Flow cytometric analysis of platelets from childrenwith the Wiskott‐Aldrich syndrome reveals defects in platelet development, activation and structure

Abstract

Summary. The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin-and ADPinduced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.

Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

“…These mechanisms could result in the type of episodic thrombocytopenia seen in some WAS patients at presentation [9] and in others after splenectomy [10]. They could also contribute to the chronic thrombocytopenia of WAS patients, given that the latter show increased levels of platelet associated antibodies (seen in 13 of 14 cases in one study [52], and corroborated in several other reports [23,53,54]). Augmented host antigen presentation has also been suggested to underlie the development of autoimmune hemolytic anemia in the context of CD47 deficiency [55].…”

“…Structural and biochemical abnormalities that have been demonstrated in WASP(−) platelets include reduced numbers of both α and dense granules, increased microparticle release, and increased surface phosphatidyl serine [12][13][14]19]. However, no consistent abnormalities in overall actin polymerization, platelet shape change, or Arp2/3 complex activation after platelet stimulation have been observed [20][21][22][23]. Murine knockout models of WAS have been independently derived by two laboratories [24,25].…”

Reticulated platelets: analytical aspects and clinical utility

“…Kienast and Schmitz initiated a breakthrough in the field when they described a flow cytometric technique for analyzing retPLT, based on RNA staining by thiazole orange [15]. In subsequent years, several research groups published their findings in a wide variety of conditions like thrombocytopenia [16][17][18][19][20][21], thrombocytosis [22,23], after stem cell transplantation [24][25][26][27], hereditary platelet diseases [28,29], thrombo-embolic disorders [30,31], kidney disease [32][33][34], preeclampsia [35], hyperthyroidism [36] and in healthy and thrombocytopenic neonates [37,38]. The overall conclusion from these studies is that retPLT in blood represent a useful non-invasive marker of megakaryopoietic activity in the bone marrow.…”

Platelet-associated IgAs and impaired GPVI responses in platelets lacking WIP

“…9 Platelet-associated antibodies could be responsible for the fast clearance of WAS platelets because antibodies are no longer detectable and circulating platelet number and size increase after WAS patients undergo splenectomy. [10][11][12] Megakaryocytes isolated from WAS patients form proplatelets normally and produce platelets of normal size in vitro. 13 WASp knockout (KO) mice have a moderate thrombocytopenia.…”