Neutrophil-Mediated Proteolysis of Thrombospondin-1 Promotes Platelet Adhesion and String Formation

Abstract

Thrombospondin-1 (TSP-1) is primarily expressed by platelets and endothelial cells (ECs) and rapidly released upon their activation. It functions in haemostasis as a bridging molecule in platelet aggregation, by promoting platelet adhesion to collagen and by protecting von Willebrand factor strings from degradation. In blood of patients undergoing surgery and in co-cultures of neutrophils with platelets or ECs, we observed proteolysis of the 185 kDa full-length TSP-1 to a 160-kDa isoform. We hypothesized that TSP-1 processing may alter its haemostatic properties. Selective enzyme inhibitors in co-cultures revealed that neutrophil proteases elastase and cathepsin G mediate TSP-1 processing. The cut site of cathepsin G was mapped to TSP-1 amino acids R237/T238 by Edman sequencing. Formation of neutrophil extracellular traps protected TSP-1 from complete degradation and promoted controlled processing to the 160-kDa isoform. Haemostatic properties were tested by platelet aggregation, adhesion, coagulation and string formation under flow. Platelets from TSP-1 deficient mice did not differ from wild-type in platelet aggregation but showed severe impairment of platelet adhesion to collagen and string formation under flow. Reconstitution experiments revealed that the 160-kDa TSP-1 isoform was markedly more potent than the 185-kDa full-length molecule in restoring function. Thus, TSP-1 processing by neutrophil proteases yields a 160-kDa isoform which shows enhanced potency to promote platelet adhesion and string formation. This finding reveals a novel mechanism of neutrophil-mediated thrombus formation and provides first evidence for the impact of TSP-1 proteolysis on its haemostatic properties.

Fig. 1

Thrombospondin-1 (TSP-1) isoforms circulating in human plasma as compared with TSP-1 proteins secreted by isolated platelets and endothelial cells (ECs). ( A ) Blood samples of two colorectal cancer patients were retrieved immediately before (pre) and 1 day after (post) resection of liver metastases and plasma was processed as previously described. ( B ) Human platelets were isolated from whole blood of healthy volunteers by size exclusion chromatography in the presence of 100 nM prostaglandin E1 (PGE ₁ ). They were then either left untreated (w/o), further inhibited with 100 nM PGE ₁ or activated with different concentrations A23187 (1–40 µM) for 30 minutes at 37°C before collection of the supernatant. ( C ) TSP-1 released into the culture medium of human dermal microvascular endothelial cells after 24 or 48 hours was analysed for confluent and untreated, tumor necrosis factor-α (TNFα) (100 ng/mL) or lipopolysaccharide (LPS) (1 µg/mL) stimulated cultures and compared with untreated sub-confluent (proliferating) ECs. Shown are representative immunoblots (Ab11) with reduced protein samples of culture supernatants (without further concentration or dilution). Please refer to Supplementary Fig. S1 (available in the online version) for quantitation of immunoblots. Experiments were repeated 3 to 4 times. M, biotinylated protein marker.

Fig. 2

Thrombospondin-1 (TSP-1) isoforms generated in co-cultures of vascular cell populations. Confluent endothelial cell (EC) cultures grown in serum-free medium for 48 hours were either left untreated (ECs) or were supplied with freshly isolated human ( A ) peripheral blood mononuclear cells (PBMCs), ( B ) platelets or ( D ) neutrophils. Where indicated (+) a stimulus was added to activate PBMCs (lipopolysaccharide [LPS] at 1 µg/mL), platelets (thrombin receptor activator peptide 6 [TRAP-6] at 10 µM) or neutrophils (phorbol myristate acetate [PMA] at 100 ng/ml). ( C ) Platelets were also combined with neutrophils or kept separately for control. Supernatant was collected after 0.5, 1, 2 and 4 hours of culture. Reduced protein samples were analysed by immunoblots with Ab11. Each experiment was repeated four times with blood drawn from different donors. Please refer to Supplementary Fig. S2 (available in the online version) for quantitation of immunoblots. M, biotinylated protein marker.

