John W. Weisel

Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy.

Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease.

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.

Fibrin gels and their clinical and bioengineering applications

Fibrin gels, prepared from fibrinogen and thrombin, the key proteins involved in blood clotting, were among the first biomaterials used to prevent bleeding and promote wound healing. The unique polymerization mechanism of fibrin, which allows control of gelation times and network architecture by variation in reaction conditions, allows formation of a wide array of soft substrates under physiological conditions. Fibrin gels have been extensively studied rheologically in part because their nonlinear elasticity, characterized by soft compliance at small strains and impressive stiffening to resist larger deformations, appears essential for their function as haemostatic plugs and as matrices for cell migration and wound healing. The filaments forming a fibrin network are among the softest in nature, allowing them to deform to large extents and stiffen but not break. The biochemical and mechanical properties of fibrin have recently been exploited in numerous studies that suggest its potential for applications in medicine and bioengineering.

Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled

Although much is known about fibrin polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in fibrin polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.

Altered Fibrin Architecture Is Associated With Hypofibrinolysis and Premature Coronary Atherothrombosis

Objective—Hypofibrinolysis promotes atherosclerosis progression and recurrent ischemic events in premature coronary artery disease. We investigated the role of fibrin physical properties in this particular setting. Methods and Results—Biomarkers of recurrent thrombosis and premature coronary artery disease (CAD) were measured in 33 young post–myocardial infarction patients with angiographic-proven CAD and in 33 healthy volunteers matched for age and sex. Ex vivo plasma fibrin physical properties were assessed by measuring fibrin rigidity and fibrin morphological properties using a torsion pendulum and optical confocal microscopy. The fibrinolysis rate was derived from continuous monitoring of the viscoelastic properties after addition of lytic enzymes. Young CAD patients had a significant increase in plasma concentration of fibrinogen, von Willebrand factor, plasminogen activator inhibitor type 1, and lipoprotein(a) as compared with controls (P<0.05). Fibrin of young CAD patients was stiffer (P=0.002), made of numerous (P=0.002) and shorter fibers (P=0.04), and lysed at a slower rate than that of controls (P=0.03). Fibrin stiffness was an independent predictor for both premature CAD and hypofibrinolysis. Conclusions—This first detailed study of clot properties in such a group of patients demonstrated that abnormal plasma fibrin architecture is an important feature of both premature CAD and fibrinolysis rate. The determinants of this particular phenotype warrant further investigation.

Structure of fibrin: impact on clot stability

Summary.  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.

Composition of Coronary Thrombus in Acute Myocardial Infarction

OBJECTIVES We sought to analyze the composition of coronary thrombus in vivo in ST-segment elevation myocardial infarction (STEMI) patients. BACKGROUND The dynamic process of intracoronary thrombus formation in STEMI patients is poorly understood. METHODS Intracoronary thrombi (n = 45) were obtained by thromboaspiration in 288 consecutive STEMI patients presenting for primary percutaneous intervention, and analyzed using high-definition pictures taken with a scanning electron microscope. Plasma biomarkers (TnI, CRPus, IL-6, PAI-1, sCD40 ligand, and TNF-α) and plasma fibrin clot viscoelastic properties were measured simultaneously on peripheral blood. RESULTS Thrombi were mainly composed of fibrin (55.9 ± 18%) with platelets (16.8 ± 18%), erythrocytes (11.5 ± 9%), cholesterol crystals (5.2 ± 8.4%), and leukocytes (1.3 ± 2.0%). The median ischemic time was 175 min (interquartile range: 140 to 297). Ischemic time impacted thrombi composition, resulting in a positive correlation with intracoronary thrombus fibrin content, r = 0.38, p = 0.01, and a negative correlation with platelet content, r = -0.34, p = 0.02. Thus, fibrin content increased with ischemic time, ranging from 48.4 ± 21% (<3 h) up to 66.9 ± 9% (>6 h) (p = 0.02), whereas platelet content decreased from 24.9 ± 23% (<3 h) to 9.1 ± 6% (>6 h) (p = 0.07). Soluble CD40 ligand was positively correlated to platelet content in the thrombus (r = 0.40, p = 0.02) and negatively correlated with fibrin content (r = -0.36; p = 0.04). Multivariate analysis indicated that ischemic time was the only predictor of thrombus composition, with a 2-fold increase of fibrin content per ischemic hour (adjusted odds ratio: 2.00 [95% confidence interval: 1.03 to 3.7]; p = 0.01). CONCLUSIONS In acute STEMI, platelet and fibrin contents of the occlusive thrombus are highly dependent on ischemia time, which may have a direct impact on the efficacy of drugs or devices used for coronary reperfusion.

