Akinobu Funatsu

Human recombinant tissue-factor pathway inhibitor prevents the proliferation of cultured human neonatal aortic smooth muscle cells

Tissue‐factor pathway inhibitor (TFPI) inhibits the procoagulant activity of the tissue‐factor/factor VIIa complex. It was recently reported that TFPI prevented restenosis following tissue injury in a rabbit atherosclerotic model. In order to clarify the mechanism behind this successful prevention of restenosis, we investigated the direct effect of human recombinant TFPI (h‐rTFPI) on the proliferation of cultured human neonatal aortic smooth muscle cells (hSMC). We found that h‐rTFPI exhibits inhibitory activity toward hSMC proliferation, while h‐rTFPI‐C which lacks the carboxyl (C)‐terminal region does not. Furthermore, we found that h‐rTFPI binds to hSMCs with K d=526 nM but that this binding is inhibited by the addition of the synthetic C‐terminal peptide, Lys254–Met276, to h‐rTFPI. Thus, the interaction of h‐rTFPI with hSMCs mediated via the C‐terminal region is responsible for the anti‐proliferative action of h‐rTFPI. On the basis of these results, we presume that the anti‐proliferative effect of h‐rTFPI in addition to its anticoagulant function plays a significant role in preventing restenosis following tissue injury.

Human recombinant tissue factor pathway inhibitor induces apoptosis in cultured human endothelial cells

Tissue factor pathway inhibitor (TFPI) is mainly synthesized in vascular endothelial cells and exhibits a strong and specific inhibitory activity against tissue factor‐mediated blood coagulation. In the present study, we demonstrate that human recombinant TFPI (h‐rTFPI) inhibits the growth of cultured human umbilical vein endothelial cells (HUVECs) by inducing apoptosis. In a growth‐rate assay of HUVECs, the growth of the cultured HUVECs is completely abolished by the addition of 1 μM h‐rTFPI to the culture medium containing fetal bovine serum (FBS), basic fibroblast growth factor, and epidermal growth factor. In addition, h‐rTFPI and h‐rTFPI‐C which lacks the carboxyl‐terminal basic region prevent the survival of growth‐arrested HUVECs which are starved in a medium containing 2% FBS alone, suggesting that h‐rTFPI directly induces the death of these HUVECs. This hypothesis is supported by the finding that h‐rTFPI does not inhibit the synthesis of DNA in HUVECs during proliferation, as shown by a 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay. Furthermore, Giemsa staining and a gel electrophoretic analysis of DNA fragmentation show that the HUVEC death mediated by h‐rTFPI has the typical characteristics of apoptosis. However, the apoptosis in HUVECs is considerably inhibited in the presence of 1 μg/ml of the protein synthesis inhibitor, cycloheximide. Therefore, the process of apoptosis triggered by h‐rTFPI is, at least in part, actively conducted by the cells.

THE CLEARANCE OF PROTEOGLYCAN-ASSOCIATED HUMAN RECOMBINANT TISSUE FACTOR PATHWAY INHIBITOR (H-RTFPI) IN RABBITS: A COMPLEX FORMATION OF H-RTFPI WITH FACTOR XA PROMOTES A CLEARANCE RATE OF H-RTFPI

The very rapid clearance of human recombinant tissue factor pathway inhibitor (h-rTFPI) may result from its binding to vascular proteogly can and LDL receptor-related protein (LRP). To investigate the effect of factor Xa on the clearance of h-rTFPI, we developed a specific ELISA for h-rTFPI/factor Xa complex, and compared the pharmacokinetic parameters of h-rTFPI/factor Xa complex and the clearance rate of the cellular proteogly can-associated h-rTFPI/factor Xa complex with those of h-rTFPI by itself in rabbits. We found that the h-rTFPI/factor Xa complex disappeared from circulation at a rapid rate of clearance, having pharmacokinetic parameters similar to those of non-complexed h-rTFPI. After the rapid disappearance of the h-rTFPI complex from plasma, an intravenous injection of heparin resulted in a release of h-rTFPI/factor Xa complex into plasma. However, the recovery of heparin-releasable h-rTFPI/factor Xa decreased significantly in a time-dependent manner. Therefore, we examined the half-life of proteogly can-associated h-rTFPI/factor Xa and determined it to be 51 min, which was significantly shorter than that of h-rTFPI by itself (107 min). These results suggest that a complex formation of h-rTFPI with factor Xa promotes a clearance of proteogly can-associated h-rTFPI existing in the liver and kidney.

