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Platelet functional alterations in a Bernard-Soulier syndrome patient with filamin A mutation.

Li J, Dai K, Wang Z, Cao L, Bai X, Ruan C - J Hematol Oncol (2015)

Bottom Line: Defects in filamin A (FLNA) gene could lead to low platelet counts and decreased surface expression of glycoprotein (GP) Ibα.On the other hand, abnormal responses to collagen, including the platelet aggregation, secretion, and GP VI signaling pathways, are associated with FLNA c.1582G > A mutation.Our findings confirm a central role for FLNA in platelet-adhesive functions.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, 188 Shizi Street, Suzhou, 215006, China. lijiaming007007@126.com.

ABSTRACT
Defects in filamin A (FLNA) gene could lead to low platelet counts and decreased surface expression of glycoprotein (GP) Ibα. Here, we report and investigate the FLNA genomic alteration of a case with Bernard-Soulier syndrome (BSS), a rare hereditary bleeding disorder caused by quantitative or qualitative abnormalities in the GP Ib-IX-V receptor. DNA sequencing analysis reveals the presence of a GP Ibα c.987G > A mutation and a FLNA c.1582 G > A mutation in this patient. Transient transfection studies show that GP Ibα c.987G > A mutation abolishes the surface expression of GP Ibα on the transfected CHO cells. On the other hand, abnormal responses to collagen, including the platelet aggregation, secretion, and GP VI signaling pathways, are associated with FLNA c.1582G > A mutation. Our findings confirm a central role for FLNA in platelet-adhesive functions. The interaction between FLNA and GP Ibα in platelets deserves to be investigated.

No MeSH data available.


Related in: MedlinePlus

The effect of GP Ibα c.987G > A mutation and FLNA c.1582G > A mutation on the platelet functions. a Platelet aggregation and secretion induced by collagen, convulxin (Cvx) and ADP. Aggregations of patient were expressed as the percentage change in light transmission. The levels of ATP release were expressed as the amount of ATP released (nmol). Traces were representative of at least 2 experiments. b Part of the GP Ibα gene sequence with the c.987G > A mutation and FLNA gene sequence with the c.1582G > A mutation. c Flow cytometric analysis and immunoblotting analysis of GP Ibα and GPIX expression on transfected CHO cells. (white curve) CHO cells incubated with specific MoAb against GP Ibα (SZ2) or GPIX (SZ1), (gray curve) CHO cells with vectors pcDNA 3.1(−) incubated with mouse IgG. WT means wild-type GP Ibα; MT means mutant GP Ibα. d The level and distribution of FLNA using a monoclonal antibody specific for the C-terminal FLNA. Fluorescent microscopy showed that FLNA was mostly found in a peripheral layer, which was similar with that of normal controls. These results were representative of at least 2 independent experiments. e Platelet signaling induced by convulxin (Cvx).Washed platelets in suspension were activated by Cvx (400 PM) in the absence of stirring. Tyrosine phosphorylation of Syk (Syk-P) and LAT (LAT-P) was assessed by immunoblotting with an anti–Syk-P and anti–LAT-P, respectively. These results were representative of at least 3 independent experiments. C means control; P means patient
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Fig1: The effect of GP Ibα c.987G > A mutation and FLNA c.1582G > A mutation on the platelet functions. a Platelet aggregation and secretion induced by collagen, convulxin (Cvx) and ADP. Aggregations of patient were expressed as the percentage change in light transmission. The levels of ATP release were expressed as the amount of ATP released (nmol). Traces were representative of at least 2 experiments. b Part of the GP Ibα gene sequence with the c.987G > A mutation and FLNA gene sequence with the c.1582G > A mutation. c Flow cytometric analysis and immunoblotting analysis of GP Ibα and GPIX expression on transfected CHO cells. (white curve) CHO cells incubated with specific MoAb against GP Ibα (SZ2) or GPIX (SZ1), (gray curve) CHO cells with vectors pcDNA 3.1(−) incubated with mouse IgG. WT means wild-type GP Ibα; MT means mutant GP Ibα. d The level and distribution of FLNA using a monoclonal antibody specific for the C-terminal FLNA. Fluorescent microscopy showed that FLNA was mostly found in a peripheral layer, which was similar with that of normal controls. These results were representative of at least 2 independent experiments. e Platelet signaling induced by convulxin (Cvx).Washed platelets in suspension were activated by Cvx (400 PM) in the absence of stirring. Tyrosine phosphorylation of Syk (Syk-P) and LAT (LAT-P) was assessed by immunoblotting with an anti–Syk-P and anti–LAT-P, respectively. These results were representative of at least 3 independent experiments. C means control; P means patient

Mentions: Patient’s platelet counts ranged between 20 and 30 × 109/L, and enlarged platelets were observed in her blood film. Platelet aggregation on ristocetin was . Collagen was decreased to induce platelet aggregation (0.5 μg/ml, 17 %; 1 μg/ml, 37 % versus 45 %, 70 % of controls). The GPVI-specific agonist, convulxin, was also decreased (400 PM, 10 %; 800 PM, 40 % versus 55 %, 70 % of controls). Platelets released 33–86 % less ATP than control platelets (Fig. 1a). The GP Ibα expression was practically undetectable on the platelet surface. DNA sequencing analysis revealed the presence of a new GP Ibα c.987G > A mutation in a homozygous state and a FLNA c.1582 G > A mutation in a heterozygous state (Fig. 1b).Fig. 1


Platelet functional alterations in a Bernard-Soulier syndrome patient with filamin A mutation.

