Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: J Thromb Haemost. 2019 Mar 13;17(4):596–606. doi: 10.1111/jth.14407

Human Neutrophil Peptide-1 Inhibits Thrombus Formation Under Arterial Flow via Its Terminal Free Cysteine Thiols

Jenny K McDaniel 1,2,*,†, Mohammad S Abdelgawwad 2,*,†, Audra Hargett 3, Matthew B Renfrow 3, Khalil Bdeir 4, Wenjing Cao 2, Douglas B Cines 4, X Long Zheng 2,§
PMCID: PMC6443439  NIHMSID: NIHMS1011297  PMID: 30741476

Summary

Background:

Human neutrophil peptides (HNPs), also known as α-defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored.

Objective:

This study aims to determine the role of HNP-1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity.

Methods:

Anticoagulated whole blood from wild-type or Adamts13−/− mice was incubated with a fluorescein-conjugated anti-human CD41 in the presence of increasing concentrations of a synthetic HNP-1 and perfused over a collagen surface or a tumor necrosis factor (TNF)-α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet-decorated ULVWF strings were determined using the BioFlux microfluidic system.

Results:

HNP-1 inhibited the rate and final coverage of fluorescein-labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2) in a concentration-dependent manner; the anti-adhesive activity of HNP-1 depended on its terminal free cysteine thiols; HNP-1 (20 μM) also dramatically inhibited the formation of platelets-decorated ULVWF strings on TNF-α activated murine endothelial surface under arterial flow.

Conclusions:

Our results demonstrate for the first time an anti-platelet adhesion or anti-thrombotic activity of HNP-1; this activity depends on its terminal free thiols, likely affecting VWF-VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.

Introduction

Human neutrophil peptides (HNP) 1–3, known as α-defensins, play an important role in innate immunity against invading bacteria, fungi, and viruses, and are the most abundant contents in azurophilic granules of polymorphic neutrophils [1–4]. HNP 1–3 consist of 29–30 amino acid residues (2–5 kDa) with 6 cysteine residues forming similar disulfide bonds (e.g. CI-CVI, CII-CIV, and CIII-CV). They differ from each other primarily in their first residue [3]. HNP-1, 2, and 3 are released at high concentrations in situ when neutrophils adhere and are activated at sites of injury where they contribute to microbial killing, inflammation, and the innate immune response [2, 5, 6]. Plasma HNP 1–3 levels are dramatically increased in patients with severe infections such as septic meningitis [7] and also in non-infectious inflammatory conditions such as systemic lupus erythematous (SLE) [8, 9] and thrombotic microangiopathy [10]. However, the biological effects of the released HNP 1–3 on the neighboring host cells or in the blood stream under pathophysiological conditions are not fully understood.

Previous studies have suggested that HNP 1–3 may be thrombogenic, because they can enhance the interactions between fibrinogen/thrombospondin-1 (TSP1) and platelets, stimulate platelet activation and aggregation [11], and modulate the binding of tissue type plasminogen activator (t-PA) and plasminogen to fibrin and endothelial cells, thereby inhibiting fibrinolysis [12]. More recently, HNP 1–3 were shown to inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 under static conditions [13] and their plasma levels are significantly elevated in patients with acute thrombotic thrombocytopenic purpura [10, 14]. This study aims to further investigate the effects of HNP-1 on thrombosis formation under arterial flow. We demonstrate that HNP-1 inhibits platelet adhesion and aggregation on a collagen surface and endothelium- derived ultra large VWF under arterial shear.

Materials and Methods

Materials.

HNP-1 and its mutant derivatives (A, B, and C) (>98% purity) were synthesized via Peptide 2.0 (Chantilly, VA). Aliphatic HNP-1 was synthesized by replacing all cysteine residues in HNP-1 with an alanine plus N-terminal acetylation and C-terminal amidation (Biomatik, Wilmington, DE). The transformed murine endothelial cell line SVEC4–10 was purchased from ATCC (Manassas, VA) [15, 16]. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, penicillin, and streptomycin were purchased from Life Technology (Grand Island, NY). D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK), apyrase, prostaglandin E1 (PGE1), and N-ethylmaleimide (NEM) were purchased from Sigma (St. Louis, MO). Type 1 fibrillar collagen was from Chrono-log Corporation (Havertown, PA). FITC-conjugated anti-CD41 IgG was from ThermoFisher Scientific (Grand Island, NY). Microfluidic plates with high and low shear were from BioFlux Biosciences (Alameda, CA).

