Wiskott-Aldrich Syndrome Platelets Research Articles

Accelerated death of megakaryocytes from Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder caused by WAS gene mutations resulting in haematopoietic/immune cell defects. Recent studies report accelerated death of WAS platelets and lymphocytes. Data on megakaryocyte (MK) maturation, viability and their possible role in thrombocytopenia development in WAS are limited. In this study we evaluate the MK viability and morphology in untreated, romiplostim-treated WAS patients compared with normal controls. The study included 32 WAS patients and 17 healthy donors. MKs were captured from bone marrow aspirates by surface-immobilized anti-GPIIb-IIIa antibody. Viability (by phosphatidylserine [PS] externalization), distribution by maturation stages and size of MK were determined by light microscopy. MK distribution by maturation stages in patients differed from controls. 40 ± 22% of WAS MKs versus 23 ± 11% of normal MKs were at maturation stage 3 (p = 0.02), whereas 24 ± 20% in WAS and 39 ± 14% in controls had megakaryoblast morphology (p = 0.05). Romiplostim treatment changed the MK maturation stages distribution close to normal. PS-positive (PS+) MK in WAS was significantly higher (21 ± 21%) than in healthy controls (2 ± 4%, p < 0.01). WAS patients with more damaging truncating mutations and higher disease score had higher PS+ MK fraction (Spearman r = 0.6, p < 0.003). We conclude that WAS MKs have increased cell death tendency and changes in maturation pattern. Both could contribute to thrombocytopenia in WAS patients.

Mechanisms of increased mitochondria-dependent necrosis in Wiskott-Aldrich syndrome platelets

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs. 3.9% in controls; P<0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P<0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia.

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition

AbstractAlthough the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to “self-deposit” additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.

Assessment of Immature Platelet Fraction in the Diagnosis of Wiskott-Aldrich Syndrome.

Children with Wiskott–Aldrich syndrome (WAS) are often first diagnosed with immune thrombocytopenia (ITP), potentially leading to both inappropriate treatment and the delay of life-saving definitive therapy. WAS is traditionally differentiated from ITP based on the small size of WAS platelets. In practice, microthrombocytopenia is often not present or not appreciated in children with WAS. To develop an alternative method of differentiating WAS from ITP, we retrospectively reviewed all complete blood counts and measurements of immature platelet fraction (IPF) in 18 subjects with WAS and 38 subjects with a diagnosis of ITP treated at our hospital. Examination of peripheral blood smears revealed a wide range of platelet sizes in subjects with WAS. Mean platelet volume (MPV) was not reported in 26% of subjects, and subjects in whom MPV was not reported had lower platelet counts than did subjects in whom MPV was reported. Subjects with WAS had a lower IPF than would be expected for their level of thrombocytopenia, and the IPF in subjects with WAS was significantly lower than in subjects with a diagnosis of ITP. Using logistic regression, we developed and validated a rule based on platelet count and IPF that was more sensitive for the diagnosis of WAS than was the MPV, and was applicable regardless of the level of platelets or the availability of the MPV. Our observations demonstrate that MPV is often not available in severely thrombocytopenic subjects, which may hinder the diagnosis of WAS. In addition, subjects with WAS have a low IPF, which is consistent with the notion that a platelet production defect contributes to the thrombocytopenia of WAS. Knowledge of this detail of WAS pathophysiology allows to differentiate WAS from ITP with increased sensitivity, thereby allowing a physician to spare children with WAS from inappropriate treatment, and make definitive therapy available in a timely manner.

“Self-Deposition” of Matrix Proteins from Platelet α-Granules Enable Extended Adhesion and Spreading on Micron/Submicron-Scale Fibrinogen and Collagen Substrates.

