Protease Domain Exosites Regulate Plasma Recovery and Extravascular Distribution of Factor IX(a) Variants in Hemophilic Mice

Introduction:

Among the coagulation protease, factor IX (FIX) is unique in that a substantial pool of extravascular factor exists that contributes to in vivo hemostasis. Extravascular distribution of FIX contributes to poor plasma recovery of infused FIX in hemophilia B (HB). The effect of mutations in the heparin (K126A/K132A) and antithrombin (R150A) binding exosites of FIX and FIXa on the distribution of these variants between the plasma and extravascular binding sites was examined in HB and hemophilia A (HA) mice, respectively.

Methods:

Recombinant human FIX wild type (18 U/kg) or equimolar FIX K126A/K132A was administered to HB mice (n=8) via tail vein injection with sacrifice 5 min post-injection following retro-orbital blood collection. Equimolar doses of human FIXa wild type (WT), K126A/K132A and R150A were similarly injected into HA mice (n=4-8), with blood collection and sacrifice 5 min post-injection. Plasma was isolated by centrifugation, organs (liver, spleen, kidneys, heart, lung, and brain) harvested, rinsed with PBS, weighed, with a portion made into tissue lysates and remainder frozen. FIX(a) content of tissue lysates was determined by a species-specific human FIX(a) ELISA with variant-specific standard curves. Tissue concentration was determined by total FIX(a) present in lysate (ng) divided by respective tissue weight (mg). Organ distribution was determined by extrapolating FIX(a) tissue concentration to total organ weight. The FIX(a) present in a specific organ was divided by the administered FIX(a) dose to determine the % organ distribution.

Results:

PK studies in HB mice demonstrated that FIX WT and R150A demonstrated a similar pattern and time course of elimination. In contrast, FIX K126A/K132A demonstrated ~2.4 fold higher plasma recovery relative to FIX WT. Based on plasma concentrations at 5 min post-injection (plasma volume 40 ml/kg), ~12.9% of FIX WT and 30.5% of FIX K126A/K132A was localized to the plasma compartment. Tissue lysates from the liver, spleen, kidney and brain demonstrated that liver had the highest FIX content by far, with ~41% of the FIX WT dose at 5 min post-injection. Other tissues demonstrated markedly less FIX WT content, including kidney (1%), spleen (0.1%) and brain (undetectable). FIX tissue concentration was significantly higher in the liver (0.69 ng/mg tissue) compared to other tissues, followed by kidney (0.07 ng/mg), spleen (0.02 ng/mg) and brain (undetectable). Comparison of FIX K126A/K132A tissue content to WT demonstrated reduced liver localization (25% dose) with concomitant reduction in tissue concentration (0.45 ng/mg). In contrast, FIX K126A/K132A increased localization to the kidney (1.7%) and spleen (0.37%) relative to WT.

In protease PK studies, FIXa K126A/K132A and R150A both enhanced plasma recovery (2.2-2.5 fold) in HA mice compared to FIXa WT. Based on plasma concentrations at 5 min post-injection, ~16.1% of FIXa WT and 41.1% of FIXa K126A/K132A dose localized in the plasma compartment. Similar to zymogen, tissue lysates demonstrated that liver had the highest FIXa WT content (28%). Other tissues contained markedly less FIXa, including kidney (2.4%), spleen (0.2%), heart (0.5%) and brain (undetectable). Similarly, FIXa tissue concentration was 4-fold higher in the liver (0.47 ng/mg) compared to other tissues, followed by kidney (0.12 ng/mg), spleen and heart (0.06 ng/mg) and brain (undetectable). Localization of FIXa K126A/K132A to the liver was reduced by nearly half compared to FIXa WT (16%) with reduction in liver tissue concentration (0.29 ng/mg). FIXa K126A/K132A had increased localization to the kidney (3.3%) relative to WT. In contrast, FIXa R150A (not shown) had similar liver distribution (31.9% dose) and tissue concentration (0.53 ng/mg) to FIXa WT, despite increased localization to the plasma (36.2%).

Conclusions:

Liver is the predominant organ for extravascular FIX binding and the heparin binding exosite contributes to this localization, similar to the collagen IV binding site in the Gla domain. Heparan sulfate and collagen IV co-localize in the basement membrane suggesting synergistic roles. FIXa also localizes to extravascular sites via the heparin-binding exosite, although that binding may be modestly limited by endogenous FIX in the HA mouse. Disruption of the antithrombin binding exosite re-distributes FIXa to the plasma compartment by an independent mechanism, as liver content is unchanged.