Blood collection:

Murine whole blood was obtained via cardiac puncture after mice anesthetized with ketamine and xylazine. The blood samples were anticoagulated with a thrombin inhibitor, PPACK (100 μM). PGE1 (10 μM) and apyrase (0.01 U/mL) were added to the blood samples to prevent in vitro activation of platelets prior to experimentation [17].

Platelet adhesion and aggregation on a collagen surface under flow:

The Bioflux-1000 microfluidic system (Fluxion Bioscience, Alameda, CA) was utilized in all experiments [18, 19]. Microfluidic channels on a high shear plate were coated with a type 1 fibrillar collagen (100 μg/mL) in 0.01M HCL for 10 minutes under low shear (5 dyne/cm2). The collagen was allowed to adhere to channels for 1 hour at room temperature. The channels were then washed and blocked with 0.5% BSA in phosphate-buffered saline (PBS) for 10 minutes at a medium shear (10 dyne/cm2). After incubation for 15–30 min with a FITC-conjugated anti-CD41 IgG (1:100 v/v) with varying concentrations of synthetic wild type HNP-1 or mutants of HNP-1 (mutant A, mutant B, and mutant C), the whole blood samples were perfused through the collagen-coated surface at arterial shear (100 dyne/cm2) for 80–100 seconds [17]. Fluorescent images were obtained every 3–4 seconds under an inverse fluorescent microscope equipped with a high-resolution digital camera (Fluxion Bioscience, Alameda, CA) during perfusion of channels.

Platelet adhesion and aggregation on activated endothelial surface under flow.

A transformed murine endothelial cell line SVEC4–10 that expresses VWF was cultured with DMEM with 10% fetal bovine serum (FBS) and 50 IU/mL of penicillin and streptomycin in a 10-cm culture dish at 37 °C with 5% CO2 and 95% room air. One day prior to each flow experiment, murine endothelial cells were seeded on the fibronectin (100 μg/mL)-coated microfluidic channels overnight and then treated with TNF-α (100 ng/mL) at 37 °C for 4 hours on the day of experiment. Adamts13−/− mouse whole blood was collected, processed, and fluorescein-labeled as described in the previous section. The whole blood samples were diluted (1:2) with Tyrode’s buffer and incubated with or without synthetic HNP-1 (20 μM) for 15 min. The blood samples were then perfused over the TNF-α activated endothelial surfaces at 5 dyne/cm2 initially for 15 sec to allow the first layer of platelets to adhere to the endothelial surface, and then at 20 dyne/cm2 for 120 sec. The image acquisition and processing were the same as those described in the previous section.

Immunofluorescent staining of platelets and VWF on murine endothelial cells.

The murine endothelial cells in the microfluidic channels were fixed with 4% paraformaldehyde in PBS for 10 min. After blocking with 1% BSA, the cells were incubated with Alexa 488-conjugated anti-CD41 IgG and Alexa594-conjugated anti-VWF IgG, and imaged under a confocal microscope as previously described [20].

Optical platelet agglutination assay:

The Chrono-log (model 700) aggregation system (Havertown, PA) was used to determine platelet agglutination or aggregation. Washed human platelets (450 μL) supplemented with purified VWF (10 μg/mL) were incubated with HNP-1 (0, 15, and 30 μM) in the absence or presence of ristocetin (1 mg/mL) in a Tyrode’s buffer. Various negative (VWF or HNP-1 alone) and positive (thrombin 0.5 mU) controls were included. Platelet agglutination or aggregation was determined by the rate of light transmission over time using the optical mode. The area under the curve (AUC) after the completion of the experimentation was plotted against each agonist using the GraphPad Prism 7 software.

Identification of free thiols in HNP-1 by LC-MS/MS:

Synthetic HNP-1 and purified HNPs were incubated with or without NEM (20 μM) for 4 hours. The sample was then purified with a spin gel filtration column to remove all salts and free NEM from the solution [17]. The peptides were analyzed with a monolithic silicon microchip-based electrospray source, the TriVersa™ Nanomate (Advion, Ithaca, NY), coupled to an Orbitrap Velos Pro mass spectrometer (LTQ FT, ThermoFisher Scientific, San Jose, CA). A full scan (200<m/z<2000) was acquired for ~1 min. Theoretical masses of unlabeled and NEM-labeled HNP-1 were determined by adding one NEM for each of the six free thiols and subtracting the loss of a hydrogen for any disulfide bond formations. Further characterization of the individual peptide species was performed by higher-energy collision-induced dissociation (HCD) with an isolation width of 10 m/z. Each HCD scan was acquired as an MS/MS spectrum (150<m/z<2000).

