Background/Purpose: Systemic Lupus Erythematosus ( SLE) is characterized by the formation of autoantibodies, and  over-exuberant antigen presentation may account for some of this pathology . Circulating microparticles (MP) are membrane-bound 100-1000 nM sized vesicles derived from  platelets, leukocytes, endothelial cells and red cells.  They are  immunomodulatory and participate in antigen presentation.  The protein crosslinking enzymes, transglutaminases (Tgases),  are involved in antigen presentation.   Evidence from mouse studies supports a potential role for excess Tgase activity in SLE and TGase inhibitors have been shows to ameliorate murine lupus.  Interestingly, we also noted a single report of Tgase inhibition with hydroxychloroquine (HCQ), a commonly used drug for SLE.   Previous studies have shown increased numbers of circulating  MPs in SLE patients.  We sought to characterize the cell sources of MPs and determine protein levels of  the Tgase enzymes ( type II Tgase and factor XIIIA) on MPs from SLE patients and controls.   We also measured Tgase activity on MPs from several normal donors and investigated the effect of HCQ on MP-associated Tgase activity.

Methods: We obtained 12 ml of blood from 25 SLE patients and 25 age and sex-matched normal controls after IRB approval. SLE patients satisfied ACR criteria for SLE and were excluded if they had anti-phospholipid antibody syndrome.  MPs were isolated from platelet rich plasma according to published protocols.  Flow cytometry was used to enumerate MPs and determine the percent of MPs from each cell source .  Cell markers included CD45 for leukocytes, CD41 (GPIIb) + CD42 (GPIX+GPIb) for platelets, CD144 (VEcadherin) for endothelial cells, CD235a (glycophorin A) for red cells, as well as the Tgase enzymes, type II Tgase and factor XIIIA.    Tgase activity was measured with a standard radiometric assay detecting crosslinking activity in the presence and absence of 10 mM HCQ.   The Mann-Whitney test was used to determine statistically significant differences between groups.

Results: Numbers of MPs were similar in SLE patients and controls.  SLE patients had higher numbers of MPs derived from red cells 1448 (CI 908-2640) positive particles (PP)/µl compared to  2570 ( CI 1069-9035) PP /µl  in SLE patients ( p=0.027).  Type II Tgase enzyme levels were significantly higher in the SLE cohort, which had 1531 (CI 854-4097) PP/µl, compared to the control group with 895 (CI 402-1526) PP/µl ( p=0.007).  Factor XIIIA levels were slightly higher in the SLE group, but did not reach statistical significance (p=0.068).   Tgase activity on a random sample of MPs from controls demonstrated an average Tgase activity level of 163 ± 75 mU/ mg protein ( n=3).  Activity levels fell to 112.8± 14.9 mU/mg protein with  HCQ( p<0.05). 

Conclusion: Type II Tgase enzyme levels  are significantly higher on MPs from SLE patients than controls,and activity  is inhibited with HCQ.  Because Tgase  enzymes may have active roles in antigen presentation as well as platelet aggregation, the ability of HCQ to suppress Tgase activity may be an additional mechanism of action for this effective SLE drug.  Further studies of the role of Tgase in SLE and on MPs are warranted.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/elevated-transglutaminase-levels-on-microparticles-from-systemic-lupus-erythematosus-patients/