The Effect of Thrombin Concentration on Fibrin Clot Structure Imaged by Multiphoton Microscopy and Quantified by Fractal Analysis

Abstract

Fibrin clots are an important component of chronic cardiovascular thrombosis and are associated with cardiovascular dysfunction, stroke and formation of microthrombi in systemic inflammatory states. During coagulation, thrombin selectively cleaves fibrinogen resulting in the formation of linear fibrin polymers that form by end-to-end and side-to-side association. Here we present a novel assay for real-time imaging of fibrin polymerization using fluorescence laser scanning multiphoton microscopy (MPM) and fractal analysis for quantification of clot structure.Human blood, collected in acid citrate dextrose tubes, was centrifuged (2x, for 8 minutes @ 500 x g) to produce platelet poor plasma (PPP). Fibrin polymerization was initiated at room temperature by addition of thrombin to PPP and imaged ( Fig.1) through incorporation of fluorescently labeled fibrinogen (5% vol/vol Oregon Green labeled fibrinogen to PPP fibrinogen) at 0,1,2,3,5,10 and 15 minutes. 900nm fs pulses (Ti-Sapphire laser) were focused through a 63x/1.2 NA objective (Leica AOBS SP2 microscope system) to a clot depth of 20 µm. Fluorescent images were acquired at 400Hz/3frame averages, emission bandwidth 500-650nm. At high doses of thrombin (0.1 and 0.05 units/ml), clots formed within 1-3 minutes of initiation. At lower thrombin doses (0.025 and 0.01 units/ml), clots were unstable over the first 5 minutes (generating motion artifacts), but were stable ten minutes after initiation. To quantify clot structure, images were processed using MATLAB (The MathWorks, Inc., MA) and quantified by fractal analysis (TruSoft Int'l Inc., FL). The image processing algorithm consisted of background signal subtraction, median filter, image thresholding (Otsu's method [1]) and binarization. Fractal dimension was calculated using the box dimension estimation method. The box dimension, Db, is defined as the exponent of N(d) ~ 1/d Db , where N(d) is the number of boxes of linear size (d) required to cover a set of points distributed in a 2D plane. As thrombin concentration increased the clot Db value rapidly increased then seemingly reached a plateau at a thrombin concentration of 0.1 unit/ml, see Fig 2B, indicating that fibrin clot structure was dependent on thrombin concentration and stabilized at the highest thrombin concentration tested.This MPM imaging technique of fibrin clots combined with fractal analysis of clot structure offers a rapid, reproducible and promising approach to investigate clotting mechanisms in disease states and to test the efficacy of drug treatments on clotting kinetics, clot structure and clot fibrinolysis. References[1] N. Otsu, IEEE 9(1) (1979) 62.[2] This work was supported by Heart and Stroke Foundation Canada (HSFC), Michael Smith Foundation for Health Research (British Columbia) and CIHR/HSFC IMPACT Postdoctoral Fellowships.

Modulation of 3D Fibrin Matrix Stiffness by Intrinsic Fibrinogen–Thrombin Compositions and by Extrinsic Cellular Activity

“…Much of the increase in the stiffness was directly related to increasing fibrinogen concentration, and to a lesser degree to thrombin concentration. Physiologic fibrinogen and thrombin concentration for clot formation has been measured around 2 mg/mL and 0.01 to 0.1 units/mL of thrombin 17,29. Our formulations include the low 2 mg/mL of fibrinogen and the low 2 units/mL of thrombin, and we have extended range of the formulation to a high 50 mg/mL of fibrinogen and 100 units/mL of thrombin.…”

“…It has been established that the modulation of the intrinsic factors such as fibrin constituents, fibrinogen, and thrombin can have profound effect on the mechanical properties of the fibrin microstructure. Modifying the thrombin concentration may affect the rate of polymerization, fiber size, and porosity 17–19. Similarly, modifying the fibrinogen concentration has also been shown to affect fiber size and porosity 20.…”

Real-Time Feedback of Histotripsy Thrombolysis Using Bubble-Induced Color Doppler

“…Bovine fibrinogen (F8630; Sigma-Aldrich Co., St. Louis, MO, USA) was then added to the processed plasma with a concentration of 400 mg/dL. To stimulate the clotting cascade, bovine thrombin (T4648; Sigma-Aldrich Co., St. Louis, MO, USA) and calcium chloride (21107; Sigma-Aldrich Co., St. Louis, MO, USA) were mixed with the reconstituted plasma to a final concentration of 1 IU/mL and 20mM/L respectively (Ryan et al 1999; Bateman et al 2005). The mixture was then poured into a rectangular mold and incubated in 37°C water bath for 2 hours.…”

Microgel core/shell architectures as targeted agents for fibrinolysis

“…9,16 The porosity of a fibrin clot depends on the clot formation conditions. [17][18][19] In this study, the concentration of fibrinogen used for clot formation was 3 mg/mL, which is close to the physiological range of ~2.5 mg/mL; the clots were made using 1 U/mL thrombin. Scheme 1 represents the experimental set up for the flow studies.…”