Volume 14 Supplement 1
Extravascular FIX and coagulation
© The Author(s). 2016
Published: 4 October 2016
This review summarizes the evidence that collagen IV binding is physiologically important, and that the extravascular compartment of FIX is composed of type IV collagen. As we have previously demonstrated, 7 days post-infusion, FIXWT (BeneFIX) is able to control bleeding as well as the same dosage of Alprolix in hemophilia B mice, tested using the saphenous vein bleeding model (Alprolix is a chimeric FIX molecule joined at its C terminus to a Fc domain). Furthermore, we have shown that in hemophilia B mice, doses of BeneFIX or Alprolix (up to a dose of 150 IU/kg) have increased bleeding-control effectiveness in proportion to the dose up to a certain limit: higher doses are no more effective than the 150 IU/kg dose. These studies suggest that in hemophilia B mice, tested using the saphenous vein bleeding model, three things are true: first, extravascular FIX is at least as important for coagulation as is circulating FIX; second, measuring circulating levels of FIX may not be the best criterion for designing new “longer lasting” FIX molecules; and third, trough levels are less diagnostic for FIX therapy than they are for FVIII therapy.
KeywordsFIX Collagen IV Coagulation Hemophilia B
Hemophilia B is caused by the absence or defect of an enzyme, coagulation factor IX (FIX). A plasma concentration of ~5 μg per ml of FIX, defined to be one international unit (IU), is the approximate average plasma concentration of pooled samples from normal individuals.
Familial bleeding, or hemophilia, has been known since biblical times. In 1952, several groups realized that the blood of some hemophilia patients could correct coagulation in blood from other hemophilia patients [1–3]. This observation led to the designation of two types of hemophilia: hemophilia A (FVIII deficiency) and hemophilia B (FIX deficiency), which are clinically indistinguishable. The designation of disease severity has been well defined for hemophilia A. Generally, patients who have less than 1 % of the normal circulating levels of FVIII in their plasma are designated as severe cases and have frequent occurrences of spontaneous bleeding. Patients with between 1 and 5 % activity are designated moderate cases; these patients experience bleeding primarily after injury. Patients with levels between 5 and 50 % activity are considered to have mild hemophilia and usually experience bleeds only with surgery or major trauma. Although the same designation criteria are applied to hemophilia B, the correlation between plasma levels and clinical outcome has not been rigorously proven .
Before the advent of replacement therapy, the average life span for a severe hemophilia patient was 13 years. A monograph published in 1937  examined the fate of 98 severe hemophilia patients; 83 died before age 16, and only 6 survived to age 40. Further, 24 of the 98 died from minor surgery or insignificant injuries, such as biting their tongue or cutting a finger. After the development of specific purified or recombinant FIX and FVIII molecules for replacement therapy, the average age of survival of hemophilia patients increased to about 60 years.
The standard of care has, until recently, been to treat patients for severe bleeding episodes as they occur (on demand). Recently, however, the World Federation of Hemophilia recommends that on-demand treatment be replaced by prophylaxis . The goal is to prevent osteoarthritis caused by bleeding into joints.
Currently, clinical practice guidelines for prophylactic hemophilia treatment requires 2–3 infusions per week. However, several companies have developed new, “longer lasting” FIX molecules; these have been designed to have a longer plasma half-life, and thus theoretically allow patients to go 7–10 days between injections [7–9].
Are FIX’s pharmacokinetics consistent with rates of disappearance from plasma?
How much extravascular FIX is expected?
If we assume that FIX’s binding affinity for collagen IV is 5 nM and that the FIX plasma concentration is 90 nM, then approximately 95 % of the total FIX will be bound to available type IV collagen. This predicts that about 20-fold more extravascular FIX is present outside the vasculature than is found in the circulation. This would be true unless the collagen IV were limiting. While it is difficult to estimate the total amount of collagen IV in the body, it is likely that there is much more collagen IV than would be saturated by our proposed optimal dosage of 150 IU/kg of FIX (if all of the infused 150 IU/kg FIX were retained in the plasma, the molar concentration of FIX would be about 340 nM). Saturation of the extravascular sites at concentrations of FIX of around 300 to 400 nM agrees very well with the values necessary to get kinetic constants with pharmacokinetic data. The 300 to 400 nM number also agrees with Stern’s data on the displacement of baboon FIX by bovine FIX with theoretical plots.