Data analysis:

The fluorescent intensity results (platelet adhesion and aggregation) were plotted against the time using Prizm7 GraphPad software. The means and standard error of the means (SEM) were shown for all results. Mann-Whitney test was performed to determine statistically significant differences between two groups.

Results

Microfluidic system is highly sensitive for assessing ADAMTS13-VWF-platelet function under flow.

A microfluidic system was employed to first investigate the role of ADAMTS13 in platelet adhesion and aggregation on a fibrillar collagen surface under flow. This flow system consists of a well plate coupled to a polymer-based layer that forms the structure of the microchannels. The polymer layer is bonded to a glass cover slip that serves as the bottom of the channel and imaging surface. This pattern is repeated to form up to 24 identical channels in one device (Fig. 1A, left panel). An airtight interface is attached to the BioFlux plate to allow tight pneumatic flow control with the BioFlux controller (Fig. 1A, right panel). A thrombin inhibitor, PPACK, was used as anti-coagulant to eliminate thrombin generation and activation of coagulation factors. This allows the assessment of platelet adhesion/aggregation on a collagen surface under shear without participation of the coagulation cascade. As shown, when anticoagulated blood from wild type or Adamts13−/− mouse was perfused over a fibrillar collagen-coated surface under arterial shear (~100 dyne/cm2), the rate of adhesion/aggregation (Fig. 1B) and total surface coverage (Fig. 1C) of fluoresceinated platelets from perfusion of Adamts13−/− whole blood were significantly higher than those of wild type blood (Suppl. Video 1). Additionally, recombinant human ADAMTS13 (10 μg/ml) added to wild type or Adamts13−/− mouse blood also dramatically reduced the rate of platelet accumulation (Fig. 1D) and the final surface coverage (Fig. 1E) of fluoresceinated platelets on the collagen surfaces under the same conditions. These results demonstrate that the microfluidic system is a highly sensitive tool for assessing perturbation of the ADAMTS13-VWF-platelet axis under arterial shear.

Fig. 1. ADAMTS13 inhibits platelet adhesion and aggregation on a collagen surface under flow.

Fig. 1.

Open in a new tab

A. The setup of microfluidic flow cells and parallel channels (1–2 and 3–4) (left) or one pneumatic flow chamber (right) in the BioFlux flow system. B and C. The rate of adhesion and accumulation over time and the surface coverage of fluoresceinated platelets at 80 seconds, respectively, after perfusion of wild type (WT) and Adamts13−/− (KO) murine whole blood through a fibrillar collagen-coated surface under shear (100 dyne/cm2). D and E. The rate of adhesion and accumulation over time and the surface platelet coverage at 80 seconds after perfusion of murine whole blood without (Ctrl) or with 10 μg/ml of human recombinant ADAMTS13 (rA13) under shear (100 dyne/cm2). The data in B and D are expressed as the means ± SD (n=3) and the average of two independent experiments (n=2), respectively.

HNP-1 inhibits platelet adhesion/aggregation on a collagen surface under flow.

When added to a sample of whole blood from wild-type mice, HNP-1 inhibited the rates of platelet adhesion/aggregation in a dose-dependent manner (Fig. 2A and 2B) as well as the final surface coverage (Fig. 2C and 2D) of murine platelets on the fibrillar collagen surface under arterial flow (100 dyne/cm2). HNP-2 and HNP-3 exhibited similar anti-adhesive activity under the same conditions (data not shown). These results suggest for the first time that HNP-1 to 3 may have anti-thrombotic activity under arterial shear.

Fig. 2. Synthetic HNP-1 inhibits adhesion and aggregation of wild-type murine platelets on a collagen surface under flow.

Fig. 2.

