Fibrinogen was exposed to ultrasound for 30 minutes at 37°C and then assayed by SDS-PAGE under reducing and nonreducing conditions (Figure ). Gel-electrophoresis without reduction of inter- and intramolecular disulfide bonds showed presence of one polypeptide with the molecular weight of more than 250 kDa (band on the top of the gel) in the US-treated and the control samples. The same samples separated on the SDS-PAGE gel containing β-mercaptoethanol showed three identical polypeptides corresponding to native Aα-, Bβ-, and γ-chains of fibrinogen (Figure ).
Figure 1 SDS-PAGE analysis of fibrinogen samples. 1 - sonicated (25.1 kHz, 48.4 W/cm2, duty 50%) at 37°C for 30 min; 2 - control (treated at 37°C for 30 min) samples of fibrinogen (Fg) in the presence (+βME) and absence (-βME) β-mercaptoethanol. (more ...)
The effects of the US on functional activity of the fibrinogen were investigated. For this purpose, two physiologically important processes involving fibrinogen were tested: the formation of a fibrinogen clot and fibrinogenolysis. The clotting ability was tested by adding thrombin and measuring the increase in turbidity at 350 nm. For the control sample of fibrinogen, exponential increase of the turbidity with an increase of incubation time was observed (Figure , line 1). The initial velocity and the stationary level of the absorption decreased for the sample treatment ultrasound during 5 min (Figure , line 2). A sonication of 30 minutes led to the total loss of the clotting ability (Figure , line 3).
Clotting of fibrinogen. native (1) and US-treated (25.1 kHz, 48.4 W/cm2, duty 50%) during 5 (2) and 30 (3) min fibrinogen.
The electrophoretic pattern of the fibrinogen proteolytic degradation products by plasmin after treatment with β-mercaptoethanol is shown in Figure . Identical distribution of polypeptide bands for both sonicated and control fibrinogen samples were observed. At zero time of proteolysis, three polypeptides corresponding to Aα-, Bβ-, and γ-chains of fibrinogen were present. The decrease of the content of the native chains and respectively the accumulation of proteolytically modified β''-, γ'-, γ''- and γ'''- chains of fibrinogen were observed during the time course of the fibrinogen incubation with plasminogen and t-PA (Figure ).
Figure 3 Densitograms of SDS-PAGE analysis of fibrinogen proteolysis products in the presence of β-mercaptoethanol. A - native and B - sonicated (25.1 kHz, 48.4 W/cm2, duty 50%,) at 37°C during 15 min fibrinogen, after 15 min (1) and 5 hours ( (more ...)
Figure shows the results of fibrinogen gel-permeation chromatography before and after sonication. There are two peaks on the chromatogram of native fibrinogen. First dominant peak is native fibrinogen (MW 340 kDa), while second one corresponds to proteins with MW > 600 kDa (Figure , line 1). In turn, on the chromatogram with the US-treated fibrinogen sample, there are two peaks corresponding to proteins with MW > 600 kDa (Figure , line 2). The increase in the molecular weight of proteins occurs due to the formation of aggregates, since the US does not induce the formation of covalent cross-links as demonstrated above.
Figure 4 Gel-permeation chromatography of fibrinogen samples. 1 - native; 2 - US-treatment (25.1 kHz, 48.4 W/cm2, duty 50%,) during 30 min. Column: Sepharose-4B CL (950 × 16 mm), buffer 0,05 M Na-phosphate, 0,15 M NaCl, pH 7,4, rate 0,1 ml/min, detection (more ...)
Next, the influence of the low-frequency US on the proteolytic degradation of fibrinogen in the fibrinogen: plasminogen: t-PA system was investigated. The fibrinogenolysis rate was estimated by determining the concentration of TCA-soluble peptide released during proteins incubation at 37°C. Figure shows that US has differential effects on individual proteins. The US treatment led to increase of fibrinogenolysis rate in case of fibrinogen (Figure , line 2), while it decreased fibrinogenolysis in case of plasminogen (Figure , line 3) and t- PA (Figure , line 4) in comparison to control (Figure , line 1).
Figure 5 Proteolytic degradation of fibrinogen by the mixture of plasminogen and rt-PA under different modes of US-treatment. 1 - control (without US action); 2 - sonicated during 30 min fibrinogen; 3 - sonicated during 30 min plasminogen; 4 - sonicated during (more ...)
