Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events

Abstract

Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.

Figure 1

Megakaryocytes and platelets differentially express MMP transcripts. mRNA was collected from megakaryocytes at the stage of proplatelet formation or freshly isolated, unactivated platelets and processed as described in “mRNA expression.” The left side of the figure shows RNA-seq analyses for MMP-1, MMP-2, MMP-3, and MMP-14 mRNA. Matched sequences are aligned to each target gene using the Integrated Genome Browser (IGB). Gene maps (bottom portion of each panel, oriented 5′-3′ direction) are represented by thick (exons) and thin (introns) lines. The bars above the gene (top portion of each panel) represent megakaryocyte or platelet transcripts that were fragmented, sequenced, and aligned to MMP-1, MMP-2, MMP-3, and MMP-14. If no bars are shown, then transcripts for these mRNAs were not detected under the conditions of these experiments. The right side of the figure shows semi-quantitative PCR analyses of mRNAs for MMPs 1-3 and MMP-14. From left to right, each lane represents: (1) amplification of DNA, which served as a positive control for primer efficiency; (2) DNA sample treated with DNase; (3) amplification of cDNA that was reverse transcribed from platelet (Plt) mRNA; (4) control reaction in platelet preparations that screened for residual genomic DNA contamination. In these studies, the reverse-transcription step was not performed (NoRT). (5) Amplification of cDNA that was reverse transcribed from megakaryocyte (Meg) mRNA; (6) negative control lane where the PCR was carried out without cDNA template (Blank). The PCR gels are representative of 5 or more independent experiments. The boxes on the right of each panel represent where the amplicons will migrate based on primer sequences specific for each exon.

Figure 2

Megakaryocytes and platelets differentially express TIMP transcripts. mRNA was collected from megakaryocytes at the stage of proplatelet formation or freshly isolated, unactivated platelets and processed as described in “mRNA expression.” The left side of the figure shows RNA-seq analyses for TIMPs 1-4. Matched sequences are aligned to each target gene as described in Figure 1. The right side of the figures shows semi-quantitative PCR analyses of mRNAs for TIMPs 1-4. The lanes for these PCR gels are labeled as described in Figure 1. Each PCR gel is representative of 5 or more independent experiments. The boxes on the right of each panel represent where the amplicons will migrate based on primer sequences specific for each exon.

Figure 3

Platelets differentially express and release MMP and TIMP proteins. MMP and TIMP protein expression was analyzed as described in “Protein expression.” (A) Intracellular or released protein for MMPs 1 and 2 in unactivated or thrombin-activated (0.1 U/mL, 60 minutes) platelets. The bars represent the mean ± SEM of 5 or more independent experiments. (B) Intracellular or released TIMP-1 and TIMP-3 protein in unactivated or thrombin-activated (0.1 U/mL, 60 minutes) platelets. The bars represent the mean ± SEM of 5 or more independent experiments.

Figure 4

Activated platelets synthesize TIMP-2 protein. (A) Intracellular or released TIMP-2 protein in unactivated or thrombin-activated (0.1 U/mL, 60 minutes) platelets. The bars represent the mean ± SEM of 8 independent experiments. (B) TIMP-2 protein that is secreted into the supernatants of thrombin-activated platelets over 16 hours. The bars represent the mean ± SEM of 8 independent experiments. (C) Incorporation of [³⁵S]methionine/cysteine into TIMP-2 protein (intracellular and secreted) in thrombin-activated platelets. The bars, which display the mean ± SEM of 5 independent experiments, represent the fold increase over baseline of [³⁵S]methionine/cysteine that incorporates into TIMP-2 protein.