Contributions
Abstract: O07
Type: Oral presentation
Session Topic: Inherited and acquired disorders of platelets
Presentation during EHA Scientific Conference on Bleeding Disorders:
On Thursday, September 15, 2016 from 09:00 - 10:30
Location: Rossini 1
Background
Platelet-type von Willebrand disease (PT-VWD) is a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα–von Willebrand factor (VWF) interaction and thrombocytopenia1. The bleeding tendency is considered to be due to thrombocytopenia and by the reduction of high-molecular-weight-VWF multimers consequent to the clearance of VWF-platelet complexes from the circulation but no conclusive evidence of this is available.
Aims
Aim of this work was to shed new light on the effects of the enhanced GPIbα-VWF interaction on proplatelet-formation and platelet function in PT-VWD.
Methods
We investigated megakaryocyte differentiation and proplatelet formation in PT-VWD culturing megakaryocytes from CD34+ cells obtained from peripheral blood of a PT-VWD patient expressing the M239V mutation2 and from bone marrow of a mouse model of PT-VWD expressing the G233V mutation3. Platelets from the PT-VWD patient were studied for aggregation and shape change by light transmission aggregometry; for αIIbβ3 expression and activation (PAC-1 binding), Ca2+ store release and α-granules secretion by flow cytometry; for δ-granules secretion by lumiaggregometry; moreover, platelet spreading on fibrinogen and VWF was assessed and Rap-1b activation (Rap-1b-GTP) and Src-kinase family phosphorylation were measured by Western blotting.
Results
Surface-bound VWF was detected on human PT-VWD megakaryocytes at early stages of differentiation, while only proplatelet-forming megakaryocytes from controls bound VWF. Murine PT-VWD megakaryocytes showed VWF-binding at low doses of ristocetin differently from WT mice. Human PT-VWD megakaryocytes formed long and branched proplatelets on different matrices including type I collagen that usually blocks proplatelet-formation, and showed impaired RhoA activation and myosin-light chain 2 phosphorylation triggered by collagen. Moreover, PT-VWD megakaryocytes migrated through a type I collagen matrix significantly more than megakaryocytes from healthy controls, confirming abnormal interaction with collagen. Bone marrow biopsy from the PT-VWD patient showed an increased number of extravascular platelets in bone marrow. Human platelet aggregation in response to different agonists was reduced and shape change was absent. Human and murine platelets showed defective PAC-1/JON-A binding, Ca2+ release and Rap-1b activation in response to collagen and convulxin, clues of defective αIIbβ3 activation. Platelet spreading was impaired and Src-family kinase phosphorylation was abnormal, suggesting defective αIIbβ3 -mediated outside-in signaling.
Conclusion
These results show a primary abnormality of megakaryocyte and platelet function in PT-VWD and demonstrate for the first time that αIIbβ3 activation and function in platelets are impaired, setting the basis for a full understanding of the bleeding tendency in PT-VWD.
References
1. Othman et al. J Thromb Haemost. 2016; 14:411.
2. Giannini et al. Haematologica. 2010;95:1021.
3. Suva et al. Am J Pathol. 2008;172:430
Abstract: O07
Type: Oral presentation
Session Topic: Inherited and acquired disorders of platelets
Presentation during EHA Scientific Conference on Bleeding Disorders:
On Thursday, September 15, 2016 from 09:00 - 10:30
Location: Rossini 1
Background
Platelet-type von Willebrand disease (PT-VWD) is a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα–von Willebrand factor (VWF) interaction and thrombocytopenia1. The bleeding tendency is considered to be due to thrombocytopenia and by the reduction of high-molecular-weight-VWF multimers consequent to the clearance of VWF-platelet complexes from the circulation but no conclusive evidence of this is available.
Aims
Aim of this work was to shed new light on the effects of the enhanced GPIbα-VWF interaction on proplatelet-formation and platelet function in PT-VWD.
Methods
We investigated megakaryocyte differentiation and proplatelet formation in PT-VWD culturing megakaryocytes from CD34+ cells obtained from peripheral blood of a PT-VWD patient expressing the M239V mutation2 and from bone marrow of a mouse model of PT-VWD expressing the G233V mutation3. Platelets from the PT-VWD patient were studied for aggregation and shape change by light transmission aggregometry; for αIIbβ3 expression and activation (PAC-1 binding), Ca2+ store release and α-granules secretion by flow cytometry; for δ-granules secretion by lumiaggregometry; moreover, platelet spreading on fibrinogen and VWF was assessed and Rap-1b activation (Rap-1b-GTP) and Src-kinase family phosphorylation were measured by Western blotting.
Results
Surface-bound VWF was detected on human PT-VWD megakaryocytes at early stages of differentiation, while only proplatelet-forming megakaryocytes from controls bound VWF. Murine PT-VWD megakaryocytes showed VWF-binding at low doses of ristocetin differently from WT mice. Human PT-VWD megakaryocytes formed long and branched proplatelets on different matrices including type I collagen that usually blocks proplatelet-formation, and showed impaired RhoA activation and myosin-light chain 2 phosphorylation triggered by collagen. Moreover, PT-VWD megakaryocytes migrated through a type I collagen matrix significantly more than megakaryocytes from healthy controls, confirming abnormal interaction with collagen. Bone marrow biopsy from the PT-VWD patient showed an increased number of extravascular platelets in bone marrow. Human platelet aggregation in response to different agonists was reduced and shape change was absent. Human and murine platelets showed defective PAC-1/JON-A binding, Ca2+ release and Rap-1b activation in response to collagen and convulxin, clues of defective αIIbβ3 activation. Platelet spreading was impaired and Src-family kinase phosphorylation was abnormal, suggesting defective αIIbβ3 -mediated outside-in signaling.
Conclusion
These results show a primary abnormality of megakaryocyte and platelet function in PT-VWD and demonstrate for the first time that αIIbβ3 activation and function in platelets are impaired, setting the basis for a full understanding of the bleeding tendency in PT-VWD.
References
1. Othman et al. J Thromb Haemost. 2016; 14:411.
2. Giannini et al. Haematologica. 2010;95:1021.
3. Suva et al. Am J Pathol. 2008;172:430