Induction of Acquired Factor IX Inhibitors in Cynomolgus Monkey (Macaca Fascicularis): A New Primate Model of Hemophilia B

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkeys plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.

A Kinetic Analysis of the Interaction of Human Recombinant Tissue Factor Pathway Inhibitor with Factor Xa Utilizing an Immunoassay and the Effect of Antithrombin III/Heparin on the Complex Formation

We have recently shown that a complex formation of tissue factor pathway inhibitor (TFPI) and factor Xa (Xa) promotes a clearance of proteoglycans-associated TFPI. In the current studies, the interaction between human recombinant TFPI (h-rTFPI) and Xa were kinetically analyzed by utilizing both a protease inhibitor, p-(amidophenyl) methanesulfonyl fluoride hydrochloride, and a specific enzyme-linked immunosorbent assay for the complex of h-rTFPI with Xa. We further investigated the effect of antithrombin III on the complex formation between h-rTFPI and Xa. We found that the h-rTFPI/Xa complex formed in a time-dependent manner: the second-order rate constant (K1) for the complex formation was calculated to be 0.86x10(6) M(-1)s(-1). The addition of antithrombin III to the h-rTFPI solution modestly reduced the rate of the complex formation between h-rTFPI and Xa. Heparin strikingly enhanced antithrombin IIIs inhibition of Xa and resulted in complete abrogation of the complex formation between h-rTFPI and Xa in the absence or presence of acidic phospholipids. Furthermore, antithrombin III induced dissociation of the preformed h-rTFPI/Xa complex in the presence of heparin. These results suggest that in the presence of heparin, antithrombin III interferes with the catabolism of TFPI mediated via Xa.

Expression of prothrombinase activity and CD9 antigen on the surface of small vesicles from stimulated human endothelial cells

We employed flow cytometry and monoclonal antibodies (MoAb) to study the surface membrane protein of shed particles (small vesicles, SV) that were released from vascular endothelial cells (EC) by agonists such as a Ca ionophore (A23187) and thrombin. After stimulation of EC by A23187, CD9 antigens disappeared entirely from the EC surface in a time- and concentration-dependent manner; they subsequently moved onto the SV surface. Von Willebrand factor (vWF) and P-selectin from Weibel-Palade (W-P) bodies were expressed rapidly on the EC surface after thrombin stimulation, but not on the SV surface. P-selectin may have some effect on maintenance of hemostasis on the EC surface. We demonstrated that the surfaces of SV and EC significantly supported prothrombinase activity and confirmed that A23187-induced SV from EC express binding sites for factors IXa and Xa. These results suggest that the SV are an important factor in a novel controlling mechanism of the coagulation system on the EC surface.

Measurement of Lp(a) with a two-step monoclonal competitive sandwich ELISA method

OBJECTIVE To evaluate the results of Lipoprotein (a)[Lp(a)] measurements by a competitive two-step monoclonal enzyme-linked immuno sorbent assay method comparing them with those by a conventional ELISA. METHODS Serum having various isoforms of Lp(a) and purified Lp(a) were assayed using the method described here and commercially available kits. The reference range was determined with the use of 324 normal subjects by means of calculation from Lp(a) results of logarithmic transformation. RESULTS Our method takes advantage of a competitive reaction between fixed antibody and free antibody to Lp(a), having the detection range up to 1000 mg/L with the lowest detection limit of 2 mg/L. The anti-Lp(a) monoclonal antibody employed in the assay system reacts uniformly with all phenotypes of Lp(a) but showing very low cross-reactivity for plasminogen and LDL. Within-run and between-run precisions were excellent, giving CVs of 2.9 and 4.0% with mean values of 145 and 635 mg/L, respectively. In comparison of the results by our method with those by a polyclonal method (Biopool) or a monoclonal antibody method (Terumo), they correlated well; Y (our method) = 0.99 x (polyclonal method, Biopool) - 1.9, r = 0.994 (n = 60), and Y = 0.94 X(monoclonal method, Terumo) -9.8, r = 0.97 (n = 60), respectively. The reference range was 105.9 +/- 25.4 mg/L, the difference between the sexes was not significant. CONCLUSION Our method has proven highly accurate and specific. It is applicable with auto analyzer because it does not require such a pre-dilution step as is necessary for Lp(a) determination by conventional ELISA assay. Accordingly, we can conclude that our test method is workable for both clinical laboratories and mass screening.