From blood coagulation to innate and adaptive immunity: the role of platelets in the physiology and pathology of autoimmune disorders

Blood flow forces platelets along the vessel wall, where they act as sentinels of vascular integrity. Disruption of the vessel wall results in exposure of extracellular matrix components such as subendothelial collagen, vitronectin, fibronectin, or laminin. Platelet glycoprotein complex GP Ib/IX/V and various surface integrins work as initial adhesive receptors and enable platelet binding at the site of an endothelial lesion. This binding initiates the adhesion, activation, and accumulation of platelets, which together is regarded as the first step of haemostasis. Platelets also contribute to the second step: the blood coagulation pathway [7]. Upon platelet activation, changes in the composition of the phospholipid bilayer of the plasma membrane occur. Unsaturated acyl chains are exposed on the outer leaflet. The platelet cell membrane bears a nett negative charge, and this accelerates the activation of factor X and prothrombin, by providing sites for the assembly of enzyme substrate complexes. Changes in the platelet cell membrane lead also to the generation of platelet-derived microparticles and a body of evidence points towards their role as initiators of thrombosis via the tissue factor pathway [8]. In mouse models, platelets were shown to become hyperactive after induction by microparticles derived from damaged endothelial cells [9]. This represents a positive feedback loop further enhancing the already increased coagulant activity of the platelets and putting them in the centre of thrombotic events.

However, a variety of pathological conditions, which are not strictly associated with vascular damage, manifest as a tendency towards thrombosis or as excessive bleeding [10]. This includes conditions as diverse as bacterial and parasite infections, malignant neoplasms, and autoimmune disorders. This observation has inspired a new route of research, investigating whether platelets should be regarded as immune cells [11].

An examination of platelet surface reveals it to be bristling with receptors. Quiescent platelets bear receptors intended to efficiently monitor vascular integrity. Upon activation, platelets upregulate other types of receptors, and shared with classic constituents of the immune system [12]. For example, toll-like receptors (TLRs) enable platelets to recognize pathogen- and damage-associated molecular patterns and immune complexes. TLRs also enable numerous interactions between platelets and leukocytes [12]. Ligand binging results in the initiation of intracellular signalling that converges upon significant cytoskeletal changes, involving the formation of pseudopodia and the opening of the canalicular system. These changes provide small-sized cells with a greater surface area, enable release of stored molecules, generation of microvesicles and microparticles and, most importantly, rearrangement of surface proteins [13]. Siglec receptors are involved in platelet apoptosis and down-regulation of the inflammatory response [14‐20].

Two of the platelet surface proteins should be discussed more thoroughly due to their role in predicting cardiovascular complications. Glycoprotein integrin αIIbβ3 (GPIIb/IIIa) is the most abundant platelet receptor with 50–80 thousand copies on the cell surface and an additional pool stored in the α granules [19]. An increase in the intracellular level of calcium leads to conformational changes of the molecule and subunit association, which only jointly form a functional complex. The surface density of the complex increases as well. This enables the cross-linking of plasma fibrinogen or von Willebrand Factor (vWF) and consequent platelet aggregation and intracellular communication, necessary for the recruitment of additional platelets to the site of vascular injury and thrombus formation. Platelet glycoprotein Ib alpha chain (GPIba), a component of the Glycoprotein Ib-IX-V receptor complex, is also up-regulated; it facilitates further platelet adhesion to the endothelium and supports intercellular signalling, also with neutrophils [21, 22]. P-selectin is present only on activated platelets (but also expressed on endothelial cells) and, therefore, serves as a marker of platelet activation. The counter ligand for P-selectin (PSGL-1) is a homodimeric mucin expressed on most leukocytes, but especially abundant on monocytes and neutrophils. The interaction is crucial for adaptive immunity as it induces leukocyte activation, integrin Mac-1 surface clustering (which enables other adhesion interactions between platelets and leukocytes), and neutrophil transendothelial migration. In addition, P-selectin enables the binding of complement C3b.

