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- MATERIALS AND METHODS
Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous (i.v.) and subcutaneous (s.c.) injections of heparins, and may thus contribute to the antithrombotic effect of heparins. We have recently shown that total TFPI activity, plasma free TFPI antigen, and heparin releasable TFPI were partially depleted during repeated and continuous i.v. infusion of unfractionated heparin (UFH), but not during s.c. treatment with a low molecular weight heparin (LMWH). The difference may be attributed to a different mode of action or the different mode of administration. In the present randomized cross-over study, s.c. administration of therapeutic doses of UFH was compared with s.c. administration of two LMWHs. 12 healthy male volunteers were treated for 3 d with UFH, 250 U/kg twice daily, dalteparin, 200 U/kg once daily, and enoxaparin, 1.5 mg/kg once daily. Six participants were also treated with UFH, 300 U/kg once daily. On day 5 a single dose of either drug was given. Peak levels of total TFPI activity and free TFPI antigen were detected 1 h after injection, whereas maximal prolongation of activated partial thromboplastin time (APTT) and peak levels of anti-factor Xa activity and anti-factor IIa activity were detected after 4 h. On UFH administered twice daily, free TFPI antigen decreased by 44% from baseline level before the first injection on day 1 to pre-injection level on day 5. On UFH administered once daily, basal free TFPI antigen decreased by 50%, 56% and 27% on day 2, 3 and 5 respectively, compared with day 1. Minimal depletion of TFPI was detected during treatment with LMWHs. The study demonstrates the different modes of action of LMWHs and UFH and may help to explain the superior antithrombotic efficacy of LMWHs.
Numerous randomized clinical trials have recently provided clear evidence that low molecular weight heparins (LMWHs) are of superior efficacy when compared with unfractionated heparin (UFH) for the treatment of both venous (Siragusa et al, 1996) and arterial (Cohen et al, 1997) thrombosis. Although a large number of studies in animal and human models have elucidated the main antithrombotic effects of UFH and LMWHs, the underlying antithrombotic mechanism(s) responsible for the difference in therapeutic efficacy are still not fully understood.
A major anticoagulant effect of both UFH and LMWHs is due to the potentiation of antithrombin activity, but the target proteases are different. LMWHs potentiate the inhibitory effect of antithrombin on factor Xa (FXa) activity to inhibit prothrombin activation (Hemker & Beguin, 1990), whereas UFH also inhibit thrombin generated by the prothrombinase complex (Samama et al, 1994). Another effect of both LMWHs and UFH is mediated through release and redistribution of tissue factor pathway inhibitor (TFPI) from the vascular endothelium to circulating blood (Sandset et al, 1988). TFPI inhibits FXa directly, but its main effect is to inhibit factor VIIa/tissue factor (FVIIa/TF) catalytic activity in an FXa-dependent manner (Broze & Miletich, 1987; Rao & Rapaport, 1987). The inhibition involves either the binding of preformed TFPI/FXa complexes to FVIIa/TF complexes (Girard et al, 1989) or the binding of TFPI to preformed FXa/FVIIa/TF complexes (Broze, 1995).
TFPI is a member of the Kunitz family of serine protease inhibitors. The molecule contains 276 amino acid residues with an acidic amino-terminal region followed by three tandem Kunitz-type inhibitory domains and a basic carboxyl-terminal region (Wun et al, 1988). The first and second Kunitz inhibitory domains contain the reactive sites responsible for binding to and inhibition of FVIIa and FXa, respectively (Girard et al, 1989). The third domain may be involved in the association of TFPI with lipoproteins (Abumiya et al, 1995) and is mandatory for the anticoagulant function of TFPI in clotting based (diluted prothrombin time) TFPI assays (Wesselschmidt et al, 1992; Nordfang et al, 1991). The carboxyl-terminal region contains a highly basic region which serves as a high affinity binding site for heparin (Wesselschmidt et al, 1992). A low affinity binding site for heparin is located in the third Kunitz domain (Wesselschmidt et al, 1993).
