The anticoagulant effect in heparinized blood and plasma resulting from interactions with extrinsic pathway inhibitor.

The influence of Extrinsic pathway inhibitor (EPI) on global clotting times of plasma was studied using activity-blocking IgG antibodies. Dilute tissue thromboplastin (TP) clotting times in plasma collected after intravenous injection of heparin were dramatically shortened by the addition of anti-EPI IgG. Anti-EPI IgG shortened the TP times to a lesser degree in plasma heparinized in vitro. Compared to plasma heparinized in vitro, the TP clotting times were markedly prolonged in post-heparin plasma of equal heparin concentration. Addition of anti-antithrombin IgG reduced the clotting times somewhat more than did anti-EPI IgG, particularly in normal plasma. In plasma from patients with cancer, about equal effect was obtained by blocking either EPI or antithrombin. These clotting time studies suggested that much of the anticoagulant effect caused by injection of heparin depended on EPI. This was confirmed by recording the release of fibrinopeptide A (FPA), as marker of thrombin generation, following addition of TP and CaCl2 to citrated blood. Thrombin generation was delayed and markedly reduced in post-heparin blood compared to that in normal blood. After incubating post-heparin citrated blood with anti-EPI IgG, the generation of FPA was more rapid; the amounts released 30 seconds after addition of TP were 6 times greater (36 vs 6 ng/ml) than in post-heparin blood without anti-EPI IgG. The subsequent FPA values were midway between pre-injection and post-heparin values. In conclusion, between one third and one half of the inhibition of TP-initiated coagulation in post-heparin plasma depends on EPI. This inhibition is mainly due to inactivation of the factor VIIa-TP complex. A small, but distinct contributing effect observed in the APTT assay (and hence no TP) indicates that even increased inactivation of activated factor X contributes. In cancer patients, these EPI-heparin interactions contribute even more to the anticoagulant effects of heparin.

Extrinsic pathway inhibitor (EPI) released to the blood by heparin is a more powerful coagulation inhibitor than is recombinant EPI.

EPI released to the blood after injection of heparin, as well as recombinant EPI (r-EPI) added to normal plasma prolonged both the dilute Tissue Thromboplastin (TTP) time and the Activated Partial Thromboplastin Time (APTT). It is known that EPI inhibits both factor Xa and the factor VIIa-TTP complex. The prolongation of the APTT by EPI reflects only its inhibition of factor Xa. Addition of anti-EPI immunoglobulins (IgG) to normal plasma shortened the dilute TTP time 7.3 seconds (p less than 0.001) and the APTT by 0.7 seconds (p less than 0.001). In postheparin plasma, with polybrene added to neutralize the direct effect of heparin, the TTP was about 26 seconds longer and the APTT about 9 seconds longer than baseline values. These effects were completely abolished by anti-EPI IgG, as were the effects of r-EPI. The EPI activity (chromogenic substrate-assay) of this postheparin plasma was 1.7 U/ml. The EPI activity of the plasma spiked with r-EPI to obtain comparable effects on clotting were much higher; about 22 U/ml for the TTP effect and about 5 U/ml for the APTT effect. The findings indicate that r-EPI is considerably less potent than postheparin EPI as inhibitor of plasma coagulation. This is most striking when coagulation is initiated through the extrinsic pathway. Possibly, the anticoagulant effect of r-EPI mainly depends on its Xa inhibitory effect.