Introduction: While the platelet’s role in achieving hemostasis in the context of bleeding is well-characterized, how platelets support and maintain vascular integrity during homeostatic, non-thrombotic conditions remains unclear. Previous studies have shown that single platelets fill micron/submicron-sized “gaps” of small discontinuities in the endothelium. In this context, however, as the overall force of adhesion correlates with the area of exposed subendothelial matrix, how platelets establish sufficient adhesion to resist the dynamic shear forces of the circulation are unknown. In this study, we investigate, using protein microcontact printing, how platelets sense the geometry of the matrix micro/nano-environment and transduce those cues to affect platelet function. Our results reveal that platelets, upon sensing spatially-limited (<25 µm2 area) micro/nanopatterns of fibrinogen or collagen, spatially regulate actin-rich filopodia extension, via Rac1, beyond the geometric constraints of the matrix pattern, redistribute α-granules to enable “self-deposition” of fibrinogen/fibronectin matrix on those regions, and spread onto those newly deposited matrix via integrin αIIbβ3-mediated interactions. Furthermore, this phenomenon is markedly attenuated in Gray Platelet Syndrome platelets, which lack α-granules, and Wiskott-Aldrich Syndrome platelets, which have impaired integrin αIIbβ3-mediated activation, decreased α-granules, and cytoskeletal defects.Materials and Methods: Human blood was collected in ACD and platelets were isolated via two-step centrifugation or gel filtration. Platelets were suspended in Tyrode’s buffer containing 2 mM MgCl2 and exposed to various pharmacologic agents such as eptifibatide, Y27632, NSC23766. Fibrinogen and collagen micro/nanostamps were prepared on glass coverslips after which platelets were incubated onto. A plasma membrane dye, phalloidin, anti-P-selectin, anti-fibronectin and PAC-1, were used to visualize platelet morphology, f-actin, α-granule secretion, matrix depositions, and activated integrin αIIbβ3, respectively.Results and Discussion: Upon contact with the protein microstamps, platelets formed filopodia and lamellipodia and spread to conform to the micropattern boundaries with high fidelity. However, on micropatterned circles (microdots) of less than 25 µm2 area, platelets spread beyond the geometric constraints (Fig.1A). Anti P-selectin staining revealed α-granule secretion at the extended lamellipodia (Fig.1B left), and interestingly, this phenomenon was not detected with GPS platelets. Also we observed direct deposition of fibronectin from the platelets granules via anti-fibronectin staining (Fig.1B center). In addition, immunostaining with PAC-1 revealed a predominance of activated αIIbβ3 on regions of platelets at and beyond the borders of the microdots (Fig.1B right). When exposed to eptifibatide, an αIIbβ3 antagonist, platelet spreading beyond the microdot boundaries was completely eliminated. Various cytoskeleton inhibition experiments provided insight into how actin rearrangement is involved in this processes of platelet extension beyond matrix boundaries. Interestingly, disruption of cytoplasmic stress fibers by inhibiting Rho kinase with Y25632 facilitated platelet extension beyond the microdot boundaries and strong actin polymerization at the margins. On the other hand, platelet extension beyond the microdot boundaries was completely inhibited with NSC23766, a Rac-1 inhibitor (Fig.1C). We also confirmed that WAS platelets had significantly less f-actin, α-granule secretion, and overall extension beyond microdot boundaries. Overall, our results indicate that the matrix geometry, in and of itself, dictates platelet α-granule secretion, αIIbβ3 distribution and activation, and Rac1 activation, all of which work in concert to extend adhesion and spreading on matrix substrates of <25 µm2. In addition, we show that during this process, directionally directed α-granule secretion enables a platelet to “self-deposit” fibrinogen/fibronectin matrix to accommodate increased spreading. This phenomenon likely endows platelets a certain amount of “spatial flexibility” to establish firm adhesion upon contact with small sites of exposed subendothelium and may provide insight into how platelets help support vascular integrity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Platelets From WAS Patients Are More Susceptible Than Controls to Phagocytosis by Activated THP-1 Cells