Open in a new tab

A and B. The rate of adhesion and aggregation of fluoresceinated platelets in whole blood from wild type mice over time on a fibrillar collagen surface under shear (100 dyne/cm2) in the absence (Ctrl) or presence of 15 and 30 μM of HNP-1, respectively. The data represent the means ± SEM of three independent experiments (n=3). C and D. The representative images of the final surface coverage of fluoresceinated platelets on the fibrillar collagen under arterial shear in the absence (Ctrl) or presence of 15 and 30 μM of HNP-1, respectively.

HNP-1 inhibits platelet adhesion/aggregation on a collagen surface under flow, independent of ADAMTS13.

To evaluate if anti-adhesion properties of HNP-1 involve ADAMTS13, we performed similar experiments using whole blood obtained from Adamts13−/− mice. When added to the whole blood, HNP-1 was able to inhibit platelet adhesion/aggregation on a fibrillar collagen surface under arterial shear (100 dyne/cm2) at even lower concentrations. For instance, at concentrations of 15–30 μM, HNP-1 reduced the rates and the final surface coverage of fluoresceinated platelets by ∼80–90% (Fig. 3 & Suppl. Video 2). These results suggest that HNP-1 can inhibit platelet adhesion/aggregation on a collagen surface under arterial flow independent of ADAMTS13 protease.

Fig. 3. HNP-1 inhibits platelet adhesion and aggregation on a collagen surface under flow, independent of ADAMTS13.

Fig. 3.

Open in a new tab

A and B. The rates of adhesion and aggregation of fluoresceinated platelets after perfusion of whole blood samples obtained from Adamts13−/− mice over time in the absence (Ctrl) or presence of 15 and 30 μM of HNP-1, respectively. The data represent the means and SEM of three independent experiments (n=3). C and D. The final coverage of fluoresceinated platelets on a fibrillar collagen surface after perfusion of the whole blood of Adamts13−/− mice in the absence (Ctrl) or presence of 15 and 30 μM of HNP-1, respectively.

HNP-1 inhibits the formation of platelet-decorated ultra large VWF strings on endothelial surface under flow.

To determine if HNP-1 is able to directly reduce the formation of ultra large VWF strings, we took advantage of an established murine endothelial cell line which was shown to synthesize and release ultra large VWF upon stimulation with TNF-α [16]. A monolayer of murine endothelial cells was cultured in the microfluidic channels (Fig. 4A & 4B), then stimulated with TNFα in a complete culture medium for 4 hours. The ultra large VWF released and anchored on the endothelial surface captured flowing platelets upon perfusion of whole blood from Adamts13−/− mice (Fig. 4C, upper). If endothelial cells were not stimulated with TNFα, no fluoresceinated platelets adhered to the surface under flow (Fig. 4C, lower). Consistent with the previous results, addition of HNP-1 (20 μM) to a murine whole blood sample dramatically reduced the final surface coverage of fluoresceinated platelets (Fig. 4D) and the rate of platelet adhesion/aggregation (Fig. 4E) under arterial flow (20 dyne/cm2) (Suppl. Video 3). The reduction of platelet adhesion/aggregation appeared to correlate with the decrease of ULVWF strings on activated endothelial surfaces as demonstrated by the immunofluorescent staining after fixation of endothelium in the microfluidic channels (Fig. 4F). Together, these results suggest that HNP-1 may directly inhibit the formation of ULVWF strings or platelet adhesion/aggregation on ULVWF strings under arterial flow.

Fig. 4. HNP-1 inhibits the formation of platelet-decorated ultra large VWF strings on endothelial surface under flow.

Fig. 4.

Open in a new tab

A. Schematic representation of the cell culture and treatment protocol, in which murine endothelial cells were first seeded on a fibronectin-coated microfluidic channel overnight. On the day of experiment, the murine endothelial cells were treated with or without TNF-α (100 ng/mL) in DMEM with 10% FBS at 37 °C for 4 hours. Whole blood obtained from Adamts13−/− mice labeled with FITC-conjugated anti-CD41 IgG in the presence or absence of an agent of interest was perfused over the endothelial cell surface at 5 dyne/cm for 15 sec, followed by 20 dyne/cm2 for 120 sec. B. Representative images of murine endothelial cell monolayers in the upper and lower channels prior to blood perfusion. C. The final surface coverage of fluoresceinated murine platelets from Adamts13−/− mice on the endothelial surface after being stimulated with TNFα (100 ng/mL) or without (Ctrl) for 4 hours. D and E. The formation of platelet-decorated ultra large VWF strings and the rate of platelet adhesion and accumulation, respectively, on TNFα-treated endothelial surfaces after perfusion of Adamts13−/− murine whole blood in the absence (Ctrl) or presence of HNP-1 (20 μM). The data in E represent the mean ± SEM of three independent experiments (n=3). Mann-Whitney test determined the statistical significance of the difference in platelet accumulation signals over time between two groups (p<0.0001). F. Confocal images demonstrate the adherent murine platelets (Alexa488 anti-CD41) and ULVWF (Alexa594 anti-VWF) on activated endothelial surface in the absence (Ctrl) or the presence of HNP-1 (20 μM) at the end of 2-min perfusion under flow (20 dyne/cm2).

