Phenotype of Platelets Are Altered and Activated in Circulation of Patients with Systemic Sclerosis
Authors: Yasuoka Hidekata , Sakata Komei , Yoshimoto Keiko , Takeuchi Tsutomu 
Published: October 2019

Systemic sclerosis (SSc), a systemic, connective tissue disease, is characterized by excessive fibrosis of skin and multiple internal organs associated with microvascular injury and autoantibody production. However, the disease process is still under investigation. Among circulating blood cells, the roles of lymphocytes and monocytes were well-examined, however, other cellular components are not well-focused. Platelets play a significant role in hemostasis physiologically. However, recent studies have revealed that platelets contain humoral factors such as cytokines, chemokines and growth factors and can distribute systemically through circulation. We hypothesized that platelets could contribute to the pathogenesis through the release of these factors. To elucidate the role of platelets in SSc, activation status and phenotype of circulating platelets in patients and association with clinical characteristics were examined.

Twenty-one patients with SSc who fulfilled 2013 classification criteria of American College of Rheumatology and European League Against Rheumatism and 16 healthy controls were involved. Platelets or platelet-derived microparticles (MPs) were defined as vesicles in platelet-rich plasma which is more or less than 1mm in diameter by forward and side scatter, respectively, and positive staining with anti-CD41 antibody using flow cytometry. Activation status of platelets was examined by the expression of activation markers on platelets such as P-selectin (CD62P) or activated glycoprotein IIb/IIIa (PAC1). Production of microparticles (MPs) is defined as ratio of proportion of MP to that of platelets. Association or correlation between proportion of activated platelets and clinical characteristics or parameters of patients with SSc was also examined. For the further phenotypic analysis of platelets, expression of selected surface markers on platelets such as membrane-bound transforming growth factor (TGF)-beta, CD147 (emmprin), CD142 (tissue factor), CD31 (platelet endothelial cell adhesion molecule (PECAM)-1) was examined using flow cytometry. Furthermore, release reaction of platelets was evaluated by release of platelet factor 4 (PF4) in culture supernatant of coculture with skin fibroblasts using enzyme-linked immunosorbent assay (ELISA).

As for the characteristics of 21 patients, male was 14 %, proportion of diffuse cutaneous SSc was 24 %, mean age was 63 ± 13 years, and mean disease duration was 16 ± 13 years. In SSc, both proportion of CD62P+ or PAC1+ activated platelets (P < 0.05, P < 0.05, respectively) and production of MP were higher in SSc (P < 0.05) compared to those in healthy controls. Of these, proportion of CD62P+ platelets and MP production were correlated each other (r = 0.88, P < 0.05). When activation status of platelets was compared to clinical parameters, only proportion of CD62P+ platelets was higher in diffuse cutaneous SSc and correlated with modified Rodnan skin score (mRSS) (P < 0.05). Phenotype of platelets was also altered based on the upregulation of expression of selected surface markers on platelets, such as membrane-bound TGF-beta and CD147. Interestingly, proportion of membrane-bound TGF-beta+ platelets was positively correlated with that of CD62P+ activated platelets and furthermore, proportion of membrane-bound TGF-beta+ platelets was correlated with mRSS (P < 0.05). Functionally, release reaction of platelet was enhanced in platelets from patient with SSc compared to healthy controls (P < 0.05).

In conclusion, circulating platelets in SSc were activated and their phenotype is altered in circulation. Furthermore, activation status of platelets is associated with skin fibrotic phenotype, suggesting that circulating platelets reflects and can contribute to the disease process of SSc. Also, phenotype of platelets can be a novel biomarker of the disease.

Disclosures

No relevant conflicts of interest to declare.