Abstract 2222The thrombocytopenia associated with the Wiskott-Aldrich Syndrome (WAS) is thought to be due to the combined effects of impaired platelet production and accelerated platelet consumption. In a murine model of WAS, we have previously demonstrated that platelet consumption is accelerated. Also, we have shown that antibody opsonization accelerates both in vivo consumption and ex vivo phagocytosis of murine WASP(-) platelets in comparison to opsonized WT platelets. Based on these findings, we tested the susceptibility of platelets from WAS patients to ex vivo phagocytosis by activated THP-1 cells. We used a lipophilic fluorescent marker (DIO) to label platelets, and distinguished between platelet uptake and adsorption with a fluorescent anti-CD61 antibody. In comparison to previous methods using CMFDA-labeled platelets, use of DIO resulted in a tenfold reduction in the number of platelets needed per assay. This in turn allowed us to perform ex vivo phagocytosis studies with the very small number of platelets available for study in peripheral blood specimens from thrombocytopenic WAS patients. We report that untreated platelets from WAS patients are taken up more rapidly than control platelets by activated THP-1 cells. Specifically, the fraction of macrophages demonstrating DIO uptake, and showing no adsorbed platelets is consistently increased in comparison to cells showing adsorbed platelets (figure 1). Using a numerical analysis method, we distinguish the effect of WASP deficiency on the probability of phagocytosis per adsorbed platelet (p) from effects on the fluorescence intensity imparted to the macrophage per internalized platelet (alpha) and from variation in the platelet/macrophage ratio (m). This was done by predicting the type of results shown in figure 1 for approximately 32,000 possible combinations of p, alpha, and m, and finding among the possible combinations those for which the predicted results best fit our observations. We validated the sensitivity of the method to changes in p (via the use of opsonized vs. non-opsonized platelets), m (via changing the platelet/macrophage ratio) and alpha (via changing the mean fluorescence intensity of the platelets). Applied to the data in figure 1, the numerical analysis method demonstrates an increased p value for each of the WAS patients studied, and a reduced alpha value (as might be expected for smaller platelets) in 5 of 6 studies (figure 2). Opsonization with murine anti-CD61 antibody did not accelerate uptake of WAS platelets in comparison to controls. However, we observed significantly increased levels of surface IgM, and possibly IgG, on platelets from WAS patients. In addition to inhibiting opsonization ex vivo, this level of surface antibody could contribute to accelerated phagocytosis of platelets in clinical WAS in the same way that ex vivo opsonization augments the in vivo clearance and ex vivo phagocytosis of murine WASP(-) platelets. Our results provide additional support for the role of accelerated platelet consumption in generating the thrombocytopenia of WAS. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increased Extra-Splenic Platelet Turnover Contributes to the Thrombocytopenia of Murine WAS.

The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive condition manifesting as variable degrees of immunodeficiency and thrombocytopenia. The strong clinical improvement in platelet count seen in WAS patients after splenectomy suggests that increased platelet consumption is a major contributor to the thrombocytopenia. Here we show that splenectomy improves but does not normalize platelet count in a murine (C57Bl/6) model of WAS, suggesting (a) that murine WAS parallels clinical WAS in this regard, and (b) that either increased extrasplenic consumption or diminished platelet production (or both) contributes to the thrombocytopenia. We quantified platelet turnover by labeling WAS and WT platelets with CMFDA and following their disappearance after injection into WT recipients. Platelet loss rates in this model fit best to a simple exponential decay curve. We found that WAS platelets turn over significantly faster than WT (survival times: 56 hr (WT) vs 40 hr (WAS), p <.01). By comparison, we have observed a 42% reduction in platelet count in WAS mice (508K/ul, vs 881K/ul for age matched WT littermates, p<.01). WAS mice also show increased splenic extramedullary hematopoiesis, and increased megakaryocyte counts in bone marrow as assayed by flow cytometry. These results suggest that both increased platelet consumption and reduced thrombopoiesis contribute to the thrombocytopenia of WAS in this system, while megakaryopoiesis may not be inhibited. To determine the site of increased consumption of WAS platelets, we repeated the turnover studies in splenectomized C57Bl/6 mice. The results fit a two-hit gamma function, with estimated lifespans of 65 hr (WT) vs 53 hr (WAS)(p <. 01), indicating that splenectomy improved but did not normalize turnover of WAS platelets. The data suggests that the spleen is not the site of increased platelet consumption in WAS mice. To further delineate the role played by splenic platelet consumption in the thrombocytopenia of WAS, we crossed C57Bl/6 WAS mice for three generations onto the reportedly hypersplenic mouse strain C3H. Suprisingly, WAS (C3H) males showed no significant thrombocytopenia. Nonetheless, we found that WAS (C3H) platelets turn over more rapidly than WT platelets in C3H mice (estimated lifespans 61 hr (WT) vs 42 hr (WAS), p <.01), despite the fact that in comparison to C57Bl/6J (but not C57Bl/6Nhsd) mice, C3H mice do not in fact have significantly larger spleens. These results suggest that WAS (C3H) mice are able to compensate for increased platelet turnover rates by increasing platelet production appropriately, and imply that WAS (C57Bl/6) mice are not. Taken together, these studies suggest that the thrombocytopenia of murine WAS (C57Bl/6) is due to both increased extrasplenic platelet consumption and deficient platelet production. The inter-strain variation in WAS phenotype we report here reproduces the marked inter- and intra-kindred phenotypic variation seen in clinical WAS. [Display omitted]