Together with the changes occurring in the composition and quantity of their surface proteins, activated platelets also release the contents of their granules. Platelets contain almost 4000 unique proteins, more than 300 of which have been detected in platelet releasates. Many of them are megakaryocyte-preformed and stored in one of the three types of granules: dense granules, alpha granules, or lysosomes. Dense granules contain mostly small molecules, such as adenosine diphosphate (ADP), ionised calcium, or serotonin (5-HT). Lysosomes encapsulate a variety of hydrolytic enzymes. Alpha granules contain a plethora of cytokines, whose immune functions range from unquestionably pro-inflammatory to serving as indispensable anti-inflammatory agents [23]. Table 1 presents some of the platelet releasates stored in alpha granules. It is unlikely that such a multitude of bioactive substances with contradictory effects is secreted randomly: it is currently believed that platelets are capable of stimulus-dependent packaging and release of granule content.

A secreted molecule that deserves particular attention in the context of autoimmunity is CD40L (also known as CD154). It is a membrane glycoprotein expressed by activated platelets [32]. Over a period of minutes to hours, surface-expressed CD40L is cleaved and released in soluble form (sCD40L). It is estimated that over 95% of sCD40L is of platelet origin [33]. Its binding to CD40 on endothelial cells triggers an inflammatory response, such as the release of leukocyte-attracting mediators [34]. It is involved in regulating T-cell function, the activation of dendritic cells, and the regulation of T-dependent antibody isotype switching; it also provides a novel mechanism for platelet autoactivation and the formation of homotypic platelet aggregates [35].

However, platelets are not just storage vesicles that degranulate upon activation. Recent studies revealed that the molecules present in platelets may come from sources other than megakaryocytes. They can be absorbed from the plasma or generated de novo [36]. Surprisingly, these anucleate cytoplasts possess functional spliceosome and transcriptome, accompanied by a range of ribonucleic acids and all the molecular machinery needed to autonomously produce proteins. This was a fascinating discovery, since spliceosome has never been described outside the nuclear boundaries [37]. Spliceosome allows platelets to synthesize proteins de novo in a stimulus-tailored manner. The process was well depicted for a potent pro-inflammatory factor interleukin 1-β (IL1-β) [38]. mRNA for IL1-β is one of the constitutive transcripts in unstimulated platelets [39]. It is located in the polysomes of resting and activated platelets. Accumulation of pro-IL1-β is sustained over hours after platelet activation and followed by processing the precursor into its mature, active form [37]. Integrated post-transcriptional control mechanisms regulate the initiation and resolution of inflammation and give explanation to the role of platelets in immune response and tissue injury [40].

A fascinating discovery revealed that platelets possess significant amounts of small non-coding RNA, of which around 80% accounts for microRNA (miRNA) [41]. miRNA molecules are thought to post-transcriptionally regulate the expression of over 60% of human genes. The miRNA present in platelets not only influences the functioning of the platelets themselves but also other immune cells, and can both restrain and promote autoimmunity [42]. For instance, miR-146a contributes to controlling the overproduction of cytokines, such as TNF-α, functions as a negative feedback control of innate immunity in TLR signalling and is critical for the suppressor functions of regulatory T lymphocytes. miR-155 promotes the development of pro-inflammatory Th17 and Th1 cell subsets [42].

Another sign of platelet activation is the generation of microparticles. A range of cells release these small membrane vesicles in both quiescent state and upon activation, but it is platelets that account for over 90% of plasma microparticles found in healthy individuals [9, 43]. Thanks to their size, platelet-derived microparticles (PMP) can easily infiltrate tissues and work as highly efficient carriers of bioactive molecules [44, 45]. They perform this task instead of activated platelets, which acquire binding properties that make it difficult for them to travel through the circulatory system. Microparticles may affect target cells by stimulating them directly via surface-expressed ligands or by transferring surface receptors, which has been reported for both physiological and malignant cells [46]. They modify recipient cell function also by the delivery of cytoplasmic proteins and miRNA [45, 47‐49]. They have been shown to increase the expression of adhesion molecules on endothelial and immune cells, enhance cytokine release, and consequently induce angiogenesis [50]. Moreover, PMPs are highly procoagulant and play important role in haemostasis and thrombosis [8, 51]. PMPs provide a surface for complement deposition and participate in complement activation [52, 53]. The fact that circulatory levels of PMPs are significantly elevated in infections, AD, cardiovascular disorders and other pathologic conditions points to their possible role as actual effectors of platelet immune functions [5].