HNP-1 does not inhibit ristocetin-induced platelet agglutination.

To determine if HNP-1 interferes with VWF and platelet interaction by binding either to the VWF-A1 domain, to platelet GP1b, or to both, we determined the effect of HNP-1 on ristocetin-induced platelet agglutination in the presence of purified VWF. As shown, isolated and washed platelets did not spontaneously agglutinate in Tyrode’s buffer over 5–7 min of incubation at 37 °C under a stirring condition (Fig. 5A); addition of thrombin (0.5 mU) activated platelets and induced robust aggregation (Fig. 5B). Addition of VWF (10 μg/mL) alone (Fig. 5C) or VWF (10 μg/mL) and HNP-1 30 μM (Fig. 5D) without ristocetin did not induce VWF-platelet agglutination. Ristocetin 1 mg/mL induced robust platelet agglutination (Fig. 5E), but addition of HNP-1 at the final concentrations of 15 μM (Fig. 5F) and 30 μM (Fig. 5G), which were shown to have dramatic effects on shear-induced platelet adhesion/aggregation to a collagen surface or endothelial ULVWF, did not inhibit ristocetin-induced VWF-platelet agglutination under the assay conditions.

Fig. 5. A. HNP-1 has no effect on ristocetin-induced platelet agglutination.

Fig. 5.

Open in a new tab

Washed human platelets were incubated at 37 °C with a Tyrode’s buffer (A), 0.5 mU/mL of thrombin (B), 10 μg/mL of purified VWF (C), 10 μg/mL of VWF and 30 μM of HNP-1 (D), 10 μg/mL of VWF and 1 mg/mL of ristocetin (E), 10 μg/mL of VWF and 1 mg/mL of ristocetin plus at final concentration of 15 μM (F) or 30 μM (G) of HNP-1. Platelet agglutination was determined by the percentage of light transmission as a function of time (min). Representative tracings are shown in A-G. The area under the curve (AUC), indicative of the degree of platelet agglutination/aggregation or amount of light transmission with each agonist added, is shown as the average of 2–3 independent experiments (H).

Free cysteine thiols in HNP-1 are required for anti-adhesion activity.

To further elucidate the potential mechanism underlying HNP-1 anti-platelet adhesion activity under shear, we determined the presence of free cysteine residues (i.e. free thiols) in HNP-1 by LC-mass spectrometry following N-ethyl maleimide (NEM) labeling as described previously [13]. There were 6 cysteines in HNP-1 that could potentially be labeled with NEM if they were not participating in disulfide bond formation [3]. HNP-1 predominantly accepted 3 or 4 molecules of NEM, but could also accept 6 NEM molecules at one time (Fig. S1), suggesting the presence of multiple free cysteine thiols in synthetic HNP-1.

To determine whether free cysteine thiols in HNP-1 were required for its anti-adhesion activity, we performed shear-induced platelet adhesion/aggregation assays using chemically modified HNP-1. As shown, NEM-treated HNP-1 completely lost its anti-platelet adhesion activity (Fig. 6A) as did an aliphatic HNP-1 in which all 6 cysteines had been replaced with alanines during the synthetic process (Fig. 6B). To try to identify the specific cysteine residues responsible for anti-platelet adhesion activity, 3 mutants of HNP-1 were utilized (Fig. 6C). Using similar flow experiments, the replacement of two N-terminal cysteine residues with a serine residue (i.e. Mutant A) (Fig. 6D) or two C-terminal cysteine residues with a serine residue (i.e. Mutant B) (Fig. 6E) did not alter the anti-adhesion activity of HNP-1. However, a replacement of two N-terminal cysteine residues and one C-terminal cysteine residue in HNP-1 (i.e. Mutant C) abolished its anti-adhesion activity (Fig. 6F). These results indicate that at least 3 terminal free cysteine thiols in HNP-1 may be required for the anti-platelet adhesion activity under arterial flow.