Li J, Dai K, Wang Z, Cao L, Bai X, Ruan C - J Hematol Oncol (2015)

The effect of GP Ibα c.987G > A mutation and FLNA c.1582G > A mutation on the platelet functions. a Platelet aggregation and secretion induced by collagen, convulxin (Cvx) and ADP. Aggregations of patient were expressed as the percentage change in light transmission. The levels of ATP release were expressed as the amount of ATP released (nmol). Traces were representative of at least 2 experiments. b Part of the GP Ibα gene sequence with the c.987G > A mutation and FLNA gene sequence with the c.1582G > A mutation. c Flow cytometric analysis and immunoblotting analysis of GP Ibα and GPIX expression on transfected CHO cells. (white curve) CHO cells incubated with specific MoAb against GP Ibα (SZ2) or GPIX (SZ1), (gray curve) CHO cells with vectors pcDNA 3.1(−) incubated with mouse IgG. WT means wild-type GP Ibα; MT means mutant GP Ibα. d The level and distribution of FLNA using a monoclonal antibody specific for the C-terminal FLNA. Fluorescent microscopy showed that FLNA was mostly found in a peripheral layer, which was similar with that of normal controls. These results were representative of at least 2 independent experiments. e Platelet signaling induced by convulxin (Cvx).Washed platelets in suspension were activated by Cvx (400 PM) in the absence of stirring. Tyrosine phosphorylation of Syk (Syk-P) and LAT (LAT-P) was assessed by immunoblotting with an anti–Syk-P and anti–LAT-P, respectively. These results were representative of at least 3 independent experiments. C means control; P means patient
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496858&req=5

Fig1: The effect of GP Ibα c.987G > A mutation and FLNA c.1582G > A mutation on the platelet functions. a Platelet aggregation and secretion induced by collagen, convulxin (Cvx) and ADP. Aggregations of patient were expressed as the percentage change in light transmission. The levels of ATP release were expressed as the amount of ATP released (nmol). Traces were representative of at least 2 experiments. b Part of the GP Ibα gene sequence with the c.987G > A mutation and FLNA gene sequence with the c.1582G > A mutation. c Flow cytometric analysis and immunoblotting analysis of GP Ibα and GPIX expression on transfected CHO cells. (white curve) CHO cells incubated with specific MoAb against GP Ibα (SZ2) or GPIX (SZ1), (gray curve) CHO cells with vectors pcDNA 3.1(−) incubated with mouse IgG. WT means wild-type GP Ibα; MT means mutant GP Ibα. d The level and distribution of FLNA using a monoclonal antibody specific for the C-terminal FLNA. Fluorescent microscopy showed that FLNA was mostly found in a peripheral layer, which was similar with that of normal controls. These results were representative of at least 2 independent experiments. e Platelet signaling induced by convulxin (Cvx).Washed platelets in suspension were activated by Cvx (400 PM) in the absence of stirring. Tyrosine phosphorylation of Syk (Syk-P) and LAT (LAT-P) was assessed by immunoblotting with an anti–Syk-P and anti–LAT-P, respectively. These results were representative of at least 3 independent experiments. C means control; P means patient
Mentions: Patient’s platelet counts ranged between 20 and 30 × 109/L, and enlarged platelets were observed in her blood film. Platelet aggregation on ristocetin was . Collagen was decreased to induce platelet aggregation (0.5 μg/ml, 17 %; 1 μg/ml, 37 % versus 45 %, 70 % of controls). The GPVI-specific agonist, convulxin, was also decreased (400 PM, 10 %; 800 PM, 40 % versus 55 %, 70 % of controls). Platelets released 33–86 % less ATP than control platelets (Fig. 1a). The GP Ibα expression was practically undetectable on the platelet surface. DNA sequencing analysis revealed the presence of a new GP Ibα c.987G > A mutation in a homozygous state and a FLNA c.1582 G > A mutation in a heterozygous state (Fig. 1b).Fig. 1

Bottom Line: Defects in filamin A (FLNA) gene could lead to low platelet counts and decreased surface expression of glycoprotein (GP) Ibα.On the other hand, abnormal responses to collagen, including the platelet aggregation, secretion, and GP VI signaling pathways, are associated with FLNA c.1582G > A mutation.Our findings confirm a central role for FLNA in platelet-adhesive functions.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, 188 Shizi Street, Suzhou, 215006, China. lijiaming007007@126.com.

ABSTRACT
Defects in filamin A (FLNA) gene could lead to low platelet counts and decreased surface expression of glycoprotein (GP) Ibα. Here, we report and investigate the FLNA genomic alteration of a case with Bernard-Soulier syndrome (BSS), a rare hereditary bleeding disorder caused by quantitative or qualitative abnormalities in the GP Ib-IX-V receptor. DNA sequencing analysis reveals the presence of a GP Ibα c.987G > A mutation and a FLNA c.1582 G > A mutation in this patient. Transient transfection studies show that GP Ibα c.987G > A mutation abolishes the surface expression of GP Ibα on the transfected CHO cells. On the other hand, abnormal responses to collagen, including the platelet aggregation, secretion, and GP VI signaling pathways, are associated with FLNA c.1582G > A mutation. Our findings confirm a central role for FLNA in platelet-adhesive functions. The interaction between FLNA and GP Ibα in platelets deserves to be investigated.

No MeSH data available.


Related in: MedlinePlus