The Antithrombotic Activity of Kininogen Is Mediated by Inhibitory Effects of Domain 3 on Thrombin-Induced Platelet Activation, In Vivo.

Background: High molecular weight kininogen (HK) exhibits anticoagulant and profibrinolytic properties and can inhibit platelet activation at low thrombin concentrations. HK acting through domain 3 (D3) is antithrombotic by interfering with the ability of thrombin to bind to the platelet GPIb complex in vitro. We hypothesize that rapid occlusive thrombosis due to de-endothelization of the aorta is due to enhanced platelet activation by thrombin and will be reversed by infusion of D3 or D3-derived peptides.

Methods: F6HKd (deficient in HK) and F6WT (wild-type) strains were produced from offspring of kininogen-deficient Brown-Norway rats by backcrossing to Lewis rats for 6 generations to ensure homogenous genetic background. In the present study, we have expressed kininogen domain 3 (D3) and a fragment of D3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. GST-D3 and either GST-E7P or GST alone was infused intravenously 10 min before inducing the endothelial injury to the abdominal aorta. (Each group n = 5)

Results: Despite an equivalent vascular injury, the mean time (± SEM) for a 90% decrease in flow measured by an ultrasonic probe was 28±5.2 minutes in GST-treated F6HKd rats compared with >240 minutes in the GST-D3 and 233±6.7 in GST-E7P-treated animals. Differences between GST and either GST-D3 or GST-E7P pretreatments were highly significant (p<0.0002). F6WT showed no occlusion at 240 min after injury. In F6HKd rats, TAT concentrations were increased from 32±5.1 to 52±4.1 ng/ml (p<0.007). Similarly D-dimer concentrations were increased from 94±16.3 to 145±9.6 ng/ml (p<0.01) after endothelial injury with only GST pretreatment. With GST-E7P pretreatment, TAT and D-dimer levels were significantly decreased from the GST-treated group (TAT from 52±4.1 to 36±4 ng/ml, p<0.01 and D-dimer from 145±9.6 to 69±11.9 ng/ml, p<0.0007). No significant difference was observed between the GST-E7P pretreated injured and non-injured F6HKd rats or between the F6WT rats which were injured and non-injured.

Conclusions: The results suggest that F6HKd rats are prothrombotic. After endothelial injury thrombotic occlusion occurs and is characterized by thrombin formation with secondary fibrinolysis. Moreover, thrombin-induced platelet activation is inhibited by D3 or the polypeptide coded for by exon 7 product in vivo. These results indicate the importance of HK in modulating arterial thrombosis and the possibility of therapy with HK fragments.