Activated platelets have been shown to adhere to circulating neutrophils and via the release of pro-inflammatory ILβ1, HMGB1, PDGF recruit them to the site of vascular injury [55]. P-selectin expressed on the surface of both activated platelets and endothelial cells promotes the generation of reactive oxygen species, activation of β2 integrins and leukocyte tissue factor, release of pentraxin 3, and proteolytic enzymes [33]. Platelets have an important effect on neutrophils: the induce neutrophil-extracellular trap (NET) formation [56]. This is a form of cell autophagy, distinctive from apoptosis, which is based on the fusion of primary granules with nuclear membrane. It results in the destruction of neutrophil genetic material and its subsequent expulsion to the extracellular environment, where the NET engulfs pathogens. NETs reversely activate platelets, what may lead to a vicious loop of NET formation and platelet activation [57], resulting in tissue injury, prothrombotic phenotype, and propagation of autoimmunity [58]. Interactions with neutrophils also enable platelet extravasation and the targeting of other tissues, as reported in several autoimmune disorders [59]. Platelets support reactive oxygen species generation by monocytes and promote neutrophil oxidative burst and prothrombotic phenotype of monocytes [55, 60]. At the same time, platelet–neutrophil conglomerates enable transcellular synthesis of bioactive lipid compounds, including molecules required for inflammation resolution such as lipoxins, maresins, and resolvins [61, Fig. 1].

In healthy subjects, about 3% of circulating lymphocytes is bound to platelets. This number increases significantly upon platelet activation and homeostasis deregulation [62, 63]. The proportion varies between lymphocyte subpopulations and depends on the level of platelet activation and the form of stimulus [64]. Larger, activated lymphocytes are more prone to bind to platelets. Surface proteins P-selectin, GPIIb/IIIa, CD40, and CD11b all contribute to the interaction. The presence of the scavenger receptor CD36 on lymphocytes was recognized as proof of interaction with platelets [65].

The co-culturing of autologous platelets and CD4 + T cells enhanced the production of IL-10 and cytokines characteristic for Th1 and Th17 cells: IFNγ, IL-17. Moreover, the T cells were more prone to differentiate towards types Th1 and Th17, which are associated with autoimmunity [66, 67]. IL-17 is an independent predictor of vascular function in rheumatic diseases [68]. At the same time, platelet binding reduces the capability of lymphocytes to proliferate and produce Th1/Th17 cytokines [69]. This suggests that platelets may have a different effect on leukocytes depending on the stage of inflammatory response. Platelets alter immunoglobulin production also through direct contact with lymphocytes B. They are capable of delivering CD40L signalling that is crucial to immunoglobulin affinity maturation and isotype switching and T-cell-dependent humoral immune response [70]. The co-incubation of differentiated B cells with activated platelets was associated with increased in vitro production of IgG1, IgG2, and IgG3 [70]. Moreover, platelets were demonstrated to directly activate naïve T cells by presenting antigens in the context of MHCI and delivering co-stimulatory molecules [71].

Adhesive platelet–heterotypic cell interactions enable the process of transcellular production of bioactive lipids that act as autacoids [72]. Eicosanoids have gained recognition as regulators of two active, contrary processes: the evolution and resolution of inflammation. The fact that these molecules are unstable and highly bioactive first implied that they must be independently synthesized by different cells. However, only a few cell types are capable of performing the complete biosynthetic process of the limited range of eicosanoids due to lack of key enzymes. Cells must, therefore, share their enzymes and coordinate efforts to produce bioactive lipid compounds in the process of transcellular synthesis [73]. The omnipresence of platelets in blood makes them perfect candidates for this phenomenon. Indeed, platelets are unique cells equipped with a range of enzymes including several forms of phospholipase A2 (PLA2) and 12-lipooxygenase (12-LOX) [9].

Platelets participate in both intensifying inflammation and in its resolution. Upon activation, platelet membrane phospholipid bilayer composition undergoes changes, and substrates are made available for bioactive species production [74]. Platelets can autonomously generate some eicosanoids: thromboxane A2 (TXA2), 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyheptadecatrienoic acid (12-HHT), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), and isoprostanes. The molecules have generally proaggregatory and pro-inflammatory properties. Moreover, platelet–neutrophil transcellular biosynthesis of leukotrienes accounts for significant physiological changes associated with an ongoing inflammatory response [75]. Platelets may deliver both arachidonic acid (AA) and leukotriene A4 (LTA4) to neutrophils, as well as other leucocytes and endothelial cells. Prostaglandins have dual role: they initiate inflammatory responses [76], but also induce the transcriptional regulation of neutrophil 15-lipooxygenase (15-LOX). This is the first step in the class switch of lipid mediators [61, 77].

