Extravascular FIX and coagulation

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 [4].

Before the advent of replacement therapy, the average life span for a severe hemophilia patient was 13 years. A monograph published in 1937 [5] 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 [6]. 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].

While the published half-life data on these “longer lasting” molecules is impressive, the extravascular role of FIX has not been considered. It is standard clinical practice to treat hemophilia as though the plasma concentration of FIX is the only significant criterion. However, this ignores the significant evidence for the existence of extravascular FIX and the role that it plays in coagulation. The following observations provide evidence that the naturally occurring extravascular stores of FIX are physiologically important. First, FIX binds reversibly and saturably to type IV collagen [10]. Second, a mouse expressing human FIX (FIX_K5A, which has a specific activity in vitro of up to twofold greater than that of FIX_WT, but a reduced affinity for type IV collagen) has a bleeding diathesis, even though its circulating level of FIX_K5A is about 20 % higher than normal [11]; the mouse exhibits this bleeding diathesis although both the plasma levels and specific activity are higher for the FIX in the FIX_K5A mouse. Third, we infused hemophilia B mice with a FIX variant (FIX_K5R) that binds more tightly to type IV collagen than does FIX_WT. The mice dosed with FIX_K5R clotted more efficiently mice infused with FIX_WT; this occurred despite the fact that circulating FIX levels were unmeasurable 3 days post-infusion [12] . There is also excellent evidence of an extravascular reservoir of FIX in baboons. Stern et al. demonstrated quite clearly that administering increasing amounts of bovine FIX into the baboon rapidly displaced endogenous stores of extravascular baboon FIX into the plasma [13]. In Fig. 1, Stern et al. infused baboons with bovine FIX and then followed the release of baboon FIX into the plasma. This was possible due to a specific antibody for bovine FIX that allows the measurement of baboon FIX with minimal cross-reactivity with bovine FIX. Clearly, in the baboon experiments, at least 3 times more FIX is released, presumably from an extravascular collagen IV compartment, than is found in the baboons’ plasma.

Figure 2 shows that, at doses of 50, 100 and 150 IU/kg, Alprolix and BeneFIX respond almost identically in hemophilia B mice tested using a saphenous vein bleeding model, and that maximum efficacy is achieved at a dose of about 150 IU/kg. Figure 2 shows that, for optimal long-term therapy, 150 IU/kg is best, at least in mice. In Fig. 2, hemophilia B mice were infused with BeneFIX at doses of 50, 100, 150, 200, 250, and 500 IU/kg and Alprolix at doses of 50, 100, 150 and 250 IU/kg. These data were tested using a saphenous vein bleeding model and were, for each dose and molecule, observed 7 days post-infusion.

We utilized homologous recombination to replace defective mouse FIX in our hemophilia B mice [14], creating the FIX_K5A mouse, which has a reduced FIX affinity for collagen IV [15]. This FIX_K5A mouse has a mild bleeding diathesis, although it has 120 % of the circulating FIX_K5A of WT mice—even though the FIX_K5R displays about a twofold increase in specific activity over the FIX_WT mouse in an APTT assay [16, 17]. Figure 3 shows the efficacy of FIX_WT and FIX_K5A in mice (data taken using the saphenous vein model). Clearly there is a bleeding diathesis associated with reduced FIX affinity for collagen IV, even though the FIX plasma activity in these mice is higher than in the wild-type mice.

What causes this cessation of bleeding when FIX is at immeasurable levels in the plasma? Is it residual FIX circulating FIX that is below the limits of detection but still effective? Is it collagen IV-bound, extravascular FIX? We examined this question recently in a paper published in Blood [18]. First we performed a saphenous vein injury on one side of the mouse and measured how many times bleeding could be terminated. Any unmeasurable, residual circulating FIX should have been exhausted when the first vein lost the ability to clot. We then injured the contralateral vein and measured the number of times that clotting from the injury ceases the bleeding. The clotting efficacy for the contralateral vein was essentially identical to that of the initial injury (Fig. 4). This appears to mean that the local extravascular FIX, not the circulating FIX, is critical for the cessation of bleeding—at least in this model of coagulation efficacy.