In Vitro and in Vivo Antithrombotic Effect of a Collagen-Derived Octapeptide
The interaction of platelets with collagen fibers is linked to the recurrence of “active” peptides, which, in type III collagen, share a common sequence of eight amino acids. An analogue of this sequence, Lys-Pro-Gly-Glu-Pro-Gly-Pro-Lys, has been synthesized, and its effects investigated on platelet activation and in vitro and in vivo venous thrombus formation. After preincubation with platelets between 0.5 and 1 mM, the octapeptide specifically inhibited collagen-induced platelet aggregation and serotonin release; it also affected the collagen-dependent exposure of a procoagulant activity and the expression of a fibrinogen receptor on the platelet surface. Its specificity on the collagen-platelet interaction was deduced from its lack of effect on the action of other platelet agonists, including thrombin. When the octapeptide was preincubated with thrombin and not with platelets, a dose-and time-dependent inhibition of the thrombin-induced platelet aggregation was then observed. This fact led us to study a possible interference by the octapeptide with the thrombin-induced coagulation of plasma or fibrinogen. It effectively provoked a dose-and time-dependent prolongation of the thrombin-induced plasma or fibrinogen clotting time and inhibited the polymerization of fibrin generated from fibrinogen by thrombin. In contrast. the octapeptide did not affect the polymerization of dissociated preformed fibrin monomers. The reptilase-induced plasma or fibrinogen coagulation was not inhibited by the octapeptide, indicating that in the in vitro coagulation system, its effect was specific for thrombin. The octapeptide also interfered with the last step of coagulation, which is the stabilization of fibrin by activated factor XIII: fibrin clots were formed by thrombin and stabilized by factor XIII plus calcium in absence and presence of octapeptide (2 and 5 mM) before being dissolved in urea plus mercaptoethanol. Polyacrylamide gel electrophoresis patterns revealed that the octapeptide inhibited the polymerization of the α chains and dimerization of the 7 chains. The in vitro “anticoagulating” effect of the octapeptide seems related to the charged amino acids, as the replacement of lysine by norleucine and of glutamic acid by alanine resulted in noninhibitory peptides. The anticoagulant effect of the octapeptide was also observed in vivo in a modified stasis thrombosis rabbit model using different thrombogenic triggering agents. Octapeptide (0.25 to 2.5 mg/kg) was administered to rabbits between 1 and 4 hr (subcutaneous route) or 5 min (intravenous route) before administration of a thrombogenic challenge such as activated prothrombin complex (FEIBA). A marked inhibition of the in vivo clot formation was effectively observed, with ED50 =1.8 mg/kg (subcutaneous route) and 0.1 mg/kg (intravenous route). Other thrombogenic challenges such as normal human serum, factor Xa, and thrombin were also administered and found to be affected by the octapeptide.
The results of these in vitro and in vivo studies suggest that the collagen-derived octapeptide has an antithrombitic effect by affecting various different mechanisms of the hemostatic system, involving both the collagen-platelet interaction and coagulation.
KeywordsFactor Xiii Fibrin Clot Intravenous Route Normal Human Serum Procoagulant Activity
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- Legrand, Y. L., Karniguian, A., and Fauvel, F., 1983, Nonapeptide du collagène: Leurre pour les plaquettes? Presse Med. 37:2327–2330.Google Scholar