Fig. 3

Thrombospondin-1 (TSP-1) processing by neutrophil-derived proteases. Freshly isolated human neutrophils were added to ( A ) confluent, serum-free cultures of endothelial cells or ( B ) platelets isolated from the same donor and incubated for 30 minutes at 37°C in the absence or presence of elastase inhibitor II (2.1 mM) and/or cathepsin G inhibitor I (0.1 mM). ( C–E ) Serum-free endothelial cell (EC) supernatant containing 185 kDa TSP-1 (w/o) was incubated with the purified proteases ( C ) cathepsin G at 10 mU/mL or ( D ) elastase at 20 mU/mL for 30 minutes to 4 hours, or ( E ) was exposed to increasing concentrations of elastase (10–50 mU/mL) and cathepsin G (2–50 mU/mL) for 30 minutes at 37°C. ( F ) The 160-kDa TSP-1 proteins generated either by co-culture of ECs with neutrophils or by 30-minute elastase digest of EC supernatant were compared with the smaller TSP-1 isoform prevalent in human plasma post-surgery. TSP-1 protein was detected in supernatants by immunoblotting with Ab11. Experiments were performed at least three times with blood from different donors. Please refer to Supplementary Fig. S3 (available in the online version) for quantitation of immunoblots. M, biotinylated protein marker.

Fig. 4

Molecular characterization of the 160-kDa thrombospondin-1 (TSP-1) isoform. Serum-free endothelial cell (EC) supernatant was either left untreated (w/o) or was digested with elastase (20 mU/mL) or cathepsin G (10 mU/mL) for 30 minutes at 37°C. ( A ) TSP-1 was detected in reduced protein samples by immunoblotting with two distinct antibodies, specific for the N-terminal or the C-terminal domain of TSP-1. ( B ) Reduced and non-reduced protein samples were compared on a gradient gel (4–20%) and TSP-1 was immunostained with Ab11, a combination of three monoclonal antibodies covering N- and C-terminal epitopes. ( C ) Illustration of the cathepsin G cut site (arrow) in the TSP-1 protein sequence as determined by mass spectrometry and Edman sequencing refers to amino acid numbering of secreted TSP-1 (not including the signal peptide); asterisks mark the cysteines involved in inter-chain disulphide bonds for trimerization. TSP-1 cleavage by cathepsin G results in the release of the monomeric N-terminal HBD (25 kDa) and a trimeric C-terminal core fragment of 160 kDa chains. ( D ) Silver staining of a synthetic peptide (comprising TSP-1 amino acids 208–247, bold letters in panel C) without or with digest by cathepsin G (50 mU/mL for 45 minutes).

Fig. 5

Impact of neutrophil extracellular trap (NET) formation on neutrophil-mediated proteolysis of thrombospondin-1 (TSP-1). Co-cultures of neutrophils and platelets were left untreated or stimulated with 4 µM A23187 in the absence or presence of the NETosis inhibitor GSK484 at 2 mM. ( A ) Release of neutrophil deoxyribonucleic acid (DNA) was assessed by incorporation of Sytox Green dye and measurement of relative fluorescence units (RFUs) over 5 hours. ( B ) Elastase was evaluated by activity assay. Supernatant was retrieved after 0, 30, 60 and 120 minutes. ( C ) Occurrence of TSP-1 isoforms (upper panel) and citrullination of histone H3 (lower panel) was determined at 120 minutes by immunoblotting. ( D ) Co-cultures seeded on cover slips were left untreated (w/o), stimulated by 4 µM A23187 for NET formation or exposed to A23187 in the presence of 2 mM GSK484 to block NETosis. After fixation and permeabilization, cultures were stained for TSP-1 (red), the granulocyte marker CD66b (green) and DNA (blue). Scale bar: 20 µm. All experiments were repeated at least three times with cells isolated from different human donors. Immunoblots and fluorescence images depict one representative experiment, while DNA release and elastase activity data are given as mean and standard deviation of three independent experiments.

Fig. 6

Comparison of thrombospondin-1 (TSP-1) isoforms in promoting platelet adhesion and string formation on collagen under flow. Blood retrieved from wild-type (WT) or TSP-1 knockout (KO) mice was supplied with anti-GPIbβ antibody to fluorescently label platelets and was perfused over collagen-coated slides in an ibidi flow chamber for 7 minutes at 7 dyne/cm ² . TSP-1 KO blood was either left untreated or was substituted with 1 or 4 µg/mL of purified 160 or 185 kDa TSP-1 protein. ( A ) Representative microscopic images of the different treatment groups were taken after 7 minutes (under continuing flow). ( B ) The number of adherent platelet aggregates and ( C ) the mean length of formed platelet strings were analysed with Fiji software as outlined in Supplementary Fig. S7 ( available in the online version). Boxplots illustrate the data distribution of 5 to 9 independent experiments (representing blood from individual mice); statistically significant differences between groups were assessed by Mann–Whitney U test (SPSS 23.0).