Fig. 6. Terminal free cysteine thiols are required for anti-thrombotic activity of HNP-1 under flow.

Fig. 6.

Open in a new tab

A and B. The rates of adhesion of fluoresceinated platelets after perfusion of a wild type murine whole blood in the absence (Ctrl) or presence of 15 μM of NEM-treated and aliphatic HNP-1, respectively. C. Amino acid sequences of the wild type (wt) and mutant derivatives (Ma, Mb, and Mc) of HNP-1, as well as the ribbon representation of the potential disulfide bond bridges in a native HNP-1. D, E, and F. The rates of adhesion and aggregation of fluoresceinated platelets after perfusion of a WT murine whole blood in the absence (Ctrl) or presence of 30 μM of Ma, Mb, and Mc, respectively. The data represent the mean and SEM of three independent experiments (n=3).

Discussion

The present study demonstrates that synthetic HNP-1 may be a potent inhibitor of platelet adhesion/aggregation onto a fibrillar collagen surface or activated endothelium under arterial flow. These findings were somewhat unexpected as our previous study demonstrates that HNP 1–3 inhibit the proteolytic cleavage of VWF by ADAMTS13 [13] and our initially hypothesis was that the addition of HNP 1–3 to a whole blood containing ADAMTS13 would enhance platelet adhesion/aggregation on a VWF-collagen surface under arterial shear owing to the expected reduction of VWF proteolysis by ADAMTS13. However, the opposite result was obtained reproducibly. Such a novel anti-platelet adhesion property of HNP-1 was independent of ADAMTS13, suggesting other mechanisms underlying HNP-1 action.

Additional experiments demonstrated that HNP-1 can dramatically inhibit platelet adhesion and aggregation on TNF-α activated endothelium under arterial flows. TNF-α stimulates the expression and release of ULVWF from cultured endothelial cells and in vivo [21–23]. Under arterial flow, soluble VWF multimers in the flowing blood or newly released endothelial ULVWF may undergo a conformational change, which exposes the free cysteine thiols; this results in the formation of new covalent disulfide bonds (-S-S-) through an interaction known as the VWF-VWF lateral association [24, 25]. The resulting ultra-long and ultra-thick VWF fibers enhance platelet adhesion and aggregation under flow [26, 27].

In the presence of HNP-1, the free thiols on VWF may be blocked, resulting in the reduction of ULVWF string formation and subsequent platelet adhesion/aggregation under arterial flow. Other mechanisms underlying HNP-1 action are possible, including activation of platelets prior to perfusion that may result in platelet exhaustion, inhibition of VWF A1 and platelet GP1 interaction, and inhibition of VWF and collagen binding, etc. However, our ristocetin-induced platelet agglutination assay demonstrates that HNP-1 does not inhibit nor enhance VWF binding to platelets; also, immunoassay results demonstrate that HNP-1 does not inhibit VWF binding to a fibrillar collagen immobilized on a microtiter plate (not shown); no platelet clumping or thrombocytopenia is observed after an incubation of whole blood with 30 μM of HNP-1 for 15–30 min (not shown). These results do not support alternative explanations as to how HNP-1 would inhibit platelet adhesion and aggregation under arterial flow.

We therefore conclude that synthetic HNP-1 inhibits platelet adhesion and aggregation on collagen surface or endothelial ULVWF, likely through interfering the VWF-VWF lateral association and the formation of ULVWF strings on activated endothelial surface under flow via blocking terminal free cysteine thiols. The findings suggest a potentially novel therapeutic strategy for arterial thrombosis. A similar thiol-mediated reductase activity toward VWF has been described in the literature with N-acetylcysteine [28, 29], C-terminal ADAMTS13 fragments [17], and complement factor H protein [30]. Further investigation of plasma concentrations of various forms of HNPs and their redox states under pathological conditions may shed light on how inflammation, neutrophil activation, and HNPs may impact hemostasis and thrombosis in vivo.

Supplementary Material

Supp VideoS1
Download video file (515.9KB, mov)
Supp VideoS2
Download video file (511.4KB, mov)
Supp VideoS3
Download video file (761.5KB, mov)
Supp figS1

Essentials.

