Antithrombin significantly influences platelet adhesion onto immobilized fibrinogen in an in-vitro system simulating low flow

Abstract

Background: Adhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system.

Methods: Platelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s-1 to 1500 s-1). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated.

Results: Within a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s-1 and 50 s-1 (r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 +/- 3.3%) and high (117 +/- 4.1%) antithrombin activity (1786 +/- 516 U vs. 823 +/- 331 U, p < 0.05) at low flow rate (13 s-1, within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 +/- 0.17 vs. 1.95 +/- 0.4 p = 0.008, respectively).

Conclusion: It appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance.

Figure 1

Platelet adhesion onto immobilized fibrinogen under different shear rate conditions with regard to the perfusion time (n = 29). 13 s^-1 and 50 s^-1 mimic venous flow and 1500 s^-1 arterial flow. Platelet adhesion between 15 sec and five min of perfusion increased five-fold at shear rate of 13 s^-1 and 14-fold at arterial shear rate of 1500 s^-1. Per each subject 3 flow experiments were conducted (13 s^-1, 50 s^-1 and 1500 s^-1). Finally 87 perfusion experiments were conducted. At each time point of perfusion (15 sec, 1 min and 5 min) a stack of 5 images was collected and analyzed.

Figure 2

Microphotographs of platelets adhesion onto immobilized fibrinogen after 1 minutes (A) and 5 minutes (B) of perfusion at shear rate of 1500 s^-1.

Figure 3

Plot of plasma antithrombin activity and platelet adhesion expressed as absolute fluorescence under low shear stress (n = 29, shear rate 13 s^-1, 15 sec of perfusion). Spearman's rank correlation coefficient was -0.53, p < 0.05.

Figure 4

Platelet adhesion under different shear rates was selected according to the plasma antithromin activity of respected samples. Low activity = plasma AT activity <95% (n = 7) and high activity = plasma AT activity >105% (n = 13). Statistically significant difference in platelet adhesion with regard to the low and high AT activity was observed at 13 s^-1 and 50 s^-1 (p < 0.05). Similar trend was observed under arterial flow conditions but without statistical significance.

Figure 5

Anticoagulated blood (n = 8) was aliquoted and one aliquot was supplemented with AT (final activity 280%). Both aliquots were further perfused through rectangular flow chamber according to our experimental protocol. At low flow conditions (13 s^-1) AT supplemented blood showed significantly lower platelet adhesion rate (65% of adhesion compared with non-supplemented blood, p < 0.05) between fifth and first minute.