MEASUREMENT OF VENOUS THROMBUS FORMATION
AND MASS TRANSFER EFFECTS IN THE INITIAL STAGE
USING MICROCHANNEL FLOWYusuke Yamamoto1, Kazuya Tatsumi1, Hitoshi Shirouzu1,Hideo Hirakata2, Naoko Sugita1, Kyo Inoue1, and Kazuyoshi Nakabe1
1
Kyoto University, JAPAN, and 2Kyoto City Hospital, JAPANABSTRACT
In this study, the process of venous thrombus formation, particularly the initial stage of fibrin
network formation, was measured and analyzed using a microfluidic platform. The results showed thatthe platelet activation and mass transfer from the platelets has a strong correlation with the fibrin
network formation and pattern.
KEYWORDS: Venous thrombus, Mass diffusion, Fibrin network, Microchannel
INTRODUCTION
Venous thrombus is a significant issue in the blood circulatory system that can lead to serious
symptoms such as deep venous thrombosis and pulmonary embolism (PE) which has large number ofmortality [1]. However, compared to arterial thrombus, the mechanism, formation process, andstructure of a venous thrombus have not yet been well understood [2,3]. We have fabricated a
microchannel, modeling the vain, with an area exposing the collagen layer to the blood flow, thus
representing a condition that the wall of vein is injured (Fig. 1). When a vessel wall is wounded,platelets aggregate at the site of injury and are activated through various reactions. In this process,fibrin fibers are formed from platelets and increase the volume by capturing other blood cells. We
measured the fibrin pattern growing from the activated platelets adhered to the vessel wall. The pattern
was compared with the apparent mass diffusion from the platelets to see the correlation between thetwo phenomena.
Tube

Flow

Sigmacote coated
PDMS

Measurement areaFlow

1 2 3 4 5 6 7 8 20…

Red blood cell…

Flow

1 2 3 4 5 6 7 8 20

Cover glass

PlateletAdhesion Activation

Scale
Collagen coatedMass diffusion(Fig.3)Collagen coatedSigmacote coatedSigmacote coated

Activated platelet

time
Collagen coatedFigure 1: Schematics of the microchannel adjusted to the blood flow condition in vein. A part of the channel
wall is coated by collagen and the platelets adhere to this region and then activates. To avoid stimulatingplatelets by glass (to prevent intrinsic coagulation), whole channel wall is coated by sigmacote.

EXPERIMENTAL
The microchannel was made of cover glass and PDMS (polydimethylsiloxane) and is shown in Fig.
1. Healthy human blood, into which fibrinogens fluorescently labeled by Fibrinogen-Alexa Fluor 546(Thermo Fisher Scientific, F13192) were mixed, were supplied to the channel at the flow rate of
1.3μL/min. The wall shear rate under this condition corresponds to that in the vein. The fluorescence
image of the fibrin and the particle motion were measured using a high-sensitivity camera. The particle
velocities and positions were analyzed by the motion analyze software (Library, Move-tr/2D ver 7.90)
to measure the apparent diffusion coefficients.
RESULTS AND DISCUSSIONFigure 2 shows the visualization of the activated platelet and fibrin network in the initial ((a), (b))
and developed ((c), (d)) stages. Figure 2(d) shows the fibrin pattern in the well (stationary fluid withzero flow velocity). As time progresses in Figures (a)-(c), fibrin fibers appear from the platelets andmainly expand downstream in the microchannel flow. While the fibrin network observed in the well
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20th International Conference on Miniaturized
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shows an isotropic pattern, the fibrin network in the microchannel flow is significantly influenced by
the flow and aligned along the flow direction.
To see if the previously mentioned pattern can be attributed to the mass diffusion of the activator
released from the platelets, each fibrin fiber formed downstream of the platelets was measured and thearea was presented by the 95% inclusive area. This area was derived using the apparent diffusion
coefficient obtained from the probability density distribution of the spanwise displacement of the fibrin
growth in 5s and is shown in Fig. 3(a). Mass diffused downstream of the platelet was measured inblood and water flows using micro-particles and is shown in Fig. 3(b). Comparing the fibrin network
pattern and diffusion area, a fair correlation can be observed. The reason why the fibrin area shows
larger value than the diffusion area can be attributed to the blood cells attached to the channel bottom
wall which could interfere the flow and change the direction and amplitude.
(b)

(a)

(d)

(c )

Figure 2: Visualization of the fibrin network and platelet at different stage. (a) and (b) show the results of elapse
time of (a) 10min (b) 12min, and (c) and (d) show the developed stage. (a)~(c) are measured in the microchannel(flow direction in these figures is left to right) and (d) is measured in the well (in stationary fluid).

(a) 20(b) 20fibrin fiber
fibrin formation area1515
10y [m]y [m]

1050-550-5-10-150fibrin formation area
mass diffusion area in blood
mass diffusion area in water-1010203040-15050

1020304050

x [m]x [m]
Figure 3: (a) the distributions of fibrin fibers formed from each platelet (the platelet is located at the coordinateorigin). The dash lines depict the fibrin fibers and the square symbols are the edge of the fibrin measured every
5s. The solid line represent 95% inclusive area obtained by the apparent diffusion rate. (b) the area of massdiffusion with the released source at the coordinate origin measured by the particle motions in the water andblood flows are compared with the result of Fig. (a).

CONCLUSION
The fibrin network growing from the platelet could be visualized clearly in the microchannel. The
diffusion area of the mass assumed to be originated from the platelets showed a fair correlation withthe direction of the fibrin fibers growing, which indicated the possibility of some relationship between
activators released from the platelets and the pattern of the fibrin network.
ACKNOWLEDGEMENTS
This work was financially supported by the Ministry of Education, Culture, Sports, Science andTechnology of Japan (KAKEN: 15H03931).
REFERENCES[1] Cohen, A. T. et al, “Venous Thromboembolism (VTE) in Europe,” Thrombus hemostasis, 98, 756-764, 2007.
[2] Weiss, H. J. et al, “Role of Shear Rate and Platelets in Promoting Fibrin Formation on Rabbit Subendothelium,” J. Clinical Investigation, 78, 1072-1082, 1986.
[3] Collet, J. P. et al, “Influence of Fibrin Network Conformation and Fibrin Fiber Diameter on Fibrinolysis
Speed,” J. Arteriosclerosis Thrombolysis Vascular Biology, 20, 1354-1361, 2000.

CONTACT* Kazuya Tatsumi; phone: +81-75-383-3606; tatsumi@me.kyoto-u.ac.jp835