Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles

Abstract

Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41(+), CD42b(+), and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P(-) and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41(+), CD42b(+), and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.

Figure 1

Megakaryocytes produce microparticles via an active, constitutive process. (A) Video-enhanced differential-interference contrast microscopy showing 2 examples of a mouse megakaryocyte forming microparticles in vitro. Small vesicles in linear arrays were observed extending from the surface of megakaryocytes. The microparticle at the tip of this process is 0.5 μm (). Scale bar represents 1 μm. (B) Surface blebs were visualized in unstained, living mouse megakaryocytes imaged by the RTM-3 in light modality using a 100× objective lens. This representative frame shows a membrane region that exhibited active surface blebbing. Scale bar represents 1 μm. (C) Thin-section electron micrograph showing the surface of a mouse megakaryocyte. Numerous small projections were observed emanating from the surface. The width of this field is 4 μm. (D) Thin-section electron micrographs showing blebs at various stages along the surface of a mouse megakaryocyte. The surface projections are approximately 300 nm in diameter.

Figure 2

Mouse megakaryocytes produce microparticles that express phosphatidylserine and multiple platelet markers on their surface. (A) Mouse megakaryocytes were cultured for 3 days and analyzed by flow cytometry. The middle gate (green) indicates particles with FSC and SSC characteristics of mouse platelets. The gate to the left (pink) indicates microparticles. The gate on the right (aqua) indicates megakaryocytes. (B) Microparticles isolated from megakaryocyte cultures were stained using anti-CD41 antibody and annexin V. (C) Microparticles isolated from megakaryocyte cultures were stained with either isotype-matched nonimmune IgG (control) or IgG directed at CD42b, GPVI, or α₂ integrin (immune). Error bars represent the standard deviation of 3 independent assays. (D) Megakaryocyte suspensions were analyzed for megakaryocytes (○) and microparticles (•) on days 3, 4, and 5 of culture. Values relative to the number of megakaryocytes and microparticles, respectively, on day 3 are indicated. Error bars indicate the standard deviation of 4 independent assays.

Figure 3

Mouse megakaryocytes cocultured with bone marrow endothelial cells produce microparticles. (A) Thin-section electron micrograph showing small projections and vesicles emanating from the surface of a megakaryocyte that has been cocultured with bone marrow–derived endothelial cells. Scale bar represents 2 μm. (B) Mouse megakaryocytes were incubated for 24 hours in the presence or absence of bone marrow endothelial cells. CD41⁺ microparticles within the supernatants were quantified by flow cytometry. Error bars indicate the standard deviation of 3 independent assays.

Figure 4

Microparticle production by megakaryocytes is a microtubule-independent process that is regulated by actin. Mouse megakaryocytes were cultured in the presence of vehicle alone (control), 0.001% DMSO, 1 μM nocodazole, 5 μM nocodazole, 1 μM latrunculin A, or 5 μM latrunculin A. Following an overnight incubation, supernatants were analyzed for CD41⁺ microparticles by flow cytometry. Values are reported as percentage of control compared with the number of microparticles detected in supernatants of megakaryocytes exposed to buffer alone. Error bars indicate the standard deviation of 4 independent assays.

Figure 5

Megakaryocyte-derived microparticles resemble plasma-derived microparticles with regard to surface markers and expression of full-length filamin A. (A) Microparticles were isolated from mouse platelets incubated with thrombin and collagen, cultured mouse megakaryocytes, or platelet-free plasma. Microparticles were subsequently labeled with nonimmune IgG, anti-CD62P antibody, or anti–LAMP-1 antibody as indicated. Error bars indicate the standard deviation of 4 independent assays. (B) Filamin A was assayed by immunoblot analysis in resting mouse platelets, mouse platelets exposed to thrombin plus collagen, and cultured megakaryocytes. Filamin A was also assayed by immunoblot analysis in microparticles isolated from activated platelets, cultured mouse megakaryocytes, or platelet-free mouse plasma. Full-length filamin A is found in megakaryocyte-derived microparticles and circulating microparticles, but not microparticles from activated platelets.

Figure 6

Analysis of CD41⁺ microparticles from human plasma demonstrates that the majority are CD62P⁻ and contain full-length filamin A. (A) Microparticles were isolated from human plasma, platelets activated with thrombin and collagen, or platelets activated by Ca²⁺-ionophore A23187 and analyzed for CD62P expression by flow cytometry. The percentage of CD62P⁺ (□) and CD62P⁻ (■) microparticles is indicated. Error bars indicate the standard deviation of 4 independent assays. (B) Filamin A was assayed by immunoblot analysis in resting human platelets, human platelets exposed to thrombin and collagen, microparticles isolated from platelet-free plasma, or microparticles isolated from activated platelets.

Figure 7

Microparticle generation from human megakaryocytes. (A) Thin-section electron micrograph showing the surface of an entire human megakaryocyte. Numerous small projections were observed emanating from the surface. Scale bar represents 10 μm. (B) Thin-section electron micrograph showing the megakaryocyte surface at higher magnification. Blebs at various stages are visualized along the surface of this human megakaryocyte. The surface projections are approximately 300 nm in diameter. Scale bar represents 1 μm. (C,D) Microparticles isolated from human megakaryocyte cultures were stained using anti-CD41 antibody and either annexin V or IgG directed at CD42b. This analysis demonstrates that the majority of human megakaryocyte-derived microparticles express PS on their surface and are CD42b⁺. Error bars indicate the standard deviation of 4 independent assays.