Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation

Abstract

Aims: In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor.

Methods and results: Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbβ3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation.

Conclusion: These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

Keywords: Aggregation; Endothelial nitric oxide synthase; Flow cytometry; Nitric oxide; Platelet subpopulations.

Figure 1

Identification of NO-producing and non/low-NO-producing Platelet Subpopulations. (A) Histogram overlay of unstained and DAF-FM-stained resting platelets (RP). (B) Platelet aggregometry traces and (C) histogram overlay of DAF-FM-stained unaggregated and collagen (10 μg/mL)-aggregated platelets. (D) Dot plots demonstrating a proportion of platelets remain low/non-NO-producers (Gate R5–middle panel) even when incubated with L-arg (30 μmol/L). FL-1 X = mean fluorescence. (E) Representative non-stained and DAF-FM-stained platelet fluorescence concentration histograms and summary data quantifying the percentage of low/non-NO producing to NO-producing platelets. N = 4. *P < 0.05. (F) Summary data of platelet DAF-FM flow cytometry confirming the specificity of DAF-FM for NO. The NOS inhibitor L-NAME reduces DAF-FM fluorescence, while SOD and a cell permeable SOD mimetic (TEMPOL) do not. N = 4. **, P < 0.01 vs. L-arg. All representative dot plots and histograms are from at least N = 3 independent experiments.

Figure 2

Platelet-derived NO Limits Aggregate Formation. (A) Representative traces and summary aggregation data of platelets stimulated by a high concentration of collagen (Coll 3 μg/mL) demonstrating that the NOS inhibitor L-NAME reverses the aggregation inhibitory effects of L-arginine. N = 5. *, P < 0.05. (B) Representative traces and summary aggregation data of platelets stimulated by a lower concentration of collagen (1 μg/mL) demonstrating that the selective iNOS inhibitor 1400 W fails to reverse the aggregation inhibitory effects of L-arginine. N = 3. P > 0.05. (C) Representative traces and summary aggregation data of platelets stimulated by low (0.01 U/mL) and high (0.03 U/mL) concentrations of thrombin demonstrating that platelet NO production inhibits aggregation at low thrombin concentrations. N = 5. *, P < 0.05.

Figure 3

Identification of eNOS-based Platelet Subpopulations. (A) Histogram overlay of CD42b-positive events (left histogram) further sub-gated (middle histogram) demonstrating eNOS^neg and eNOS^pos platelets (isotype control IgG–black; eNOS–red) and platelet eNOS fluorescence surface density (right histogram). (B) Summary data comparing percentage of eNOS^neg to eNOS^pos platelets. N = 10. ***P < 0.0001. (C) Confocal microscopy of eNOS^neg (small arrows) and eNOS^pos (large arrows) platelets. (D) (i-iii) confocal microscopy of eNOS within cardiac-derived human microvascular endothelial cells (HMVEC-C). Scale bars = 20 μm. All representative images from at least N = 3 independent experiments. (E) An immunoblot comparing eNOS in HMVEC and human platelets (Plt) from 3 donors. (F) (i) Representative platelet DAF-FM and eNOS^neg/eNOS^pos fluorescence histograms prior to and following FACS and (ii) summary data demonstrating that DAF-FM based sorting changes levels of eNOS^neg to eNOS^pos platelets. N = 3. *P < 0.05.

Figure 4

eNOS-negative platelets have a down-regulated sGC-PKG signalling pathway. (A) Summary data and representative histograms of sGC levels in eNOS-based platelet subpopulations, respectively. N = 4 independent experiments. *P < 0.05. (B) A comparison of cGMP generation by DAF-FM-negative vs. DAF-FM-positive FACS sorted platelets upon stimulation by collagen (10 μg/mL). N = 4. (C–D) Summary data and representative histograms of PKG, and VASP levels in eNOS-based platelet subpopulations, respectively. N = 5. *P < 0.05. Antibody specificity for platelet sGC, PKG, and VASP were confirmed in Supplementary material online, Figure S14A–F. (E) Representative histograms along with summary data measuring activated α_IIbβ₃ (PAC-1 antibody) on eNOS^neg and eNOS^pos collagen-aggregated platelets. N = 4 independent experiments. *P < 0.05 vs. eNOS^neg.

Figure 5

eNOS-negative platelets initiate adhesion and aggregation reactions. (A) Representative platelet eNOS^neg and eNOS^pos platelet flow cytometry histograms and summary data pre- and post flow chamber experiments. N = 4. *P < 0.05. (B) (i) Representative platelet aggregometry trace indicating time points at which DAF-FM-stained platelet samples were taken for fluorescence microscopy. (ii-iv) Merged brightfield-fluorescence microscopy images of non/low-NO-producing and NO-producing platelets binding to collagen strands (blue arrows) and each other (iii). Black arrows indicate platelets producing large amounts of NO. White arrows indicate platelets producing little/no NO. Images representative from N = 3 independent experiments. Bars indicate 10 μm.

Figure 6

Confirmation that non-NO-producing platelets initiate adhesion and aggregation reactions while NO-producing platelets form the bulk of an aggregate and limit its size. (A) Merged brightfield-fluorescence microscopy frames from Supplementary material online, video 1 of DAF-FM-loaded human platelets adhering to collagen-coated coverslips. White arrows indicate low/no-NO-producing platelets; black arrows indicate NO-producing platelets. Bars indicate 10 μm. (B) (i and ii) Summary data of low/none-NO-producing and NO-producing platelets binding to collagen over time from N = 3 independent experiments. *P < 0.05.

Figure 7

(A) (i) Representative platelet aggregometry trace indicating time points at which platelet releasates were taken for zymography analysis. (ii) Representative zymogram of the platelet MMP-2 secretion time course and summary densitometry data. N = 3 independent experiments. *P < 0.05. (B) Representative flow cytometer fluorescence surface density histograms and summary data of surface MMP-2 on collagen-activated (1 μg/mL) eNOS^neg and eNOS^pos platelets. N = 5. * P < 0.05. (C) Representative β-actin loading-controlled zymogram and summary data of platelet pro-MMP-2 within DAF-FM-negative and –positive FACS sorted platelet lysates. N = 4. * P < 0.05. (D) Representative intracellular flow cytometry fluorescence concentration histograms and summary data of COX-1 levels in eNOS^neg and eNOS^pos platelets. N = 5. * P < 0.05. (E) Summary LC-ESI-MS data of thromboxane generation by DAF-FM-negative and –positive FACS sorted platelets in response to collagen (10 μg/mL). N = 4. * P < 0.05.