Vascular endothelium is the primary production site for TFPI (Bajaj et al, 1990). A major pool of TFPI in vivo (50–80% of total) is native TFPI bound to the endothelial surface which may be released into circulating blood after injection of heparin (Hubbard et al, 1994; Novotny et al, 1991; Sandset et al, 1987). A smaller TFPI pool (10–50% of total) circulates in plasma, but is predominantly found as various truncated forms of TFPI bound to lipoproteins (Hansen et al, 1995; Novotny et al, 1989). Only 5–20% of plasma TFPI circulates as carrier-free TFPI molecules. Small amounts of TFPI have also been detected in platelets (Novotny et al, 1988). Heparin-releasable TFPI and plasma-free TFPI, but not lipoprotein-associated TFPI, exert strong anticoagulant effect in diluted prothrombin time assays (Hansen et al, 1997; Lindahl et al, 1992), which indicate that these forms of TFPI are physiologically active.
We have recently shown that plasma-free TFPI antigen and heparin releasable TFPI are partially depleted during repeated and continuous intravenous (i.v.) infusion of therapeutic doses of UFH in man (Hansen et al, 1996, 1998), but not during subcutaneous (s.c.) injections of therapeutic doses of a LMWH (Hansen et al, 1998). We speculated that differential effects of UFH and LMWH on depletion of TFPI could explain the apparent clinical superiority of LMWHs. However, since the mode of administration of UFH and LMWH differed, the possibility remains that the differential effect is related to different pharmacokinetics rather than a real difference of UFH and LMWH on TFPI levels. In the present study we have therefore carefully compared s.c. injections of UFH with two LMWHs, all in therapeutic doses, to determine whether the differences could be attributed to a different mode of action, i.e. a novel property of LMWHs, or to a different mode of administration.
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- MATERIALS AND METHODS
We have previously shown that intravascular TFPI is partially depleted during repeated i.v. injections (Hansen et al, 1996) and continuous i.v. infusion (Hansen et al, 1998) of UFH, but not during s.c. treatment with a LMWH (Hansen et al, 1998). The difference is of potential importance for the understanding of the apparent superior efficacy of LMWHs in the prophylaxis and treatment of thrombosis. Since the mode of administration differed, our study did not allow us to conclude whether this was a novel property of LMWHs or whether the difference simply was related to different mode of administration (Hansen et al, 1998). In the present study we have therefore compared the effects of s.c. treatment with UFH and two LMWHs on TFPI levels in plasma.
The study was designed to make administration of UFH and LMWH as similar and comparable as possible with a high peak plasma concentration and only trace amounts of UFH and LMWH detectable before the next injection. The bioavailability of therapeutic, but not prophylactic, dosages of s.c. UFH is high and comparable to that of LMWHs (Bergqvist et al, 1983). We initially considered a twice daily regimen of UFH to be comparable to a once daily regimen of LMWH. Our results showed, however, that twice daily injections of UFH caused significant accumulation as judged by prolongation of APTT and levels of anti-Xa activity and anti-IIa activity in the pre-injection and post-injection samples. Since accumulation of heparin in plasma might release more TFPI from the vessel wall to plasma and interfere with interpretation of the test results, a second series was performed with UFH injected only once daily. In this experimental group no accumulation of UFH was observed as judged by the pre-injection levels, but there was an interesting progressive increase in post-injection anti-Xa activity levels. This increase may not be explained by accumulation of heparin, since heparin did not increase in the pre-injection samples, but could be attributed to depletion of heparin-neutralizing proteins on repeated injections of heparin. We believe that the present experiments compared the different heparins as close as possible.
Our findings confirm and extend our previous observations on depletion of TFPI during i.v. UFH therapy (Hansen et al, 1996, 1998) that TFPI is also is depleted during s.c. treatment with UFH. The effect was most evident from the progressive decrease in basal free TFPI antigen levels by > 50% during s.c. UFH once daily. During UFH given twice daily we believe that the large accumulation of heparin may have released more TFPI from the vessel wall to mask a true depletion of free TFPI antigen in plasma. However, also in this group depletion was evident in the sample collected on the fifth day, i.e. 36–48 h after the last injection of UFH. At this time-point no heparin was detectable in plasma and basal free TFPI antigen was reduced by nearly 50%.
In the post-heparin samples, accumulation of heparin on UFH given twice daily may similarly have masked a true reduction of heparin-releasable TFPI. Again, the reduced response after the injection on the fifth day indicates that depletion of heparin-releasable TFPI indeed took place. The results obtained on the release of TFPI after injection of UFH once daily indicated no significant depletion of TFPI (Fig 3B). However, this regimen was associated with a peculiar progressive increase in the post-injection heparin concentration not related to accumulation (see above) and may have masked a true depletion of free TFPI antigen. The present results may therefore be taken as evidence that both plasma TFPI and endothelial-associated or heparin-releasable TFPI were depleted during s.c. UFH therapy.
