Comprehensive Analysis of the Role of Fibrinogen and Thrombin in Clot Formation and Structure for Plasma and Purified Fibrinogen.

Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.

What is the diameter of a fibrin fiber?

Background: Altered fibrin fiber structure is linked to pathologic states, including coronary heart disease, ischemic stroke, and atherosclerosis. However, several different techniques are commonly utilized for studying fibrin structures, and comparison of results obtained using different techniques can be challenging due to lack of standardization. Objectives: This study provides a path toward standardization by comparing fibrin fiber diameters for a range of physiologic fibrinogen and thrombin concentrations using multiple different complementary experimental methods. Methods: We determined fiber diameter using scanning electron microscopy (SEM), superresolution (stochastic optical reconstruction microscopy) fluorescence microscopy, and 4 commonly utilized turbidimetric approaches to determine the congruence between the results and the conditions under which each should be used. Results: We found that diameter values obtained using SEM and superresolution imaging agree within 10% for nearly all conditions tested. We also found that when a wavelength range of 500 to 800 nm was used for measurements and accounting for the wavelength dependence of the refractive index and specific refractive index increment, diameters obtained using the corrected Yeromonahos turbidimetric approach agree with SEM within 20% for most conditions. Conclusion: We performed a systematic, multitechnique survey assessing fibrin fiber diameters under a range of biochemical conditions. The similarity in the diameter values obtained using SEM and superresolution imaging suggests that drying and fixation during SEM sample preparation do not dramatically alter fiber cross-sections. Congruence, under certain conditions, between diameter values obtained using SEM, superresolution fluorescence imaging, and turbidimetry demonstrates the feasibility of a fibrin diameter standardization project.

Fluorescent microspheres can affect in vitro fibrinolytic outcomes.

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.

Fibrinolysis: an illustrated review.

In response to vessel injury (or other pathological conditions), the hemostatic process is activated, resulting in a fibrous, cellular-rich structure commonly referred to as a blood clot. Succeeding the clot's function in wound healing, it must be resolved. This illustrated review focuses on fibrinolysis-the degradation of blood clots or thrombi. Fibrin is the main mechanical and structural component of a blood clot, which encases the cellular components of the clot, including platelets and red blood cells. Fibrinolysis is the proteolytic degradation of the fibrin network that results in the release of the cellular components into the bloodstream. In the case of thrombosis, fibrinolysis is required for restoration of blood flow, which is accomplished clinically through exogenously delivered lytic factors in a process called external lysis. Fibrinolysis is regulated by plasminogen activators (tissue-type and urokinase-type) that convert plasminogen into plasmin to initiate fiber lysis and lytic inhibitors that impede this lysis (plasminogen activator inhibitors, alpha 2-antiplasmin, and thrombin activatable fibrinolysis inhibitor). Furthermore, the network structure has been shown to regulate lysis: thinner fibers and coarser clots lyse faster than thicker fibers and finer clots. Clot contraction, a result of platelets pulling on fibers, results in densely packed red blood cells (polyhedrocytes), reduced permeability to fibrinolytic factors, and increased fiber tension. Extensive research in the field has allowed for critical advancements leading to improved thrombolytic agents. In this review, we summarize the state of the field, highlight gaps in knowledge, and propose future research questions.

Systematic mapping of the conformational landscape and dynamism of soluble fibrinogen.

BACKGROUND: Fibrinogen is a soluble, multisubunit, and multidomain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin, initiating polymerization that substantially contributes to clot growth. Fibrinogen contains numerous, transiently accessible "cryptic" epitopes for hemostatic and immunologic proteins, suggesting that fibrinogen exhibits conformational flexibility, which may play functional roles in its temporal and spatial interactions. Hitherto, there have been limited integrative approaches characterizing the solution structure and internal flexibility of fibrinogen. METHODS: Here, utilizing a multipronged, biophysical approach involving 2 solution-based techniques, temperature-dependent hydrogen-deuterium exchange mass spectrometry and small angle X-ray scattering, corroborated by negative stain electron microscopy, we present a holistic, conformationally dynamic model of human fibrinogen in solution. RESULTS: Our data reveal 4 major and distinct conformations of fibrinogen accommodated by a high degree of internal protein flexibility along its central scaffold. We propose that the fibrinogen structure in the solution consists of a complex, conformational landscape with multiple local minima. This is further supported by the location of numerous point mutations that are linked to dysfibrinogenemia and posttranslational modifications, residing near the identified fibrinogen flexions. CONCLUSION: This work provides a molecular basis for the structural "dynamism" of fibrinogen that is expected to influence the broad swath of its functionally diverse macromolecular interactions and fine-tune the structural and mechanical properties of blood clots.

