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Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease

View Article: PubMed Central - PubMed

ABSTRACT

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott–Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

No MeSH data available.


Related in: MedlinePlus

ARPC1B-deficient platelets are microthrombocytes containing decreased numbers of dense granules.(a) A comparison of platelet circumferential ring diameter distributions for all three ARPC1B mutant patients (P1, P2, P3) and a WASP- patient (WAS) shows that all are significantly smaller (that is, microthrombocytes) compared to platelets from a normal donor (NC; P<0.0001, unpaired t-test; n=57 platelets, means and 95% confidence intervals shown by bars). No significant differences were observed among patients. (b) TEM of platelet whole mounts from an unaffected parent (left) and Patient 1 (right), showing dense granules detectable as dark spots (bar=500 nm; × 25,000). (c) Dense granule counts derived from whole-mount TEM imaging of platelets from Patient 1 (P1), Patient 2 (P2) and a WASP- patient (WAS) were significantly lower than for cells from an unaffected relative (F1); Mann–Whitney test P<0.0008, n=50 platelets. Means for Patient 1 and WASP  were not significantly different (P=0.07), while the mean for Patient 2 was significantly higher than for Patient 1 (P<0.0001). Means and 95% confidence intervals shown by bars.
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f5: ARPC1B-deficient platelets are microthrombocytes containing decreased numbers of dense granules.(a) A comparison of platelet circumferential ring diameter distributions for all three ARPC1B mutant patients (P1, P2, P3) and a WASP- patient (WAS) shows that all are significantly smaller (that is, microthrombocytes) compared to platelets from a normal donor (NC; P<0.0001, unpaired t-test; n=57 platelets, means and 95% confidence intervals shown by bars). No significant differences were observed among patients. (b) TEM of platelet whole mounts from an unaffected parent (left) and Patient 1 (right), showing dense granules detectable as dark spots (bar=500 nm; × 25,000). (c) Dense granule counts derived from whole-mount TEM imaging of platelets from Patient 1 (P1), Patient 2 (P2) and a WASP- patient (WAS) were significantly lower than for cells from an unaffected relative (F1); Mann–Whitney test P<0.0008, n=50 platelets. Means for Patient 1 and WASP were not significantly different (P=0.07), while the mean for Patient 2 was significantly higher than for Patient 1 (P<0.0001). Means and 95% confidence intervals shown by bars.

Mentions: Thin section transmission electron microscopy (TEM) was used to examine platelets from the ARPC1B- patient, a WASP- patient and a normal donor. All had platelets containing typical cellular structures including mitochondria and α-granules (Figs 3a and 4). ARPC1B- and WASP- platelets showed a propensity towards small size (Fig. 4), and examination via IF microscopy (Fig. 3b) confirmed these platelets to be small and dysmorphic compared to normal. A comparison of circumferential tubulin ring diameters (Fig. 3c) confirmed that both ARPC1B- and WASP- platelets are significantly smaller than normal, and do not differ significantly from each other. ARPC1B- platelets can thus be classified as microthrombocytes, as can ARPC1B-deficient platelets from Patients 2 and 3 (Fig. 5a). A significant proportion (∼20%) of ARPC1B- platelets shared dysmorphic features with WASP- platelets (Fig. 3d,e) that included odd shapes, collapse/loss of circumferential microtubule coils and highly variable P-selectin and thrombospondin-1 content (both indicators of α-granules). As has been reported for WAS platelets28, whole-mount TEM29 revealed a reduction/absence of calcium-rich platelet dense granules in ARPC1B- and -deficient platelets (Fig. 5b,c). Clinical lumi-aggregometry analysis of platelets from Patients 1 and 2 confirmed decreased dense granule ATP release (0.16 and 0.19 nmol respectively; normal range 0.29–1.93 nmol). Platelet aggregation investigations with collagen, SFLLRN, arachidonic acid, ristocetin and ADP were normal for both patients.


Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease
ARPC1B-deficient platelets are microthrombocytes containing decreased numbers of dense granules.(a) A comparison of platelet circumferential ring diameter distributions for all three ARPC1B mutant patients (P1, P2, P3) and a WASP- patient (WAS) shows that all are significantly smaller (that is, microthrombocytes) compared to platelets from a normal donor (NC; P<0.0001, unpaired t-test; n=57 platelets, means and 95% confidence intervals shown by bars). No significant differences were observed among patients. (b) TEM of platelet whole mounts from an unaffected parent (left) and Patient 1 (right), showing dense granules detectable as dark spots (bar=500 nm; × 25,000). (c) Dense granule counts derived from whole-mount TEM imaging of platelets from Patient 1 (P1), Patient 2 (P2) and a WASP- patient (WAS) were significantly lower than for cells from an unaffected relative (F1); Mann–Whitney test P<0.0008, n=50 platelets. Means for Patient 1 and WASP  were not significantly different (P=0.07), while the mean for Patient 2 was significantly higher than for Patient 1 (P<0.0001). Means and 95% confidence intervals shown by bars.
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Related In: Results  -  Collection

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f5: ARPC1B-deficient platelets are microthrombocytes containing decreased numbers of dense granules.(a) A comparison of platelet circumferential ring diameter distributions for all three ARPC1B mutant patients (P1, P2, P3) and a WASP- patient (WAS) shows that all are significantly smaller (that is, microthrombocytes) compared to platelets from a normal donor (NC; P<0.0001, unpaired t-test; n=57 platelets, means and 95% confidence intervals shown by bars). No significant differences were observed among patients. (b) TEM of platelet whole mounts from an unaffected parent (left) and Patient 1 (right), showing dense granules detectable as dark spots (bar=500 nm; × 25,000). (c) Dense granule counts derived from whole-mount TEM imaging of platelets from Patient 1 (P1), Patient 2 (P2) and a WASP- patient (WAS) were significantly lower than for cells from an unaffected relative (F1); Mann–Whitney test P<0.0008, n=50 platelets. Means for Patient 1 and WASP were not significantly different (P=0.07), while the mean for Patient 2 was significantly higher than for Patient 1 (P<0.0001). Means and 95% confidence intervals shown by bars.
Mentions: Thin section transmission electron microscopy (TEM) was used to examine platelets from the ARPC1B- patient, a WASP- patient and a normal donor. All had platelets containing typical cellular structures including mitochondria and α-granules (Figs 3a and 4). ARPC1B- and WASP- platelets showed a propensity towards small size (Fig. 4), and examination via IF microscopy (Fig. 3b) confirmed these platelets to be small and dysmorphic compared to normal. A comparison of circumferential tubulin ring diameters (Fig. 3c) confirmed that both ARPC1B- and WASP- platelets are significantly smaller than normal, and do not differ significantly from each other. ARPC1B- platelets can thus be classified as microthrombocytes, as can ARPC1B-deficient platelets from Patients 2 and 3 (Fig. 5a). A significant proportion (∼20%) of ARPC1B- platelets shared dysmorphic features with WASP- platelets (Fig. 3d,e) that included odd shapes, collapse/loss of circumferential microtubule coils and highly variable P-selectin and thrombospondin-1 content (both indicators of α-granules). As has been reported for WAS platelets28, whole-mount TEM29 revealed a reduction/absence of calcium-rich platelet dense granules in ARPC1B- and -deficient platelets (Fig. 5b,c). Clinical lumi-aggregometry analysis of platelets from Patients 1 and 2 confirmed decreased dense granule ATP release (0.16 and 0.19 nmol respectively; normal range 0.29–1.93 nmol). Platelet aggregation investigations with collagen, SFLLRN, arachidonic acid, ristocetin and ADP were normal for both patients.

View Article: PubMed Central - PubMed

ABSTRACT

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott&ndash;Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

No MeSH data available.


Related in: MedlinePlus