The present study also confirms that LMWH therapy is associated with no or only minimal depletion of TFPI (Hansen et al, 1998). Similar responses were seen for dalteparin and enoxaparin, which suggests that the effect may be a class effect of LMWHs. Release of TFPI was slightly higher for the recommended therapeutic dose of dalteparin (200 U/kg) compared with that of enoxaparin (1.5 mg/kg). The clinical significance remains questionable, since there were no signs of depletion in any of the TFPI pools and the role of different TFPI pools for the antithrombotic effect of heparins is unknown.
Our study failed to show significant effects of either UFH or LMWH on TFPI activity, which may again in part be explained by the accumulation or progressive increase in plasma concentration of UFH. TFPI activity was assayed with a chromogenic substrate assay (Sandset et al, 1991), which detects both truncated and full-length free TFPI and lipoprotein-associated TFPI (Sandset et al, 1991; Lindahl et al, 1992; Hansen et al, 1996; Sandset, 1999). Since the assay was calibrated against pooled normal plasma, which contains mostly lipoprotein-associated TFPI, it was rather insensitive to the assay of free or heparin-releasable TFPI and may therefore have not detected depletion of TFPI in the present study. Also in our previous study, depletion of TFPI was much less prominent in the activity assay compared with the free antigen assay (Hansen et al, 1996). Finally, it must be borne in mind that free TFPI and heparin-releasable TFPI exert much stronger anticoagulant activity when compared with lipoprotein-associated TFPI (Lindahl et al, 1992; Hansen et al, 1996, 1997; Sandset, 1999). These data indicate that free TFPI and heparin-releasable TFPI are biologically active in vivo, and that the assay of free TFPI antigen may be more relevant than the assay of TFPI by an end-point activity assay (Sandset, 1999).
Two recent studies have indicated increased survival in patients with venous thromboembolism and metastatic carcinoma who initially received treatment with LMWH compared with UFH (Hull et al, 1992; Prandoni et al, 1992; Leizorovicz et al, 1994). High levels of TFPI have been detected in some patients with cancer (Iversen et al, 1998), which may indicate an important physiological role of TFPI in cancer. Depletion of TFPI on UFH, but not on LMWH, could be an important mechanism to explain the increased survival, although depletion of several other plasma and vascular wall proteins of potential importance for tumour cell growth or angiogenesis, e.g. lipoprotein lipase, superoxide dismutase, and platelet factor 4, could also be involved.
Maximal release of TFPI after injection of UFH and LMWH was observed within only 1 h, in contrast to maximal prolongation of APTT and peak levels of anti-IIa and anti-Xa activities after 3–4 h. Similar discordant effects of s.c. heparin on TFPI and anti-Xa activity have also been observed by other investigators (Falkon et al, 1998; Holst et al, 1997). After i.v. injections of either UFH (Sandset et al, 1988) or LMWH (Samama et al, 1994), release of TFPI is closely related to anti-Xa activity. The rapid release of TFPI after s.c. injections of UFH or LMWH may involve rapid absorption of heparin fragments without anti-IIa or anti-Xa activities, but with retained ability to release TFPI. It could also be an effect secondary to high binding affinity of heparin to TFPI, which fits with the low concentration of heparin needed to release TFPI in cell culture studies (Iversen et al, 1996). Alternatively, rapid release of TFPI could be mediated by other components released or generated by the absorption of UFH and LMWH.
The differential effect on intravascular TFPI may reflect a real pharmacological difference between UFH and LMWH related to differences in chemical composition. Valentin et al (1994) demonstrated that the affinity of full-length TFPI for heparin increased with the charge density and chain length of heparin species. One possibility is that LMWHs, unlike UFH, do not bind with high affinity to, and hence do not deplete, plasma TFPI. Since also in this study the pharmacokinetic profiles of UFH and LMWH were not identical, we may not completely rule out that the differential effect was related different pharmacokinetics (Hansen et al, 1998). However, we may conclude that treatment with i.v. and s.c. UFH, but not with s.c. LMWH, was associated with partial depletion of plasma TFPI and heparin-releasable TFPI, which may help to explain the apparent clinical superior efficacy of LMWHs in patients with acute thrombosis.