Figure shows that the beforehand incubation of plasminogen with t- PA (Figure , line 2) for 30 min led to increase in the fibrinogenolysis rate in comparison with the control experiment (Figure , line 1). Obviously, in this case a larger quantity of already formed active plasmin was added to fibrinogen. In turn, beforehand US treatment of the mixture of plasminogen with t-PA leads to considerable reduction in the fibrinogenolysis rate (Figure , line 5). Moreover, this effect was not the sum of the inactivating actions of ultrasound on t-PA (Figure , line 3) and plasminogen (Figure , line 4), but was taken intermediate value.
Figure 6 Proteolytic degradation of fibrinogen by the mixture of plasminogen and rt-PA under different modes of US-treatment. 1-control; 2- prior incubation rt-PA with plasminogen at 37°C during 30 min; 3- sonicated during 30 min plasminogen; 4- sonicated (more ...)
The influence of ultrasound on the rate of the enzymatic hydrolysis of fibrinogen in the fibrinogen: plasminogen: t-PA system was investigated upon introduction of the plasminogen activator after the US treatment. Figure presents data analysis of reducing and non-reducing samples of fibrinogen proteolysis products. Time-dependent degradation of the native fibrinogen polypeptide chain (Aα, Bβ and γ) and accordingly the accumulation of the end products of the fibrinogen plasminolysis - fragments D1, D2, D3 through the intermediate formation of the partially degraded fragments X and Y occurred independently of the US treatment. However, the electrophoregrams of the not reducing control sample after 1 hour of the hydrolysis (Figure , right column, line 5) show the presence of only early fibrinogen degradation product - fragment X. At the same time, in the case of preliminary sonication of fibrinogen with plasminogen approximately half of all fibrinogen is converted into the fragments Y and D (Figure , right column, line 5).
Figure 7 Gel- electrophoresis of the fibrinogen degradation products in the model systems. A - fibrinogen:plasminogen:t-PA, B - preliminary US treatment (25.1 kHz, 48.4 W/cm2, duty 50%) mixture of fibrinogen with plasminogen during 15 min. with the subsequent (more ...)
The following part concerns the investigation of the influence of ultrasound on fibrinogenolysis with the US treatment of all three proteins (Figure ). It is evident that in the case of combined US-treatment (Figure , line 3), an increase of fibrinogenolysis rate occurs as in case of fibrinogen.
Figure 8 Proteolytic degradation of fibrinogen by the mixture of plasminogen and rt-PA under different modes of US-treatment. US - 25.1 kHz, 48.4 W/cm2, duty 50%. 1-control; 2- sonicated during 30 min fibrinogen; 3- US-treatment mixture of fibrinogen, plasminogen (more ...)
For the investigation of US effects on fibrinolysis, the model of fibrin clot, prepared by coagulation of pure fibrinogen by thrombin in presence of Ca2+ ions, was used. Initial fibrinogen contained sufficient quantity of factor XIII for complete covalent cross-linking of fibrin monomers. Plasminolysis was initiated by addition of the plasminogen and t-PA to the fibrinogen solution. The process of the fibrinolysis, like fibrinogenolysis, was controlled by the determination of the concentration of TCA-soluble peptide formation. As in the case of fibrinogen, US-treatment leads to the increased rate of proteolysis in both cases, after treating the clot itself and clot in the presence of plasminogen and/or rt-PA. SDS-PAGE of fibrin degradation products revealed that already after 2 hour of hydrolysis mainly D-dimers were present. At the same time, in the degradation products of previously US-treated fibrin parallel with D-dimers early fibrin degradation products - fragments X and Y were present (data not shown). Analysis by the scanning electron microscopy (Figure ) showed that clot revealed many deepening of 4 - 10 μm in size as a result of the US action on the surface of fibrin. The analysis of the internal structure of the clot showed that multiple channels appeared near the clot surface. These channels were 1-2 μm in diameter, and 20-45 μm in length (Figure . c). Changes were also observed in the fibrin network that acquired fine-mesh shape due to mach irregular disruptions of fibers (Figure . d). The quantitative analysis of the image showed substantial changes in parameters of the US-treated fibrin network compared to control. The ultrasound caused decrease of length and diameter of fibrin fibers, but at the same time increase in pore size.
Images of control (a, b) and US-treated (c, d) fibrin clot obtained by scanning electron microscopy at different magnifications.