Normal Arp2/3 complex activation in platelets lacking WASp

AbstractArp2/3 complex is believed to induce de novo nucleation of actin filaments at the edge of motile cells downstream of WASp family proteins. In this study, the signaling pathways leading to Arp2/3 complex activation, actin assembly, and shape change were investigated in platelets isolated from patients with Wiskott-Aldrich Syndrome (WAS), that is, who lack WASp, and in WASp-deficient mouse platelets. WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, identical to control WASp-expressing platelets. Human platelets contain 2 μM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the lamellae and to the Triton X-100–insoluble actin cytoskeleton of activated WASp-deficient platelets. Furthermore, the C-terminal CA domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the actin nucleation capacity of octylglucoside-permeabilized and activated WAS platelets, similar to its effect in WASp-expressing cells. Along with WASp, platelets express WAVE-2 as a physiologic activator of Arp2/3 complex and a small amount of N-WASp. Taken together, our findings show that platelets activate Arp2/3 complex, assemble actin, and change shape in the absence of WASp, indicating a more specialized role for WASp in these cells.

Normal Arp2/3 complex activation in platelets lacking WASp

Arp2/3 complex is believed to induce de novo nucleation of actin filaments at the edge of motile cells downstream of WASp family proteins. In this study, the signaling pathways leading to Arp2/3 complex activation, actin assembly, and shape change were investigated in platelets isolated from patients with Wiskott-Aldrich Syndrome (WAS), that is, who lack WASp, and in WASp-deficient mouse platelets. WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, identical to control WASp-expressing platelets. Human platelets contain 2 μM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the lamellae and to the Triton X-100–insoluble actin cytoskeleton of activated WASp-deficient platelets. Furthermore, the C-terminal CA domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the actin nucleation capacity of octylglucoside-permeabilized and activated WAS platelets, similar to its effect in WASp-expressing cells. Along with WASp, platelets express WAVE-2 as a physiologic activator of Arp2/3 complex and a small amount of N-WASp. Taken together, our findings show that platelets activate Arp2/3 complex, assemble actin, and change shape in the absence of WASp, indicating a more specialized role for WASp in these cells.

Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Pathological events in platelets of Wiskott-Aldrich syndrome patients.

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked platelet/immunological disorder arising from mutations of the gene WASP. At the clinical level, the major platelet abnormalities are small size and low number, both partially correctable by splenectomy. To identify underlying pathological events, we examined WAS platelets at various stages of their lifetime. In spleen sections from WAS patients, fluorescence microscopy showed dramatic co-localization of markers of platelets (CD41) and macrophages (CD68) compared to non-thrombocytopenic controls, suggesting that WAS splenic macrophages are involved in platelet removal. Study of isolated WAS blood platelets by flow cytometry showed substantial enhancement of surface exposure of phosphatidylserine (PS), a signal for engulfment by macrophages. Isolated resting WAS platelets were also aberrantly susceptible to microparticle release, and plasma samples of WAS patients contained > 5 times normal numbers of platelet-derived microparticles which may explain the small size of circulating platelets. Measurements with the Ca2+ sensitive dye fluo-3 revealed significantly increased Ca2+ levels, 310 +/- 13 nmol/l for WAS platelets versus 106 +/- 12 nmol/l for normal platelets, and also prolongation of agonist-induced Ca2+ flux. Cumulatively, these studies identify abnormal events occurring in WAS platelets: increased Ca2+ levels and enhancement of two Ca2+ dependent processes, PS exposure and microparticle release; these abnormal events may contribute to the in vivo decrease of platelet number and reduction of platelet size in this disease.

