The Application Of Thrombin In Preparation Of Thrombus In Vitro:the Effect On Degradation Time And Its Mechanism

Objective: Thrombolysis essay in vitro is an important way to test the efficacy of thrombolytic drugs.There are a variety of factors influencing the efficacy,of which the composition of thrombus is an important one i.e. the efficacy decreases with the content of cross-linked fibrin while increases with the proportion of erythrocytes within the thrombus and, by comparison, the effect of the cross-linked fibrin content might be more prominent. In the previous reports, the thrombi used in thrombolysis essays were commonly prepared by autologous coagulation of whole blood of human or animals consisting of packed erythrocytes within a sparse fibrin net. The thrombi prepared in such a way are soft and fragile, readily undergoing autolysis even in saline, or being degraded so fast by thrombolytic agents that prevents the effective assess the efficacy of the agents, a fact that may be responsible for lower efficacies observed in clinical practice than that assayed in laboratory conditions. Recently, some investigators found that application of thrombin in preparation of the thrombus in vitro conferred the thrombus with more tenacity and prolonged degradation time, thus overcoming the above shortcoming. The responsible mechanism may relate to increasing the fibrin content of thrombus by thrombin, however, the direct evidence has yet been lacking. Noticeably, there are only a few reports that apply thrombin to preparation of thrombi up to now and, furthermore, the concentration used varies greatly from each other; it is warranty to standardize the preparation by searching an optimal concentration of thrombin, which would conceivably improve the practicability and comparability of thrombolysis essay in vitro. The present study is to observe the effect of thrombin concentrations on the lysability of the thrombus, and clarify whether the underline mechanism involves the change in cross-linked fibrin content of the thrombus; furthermore and on such basis, the relationship between the level of plasma fibrinogen and the content of cross-linked fibrin of the thrombus was analyzed to determine the predictive value of the plasma fibrinogen level for the lysability of the thrombus. Intriguingly, we report here a novel method that allows to precisely determine the content of cross-linked fibrin in thrombus, expecting to be recognized by the thrombosis-related study.Methods: 1.Venous blood of 10ml was drawn from healthy volunteers with erythrocrits being between 40% and 50%, and the sample from an individual was divided equally with syringes into five portions. Of the five portions, three were treated with low(25U/mL),medium(50U/mL)and high(75U/mL)concentration of thrombin-containing solutions, respectively, and one portion was treated with saline. After vortex, the reaction systems were placed in water bathes at 37℃for three hours during which the thrombi were formed by coagulation process.2.The plasma fibrinogen level was measured with Clauss method using the remained one portion of the blood sample.3.After removing the formed thrombi from syringes, the mechanical properties of the thrombi, including elasticity and plasticity, were compared.4.A portion of each thrombus was cut and trimmed into cubes weighting around 150 mg. After washing with phosphoric acid buffer solution, the cubes were incubated in plasmin-containing solutions at 37℃with gentle rocking, and the degrading processes were observed with naked eyes.5.The degradation times of thrombi, including the disappearing times for both mass and fibrin floss of thrombi, were recorded. After centrifuging, the supernatant was collected and stored at -20℃.6.After the iced supernatants were thawed and diluted,D-dimmer concentrations were measured with ELISA.7.The cross-linked fibrin content of thrombi was calculated from D-dimmer concentrations in reaction systems, a method that is proposed for the first time to our knowledge.8.Variance analysis of fibrin content,rank test of degradation time of the thrombi prepared with four concentrations of thrombin and correlation analysis between the fibrinogen and fibrin content were performed.Results: The thrombi produced in the absence of thrombin had less tenacity and easily to be fragile; in contrast, all the thrombi produced in the presence of three concentrations (low, medium and high) of thrombin acquired satisfied tenacity. Compared with the thrombi produced in the absence of thrombin, both the degradation time and cross-link fibrin content increased significantly in all the thrombi produced in the presence of the three concentrations of thrombin (P<0.01 in all cases). Greater increases in both the degradation time and cross-link fibrin content were also observed in the thrombi produced in the presence of medium and high concentrations of thrombin compared with that produced in the presence of low concentration of the enzyme (P<0.01 in all cases); however, there are no differences in both the degradation time and cross-link fibrin content when the comparison was made between the thrombi produced in the presence of medium and high concentration of the enzyme (P>0.05 in both cases). There was a linear correlation between the plasma fibrinogen levels and cross-link fibrin contents of the thrombi (r=0.825,P<0.001).Conclusions: In experiment essaying the efficacy of thrombolysis, application of thrombin to prepare the thrombus in vitro increases the tenacity and cross-link fibrin content of the thrombus, which allows the degradation times for the thrombus to be prolonged thus optimizing the essay conditions. The appropriate concentrations of thrombin appears to be between 25U/mL to 50U/mL in preparation of the thrombus; applying higher concentration does not result in further increases in tenacity and cross-link fibrin content of the thrombus. The higher the plasma fibrinogen level, the more the cross-link fibrin content of the thrombus from which is made is. Consequently, the plasma fibrinogen level is justified to be a predictive factor for the lysability of the thrombus. Noticeably, the method proposed by this study to measure the cross-link fibrin content of the thrombus is a novel finding and expected to have widely applications.