Jo Franssen

Inhibition of Tissue Factor-Factor VIIa-catalyzed Factor X Activation by Factor Xa-Tissue Factor Pathway Inhibitor A ROTATING DISC STUDY ON THE EFFECT OF PHOSPHOLIPID MEMBRANE COMPOSITION

The physiological inhibitor of tissue factor (TF)·factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPIFL) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF·FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF·PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF·PSPC) was measured in the presence of preformed complexes of FXa·TFPIFL or FXa·TFPI1–161 (TFPI lacking the third Kunitz domain and C terminus). At TF·PC, FXa·TFPIFL and FXa·TFPI1–161 showed similar rate constants of inhibition (0.07 × 108 m −1 s−1 and 0.1 × 108 m −1 s−1, respectively). With phosphatidylserine present, the rate constant of inhibition for FXa·TFPIFL increased 3-fold compared with a 9-fold increase in the rate constant for FXa·TFPI1–161. Incubation of TF·PSPC with FXa·TFPIFL in the absence of FVIIa followed by depletion of solution FXa·TFPIFL showed that FXa·TFPIFL remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa·TFPI1–161 or at TF·PC membranes. These data suggest that the membrane-bound pool of FXa·TFPIFL may be of physiological importance in an on-site regulation of TF·FVIIa activity.

Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III

We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute to the action of this drug in blood coagulation.

NEUTRALIZATION OF HEPARIN BY PROTHROMBIN ACTIVATION PRODUCTS

The neutralization of heparin by active site blocked meizothrombin and thrombin, prothrombin fragment 1.2, fragment 1 and fragment 2 was probed by the heparin-dependent factor Xa inactivation by antithrombin III (AT III). Meizothrombin had no effect on the inactivation of factor Xa, whereas thrombin had an inhibitory effect (IC50 = 700 nM). After factor Xa catalyzed cleavage of meizothrombin, the resulting products, prothrombin fragment 1.2 plus thrombin, did not show any heparin neutralizing properties. However, after isolation of the reaction products, both thrombin and prothrombin fragment 1.2 exhibited heparin neutralizing properties in the factor Xa inactivation reaction. The IC50-values were 700 nM and 100 nM, respectively. Prothrombin fragment 1, when present at 125 nM, caused a 50% reduction of the heparin-dependent rate of inactivation of factor Xa and prothrombin fragment 2 had no effect at all. From this we conclude that, in addition to the thrombin part of the prothrombin molecule, the fragment 1 region also exhibits a rather high affinity for heparin.