The cytoskeletal linker protein moesin: decreased levels in Wiskott-Aldrich syndrome platelets and identification of a cleavage pathway in normal platelets.

The Wiskott-Aldrich syndrome (WAS) is a severe disease of platelets (small size, thrombocytopenia) and lymphocytes (immunodeficiency) arising from mutations of the X-chromosome gene WASP. Because of the prominent role of cytoskeletal abnormalities, particularly the paucity of surface microvilli, in the cellular pathology of this disease, blood cells from WAS patients were examined for moesin, a cytoskeletal linker protein that stabilizes cell surface microvilli, filopodia and lamellipodia. Comparison of patient and normal lymphocytes by immunofluorescence microscopy and immunoblotting showed normal levels and distribution of moesin in lymphocytes of WAS patients. In contrast, platelets from WAS patients stained only dimly for moesin relative to normal platelets. Quantitation by immunoblot revealed significantly decreased moesin levels in WAS patient platelets relative to normal platelets (63.5 +/- 4.9% of normal levels, n = 8, P < 0.0001). A novel reaction of normal platelets was discovered that may play a role in the depletion of moesin in patient platelets, namely the cleavage of moesin as a late event in platelet activation in response to certain platelet agonists.

Wiskott-Aldrich syndrome: current research concepts.

Wiskott-Aldrich syndrome: current research concepts.

Evidence implicating calpain (Ca(2+)-dependent neutral protease) in the destructive thrombocytopenia of the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is an inherited platelet/T-lymphocyte disease characterized by small platelets, thrombocytopenia and immunodeficiency. Because degradative events have a significant role, we directly examined calpain (Ca(2+)-dependent neutral protease), a prominent protease in the affected cells, by functional and antigenic quantitation. Calpain activity in platelets of seven WAS patients was decreased to 59 +/- 3.7% (P < 0.01) relative to platelets of 11 normals. Platelets of two patients with immune thrombocytopenia had normal calpain activity. By immunoblotting, mu-procalpain, the mu-calpain species in resting (unstimulated) blood cells, was decreased in platelets of nine WAS patients to 58 +/- 14.6% (P < 0.01) relative to paired normals. In contrast, mu-procalpain levels in lymphocytes of seven WAS patients did not differ from normal lymphocytes. Normal platelets and lymphocytes have different mechanisms for Ca(2+)-dependent mu-procalpain activation. On addition of ionophore and Ca2+ to stirred platelets, 80kD mu-procalpain was rapidly (0.5 min) and quantitatively converted to 76 kD active mu-calpain; this process was the same in WAS platelets. In lymphocytes, mu-procalpain activation was slow, only partially complete (40 min), and the active species was 78 kD. The marked depletion of calpain in WAS platelets demonstrated in this study may result from inappropriate stimulation of platelets and be related to the severe thrombocytopenia that characterizes this disease.