The Applicability of Current Turbidimetric Approaches for Analyzing Fibrin Fibers and Other Filamentous Networks.

Turbidimetry is an experimental technique often used to study the structure of filamentous networks. To extract structural properties such as filament diameter from turbidimetric data, simplifications to light scattering theory must be employed. In this work, we evaluate the applicability of three commonly utilized turbidimetric analysis approaches, each using slightly different simplifications. We make a specific application towards analyzing fibrin fibers, which form the structural scaffold of blood clots, but the results are generalizable. Numerical simulations were utilized to assess the applicability of each approach across a range of fiber lengths and diameters. Simulation results indicated that all three turbidimetric approaches commonly underestimate fiber diameter, and that the "Carr-Hermans" approach, utilizing wavelengths in the range of 500−800 nm, provided <10% error for the largest number of diameter/length combinations. These theoretical results were confirmed, under select conditions, via the comparison of fiber diameters extracted from experimental turbidimetric data, with diameters obtained using super-resolution microscopy.

Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker.

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.

Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen.

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin's soluble precursor, would advance the ability to construct mutational libraries that would enable structure-function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8-12 mg/L using suspension HEK Expi293TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin's knob 'A' sequence enables the purification of 50-75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial.

Microscale structural changes of individual fibrin fibers during fibrinolysis.

Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been debated and obtaining detailed structural knowledge of these processes is important for developing effective clinical approaches to treat ischemic stroke and pulmonary embolism. Using dynamic fluorescence microscopy, we studied the time-resolved digestion of individual fibrin fibers by the fibrinolytic enzyme plasmin. We found that plasmin molecules digest fibers along their entire lengths, but that the rates of digestion are non-uniform, resulting in cleavage at a single location along the fiber. Using mathematical modeling we estimated the rate of plasmin arrival at the fiber surface and the number of digestion sites on a fiber. We also investigated correlations between local fiber digestion rates, cleavage sites, and fiber properties such as initial thickness. Finally, we uncovered a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. Taken together these results promote a paradigm shift in understanding mechanisms of fibrinolysis and underscore the need to consider fibrin tension when assessing fibrinolytic approaches. STATEMENT OF SIGNIFICANCE: We developed a method for interrogating lysis of individual fibrin fibers, enabling the time-resolved observation of individual fiber digestion for the first time. Our results resolve longstanding disagreements about fibrinolytic processes and reveal previously unknown mechanisms that also play a role. Also, we developed the first microscale mathematical model of plasmin-fibrin interaction, which predicts the number of plasmin molecules on each fiber and can serve as a framework for investigating novel therapeutics.

The Utility and Potential of Mathematical Models in Predicting Fibrinolytic Outcomes.

The enzymatic degradation of blood clots, fibrinolysis, is an important part of a healthy hemostatic system. If intrinsic fibrinolysis is ineffective, thrombolysis - the clinically-induced enzymatic degradation of blood clots - may be necessary to treat life-threatening conditions. In this review we discuss recent models of fibrinolysis and thrombolysis, and open questions that could be resolved through modeling and modeling-experimental collaboration. In particular, we focus on 2- and 3-dimensional models that can be used to study effects of fibrin network structure and realistic blood vessel geometries on the phenomena underlying lytic outcomes. Significant open questions such as the role of clot contraction, network and inherent fiber tension, and fibrinolytic inhibitors in lysis could benefit from mathematical models aimed at understanding the underlying biological mechanisms.

Inherent fibrin fiber tension propels mechanisms of network clearance during fibrinolysis.