Multiscale Mechanics of Fibrin Polymer: Gel Stretching with Protein Unfolding and Loss of Water

<< Focusing on Fibrin Vascular injury initiates biochemical reactions that cause the blood protein, fibrin, to polymerize and help to stop bleeding and support wound healing. Fibrin can also be a scaffold for thrombi that lead to cardiovascular diseases. To maintain homeostasis, fibrin clots must be stiff, plastic, and, so that the network can be decompsed, permeable. Brown et al. (p. 741) investigated the behavior of fibrin clots at the macroscopic, single-fiber, and molecular scale. At relatively low strains, fibers aligned and formed bundles, and at higher strains, protein unfolding occurred. An integrated model provides a molecular basis for fibrin elasticity and extensibility. Protein unfolding in stretched fibrin blood clots creates porous gels that can withstand high strains. Blood clots and thrombi consist primarily of a mesh of branched fibers made of the protein fibrin. We propose a molecular basis for the marked extensibility and negative compressibility of fibrin gels based on the structural and mechanical properties of clots at the network, fiber, and molecular levels. The force required to stretch a clot initially rises linearly and is accompanied by a dramatic decrease in clot volume and a peak in compressibility. These macroscopic transitions are accompanied by fiber alignment and bundling after forced protein unfolding. Constitutive models are developed to integrate observations at spatial scales that span six orders of magnitude and indicate that gel extensibility and expulsion of water are both manifestations of protein unfolding, which is not apparent in other matrix proteins such as collagen.

Mechanisms of fibrin polymerization and clinical implications

Research on all stages of fibrin polymerization, using a variety of approaches including naturally occurring and recombinant variants of fibrinogen, x-ray crystallography, electron and light microscopy, and other biophysical approaches, has revealed aspects of the molecular mechanisms involved. The ordered sequence of fibrinopeptide release is essential for the knob-hole interactions that initiate oligomer formation and the subsequent formation of 2-stranded protofibrils. Calcium ions bound both strongly and weakly to fibrin(ogen) have been localized, and some aspects of their roles are beginning to be discovered. Much less is known about the mechanisms of the lateral aggregation of protofibrils and the subsequent branching to yield a 3-dimensional network, although the αC region and B:b knob-hole binding seem to enhance lateral aggregation. Much information now exists about variations in clot structure and properties because of genetic and acquired molecular variants, environmental factors, effects of various intravascular and extravascular cells, hydrodynamic flow, and some functional consequences. The mechanical and chemical stability of clots and thrombi are affected by both the structure of the fibrin network and cross-linking by plasma transglutaminase. There are important clinical consequences to all of these new findings that are relevant for the pathogenesis of diseases, prophylaxis, diagnosis, and treatment.

Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk

BACKGROUND Polymorphisms in the fibrinogen and factor XIII genes are associated with atherothrombotic risk, but clinical studies have produced inconsistent results and laboratory studies have not explained these findings. We aimed to investigate interactions between polymorphisms in the factor XIII and fibrinogen genes, fibrinogen concentrations, and other cardiovascular risk factors in relation to fibrin structure and function. METHODS We used permeation analysis and electron microscopy to investigate interactions between fibrin structure, factor XIII Val34Leu, fibrinogen Aalpha Thr312Ala, fibrinogen Bbeta Arg448Lys, and fibrinogen concentrations in plasma and purified systems. FINDINGS Increased fibrinogen concentrations were associated with decreases in permeability, with tighter clot structures in the presence of factor XIII 34Val alleles compared with those in the presence of 34Leu alleles. Findings were confirmed by scanning electron microscopy of fibrin. Similar changes in permeability were noted for Aalpha fibrinogen 312Ala compared with that for 312Thr. INTERPRETATION Our results show interactions between coding polymorphisms in fibrinogen and factor XIII and fibrinogen concentrations that modify fibrin and explain the apparent paradox between epidemiological studies of factor XIII 34Leu and reported in-vitro effects on fibrin structure and function. We suggest a potential complexity of gene-gene and gene-environment interactions in determining cardiovascular risk.