  • Biological activity of human neutrophil peptide (HNP)-1 in hemostasis under physiological conditions is not fully understood.

  • HNP-1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow.

  • The anti-adhesion activity appears to depend on the terminal free thiols of HNP-1, which may inhibit VWF-VWF lateral associations.

  • Our results suggest a protective role and potential novel therapeutic use of HNP-1 for arterial thrombosis.

Acknowledgements

Authors thank Dr. David Ginsburg for providing us with Adamts13−/− mice. We also thank Dr. Prabha Nagareddy at the University of Alabama at Birmingham for providing murine endothelial cells line. This study was supported in part by grants from the National Institutes of Health (HL126724 and HL115187), Answering T.T.P. foundation, and institutional funds from the University of Alabama at Birmingham.

Footnotes

Conflict-of-interest statement

XLZ serves in the speaker’s bureau of Alexion and is a consultant for Ablynx/Sanofi, BioMedica, and Shire. XLZ is the founder of Clotsolution, Inc. at Birmingham, AL. All other authors declare no relevant conflict of interest associated with this work.

References

  • 1.Quinn K, Henriques M, Parker T, Slutsky AS, Zhang H. Human neutrophil peptides: a novel potential mediator of inflammatory cardiovascular diseases. Am J Physiol Heart Circ Physiol 2008; 295: H1817–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K, Bainton DF, Lehrer RI. Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 1985; 76: 1427–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Selsted ME, Harwig SS, Ganz T, Schilling JW, Lehrer RI. Primary structures of three human neutrophil defensins. J Clin Invest 1985; 76: 1436–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Rice WG, Ganz T, Kinkade JM Jr., Selsted ME, Lehrer RI, Parmley RT. Defensin-rich dense granules of human neutrophils. Blood 1987; 70: 757–65. [PubMed] [Google Scholar]
  • 5.Zhao L, Lu W. Defensins in innate immunity. Curr Opin Hematol 2014; 21: 37–42. [DOI] [PubMed] [Google Scholar]
  • 6.Kagan BL, Ganz T, Lehrer RI. Defensins: a family of antimicrobial and cytotoxic peptides. Toxicology 1994; 87: 131–49. [DOI] [PubMed] [Google Scholar]
  • 7.Panyutich AV, Panyutich EA, Krapivin VA, Baturevich EA, Ganz T. Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis. J Lab Clin Med 1993; 122: 202–7. [PubMed] [Google Scholar]
  • 8.Froy O, Sthoeger ZM. Defensins in systemic lupus erythematosus. Ann N Y Acad Sci 2009; 1173: 365–9. [DOI] [PubMed] [Google Scholar]
  • 9.Sthoeger ZM, Bezalel S, Chapnik N, Asher I, Froy O. High alpha-defensin levels in patients with systemic lupus erythematosus. Immunology 2009; 127: 116–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Cao W, Pham HP, Williams LA, McDaniel J, Siniard RC, Lorenz RG, Marques MB, Zheng XL. Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura. Haematologica 2016; 101: 1319–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Horn M, Bertling A, Brodde MF, Muller A, Roth J, Van Aken H, Jurk K, Heilmann C, Peters G, Kehrel BE. Human neutrophil alpha-defensins induce formation of fibrinogen and thrombospondin-1 amyloid-like structures and activate platelets via glycoprotein IIb/IIIa. J Thromb Haemost 2012; 10: 647–61. [DOI] [PubMed] [Google Scholar]
  • 12.Higazi AA, Ganz T, Kariko K, Cines DB. Defensin modulates tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells. J Biol Chem 1996; 271: 17650–5. [DOI] [PubMed] [Google Scholar]
  • 13.Pillai VG, Bao J, Zander CB, McDaniel JK, Chetty PS, Seeholzer SH, Bdeir K, Cines DB, Zheng XL. Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP. Blood 2016; 128: 110–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Staley EM, Cao WJ, Pham HP, Kim CH, Kocher NK, Zheng L, Gangaraju R, Lorenz RG, Williams LA, Marques MB, Zheng XL. Clinical factors and biomarkers predicting outcome in patients with immunemediated thrombotic thrombocytopenic purpura. Haematologica 2018; In press [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.O’Connell KA, Edidin M. A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells. J Immunol 1990; 144: 521–5. [PubMed] [Google Scholar]
