Bonnie J. Warn-Cramer

Purification of tissue factor pathway inhibitor (TFPI) from rabbit plasma and characterization of its differences from TFPI isolated from human plasma

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that is thought to function as a natural anticoagulant to regulate tissue factor-induced coagulation (Proc. Natl. Acad. Sci. U.S.A. 88, 708, 1991). TFPIs mechanism of action has been well characterized as a two step reaction in which TFPI combines with factor Xa and subsequently TFPI/factor Xa combines with and effectively neutralizes factor VIIa/tissue factor. In human plasma, TFPI occurs in two major molecular weight forms of 34 and approximately 40 kDa. The 40 kDa form is a heterodimer of TFPI in covalent disulfide linkage to human apolipoprotein AII. TFPI circulates in human plasma primarily in association with the plasma lipoproteins. We have now isolated and partially characterized TFPI from rabbit plasma and find that, although functionally and immunologically related to TFPI isolated from human plasma, it differs from human TFPI in some of its physical properties. Rabbit TFPI is larger (approximately 45 kDa) and more extensively glycosylated than human TFPI, does not form mixed disulfides with other proteins in plasma, and unlike its human counterpart, does not circulate in plasma associated with lipoproteins.

A sulfated rabbit endothelial cell glycoprotein that inhibits factor VIIa/tissue factor is functionally and immunologically identical to rabbit extrinsic pathway inhibitor (EPI)

Colburn and Buonassisi (In Vitro Cell Dev. Biol. 24, 1133-1136, 1988) have isolated a single chain sulfated glycoprotein inhibitor of factor VIIa/tissue factor-catalyzed activation of factor X from conditioned media of an established rabbit endothelial cell line. We report herein that their endothelial cell-derived inhibitor and extrinsic pathway inhibitor (EPI) isolated from rabbit plasma have identical functional properties with respect to their interactions with factor Xa and with factor VIIa/tissue factor. In addition, the endothelial cell inhibitor and rabbit plasma EPI migrate with the same apparent molecular weights on non-reduced SDS-PAGE and contain similar amounts of N-linked carbohydrate. Like the endothelial cell inhibitor the EPI of rabbit plasma exists as a single chain molecule. Furthermore, the endothelial cell inhibitor is recognized and neutralized by a polyclonal antibody raised against rabbit plasma EPI. We therefore conclude that cultured rabbit endothelial cells produce an inhibitor of factor VIIa/tissue factor activity that is functionally and immunologically identical to rabbit plasma EPI.

Evidence Suggestive of Activation of the Intrinsic Pathway of Blood Coagulation After Injection of Factor Xa/Phospholipid Into Rabbits

The present study was carried out to extend an earlier observation from this laboratory that mean plasma factor X levels fell by about 15% after the injection into rabbits of a formed factor Xa/phospholipid complex that caused only minimal intravascular coagulation. We have now injected rabbits with formulations of factor Xa/phospholipid that caused considerable intravascular coagulation, as documented by substantial falls in fibrinogen, factor V, and factor VIII and a fall in plasma prothrombin activity of about 15% to 20% of the initial level. Mean plasma factor X activity fell by about 30% of the initial level. Factors participating in the intrinsic coagulation pathway--XII, XI, and IX--all fell by about 50% after injection of a complex made with 16.3 pmol factor Xa and 80 nmol phospholipid per 1 kg body wt and by about 35% after injection of a complex made with 32.6 pmol factor Xa and 40 nmol phospholipid per 1 kg body wt. In contrast, total plasma factor VII activity did not change, and specific plasma factor VIIa levels, which were lower than those measured in human plasma, did not rise after injection of factor Xa/phospholipid. The data are compatible with the hypothesis that factor Xa/phospholipid-induced generation of thrombin in vivo leads to factor XII-dependent activation of the intrinsic pathway of coagulation that results in significant activation of factor X. Further testing of this hypothesis appears warranted.

Studies of factor Xa/phospholipid-induced intravascular coagulation in rabbits : effects of immunodepletion of tissue factor pathway inhibitor

In earlier studies from this laboratory evidence was obtained for a physiological function of tissue factor pathway inhibitor (TFPI) as a regulator of hemostasis capable of preventing thrombotic complications that might otherwise result from exposure of blood to trace amounts of tissue factor (TF). However, it was not possible to conclude that the protective effect of TFPI stemmed solely from inhibition of factor VIIa/TF catalytic activity, since TFPI neutralizes stoichiometric amounts of factor Xa in forming an inhibited factor Xa/TFPI/factor VIIa/TF complex. Therefore, we examined the effects of immunodepletion of TFPI on the extent of coagulation initiated in rabbits by exposure to factor Xa and phospholipid in the absence of TF. In one experimental approach, factor Xa was generated endogenously with the factor X-activating fraction of Russells viper venom (0.33 microgram/kg) in rabbits receiving an infusion of phosphatidylcholine/phosphatidylserine (PCPS) vesicles, 1 mg/kg over 2 hours. In a second approach, rabbits were injected with a complex of factor Xa (0.75 microgram/kg) and PCPS (12.5 micrograms/kg). In contrast with the observed sensitization of TFPI-depleted rabbits to TF-induced coagulation, TFPI-depleted rabbits were not sensitized to coagulation initiated by factor Xa and phospholipid in the absence of TF. These data support the conclusion that the physiological function of TFPI in regulating TF-dependent coagulation stems primarily from its ability to inhibit factor VIIa/TF catalytic activity.

Stoichiometry of binding of high molecular weight kininogen to factor XIXIa

Factor XI is a dimeric protein and circulates in plasma complexed with high molecular weight kininogen (HMWK). We investigated the binding of HMWK to factor XIa utilizing two active site directed fluorescent probes: nitrobenzoxadiazole aminopentyl methylphosphonofluoridate for serine and dansyl-glu-gly-arg-chloromethyl ketone for histidine. In the presence of saturating amounts of HMWK, the fluorescence of factor XIa-fluorophore was quenched by approximately 28% for each probe. Titrations of the fluorescent factor XIa with HMWK revealed that each subunit of factor XIa binds one molecule of HMWK with a Kd approximately 3.4 X 10(-8)M.

The Effect of Immunodepletion of Antithrombin III on the Response of Rabbits to Russell's Viper Venom–Induced Activation of Factor X

Many years ago it was shown that an infusion of tissue factor (TF) into rabbits causing only limited consumption of factor X and prothrombin resulted in extensive consumption of fibrinogen. More recently it was shown that an injection of a concentration of the factor X-activating fraction of Russells viper venom (RVV-X) depleting rabbits of factor X resulted in only minimal consumption of both plasma prothrombin and fibrinogen. We report here experiments in which rabbits depleted of antithrombin III (ATIII) to different degrees were infused over 4 hours with a concentration of RVV-X, causing consumption of about 60% of plasma factor X. Similar minimal mean falls in plasma prothrombin and fibrinogen levels were observed in control rabbits given nonimmune goat IgG and in rabbits immunodepleted with goat anti-rabbit ATIII IgG to about 40% of normal plasma ATIII activity. However, if rabbits were immunodepleted to about 10% to 20% of normal plasma ATIII, then mean consumption of prothrombin was increased modestly and, more impressively, mean consumption of plasma fibrinogen was increased markedly. Whereas limited amounts of thrombin generated on the surface of phospholipid vesicles by factor VIIa/ TF can trigger extensive intravascular coagulation in rabbits with normal plasma ATIII levels, limited amounts of thrombin generated by reactions triggered by factor Xa formed in fluid phase did so only after plasma ATIII levels were markedly depleted. A possible reason for this difference is discussed.