The resolution of inflammation is an active process initiated by a switch in the class of synthesized mediators, from the aforementioned thromboxanes, leukotrienes, and prostaglandins to endogenous pro-resolution lipid mediators: resolvins, protectins, maresins, and lipoxins. Lipoxins may be AA derivatives, but others are derived solely from dietary polyunsaturated fatty acids (PUFAs) [77]. Lipoxins themselves are produced in only three pathways, one of which is performed by platelet–neutrophil aggregates, whereby the neutrophils deliver LTA4, which is consequently transformed into lipoxin B4 (LB4) with platelet 12-LOX. LB4 has potent anti-inflammatory properties: it counter-regulates LT and cytokine production (including TNF), decreases vascular permeability, inhibits neutrophil tissue infiltration, and regulates platelet–neutrophil interactions [61]. These are also affected by ASA-triggered lipoxin (ATL), a molecule synthesized in a pathway enabled by acetylation of cyclooxygenase 2 (COX-2) enzymes [78]. Platelets deliver prostaglandin H2 (PGH2), a COX product, to endothelial cells which transform it into prostacyclin (PGI2). PGI2 works as a counterweight to TXA2. An imbalance between these two molecules may result in the formation of a prothrombotic phenotype [9].

Along with macrophages, monocytes and neutrophils, platelets transform omega-3 essential PUFAs to resolvins, protectins, and maresins. These molecules promote wound healing, deactivate leukotrienes, and attenuate nuclear factor NFkB [79]. They also intensify phagocytosis of apoptotic cells and phagocyte removal via lymphatic vessels. Their anti-inflammatory effect on platelets is based on reducing ADP-dependent platelet aggregation and redirection molecule production onto growth factors and pro-resolving mediators [77]. Platelet–neutrophil aggregates form maresin 1, a compound that is organ-protective [80], and influences blood cells by promoting a pro-resolving platelet phenotype and reposing macrophage functions [81, 82].

The platelet indices such as a mean platelet volume (MPV) and platelet distribution width (PDW) are currently under investigation as possible new indicators of platelet activation. Since they are routinely evaluated as a part of a complete blood count test, they could become a cheap and highly accessible measurement of platelet parameters. The hypothesis is based on the fact that upon activation platelets increase volume and form pseudopodia. Several research teams have attempted to correlate MPV with the RA, SSc, and SLE disease severity and cardiovascular risk; however, they have reported contradictory results [63, 90, 92, 128, 140‐142]. In SLE, smaller platelet size was proved to correlate with greater platelet activation, PMP formation, the presence of aCL antibodies, and secondary APS [143]. Changes to MPV have also been investigated in response to the RA treatment with anti-TNFα agent. A recent study showed that platelet count decreased and MPV increased at the end of the treatment period [144]. These discrepancies may be due to a magnitude of variables influencing MPV with age and sex being just the tip of the iceberg. There is an inverse correlation between platelet count and MPV and there are large interindividual differences between both the indices. MPV is also a parameter prone to change upon pre-analytical variabilities [145]. Due to MPV variability, one research team has identified PDW as a more specific marker of platelet activation of coagulation [146]. Even then, there are significant differences in measuring MPV and PDW among blood counters [147]. It remains controversial whether platelet indices correlate with the optical aggregometry results [148].

Another challenge in the interpretation of platelet indices in the systemic AD is the vast prevalence of atherothrombosis among patients. MPV is a well-established predictor of cardiovascular risk [149]. Increased MPV was associated with an acute myocardial infarction, ischemic stroke, and atrial fibrillation [150]. High MPV predicts a poor outcome of these events [151]. As rheumatic disorders are known to increase the risk of atherosclerosis and its complications, it cannot be excluded that increased MPV in AD reflects the existing subclinical atherosclerosis. Therefore, altered MPV value in AD patients could be an expression of an overlapping cardiovascular disease. At the same time, both systemic AD and chronic inflammatory diseases (including cardiovascular disease) are characterized by a high level of IL6. IL6 may stimulate the bone marrow to an increased release of younger, bigger platelets [152].

Future studies could investigate these issues further by designing research protocols including several different methods of platelet reactivity assessment. This includes well documented methods such as light transmission aggregometry, flow cytometry, and quantification of platelet-derived soluble proteins in blood plasma.