Effect of platelet calpain on normal T-lymphocyte CD43: hypothesis of events in the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an inherited disease involving defects of platelets (small size, severe thrombocytopenia due to accelerated destruction) and T lymphocytes (progressive immunodeficiency, lymphopenia). The best-characterized molecular defect is the deficiency and, in some cases, abnormal forms of the T- lymphocyte surface mucin molecule CD43; deficiency of the platelet surface mucin GPIb was observed previously in two of four patients. Neither of these defects is primary, since CD43 and GPIb are encoded by autosomal genes and the disease is X-linked. This study uses cellular biological approaches to explore the possibility that destruction of structurally defective WAS platelets, mimicked experimentally by sonication of normal platelets, plays a role by releasing protease and generating other cellular defects. We show that a protease of normal platelets, identified as Ca(2+)-dependent neutral protease (calpain), which is known to cleave platelet GPIb, also specifically cleaves CD43 on the surface of neighboring desialylated T lymphocytes. The identification of the CD43 cleaving protease was based on its requirement for Ca2+ and inhibition by leupeptin, but not by diisopropylfluorophosphate (DFP). The approximate site of CD43 cleavage was identified by the use of a rabbit antibody. Sensitivity of GPIb to calpain is shown to be sialylation-independent and that of CD43 to be sialylation-dependent, and these findings are explained in terms of molecular structures. These and previous findings are incorporated into a putative mechanism, which explains most of the defects in the WAS. The mechanism suggests that the primary defective molecule in the WAS is unlikely to be a surface glycoprotein, but rather a cytoplasmic molecule with a function in cytoskeletal interactions and/or calcium ion regulation and calpain activation.

Effect of platelet calpain on normal T-lymphocyte CD43: hypothesis of events in the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an inherited disease involving defects of platelets (small size, severe thrombocytopenia due to accelerated destruction) and T lymphocytes (progressive immunodeficiency, lymphopenia). The best-characterized molecular defect is the deficiency and, in some cases, abnormal forms of the T-lymphocyte surface mucin molecule CD43; deficiency of the platelet surface mucin GPIb was observed previously in two of four patients. Neither of these defects is primary, since CD43 and GPIb are encoded by autosomal genes and the disease is X-linked. This study uses cellular biological approaches to explore the possibility that destruction of structurally defective WAS platelets, mimicked experimentally by sonication of normal platelets, plays a role by releasing protease and generating other cellular defects. We show that a protease of normal platelets, identified as Ca(2+)-dependent neutral protease (calpain), which is known to cleave platelet GPIb, also specifically cleaves CD43 on the surface of neighboring desialylated T lymphocytes. The identification of the CD43 cleaving protease was based on its requirement for Ca2+ and inhibition by leupeptin, but not by diisopropylfluorophosphate (DFP). The approximate site of CD43 cleavage was identified by the use of a rabbit antibody. Sensitivity of GPIb to calpain is shown to be sialylation-independent and that of CD43 to be sialylation-dependent, and these findings are explained in terms of molecular structures. These and previous findings are incorporated into a putative mechanism, which explains most of the defects in the WAS. The mechanism suggests that the primary defective molecule in the WAS is unlikely to be a surface glycoprotein, but rather a cytoplasmic molecule with a function in cytoskeletal interactions and/or calcium ion regulation and calpain activation.

956 THE WISKOTT-ALDRICH SYNDROME: A MEMBRANE DEFECT OF THE LYMPHO-HEMATOPOIETIC SYSTEM

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, decreased T lymphocyte function, and decreased platelet count with small, poorly functioning platelets. A unitarian hypothesis to explain the various immune and hematological manifestations of WAS has not existed. The size of WAS platelets normalized and their in vitro function improves following splenectomy suggesting the WAS platelet defect is an acquired rather than an intrinsic defect. To determine if the basic defect in WAS patients is a membrane defect present in both platelets and lymphocytes, the size of lymphocytes of a WAS patient was determined before and after splenectomy. The volume of the patient's lymphocytes was markedly reduced before splenectomy (1264 ± 20μ3) as compared to normal lymphocytes (2320 ± 228μ3, n=6). Seven days after splenectomy two populations of peripheral lymphocytes were identified, one of which was slightly smaller than the patient's original lymphocytes (1114 ± 76μ3) while the second was normal size (2288 ± 64μ3). Fourteen days after splenectomy only normal sized lymphocytes were detected. No change in lymphocyte size was seen in patients with ITP following splenectomy (n=2). No change in the normal blastogenic response of the patient's T lymphocytes was seen after splenectomy. These results suggest that the basic defect in WAS may be a membrane defect which leads to the production of small platelets and lymphocytes (microplatelets and microlymphocytes) by the spleen.