Proper wound healing necessitates both coagulation (the formation of a blood clot) and fibrinolysis (the dissolution of a blood clot). A thrombus resistant to clot dissolution can obstruct blood flow, leading to vascular pathologies. This study seeks to understand the mechanisms by which individual fibrin fibers, the main structural component of blood clots, are cleared from a local volume during fibrinolysis. We observed 2-D fibrin networks during lysis by plasmin, recording the clearance of each individual fiber. We found that, in addition to transverse cleavage of fibers, there were multiple other pathways by which clot dissolution occurred, including fiber bundling, buckling, and collapsing. These processes are all influenced by the concentration of plasmin utilized in lysis. The network fiber density influenced the kinetics and distribution of these pathways. Individual cleavage events often resulted in large morphological changes in network structure, suggesting that the inherent tension in fibers played a role in fiber clearance. Using images before and after a cleavage event to measure fiber lengths, we estimated that fibers are strained ~23% beyond their equilibrium length during polymerization. To understand the role of fiber tension in fibrinolysis we modeled network clearance under differing amounts of fiber polymerized strain (prestrain). The comparison of experimental and model data indicated that fibrin tension enables 35% more network clearance due to network rearrangements after individual cleavage events than would occur if fibers polymerized in a non-tensed state. Our results highlight many characteristics and mechanisms of fibrin breakdown, which have implications on future fibrin studies, our understanding of the fibrinolytic process, and the development of thrombolytic therapies. STATEMENT OF SIGNIFICANCE: Fibrin fibers serve as the main structural element of blood clots. They polymerize under tension and have remarkable extensibility and elasticity. After the cessation of wound healing, fibrin must be cleared from the vasculature by the enzyme plasmin in order to resume normal blood flow: a process called fibrinolysis. In this study we investigate the mechanisms that regulate the clearance of individual fibrin fibers during fibrinolysis. We show that the inherent tension in fibers enhances the action of plasmin because every fiber cleavage event results in a redistribution of the network tension. This network re-equilibration causes fibers to buckle, bundle, and even collapse, leading to a more rapid fiber clearance than plasmin alone could provide.

Biophysical Mechanisms Mediating Fibrin Fiber Lysis.

The formation and dissolution of blood clots is both a biochemical and a biomechanical process. While much of the chemistry has been worked out for both processes, the influence of biophysical properties is less well understood. This review considers the impact of several structural and mechanical parameters on lytic rates of fibrin fibers. The influences of fiber and network architecture, fiber strain, FXIIIa cross-linking, and particle transport phenomena will be assessed. The importance of the mechanical aspects of fibrinolysis is emphasized, and future research avenues are discussed.

ß-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbß3 Headpiece.

The platelet integrin αIIbß3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and ß-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the ß3-subunit. Ligand binding induces headpiece opening, with conformational change in the ß-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the ßI-domain ß1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with ß3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbß3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbß3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the ß3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required.

Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases.

Mutations in the ultralong vascular protein von Willebrand factor (VWF) cause the common human bleeding disorder, von Willebrand disease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding. Gain-of-function mutations in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster on-rate, providing a mechanism for activating VWF binding to platelets. Switching occurs near 10 pN, a force that also induces a state of the receptor-ligand complex with slower off-rate. Force greatly increases the effects of VWD mutations, explaining pathophysiology. Conversion of single molecule kon (s(-1)) to bulk phase kon (s(-1)M(-1)) and the kon and koff values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.

Physical determinants of fibrinolysis in single fibrin fibers.

Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

Structural determinants of integrin ß-subunit specificity for latent TGF-ß.

Eight integrin α-ß heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the structural mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVß6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-ß1 and TGF-ß3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the ß6 subunit. Elucidation of the basis for ligand binding specificity by the integrin ß subunit reveals contributions by three different ßI-domain loops, which we designate specificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire ß subunit in integrin evolution, thus suggesting a paradigmatic role in overall ß-subunit function.

Submillisecond elastic recoil reveals molecular origins of fibrin fiber mechanics.

Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin's elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin's mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers.

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers.

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.

Stiffening of individual fibrin fibers equitably distributes strain and strengthens networks.

As the structural backbone of blood clots, fibrin networks carry out the mechanical task of stemming blood flow at sites of vascular injury. These networks exhibit a rich set of remarkable mechanical properties, but a detailed picture relating the microscopic mechanics of the individual fibers to the overall network properties has not been fully developed. In particular, how the high strain and failure characteristics of single fibers affect the overall strength of the network is not known. Using a combined fluorescence/atomic force microscope nanomanipulation system, we stretched 2-D fibrin networks to the point of failure, while recording the strain of individual fibers. Our results were compared to a pair of model networks: one composed of linearly responding elements and a second of nonlinear, strain-stiffening elements. We find that strain-stiffening of the individual fibers is necessary to explain the pattern of strain propagation throughout the network that we observe in our experiments. Fiber strain-stiffening acts to distribute strain more equitably within the network, reduce strain maxima, and increase network strength. Along with its physiological implications, a detailed understanding of this strengthening mechanism may lead to new design strategies for engineered polymeric materials.