Alessandro Malara

Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes

Summary.  Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. Methods: Human megakaryocytes were obtained by differentiation of cord blood‐derived CD34+ cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: We found that human megakaryocytes extended proplatelets in a time‐dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin αIIbβ3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb‐IX‐V, or upon cleavage of GPIbα by the metalloproteinase mocarhagin. Membrane‐associated VWF was detected exclusively on proplatelet‐forming megakaryocytes, but not on round mature cells that do not extend proplatelets. Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio‐temporal regulation orchestrated by adhesive proteins, GPIb‐IX‐V and myosin IIA.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

Megakaryocytes of patients with MYH9-related thrombocytopenia present an altered proplatelet formation

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations of MYH9, the gene for the heavy chain of myosin-IIA. Pathogenesis of thrombocytopenia of MYH9-RD is unknown. Recent studies in mice demonstrated that myosin-IIA is an inhibitor of proplatelet formation (PPF), and suggested that it could be involved in the suppression of PPF exerted by megakaryocyte adhesion to type I collagen, which regulates the timing of platelet release within bone marrow. However, the consequences on PPF of the heterozygous mutations causative of the MYH9-RD have never been investigated. We studied the in-vitro PPF by megakaryocytes obtained from four patients carrying the p.D1424N or the p.R1933X mutations. We demonstrated that MYH9-RD megakaryocytes completely lose the physiologic suppression of proplatelet extension exerted by interaction with type I collagen, thus supporting the hypothesis that a premature platelet release within bone marrow contributes to pathogenesis of MYH9-related thrombocytopenia. Moreover, proplatelets extended by MYH9-RD megakaryocytes presented a significant defect in branching in secondary processes (p=0.001) and formed a significantly lower number of proplatelet tips (p=0.005). Since platelets are assembled at the level of proplatelet tips, this defect could further contribute to pathogenesis of thrombocytopenia of MYH9-RD patients.

Megakaryocytes contribute to the bone marrow-matrix environment by expressing fibronectin, type IV collagen, and laminin

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin‐treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4‐fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5‐fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM‐producing cells for the establishment of cell niches during bone marrow regeneration. Stem Cells 2014;32:926–937

Proplatelet formation in heterozygous Bernard‐Soulier syndrome type Bolzano

Summary.  Background: Although mutations of GPIbα are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined. Objective: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIbα (Bolzano mutation). Methods: Human megakaryocytes were obtained by differentiation of patient cord blood‐derived CD34+ cells and peripheral blood‐derived CD45+ cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients’ blood platelets. Moreover, α‐tubulin distribution within proplatelets was severely deranged. Conclusions: Megakaryocytes from patients carrying a Bolzano allele of GPIbα display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIbα in proplatelet formation.

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control

Megakaryocytes are rare cells found in the bone marrow, responsible for the everyday production and release of millions of platelets into the bloodstream. Since the discovery and cloning, in 1994, of their principal humoral factor, thrombopoietin, and its receptor c-Mpl, many efforts have been directed to define the mechanisms underlying an efficient platelet production. However, more recently different studies have pointed out new roles for megakaryocytes as regulators of bone marrow homeostasis and physiology. In this review we discuss the interaction and the reciprocal regulation of megakaryocytes with the different cellular and extracellular components of the bone marrow environment. Finally, we provide evidence that these processes may concur to the reconstitution of the bone marrow environment after injury and their deregulation may lead to the development of a series of inherited or acquired pathologies.

Extracellular matrix structure and nano-mechanics determine megakaryocyte function.

Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2β1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

Megakaryocytes derived from patients with the classical form of Bernard-Soulier syndrome show no ability to extend proplatelets in vitro

To the editor Bernard-Soulier syndrome (BSS) is an inherited macrothrombocytopenia induced by mutations in glycoprotein (GP) Ib , GPIb or GPIX resulting in quantitative (classical BSS) or qualitative (variant BSS) abnormalities of the GPIb/IX/V complex that affect platelet-von Willebrand factor (vWF) interaction. Patients with homozygous or heterozygous biallelic mutations always present a severe form with greatly reduced platelet counts, giant platelets, defective ristocetin induced platelet aggregation and recurrent episodes of spontaneous bleeding, while subjects with monoallelic mutation have normal phenotypes [1]. The Ala156Val mutation in the GPIb (Bolzano mutation) represents an exception, in that in the monoallelic status induces a slightly reduced platelet count with platelet macrocytosis and mild bleeding tendency [2]. While the altered GPIb -vWF interaction explains the platelet functional defects in BSS, new insights need to be addressed to explain the mechanisms that lead to macrothrombocytopenia. However, we recently investigated in vitro megakaryopoiesis in six subjects with monoallelic Bolzano mutation and observed that megakaryocyte (Mk) differentiation and maturation were similar to controls, while proplatelet formation was severely affected. Thus, we concluded that the GPIb functional defect induced by Ala156Val monoallelic mutation affects in vitro platelet release [2]. We are now reporting that a severe defect of proplatelet formation is also responsible for thrombocytopenia in patients with classical forms of BSS deriving from homozygous mutations in GPIb or GPIX. Three adult patients from two unrelated families have been investigated. Patient 1 is a 30 year old woman who suffered from recurrent epistaxis, ecchymosis, and gum bleedings since childhood. She received transfusions of both red blood cells and platelets for menorrhagia until estroprogestinic therapy was started. Platelet concentrates were also given at each dental prophylaxis and extractions. Patients 2A and 2B are brothers born from parents who are second cousins. Thrombocytopenia was incidentally detected in patient 2A when he was 2 years old and experienced a severe epistaxis after a mild nasal trauma. A complete blood cell count performed in the brother (patient 2B) revealed that he was similarly affected. Both had a mild bleeding tendency with easy bruising and sporadic gum bleedings. Patient 2A received prophylactic platelet transfusion before dental extraction. Laboratory investigations [3, 4] revealed manual platelet counts and mean platelet diameters ranging between 38 and 52 10/L and 3,7 and 4,1 mm, respectively. Platelet aggregation induced by ristocetin 3 mg/ml was absent in patient 1 and severely reduced in both patients 2A and 2B (7 and 11%) while it was normal after ADP 5 mm and collagen 4 mg/ml (data not shown). In all the patients the components of the GPIb/IX/V complex were lower than 10% of control while the expression of GPIIb-IIIa was normal (data not shown). Mutation screening of GP1BA,

Constitutively released adenosine diphosphate regulates proplatelet formation by human megakaryocytes

Background The interaction of adenosine diphosphate with its P2Y1 and P2Y12 receptors on platelets is important for platelet function. However, nothing is known about adenosine diphosphate and its function in human megakaryocytes. Design and Methods We studied the role of adenosine diphosphate and P2Y receptors on proplatelet formation by human megakaryocytes in culture. Results Megakaryocytes expressed all the known eight subtypes of P2Y receptors, and constitutively released adenosine diphosphate. Proplatelet formation was inhibited by the adenosine diphosphate scavengers apyrase and CP/CPK by 60-70% and by the P2Y12 inhibitors cangrelor and 2-MeSAMP by 50-60%, but was not inhibited by the P2Y1 inhibitor MRS 2179. However, the active metabolites of the anti-P2Y12 drugs, clopidogrel and prasugrel, did not inhibit proplatelet formation. Since cangrelor and 2-MeSAMP also interact with P2Y13, we hypothesized that P2Y13, rather than P2Y12 is involved in adenosine diphosphate-regulated proplatelet formation. The specific P2Y13 inhibitor MRS 2211 inhibited proplatelet formation in a concentration-dependent manner. Megakaryocytes from a patient with severe congenital P2Y12 deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. Conclusions This is the first demonstration that adenosine diphosphate released by megakaryocytes regulates their function by interacting with P2Y13. The clinical relevance of this not previously described physiological role of adenosine diphosphate and P2Y13 requires further exploration.