Calcium Prevents Enhanced Degradation of Factor VIII in the Condition of Motion

Abstract

Background: Hemophilia A and B induce recurrent bleeding episodes, mainly in skeletal muscles and joints that are in intermittent motion. We have previously demonstrated that intermittent motion contributes to increased degradation of factors VIII and IX. Objectives: Given that calcium ions are known to enhance factor VIII-von Willebrand factor (vWF) interaction, the present study has investigated the role of these ions on factors VIII and IX in the condition of motion. Methods: The effects of calcium ions were assessed using purified proteins via Western blot, factor VIII activity, immunocytochemistry, and in Institute of Cancer Research (ICR) mice with no specific genetic background. Results: Calcium was found to prevent degradation of plasma-derived factor VIII but not that of factor IX, during intermittent motion. Calcium levels in the microcirculation of mouse striated muscles were elevated following movement, enabling prevention of factor VIII degradation in normal physiology. Calcium supplementation in drinking water increased factor VIII levels in blood and striated muscles of ICR mice during movement. Conclusions: calcium ions decrease factor VIII degradation in the condition of motion. Further research on the impact of calcium salt oral supplementation on hemophilia patients is warranted.

Keywords: calcium ions; calcium supplement; degradation of factor VIII; factor VIII; hemophilia; movement.

Figure 1

Calcium prevents degradation of factor VIII during motion. (A). Factor VIII, which included thrombin dissolved in the indicated salt solutions, was set in motion via 10 min centrifugation (500 g) at room temperature, and then the level of factor VIII was measured via Western blot. Comparison of the effects elicited by each of the above salts, dissolved at the same concentration in double-distilled water (DDW), demonstrated that only calcium prevented the degradation of factor VIII. The protective effect was preserved even when the solutions were centrifuged. As all the assessed salts have Cl⁻ ions, the comparison indicates that Ca⁺² ions and not Cl⁻ ions are responsible for the stabilization effect. Calcium dissolved in DDW at a physiologic plasma concentration (1.5–3 mM) still prevented degradation of factor VIII in a dose-dependent manner, and the effect was maintained following agitation for 10 min at 30 RPM (B). Relative protein levels in Western blots were quantified via densitometry analysis (upper panel). Assays were conducted in triplicate. The results represent mean and range. * p < 0.05, ** p < 0.005, *** p < 0.0005. Significance was determined using the Mann–Whitney U-test, an agitator-horizontal platelet shaker LPS-A20 (LABTRON, Comberlay, UK), and a Centrifugation–Megafuge 16R (Thermo Fisher Scientific, Waltham, MA, USA).

Figure 2

Calcium levels in the microcirculation of skeletal striated muscles are elevated following movement. ICR mice (no specific genetic background) were put to sleep following isoflurane anesthesia for 15 min. The study group (n = 5) continued regular activity in the cage. Both groups were sacrificed, and the hind legs’ thigh striated muscles were studied using Alizarin Red staining specific for calcium ions. A significant increase in the calcium level in the microcirculation was observed in mice in the condition of movement relative to resting mice ((A,B) black arrows, (C)-statistical evaluation), enabling the prevention of factor VIII degradation in normal physiology. Representative images were captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), original magnification, ×10. Contiguous table shows calcium staining intensity in striated muscles. Significance was determined using the Mann–Whitney U-test (absence + week groups were compared to moderate + strong groups).

Figure 3

Calcium supplementation in drinking water raises the level of Factor VIII in the blood and skeletal striated muscles of mice. Seven- to eight-week-old male ICR mice were divided into two groups. One group received the recommended daily dose of calcium gluconate (Solgar, water-soluble) supplementation in drinking water (7.5 mM), and the control group received drinking water without additives. The experiment was conducted twice with two different mouse groups. The first experiment lasted for two days and the second for one month. At the end of the experiments, blood was drawn, and the mice were sacrificed. In mice that drank water with calcium, factor VIII levels in plasma were higher than in mice that drank water without calcium supplementation (A). In addition, in mice that drank water with calcium, factor VIII levels in the hind legs’ thigh striated muscle microcirculation were also higher compared to levels in the control group ((B,C) black arrows, (D)-statistical evaluation). The result of (A). represents mean ± SEM. Significance was determined using the Mann–Whitney U-test (* p < 0.05). The significance of (B,C) was determined using the U-test (absence + week groups were compared to moderate + strong groups). Results were similar after two days and after 1 month of experiments (A–C).

Figure 4

Calcium solution does not prevent factor IX degradation in movement. Factor IX in the indicated solution was placed in the agitator for 10 min (30 RPM) at room temperature, and then the level of factor IX was measured using Western blot. Addition of factor VIII prevented factor IX degradation, while movement enhanced its degradation. Although calcium solution in comparison to NaCl solution prevented degradation of factor IX in the resting condition (thick horizontal lines), the effect was similar in movement and did not prevent factor IX degradation (thin horizontal lines). Relative protein levels in Western blots were quantified via densitometry analysis (upper panel). Assays were conducted in triplicate. The results represent mean and range. ** p < 0.005, *** p < 0.0005. Significance was determined by the Mann–Whitney U-test and Agitator-horizontal platelet shaker LPS-A20 (LABTRON, Comberlay, UK).