Impact of Anticoagulant Function By the Addition of Heparinoid in Vitro in the Plasmas from Patients with Hemophilia and Von Willebrand Disease

Background: Heparinoid (Hirudoid ®) is a local anticoagulant preparation as skin application that relieves pain and inflammation with addition to promoting healing in superficial thrombophlebitis and bruising. Heparinoid is mainly composed of chondroitin sulfate and has high moisturizing ability due to the large number of hydrophilic group. Thus, it is used as topical treatments for sebum deficiency in Japan. Since heparinoid has a structure similar to heparin, the package insert states the contraindication for hemorrhagic diseases such as hemophilia and von Willebrand disease (VWD) in Japan. Hemophilia A and B (HA/HB) are inherited bleeding disorders caused by a defect in factor (F) VIII and FIX coagulant protein, respectively. Based on its activity levels, the clinical severity is classified as severe (<1 IU/dL), moderate (1-5 IU/dL), and mild (>5-40 IU/dL). VWD is the common congenital bleeding disorders and is caused by a deficiency of von Willebrand factor (VWF). VWD is classified into partial deficiency (type 1), qualitative deficiency (type 2), and complete quantitative deficiency (type 3) of VWF, based on the VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) level. In the clinical practice, there are cases of hemorrhagic patients using heparinoid at the discretion of their families. However, the influence of heparinoid on coagulant function in human subjects remains to be investigated, and it is unclear if the patients with hemophilia and VWD can use heparinoid safely.

Aim: To examine the impact of anticoagulant function of heparinoid in vitro in the plasmas from patients with hemophilia and VWD.

Methods: The maximum blood concentration from absorbed heparinoid was estimated to be 2.5 µg/mL, based on the previous report, when heparinoid was applied to the skin as stated in package insert. Heparinoid (0-6.5 µg/mL) or unfractionated heparin (0-0.6 IU/mL) were added to normal plasma (NP), followed by measuring activated partial thromboplastin time (aPTT) by one-stage clotting assay. A modified clot waveform analysis (CWA) using an aPTT reagent were performed on the CS-2400™ (Sysmex), followed by measuring clotting time (CT) and adjusted maximum coagulation velocity (Ad|min1|). Heparinoid was also added to the patients' plasmas with HA, HB, or VWD, followed by CWA assessment. The CT and Ad|min1| of 20 healthy individuals were 31.1±0.1 sec and 6.90±0.05 (median±SEM).

Results: aPTT and CT values in NP by the spike of heparinoid were prolonged and Ad|min1| was decreased dose-dependently. The addition of 2.5 µg/mL heparinoid showed that aPTT (34.3 sec; 1.2-fold of control (no heparinoid)), CT (36.3 sec; 1.2-fold of control), and Ad|min1| (9.5; 0.96-fold of control) were comparable to the effect of ~0.2 IU/mL heparin, suggesting that heparinoid exhibited slight anticoagulant effect for NP. To examine the effect of heparinoid on HA/HB plasmas, recombinant (r) FVIII or rFIX (0-50 IU/dL) were added to FVIII- or FIX-deficient plasmas (3 lots each) together with heparinoid (0-6.3 µg/mL), respectively. The addition of heparinoid had prolonged the CT and decreased the Ad|min1| at each concentration of FVIII and FIX dose-dependently. The addition of 2.5 µg/mL heparinoid showed modest anticoagulant effect for HA and HB at each concentration of FVIII and FIX (CT; 1.3- and 1.5-fold of control, Ad|min1|; 0.82- and 0.87-fold of control). Next, the effect of heparinoid on the plasma from one VWD patient each with type 1 (VWF:Ag/VWF:RCo 28 IU/dL/26 IU/dL), type 2 (20 IU/dL/9 IU/dL), and type 3 (<2 IU/dL/<3 IU/dL) was assessed. The addition of 2.5 µg/mL heparinoid showed slight anticoagulant effect for each type of VWD (CT; ~1.3-fold of control, Ad|min1|; ~0.91-fold of control).

Conclusion: The presence of heparinoid demonstrated mild anticoagulant effect in vitro in the plasmas of patients with hemophilia and VWD. It might be possible to apply the heparinoid-contained product as skin treatment for these patients.