How can we explain the lack of binding of FIX to much of the available collagen IV?
Much of the body’s collagen IV is probably not normally in contact with plasma-borne FIX. But the sinusoids, which do not bind FIX, clearly are exposed to the circulation. A possible explanation for this discrepancy is that post-translational modification of collagen IV is required to prevent binding of FIX to regions of collagen IV that are readily accessible to blood. An example is provided recently by a report that a specific prolyl hydroxylase is required to prevent lethal platelet aggregation via the platelet receptor GPVI . A similar post-translational modification could render the collagen IV lining the sinusoids incapable of binding with FIX.
Thrombosis at higher doses
The best evidence against a slight elevation of FIX causing a significant risk of thrombosis comes from a particular group of patients whose FIX—FIX Padua (FIXR333L)—has a specific activity that is about eight times greater than that of FIXWT . Affected males develop thromboses at around 16–20 years of age, but the heterozygous mother, whose FIX activity is increased to almost 4 times that of FIXWT, has had no problems with thrombosis. The FIX Padua patients in the Simioni study are chronically exposed to FIX that, while present at normal doses, has an eightfold increase in specific activity, which should be equivalent to the activity of 8 times the normal amount of circulating FIX. By contrast, hemophilia B patients infused with 150 IU/kg of FIX would be only transiently exposed to higher doses of circulating FIX. For example, in our experiments that injected different amounts of FIX into hemophilia B mice, we found that at a dose of 150 IU/kg, the increased level of plasma exposure lasts for less than 2 min. Even at doses of 320 IU/kg, the plasma level of FIX decreases to normal levels in mice by the 30-min mark. More importantly, the studies that correlate circulating FIX activity to thrombosis risk do not consider the relatively large extravascular reservoir of FIX. For example, a plasma level of 5 mcg/mL would imply a total dose of FIX of at least 22.5 mcg/mL—equivalent to a dose of about 180 IU/kg of FIX. So a dose of 150–200 IU/kg given to a patient with no circulating levels of FIX antigen is very nearly the same (or less) than the total amount present in normal individuals. Clearly, not all the loss of FIX from circulation is caused by clearance in the traditional sense, because infused hemophilia B mice continue to exhibit relatively good function—even 7 days post-infusion, when no FIX has been detectible in the circulation since day 3 post-infusion. In mice, Alprolix and BeneFIX protect from bleeding for 7 days with essentially identical efficacy.
The cumulative thrust of these studies sheds light on the reports that, at the same plasma levels of activity, FVIII mutations appear to be more severe than FIX mutations expressing the same levels of FIX activity [22, 23]. This difference may be explained by the role of extravascular FIX in coagulation—a significant proportion of FIX is extravascular, and this extravascular FIX remains active. Presumably, FVIII does not have significant extravascular stores.
The assumption that plasma FIX concentrations are the best marker of FIX supplementation efficacy is thus not completely true. FIX is also found distributed around the extra-vasculature throughout the body. The sites that react with antibodies to FIX also react with antibodies to type IV collagen (although not all of the apparently readily available collagen IV in the body reacts with FIX antibodies).
Accumulating data suggest that the extravascular compartment approaches saturation at around 150 IU/kg; therefore we suggest that, an appropriate dose of FIX would be around 150 IU/kg. Longer time points should also be tested in future experiments, since, at this dose, there is still rather robust control at 7 days post-infusion.
- Hemophilia B:
an individual or animal lacking FIX
The work discussed here was a result of numerous NIH grants and a generous grant from Pfizer corporation.
Publication fees for this article have been funded by APSTH 2016.
This article has been published as part of Thrombosis Journal Volume 14 Supplement 1, 2016. The full contents of the supplement are available at https://thrombosisjournal.biomedcentral.com/articles/supplements/volume-14-supplement-1.
Availability of data and materials
This review was written entirely by the Author. A professional editor, Heath Sledge checked for accuracy.
Darrel Stafford is a founding member of a small biotechnology firm, ChimeraTech. ChimeraTech is developing a chimera between FIX and FVII for coagulation purposes. I also receive royalties for use of VKOR in producing FIX.
Consent for publication
Darrel Stafford gives consent for publication.
Ethics approval and consent to participate
All the experiments described in this review article were done under supervision of an Institutional Animal Care Committee.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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