Anti‐phosphatidylserine/prothrombin complex antibodies in patients with cutaneous vasculitis: Possible involvement in the pathogenesis

We assessed the IgG and IgM prevalence of anti‐phosphatidylserine/prothrombin complex (aPS/PT) antibodies (Abs) in patients with vasculitis using a novel commercial ELISA kit. To examine whether aPS/PT Abs were involved in the pathogenesis of cutaneous vasculitis, inbred wild‐type rats were intravenously administered with a rat IgM class aPS/PT monoclonal Ab established previously or with rat immunoglobulins as controls. To express PS on the surface of vascular endothelium, these rats were given a subcutaneous injection of cell‐free histones in advance. Serum IgM aPS/PT Ab levels were elevated in patients with systemic vasculitis with skin involvement and cutaneous arteritis compared to those in patients with systemic vasculitis without skin involvement and healthy controls. There was no significant difference in the serum levels of IgG aPS/PT Abs between the patients and healthy controls. Correspondingly, inbred wild‐type rats intravenously administered with the aPS/PT monoclonal IgM Ab after appropriate priming‐subcutaneous histone injection developed cutaneous vasculitis. Some rats given rat IgM instead of the aPS/PT monoclonal Ab also developed cutaneous vasculitis, whereas vasculitis did not occur in rats given IgG or only priming by histones. We suggested that IgM aPS/PT Abs could be involved in the pathogenesis of cutaneous vasculitis based on these findings.

monoclonal Ab established previously or with rat immunoglobulins as controls. To express PS on the surface of vascular endothelium, these rats were given a subcutaneous injection of cell-free histones in advance. Serum IgM aPS/PT Ab levels were elevated in patients with systemic vasculitis with skin involvement and cutaneous arteritis compared to those in patients with systemic vasculitis without skin involvement and healthy controls.
There was no significant difference in the serum levels of IgG aPS/PT Abs between the patients and healthy controls. Correspondingly, inbred wild-type rats intravenously administered with the aPS/PT monoclonal IgM Ab after appropriate priming-subcutaneous histone injection developed cutaneous vasculitis. Some rats given rat IgM instead of the aPS/PT monoclonal Ab also developed cutaneous vasculitis, whereas vasculitis did not occur in rats given IgG or only priming by histones. We suggested that IgM aPS/PT Abs could be involved in the pathogenesis of cutaneous vasculitis based on these findings.

K E Y W O R D S
anti-phosphatidylserine/prothrombin complex antibodies, cutaneous arteritis, cutaneous polyarteritis nodosa, histones, IgM histones was employed as priming to express PS on the surface of vascular endothelial cells.

| E XPERIMENTAL PROTOCOL S
1. Rats (n = 4) were given a subcutaneous injection of 0, 2.5, 25, and 250 µg/mL cell-free histones (calf thymus-derived histones containing unfractionated whole histones; Sigma-Aldrich, St. Louis, MO, USA) on the back (300 µL/site). Two hours later, the skin was resected for immunostaining using a rat IgM class aPS/PT monoclonal Ab 6 . Formalin-fixed, paraffin-embedded skin tissues were cut into 4-µm sections, and the sections were allowed to react with 1 µg/mL aPS/PT monoclonal Ab (rat IgM) followed by 2 µg/mL Alexa Fluor 594-conjugated goat anti-rat IgM Ab histone isc with aPS/PT iv), Group B with rat IgM (eBioscience, San Diego, CA, USA; n = 4; histone isc with IgM iv), Group C with rat IgG (eBioscience; n = 5; histone isc with IgG iv), and Group D without immunoglobulin administration (n = 5; histone isc). One week later, all rats were euthanized for histopathological analyses.

| aPS/PT Abs in patients
Patients in Group 1 comprised five females and three males with a mean age of 48.9 ± 18.70 years (Table S1). They consisted of three patients with IgA vasculitis, two patients with eosinophilic granulomatosis with polyangiitis (EGPA), and one patient each with CA, microscopic polyangiitis (MPA), and granulomatosis with polyangiitis (GPA). Patients in Group 2 with no cutaneous vasculitis comprised five females and two males with a mean age of 67.71 ± 21.37 years. They consisted of two patients with MPA, two patients with EGPA, and onw patient each with polyarteritis nodosa (PAN), GPA, and rheumatoid vasculitis. Healthy patients comprised six females and four males with a mean age of 58.5 ± 18.73 years. There was not a significantly higher presence of IgG aPS/PT Abs among Group 1, Group 2, and the normal controls (11.26 ± 1.78 U/mL vs 10.75 ± 1.64 U/mL vs 10.48 ± 0.65 U/mL; Figure 1a, Table S2). In contrast, the mean IgM aPS/PT Ab level in Group 1 was significantly higher than that in Group 2 (21.52 ± 5.64 U/mL vs 12.85 ± 1.86 U/mL, P < 0.01; Figure 1b).
There was a significantly higher presence of IgM aPS/PT Abs in Group 1 compared to the normal controls (21.52 ± 5.64 U/mL vs 9.74 ± 1.03 U/mL, P < 0.001). There was not a significantly higher presence of IgG aPS/PT Abs between Group 2 and the normal controls (Table S3).

| Binding of aPS/PT Ab with vascular endothelium
To bind aPS/PT Abs in the serum with vascular endothelium, endothelial cells have to express PS on the surface. In the preceding study, cellfree histones (more than 12.5 µg/mL) induced apoptosis of cultured rat vascular endothelial cells. 8 To determine the concentration of histones necessary for the cell surface expression of PS on vascular endothelium in the skin, inbred wild-type rats were given a subcutaneous injection of histones at varied concentrations, and then the skin was subjected to immunostaining using an aPS/PT monoclonal Ab (Figure 2a). Although the aPS/PT monoclonal Ab bound with the subcutaneous connective tissues of rats given a histone injection (more than 2.5 µg/mL), the binding of aPS/PT monoclonal Ab with vascular endothelium was observed only when 250 µg/mL histones were injected. These findings suggest that the subcutaneous injection of histones (250 µg/mL) induced PS expression on vascular endothelial cells and subsequent formation of the PS/PT complex with PT in the serum. Based on these findings, this condition was employed as priming for the following experiments.

| Development of cutaneous vasculitis in rats given an intravenous administration of aPS/PT Ab
To determine the pathogenicity of aPS/PT Abs, inbred wild-type rats were intravenously administered with or without 1.25 µg/g weight of a rat IgM class aPS/PT monoclonal Ab 2 h after priming by histones. For controls, rat IgM and IgG were administered instead of the  (Figure 2b). Some rats in Group B (histone isc with IgM iv) also developed cutaneous vasculitis, whereas no rat in Group C (histone isc with IgG iv) and Group D (histone isc) did. Vasculitis did not occur in the systemic organs other than the skin. The occurrence of vasculitis in Group A was significantly higher than in other groups (Table 1).

| DISCUSS ION
We cialists on cases of CA demonstrated that it is important for dermatologists to detect the presence of vasculitis-related symptoms early in order to establish an accurate diagnosis and a timely treatment. 10 We suggest that patients presenting as IgM aPS/PT Abs positive could be originally diagnosed as having CA, and should be carefully followed to ensure this condition does not to progress from CA to systemic PAN.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The study was approved by the ethics committee of Tohoku Medical