Gel point and fractal microstructure of incipient blood clots are significant new markers of hemostasis for healthy and anticoagulated blood

Abstract

IntroductionCoagulation pathway changes play an important role in the outcome of both clot propagation and fibrinolysis. The structurefunction relationship of the developing fibrin clot is known to be affected by many factors, such as environment, therapy, and disease, compared with normal clot growth. 1,2 A fibrin clot's primary microstructure consists of a disordered network of entangled, branching fibrin fibers. Thinner fibers are associated with networks that display an increased number of branch points, creating denser, less permeable clots that have a known association with thromboembolic disease. [3][4][5][6] More open/permeable networks are formed from thicker fibers, the latter displaying a reduced number of branch points for a given amount of fibrinogen and producing a more porous system. [7][8][9][10] Clots with altered fibrin microstructure exhibit different susceptibility to fibrinolysis, 8,10,11 with clot permeability being the rate-limiting factor for the activity of the fibrin network degradation enzyme plasmin. The permeability will aid or hamper the ability of tissue plasmin activator, tPA, and/or urokinase plasmin activator to move through the 3-dimensional fibrin network and activate the zymogen plasminogen to fibrinolytic plasmin. The effect of anticoagulants such as heparin in the therapeutic manipulation of fibrin clot microstructure by thrombin inhibition increases clot permeability/porosity and produces clots with thicker fibers. 12,13 The evolution of clot microstructure is associated with significant changes in blood viscoelasticity (a measure of a material's viscous and elastic properties). Viscoelastic properties are among the most sensitive measures of fibrin polymerization and blood clot structure. 7,14 In the present study, we focused on the formation of the incipient clot, which provides the microstructural template that determines the future clot morphology, 15-17 by measuring the incipient clot's viscoelastic properties with an oscillatory shear technique known as Fourier transform mechanical spectroscopy (FTMS). [18][19][20] This technique provides an accurate determination of the gel point (GP) of coagulating blood and allows the microstructure of the incipient clot to be quantified by fractal analysis, a technique widely used in medicine and biology to characterize nonlinear growth in branching network structures. 21 The authors of previous studies of clot structure on the basis of techniques such as scanning electron microscopy have reported qualitative descriptions of clot microstructure (involving terms such as "rigid clot structures," "open/porous/dense/loose," etc 1 ), whereas studies of the fractal properties of fibrin gels on the basis of light-scattering techniques have been restricted to dilute solutions of fibrinogen, at concentrations less than those of physiologic relevance in whole blood. 22 A recent study of fibrin clot structure suggests there is a definitive diagnostic potential of characterizing clot structure and the modulation of clot architecture as a pos...

A new structural biomarker that quantifies and predicts changes in clot strength and quality in a model of progressive haemodilution

“…The method detects the Gel Point (GP), by measuring the change in at the different frequencies with respect to time, and recording the attainment of a frequency independent  (see Figure 2). [2,3,16] The GP marks the first time that the fluid transitions to a solid. In coagulating blood this transition corresponds to the formation of the incipient clot, whose sample-spanning fibrin network provides the mechanical basis for attaining a haemostatic function.…”

“…[3] Briefly a 6.6 ml aliquot of diluted blood was immediately loaded into a double-gap concentric cylinder of a TA Instruments AR-G2 controlled-stress rheometer at 37 ºC (± 0.1ºC). Immediately after sample loading, small amplitude oscillatory shear measurements were performed at test frequencies 2Hz, 0.93Hz, 0.43Hz and 0.2Hz with a peak stress amplitude of 0.03Pa.…”

“…In coagulating blood this transition corresponds to the formation of the incipient clot, whose sample-spanning fibrin network provides the mechanical basis for attaining a haemostatic function. [3][4][5][6] A recent study involving analysis of GP and imaging data has established that the incipient clot acts as a template for ensuing clot development. [5,6] The ability to detect the GP allows us to quantify how the fibrin clot is organised using fractal analysis.…”

“…[1][2][3][4] In the present study we exploit a novel haemorheological technique that uses oscillatory shear to detect the establishment of the incipient clot (the first point at which a solid clot is formed that can achieve its haemostatic function). [3][4][5][6] This technique provides three separate but related biomarkers of clot properties, the rate of coagulation (TGP), clot elasticity/strength (G`GP) and clot microstructure (df), which are all taken from one measurement, the Gel Point (GP). The df represents a quantitative measure of clot microstructure, a decrease in df corresponding to more permeable, less branched and mechanically weaker clot and a raised df giving a tightly packed, highly branched clot that is less permeable and stronger.…”

“…The df represents a quantitative measure of clot microstructure, a decrease in df corresponding to more permeable, less branched and mechanically weaker clot and a raised df giving a tightly packed, highly branched clot that is less permeable and stronger. [3][4][5][6] Previous studies have highlighted the importance of clot microstructure and how it affects the rate of clot lysis, where densely organised clots have been linked to thromboembolic disease. [7][8][9][10] This study investigates the in-vitro effect of dilution with saline on the organization and arrangement of clot microstructure and how this affects the mechanical properties of the developing clot.…”