Figure 1.
Megakaryocyte differentiation and proplatelet formation.
(A) Megakaryocyte maturation stages of patients did not differ from those of controls. (B) Proplatelet formation (PPF) from patient megakaryocytes in suspension was drastically reduced. When megakaryocytes were plated on type I collagen proplatelet formation was absent, similar to controls, while on fibrinogen and von Willebrand factor the number of megakaryocytes extending proplatelets was normal. *p<0.05 vs control. (C) Representative pictures of proplatelet formation in suspension, in a control subject and a patient (20× magnification). Arrows indicate pro-platelets, only one developing proplatelet is evident in the patient sample. (D, E) Patient megakaryocytes extended a reduced number of proplatelets with abnormal characteristics: a spread shape with shorter than normal proplatelet shafts and tips significantly decreased in number and larger in size than those of controls. *p<0.05 vs control. (F) Representative images of megakaryocytes from patients and controls releasing proplatelets upon adhesion to fibrinogen.
Figure 2.
Megakaryocyte spreading on fibrinogen and αIIbβ3 expression.
(A) and (B) When plated on fibrinogen two populations of megakaryocytes are visible: half of the population spread regularly, while half showed abnormal spreading, with nuclei displayed towards cell periphery, a disordered distribution of actin and focal adhesion points more evident than stress fibres. (C) Flow cytometry showed decreased expression of αIIbβ3 on the surface of patient's megakaryocytes as compared with control megakaryocytes. *p<0.05 vs control.
Figure 3.
Integrin αIIbβ3 activation and outside-in signalling.
(A) Flow cytometry analysis of PAC-1 binding to resting megakaryocytes is significantly increased in patients as compared with controls. *p<0.05 vs control. (B) Western blotting showed Src and FAK phosphorylation in patient megakaryocytes in suspension as well as after adhesion onto fibrinogen. (C) Differently from control cells (upper panels), patient megakaryocytes showed FAK clustering already after 1 hour of adhesion onto fibrinogen, and also in suspension (lower panels).
Figure 4.
Conversion of preplatelets into mature platelets.
In patient's peripheral blood less “figure 8” shapes are present. Two “figure 8” shapes are circled in white in control blood (left), while no “figure 8” shapes are visible in this picture of patient peripheral blood (right). Arrows show examples of preplatelets.