The interaction between components of the fibrinolytic system and GPIb/V/IX of platelets thrombus formation in mice

Abstract

The interaction of fibrinolytic components with GPIb/V/IX of platelets on thrombus formation, was investigated in mice deficient in tissue type (tPA-/-), urokinase type plasminogen activator (uPA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) and in their wild type control (tPA+/+, uPA+/+, PAI-1+/+). A thrombus was induced in the murine carotid artery using a photochemical reaction. The times to occlusion after the initiation of endothelial injury in all wild type mice was within 12 min, and no significant changes in occlusion delay were observed in uPA-/- and tPA-/- mice compared to wild type mice, whereas that of PAI-1 mice were significantly prolonged (16.9+/-2.9 min, P<0.05). When high molecular weight aurintricarboxylic acid (ATA), an inhibitor of platelet glycoprotein Ib/V/IX, was administered, the time to occlusion was prolonged in a dose-dependent manner in all types of mice. However, when this compound was injected in tPA-/- mice, the most significant changes were observed: i.e. the estimated ED(50) was 20.2 times higher than that in tPA+/+ mice, but the estimated ED(50) in uPA-/- mice was not changed as compared with that of wild type mice. On the other hand, when ATA was injected in PAI-1-/- mice, the estimated ED(50) was significantly decreased (P<0.05). Platelet aggregation induced by botrocetin was not significantly different among all types of mice. The bleeding time was prolonged in a dose dependent-manner when ATA was injected in all types of mice. In conclusion, the antithrombotic effect of inhibition of platelet GPIb/V/IX is severely affected by the absence or presence of tPA-production on thrombus formation and the inhibition of PAI-1 could enhance this antithrombotic effect.

Figure 1

Inhibitory effect of high molecular weight ATA on thrombus formation in the carotid artery of tPA−/−, uPA−/−, PAI-1−/− or wild type mice. The carotid arterial blood flow was continuously monitored for 60 min after the initiation of endothelial injury using a photochemical reaction. Time to occlusion is given as mean±s.e.mean in each experiment. Times greater than 60 min are given as 60 min for calculation of mean±s.e.mean ^*P<0.05, ^**P<0.01 versus each control (infusion of saline). #P<0.05 versus PAI-1+/+ mice.

Figure 2

Dose dependent inhibition by high molecular weight ATA of platelet aggregation in PRP of tPA−/−, uPA−/−, PAI-1−/− and their wild type mice. Platelet aggregation was induced by botrocetin (4.0 μg ml⁻¹). Data are represented as mean±s.e.mean. ^*P<0.05, ^**P<0.01 versus each control (infusion of saline).

Figure 3

Scanning electron micrographs from a wild type mouse (a and b), a mouse deficient tPA (c and d) and a mouse deficient PAI-1 (e and f). (a) thrombus formation after endothelial injury in a wild type mouse. A mural thrombus including a lot of activated platelets, fibrin net and red blood cells. (b) Intact arterial surface in a tPA−/− mice. Locally activated platelets were observed. Adherent platelets consist of microthrombus formation on non-injured endothelial surface. (c) thrombus formation after endothelial injury in a PAI-1−/− mouse. Activated platelets aggregate on injured surface with red blood cells. However fibrin networks were rarely observed in thrombus formation. d, e and f are representative of high magnification of a, b and c, respectively.