- Platelets do express MHC Class I on their cell surface.
- In fact surface MHC Class I expression on platelets was observed decades back (1, 2).
- The problem? Is platelet MHC Class I self-synthesized (de novo synthesis) or acquired (adsorbed) from plasma (as part of cellular debris indiscriminately picked up by the platelet) (3, 4, 5, 6; figure below)?
- Platelets are anucleated fragments of megakaryocytes.
- For a long time, dogma stated absence of nucleus, i.e. being anucleated, meant platelets are incapable of de novo protein synthesis. As such, platelet protein synthesis was not an active area of research.
- A pity since recent research shows platelets are not just key for blood clotting but are involved in almost every facet of immune response.
Majority of platelet surface MHC Class I is indeed adsorbed from plasma (2, 7).
Early mechanistic studies in animal model studies also suggested platelet surface MHC Class I was not physiologically relevant.
- One, allogeneic (i.e. expressing different MHC haplotype) platelets did not stimulate cytotoxic CD8+ T cell (CTL) response in vitro (3).
- Two, allogeneic platelet transfusions suppressed in vivo skin graft rejections (4). Relevance for platelet surface MHC Class I? Decades of transplantation immunology studies implicate CTLs almost exclusively in skin graft rejections. Implies platelet MHC Class I inhibits, not activates CD8 T cells.
- Three, NK (natural killer) cell-derived interferon is the critical checkpoint that determines whether anti-MHC immunity against platelets will proceed or not (8).
OTOH, platelet-associated MHC Class I induced anti-MHC Class I antibodies (6, 9). Raises the question whether platelets drive immunity directly/indirectly (6; figure below).
Well, the answer’s taken decades to sort out but now a healthy handful of papers have shown that platelets
- Are capable of expressing self-synthesized (i.e. de novo) MHC Class I on their surface (human and mouse data).
- Express mRNA for MHC Class I and assorted molecules such as beta-2 microglobulin (b-2 m) and proteasomes. In other words, an intact molecular machinery necessary for MHC Class I synthesis, assembly and surface expression (human and mouse data).
- Present peptides derived from proteins expressed by both megakaryocytes and platelets (human data).
- Platelet MHC Class I expression is physiologically important for effective CTL responses (only mouse model data).
Let’s look at this data in sequence, and finally, consider the implications.
Platelets express self-synthesized MHC Class I on their surface as well as mRNA for MHC Class I and assorted molecules such as beta-2 microglobulin (b-2 m) and proteasomes
- Retain mRNA from megakaryocytes (10),
- Including HLA Class I mRNA, observed to be translated into metabolically labeled, i.e. functional, protein (11).
- Platelets also retain rough endoplasmic reticulum and polyribosomes.
- Even intact immunoproteasomes are found in human platelets (12, 13, 14, 15). Immunoproteasome is the organelle that specializes in generating peptides destined for presentation by MHC Class I (16).
- Thus, platelets retain capacity to synthesize some, if not all, the proteins synthesized by megakaryocytes.
- In particular, platelets contain the intracellular machinery necessary and sufficient to process, assemble and present fully functional MHC Class I+peptide on their surface (15; figure below).
Platelets present peptides derived from proteins expressed by both megakaryocytes and platelets
Comparison of peptides lodged inside MHC Class I molecules present on the surface of platelets derived from either healthy volunteers or those with ITP (Idiopathic Thrombocytopenic Purpura) showed (17; figure below) that they
- Were either derived from platelet-specific proteins, ubiquitous proteins found in almost every cell or in the case of ITP, proteins indicative of ongoing inflammation.
- Were different between healthy volunteers and ITP patients.
- Originated from the MHC Class I groove because they were of the correct length (8-9 amino acids) and had the appropriate anchor residues necessary to bind to the MHC haplotype of the platelet donors.
How could an anucleated fragment, a cell that lacks a nucleus, be capable of de novo protein synthesis, especially of so many immunologically relevant proteins?
- Turns out platelets specialize in novel post-transcriptional signaling mechanism called splicing pre-mRNA (18; figure below).
- In other words, the process of differentiation of the nucleated megakaryocyte (parent cell of the platelet) into the anucleated platelet is accompanied by specific enrichment of post-transcriptional and translational machinery necessary for de novo protein synthesis.
- Such studies open the door for discovery of other novel protein synthesis machinery in anucleated platelets. Dogma overturned indeed.
Another question arises at this point. Is platelet MHC class I expression physiologically relevant? Mouse model studies suggest yes, very much so.
- In an acute skin graft model, platelets increased T-cell graft infiltrates and rejection, and platelet-derived thromboxane was found critical for this process (19). Thromboxane is specific to platelets.
- Mouse model malaria study (20).
