Lasting safe interruption of endarterectomy thrombosis by transiently infused antithrombin peptide D-Phe-Pro-ArgCH₂Cl in baboons

To evaluate the relative antithrombotic efficacy and hemostatic safety of antithrombin therapy for vascular thrombus formation at sites of mechanical vascular injury, we administered the potent and specific irreversible synthetic antithrombin D-PHE-PRO-ARG chloromethyl ketone (D-FPRCH₂Cl) after performing carotid endarterectomies in baboons. The continuous intravenous infusion of D-FPRCH₂Cl, 100 nmol/kg per minute for 1 hour, abolished acute carotid endarterectomy thrombosis for at least 48 hours. The plasma level of D-FPRCH₂Cl during the infusion was maintained steady at 7.2 ± 0.9 μmol/L, but decreased rapidly after discontinuing its infusion (T₅₀ 17 minutes). Platelet deposition, measured in real time using autologous ¹¹¹In-platelet scintillation camera imaging, was 1.51 ± 0.40 × 10⁸ platelet/cm in the 14 treated animals 90 minutes postoperatively, compared with 11.7 ± 1.16 × 10⁸ platelet/cm in 14 heparin-treated controls (P < .002). The antithrombotic benefit was equivalent for treatment begun either 5 minutes before (nine animals) or 15 minutes after (five animals) reestablishing flow in the operated vessel, ie, 1.59 ± 0.36 × 10⁸ platelet/cm versus 1.35 ± 0.51 × 10⁸ platelet/min, respectively; P > .5. Endarterectomy thrombosis remained decreased for at least 48 hours post-operatively, as determined by the ratio between net ¹¹¹Inplatelet radioactivity at the endarterectomized site versus whole blood (ratio 0.82 ± 0.25 in the treatment group v 3.03 ± 0.51 in heparin controls at 90 minutes, P < .005; and 0.85 ± 0.23 v 3.25 ± 0.48 at 48 hours, P < .002). The marked reduction in endarterectomy thrombosis in treated animals at 48 hours was confirmed by scanning electron microscopy. Thrombin activity formed rapidly and became immediately bound to thrombus on thrombogenic segments in untreated control studies; treatment with D-FPRCH₂Cl irreversibly inactivated the thrombus-bound thrombin. Hemostatic function, as measured by bleeding time (BT), activated partial thromboplastin time (APTT), and prothrombin time (PT) was impaired throughout the intravenous administration of D-FPRCH₂Cl (BT > 30 minutes, APTT > 150 seconds, PT > 50 seconds); BT, APTT, and PT values were normal 30 minutes after discontinuing the infusions. As expected, blood loss into the surgical wound was substantial in nine animals receiving therapy initiated before restoring flow in the operated vessel (mean 95 mL, range 45 to 130 mL). By contrast, beginning D-FPRCH₂Cl therapy in five animals 15 minutes after restoring arterial flow, a time when surgical hemostasis had been achieved, prevented excessive blood loss (mean 15 mL, range 10 to 35 mL; P < .01 compared with earlier treatment) without compromising the antithrombotic effects. Continuous intravenous infusion of the synthetic antithrombin D-FPRCH₂Cl (100 nmol/kg per minute) for 1 hour, beginning immediately after establishing surgical hemostasis, permanently interrupts endarterectomy thrombosis by irreversibly inactivating thrombin bound to the thrombus and retaining already formed hemostatic plugs to prevent surgical bleeding.