Page 255 - 2021_03-Haematologica-web

Letters to the Editor
bin to mature collagen I, which then binds to platelet membrane and decorates the platelet surface. The exper- imental evidence relating to the expression and process- ing of collagen I is briefly presented here; the wider impli- cations these findings can have for PCOS patients and other metabolic conditions are the subject of a separate study.
Amino acid profiling revealed that platelets from PCOS patients contained significantly more hydroxyproline than platelets from non-PCOS subjects (Figure 1A), with no consistent differences in levels of the other amino acids evaluated (see Online Supplementary Table S1 for amino acid concentrations and Online Supplementary Figure S1 for the heatmap). Hydroxyproline is a proteino- genic amino acid that is produced by the hydroxylation of proline and a major component of fibrillary collagen,5,6 making it a reliable indicator of collagen levels. As platelets are known to respond to collagen,7 but not to generate it, different techniques were used to verify platelet expression of collagen. Using an assay based on the acid hydrolysis of samples to form hydrolysates and hydroxyproline, that is specific for collagen, it was possi- ble to confirm that platelets express collagen. Moreover, platelets from the PCOS collective expressed significantly more collagen than platelets from healthy individuals (Figure 1B). Mass spectrometry identified collagen I, par- ticularly the a1 and a2 subunits, as the most abundant isoform (Table 1). This fits well with the detection of mRNA encoding pro-collagen I a1 and a2 in human platelets (Figure 1C), implying that collagen I could be synthesized de novo by platelets. Western blotting con- firmed the expression of collagen in platelets and revealed that the platelet activation associated with PCOS increased platelet collagen I expression (Figure 1D), even though mRNA levels were comparable in platelets from the healthy and PCOS donors. The anti- collagen I antibody used did not bind to samples that had been pre-treated with collagenase, confirming the specif- ic binding of the antibody to collagen I (Figure 1E). Finally, immunohistochemistry of platelets fixed on poly- L-lysine confirmed the presence of collagen I on the sur- face of non-permeabilized platelets (Figure 1F).
Collagens are synthesized from pro-collagens that are secreted into the extracellular space where they undergo proteolysis to remove the N- and C-terminal extension peptides before being cross-linked and assembled into collagen fibrils.8 We next assessed whether platelets are able to release pro-collagen I and to process it into mature collagen I. Procollagen I was detectable in the supernatant of unstimulated platelets but levels increased significantly after stimulation with thrombin (Figure 2A) whereas platelet stimulation with the thromboxane A2 analog, U46619, exerted a much weaker effect. No signif- icant differences were observed between platelets from healthy and from PCOS subjects, suggesting the storage of similar amounts of pro-collagen in platelets from healthy and PCOS patients. These findings implied that the higher levels of mature collagen I detected in platelets from PCOS patients may be linked to the accelerated conversion of pro-collagen I to mature collagen I. Indeed, in the absence of a platelet agonist, pro-collagen I was only detected in Triton X-100 permeabilized platelets, i.e., pro-collagen I was stored and sequestered intracellu- larly. However, after stimulation with thrombin, pro-col- lagen was released to decorate the platelet surface and could be detected in the absence of Triton X-100 (Figure 2B). The finding that platelets express pro-collagen I mRNA and are able to release pro-collagen I protein in response to platelet agonists convincingly demonstrates
Table 1. Collagen isoforms detected in human platelets.
Protein name
Collagen alpha-2(I) chain
Collagen alpha-1(I) chain Collagen alpha-1(XII) chain Collagen alpha-1(III) chain
Gene name Peptides
COL1A2 5
COL1A1 2 COL12A1 1 COL3A1 1
Uniprot ID
P08123
P02452 H0Y5N9 P02461
that platelets are a novel source of pro-collagen I. Although the presence of enzymes involved in collagen biosynthesis in human platelets was reported more than 40 years ago,9 this study presents the first evidence of pro-collagen I release by platelets and the presence of mature collagen on the platelet surface.
To determine whether pro-collagen I could be convert- ed to collagen I upon platelet stimulation, platelets from healthy donors were stimulated with thrombin and levels of collagen were assessed by western blotting. This revealed a clear thrombin-induced decrease in pro-colla- gen I and increase in collagen I levels suggesting that col- lagen I was generated in response to platelet activation (Figure 2C). A number of different peptidases have been implicated in the generation of collagen,10 including bone morphogenic protein (BMP)-1 which is the main pro-collagen C-proteinase.11 There is no evidence of BMP- 1 expression in platelets (PlateletWeb database12) and the BMP-1 inhibitor; UK383367, failed to affect the throm- bin-induced increase in collagen I levels. These observa- tions suggested that an alternative protease could target platelet pro-collagen I, and as thrombin was suggested to cleave pro-collagen in cultured fibroblasts,13 collagen cleavage was studied in platelets stimulated with throm- bin or the thromboxane A2 analog. Thrombin and U46619 both elicit platelet activation; however, only thrombin led to the cleavage of pro-collagen and the appearance of a ~60 kD product recognized by the pro- collagen antibody (Figure 2D). To confirm this finding, a truncated recombinant pro-collagen a1 (~80 kD) that is a substrate for procollagen N- and C-proteinases was incu- bated with thrombin in vitro. This procedure resulted in the appearance of three additional peptides (~60, ~40 and ~28 kD), and was prevented by the thrombin inhibitor, hirudin (Figure 2E).
Taken together, the results of the present study demon- strate that platelets are a source of circulating collagen. Using different techniques, i.e., mass spectrometry, a spe- cific assay for total collagen, real-time-quantitative poly- merase chain reaction, immunoblotting and immunohis- tochemistry, it was possible to show that platelets express pro-collagen I mRNA and protein. The latter is secreted in response to platelet activation and can under- go thrombin-mediated proteolysis to generate mature collagen I that binds to the platelet surface. The platelet receptor that binds pro-collagen I was not characterized here, but procollagen I very likely attaches to platelets via a2β1 integrin.14 The functional consequences of the enhanced collagen I levels on the surface of the pre-acti- vated platelets from PCOS patients remains to be demon- strated. However, the collagen-coating on activated platelets may well act as an agonist for other cells, to link platelet activation with the enhanced atherothrombosis risk in these patients.15
Anastasia Kyselova,1,2 Sven Zukunft,1,2 Deborah Puppe,1 Ilka Wittig,2,3 W. Alexander Mann,4 Imke Dornauf,4
Ingrid Fleming1,2 and Voahanginirina Randriamboavonjy1,2
1Institute for Vascular Signalling, Centre of Molecular Medicine,
haematologica | 2021; 106(3)
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