- Mice were either wild-type (i.e., normal and not genetically manipulated) or beta-2 microglobulin (b2-m)-null (i.e., genetically manipulated to delete b2-m).
- B2-m null cells do not express MHC Class I, i.e. cannot present antigens to cytotoxic CD8+ T cells (CTLs).
- Mice were infected with Plasmodium berghei (malaria parasite that infects mouse).
- Activated platelets were exposed to malaria antigens.
- Normal, but not b2-m null, platelets activated T cells in vitro.
- Mice treated with normal activated and antigen-exposed platelets cleared Plasmodium berghei with 100% survival. Mice treated with b2-m null platelets didn’t and succumbed to the malaria infection.
- Caveats to the study:
- Only observed mice for 7 days post-malaria infection.
- Does not conclusively exclude role for dendritic cell presentation of MHC Class I+peptide (small chance their enriched platelets were contaminated with dendritic cells).
Thus, a combination of human and mouse model data shows that
- Platelets process and present protein antigens in MHC Class I (human and mouse data).
- Platelets express costimulatory molecules known to be essential for T cell activation (human and mouse data).
- Platelets directly activate naive CD8+ T cells (CTLs) in a MHC Class I-dependent manner and induce their differentiation and cytokine production (mouse data).
- Trägårdh, L., et al. “Isolation and Properties of Detergent‐Solubilized HLA Antigens Obtained from Platelets.” Scandinavian journal of immunology 9.4 (1979): 303-314.
- Kao, K. J., D. J. Cook, and J. C. Scornik. “Quantitative analysis of platelet surface HLA by W6/32 anti-HLA.” Blood 68.3 (1986): 627-632.
- Gouttefangeas, Cécile, et al. “Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro.” Blood 95.10 (2000): 3168-3175.
- Aslam, Rukhsana, et al. “Transfusion‐related immunomodulation by platelets is dependent on their expression of MHC Class I molecules and is independent of white cells.” Transfusion 48.9 (2008): 1778-1786.
- Chow, L. “A novel mouse model demonstrating both antibody-and T cell-mediated thrombocytopenia: differential response to therapy.” Blood 115 (2010): 1247-1253.
- Semple, John W., Joseph E. Italiano, and John Freedman. “Platelets and the immune continuum.” Nature Reviews Immunology 11.4 (2011): 264-274.
- Kao, K. J. “Plasma and platelet HLA in normal individuals: quantitation by competitive enzyme-linked immunoassay.” Blood 70.1 (1987): 282-286.
- Sayeh, Ebrahim, et al. “IgG antiplatelet immunity is dependent on an early innate natural killer cell–derived interferon-γ response that is regulated by CD8+ T cells.” Blood 103.7 (2004): 2705-2709.
- Delaflor-Weiss, Eduardo, and Paul D. Mintz. “The evaluation and management of platelet refractoriness and alloimmunization.” Transfusion medicine reviews 14.2 (2000): 180-196.
- Newman, Peter J., et al. “Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction.” Journal of Clinical Investigation 82.2 (1988): 739.
- Santoso, Sentot, et al. “The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis.” British journal of haematology 84.3 (1993): 451-456.
- Yukawa, Masao, et al. “Proteasome and its novel endogeneous activator in human platelets.” Biochemical and biophysical research communications 178.1 (1991): 256-262.
- Yukawa, Masao, et al. “Purification and characterization of endogenous protein activator of human platelet proteasome.” Journal of biochemistry 114.3 (1993): 317-323.
- Klockenbusch, Cordula, et al. “Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets.” Molecular & Cellular Proteomics 13.12 (2014): 3308-3319.
- Zufferey, Anne, et al. “Characterization of the platelet granule proteome: evidence of the presence of MHC1 in alpha-granules.” Journal of proteomics 101 (2014): 130-140.
- Kloetzel, Peter-Michael, and Ferry Ossendorp. “Proteasome and peptidase function in MHC-class-I-mediated antigen presentation.” Current opinion in immunology 16.1 (2004): 76-81.
- Hopkins, Leann M., et al. “MHC Class I–Associated Peptides Identified From Normal Platelets and From Individuals With Idiopathic Thrombocytopenic Purpura.” Human immunology 66.8 (2005): 874-883.
- Weyrich, Andrew S., et al. “Protein synthesis by platelets: historical and new perspectives.” Journal of Thrombosis and Haemostasis 7.2 (2009): 241-246.
- Swaim, AnneMarie F., et al. “Platelets contribute to allograft rejection through glutamate receptor signaling.” The Journal of Immunology 185.11 (2010): 6999-7006.
- Chapman, Lesley M., et al. “Platelets present antigen in the context of MHC class I.” The Journal of Immunology 189.2 (2012): 916-923.