamic forces of blood flow, vessel wall fluctuations, extravascular muscle contraction and other forces. There are several mechanisms that generate tension in a blood clot, of which the most well-known is the contraction/retraction caused by activated platelets. Here we show through experiments and modeling that clot tension is generated by the polymerization of fibrin. Our mathematical model is built on the hypothesis that the shape of fibrin monomers having two-fold symmetry and off-axis binding sites is ultimately the source of inherent tension in individual fibers and the clot. As the diameter of a fiber grows during polymerization the fibrin monomers must suffer axial twisting deformation so that they remain in register to form the half-staggered arrangement characteristic of fibrin protofibrils. This deformation results in a pre-strain that causes fiber and network tension. Our results for the pre-strain in single fibrin fibers is in agreement with experiments that measured it by cutting fibers and measuring their relaxed length. We connect the mechanics of a fiber to that of the network using the 8-chain model of polymer elasticity. By combining this with a continuum model of swellable elastomers we can compute the evolution of tension in a constrained fibrin gel. The temporal evolution and tensile stresses predicted by this model are in qualitative agreement with experimental measurements of the inherent tension of fibrin clots polymerized between two fixed rheometer plates. These experiments also revealed that increasing thrombin concentration leads to increasing internal tension in the fibrin network. Our model may be extended to account for other mechanisms that generate pre-strains in individual fibers and cause tension in three-dimensional proteinaceous polymeric networks.
Topics: Blood Platelets; Elasticity; Fibrin; Humans; Thrombosis
PubMed: 35803206
DOI: 10.1016/j.jmbbm.2022.105328 -
Journal of Thrombosis and Haemostasis :... Sep 2022Intra-device thrombosis remains one of the most common complications during extracorporeal membrane oxygenation (ECMO). Despite anticoagulation, approximately 35% of...
OBJECTIVES
Intra-device thrombosis remains one of the most common complications during extracorporeal membrane oxygenation (ECMO). Despite anticoagulation, approximately 35% of patients develop thrombi in the membrane oxygenator, pump heads, or tubing. The aim of this study was to describe the molecular and cellular features of ECMO thrombi and to study the main drivers of thrombus formation at different sites in the ECMO circuits.
APPROACH AND RESULTS
Thrombi (n = 85) were collected immediately after veno-arterial-(VA)-ECMO circuit removal from 25 patients: 23 thrombi from the pump, 25 from the oxygenator, and 37 from the tubing. Quantitative histological analysis was performed for the amount of red blood cells (RBCs), platelets, fibrin, von Willebrand factor (VWF), leukocytes, and citrullinated histone H3 (H3Cit). ECMO thrombi consist of a heterogenous composition with fibrin and VWF being the major thrombus components. A clustering analysis of the four major histological parameters identified two typical thrombus types: RBC-rich and RBC-poor/fibrin-rich thrombi with no significant differences in VWF and platelet content. Thrombus composition was not associated with the thrombus location, except for higher amounts of H3Cit that were found in pump and oxygenator thrombi compared to tubing samples. We observed higher blood leukocyte count and lactate dehydrogenase levels in patients with fibrin-rich thrombi.
CONCLUSION
We found that thrombus composition is heterogenous, independent of their location, consisting of two types: RBC-rich and a fibrin-rich types. We also found that NETs play a minor role. These findings are important to improve current anticoagulation strategies in ECMO.
Topics: Anticoagulants; Extracorporeal Membrane Oxygenation; Fibrin; Humans; Thrombosis; von Willebrand Factor
PubMed: 35703468
DOI: 10.1111/jth.15784