  • 16.O’Connell K, Landman G, Farmer E, Edidin M. Endothelial cells transformed by SV40 T antigen cause Kaposi’s sarcomalike tumors in nude mice. Am J Pathol 1991; 139: 743–9. [PMC free article] [PubMed] [Google Scholar]
  • 17.Bao J, Xiao J, Mao Y, Zheng XL. Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol-Dependent Manner. Arterioscler Thromb Vasc Biol 2014; 34: 397–407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Conant CG, Schwartz MA, Nevill T, Ionescu-Zanetti C. Platelet adhesion and aggregation under flow using microfluidic flow cells. J Vis Exp 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Conant CG, Nevill JT, Zhou Z, Dong JF, Schwartz MA, Ionescu-Zanetti C. Using well-plate microfluidic devices to conduct shear-based thrombosis assays. J Lab Autom 2011; 16: 148–52. [DOI] [PubMed] [Google Scholar]
  • 20.Abdelgawwad MS, Cao W, Zheng L, Kocher NK, Williams LA, Zheng XL. Transfusion of Platelets Loaded With Recombinant ADAMTS13 (A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13) Is Efficacious for Inhibiting Arterial Thrombosis Associated With Thrombotic Thrombocytopenic Purpura. Arterioscler Thromb Vasc Biol 2018; 38: 2731–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.van der Poll T, van Deventer SJ, Pasterkamp G, van Mourik JA, Buller HR, ten Cate JW. Tumor necrosis factor induces von Willebrand factor release in healthy humans. Thromb Haemost 1992; 67: 623–6. [PubMed] [Google Scholar]
  • 22.Bernardo A, Ball C, Nolasco L, Moake JF, Dong JF. Effects of inflammatory cytokines on the release and cleavage of the endothelial cell-derived ultralarge von Willebrand factor multimers under flow. Blood 2004; 104: 100–6. [DOI] [PubMed] [Google Scholar]
  • 23.Claus RA, Bockmeyer CL, Kentouche K, Sieber MW, Oberle V, Kaufmann R, Deigner HP, Losche W. Transcriptional regulation of ADAMTS13. Thromb Haemost 2005; 94: 41–5. [DOI] [PubMed] [Google Scholar]
  • 24.Choi H, Aboulfatova K, Pownall HJ, Cook R, Dong JF. Shear-induced disulfide bond formation regulates adhesion activity of von Willebrand factor. J Biol Chem 2007; 282: 35604–11. [DOI] [PubMed] [Google Scholar]
  • 25.Chung DW, Chen J, Ling M, Fu X, Blevins T, Parsons S, Le J, Harris J, Martin TR, Konkle BA, Zheng Y, Lopez JA. High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion. Blood 2016; 127: 637–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Arya M, Anvari B, Romo GM, Cruz MA, Dong JF, McIntire LV, Moake JL, Lopez JA. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 2002; 99: 3971–7. [DOI] [PubMed] [Google Scholar]
  • 27.Bernardo A, Bergeron AL, Sun CW, Guchhait P, Cruz MA, Lopez JA, Dong JF. Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation. J Thromb Haemost 2004; 2: 660–9. [DOI] [PubMed] [Google Scholar]
  • 28.Chen J, Reheman A, Gushiken FC, Nolasco L, Fu X, Moake JL, Ni H, Lopez JA. N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice. J Clin Invest 2011; 121: 593–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Tersteeg C, Roodt J, Van Rensburg WJ, Dekimpe C, Vandeputte N, Pareyn I, Vandenbulcke A, Plaimauer B, Lamprecht S, Deckmyn H, Lopez JA, De Meyer SF, Vanhoorelbeke K. N-acetylcysteine in preclinical mouse and baboon models of thrombotic thrombocytopenic purpura. Blood 2017; 129: 1030–8. [DOI] [PubMed] [Google Scholar]
  • 30.Nolasco L, Nolasco J, Feng S, Afshar-Kharghan V, Moake J. Human complement factor H is a reductase for large soluble von Willebrand factor multimers--brief report. Arterioscler Thromb Vasc Biol 2013; 33: 2524–8. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp VideoS1
Download video file (515.9KB, mov)
Supp VideoS2
Download video file (511.4KB, mov)
Supp VideoS3
Download video file (761.5KB, mov)
Supp figS1
NIHMS1011297-supplement-Supp_figS1.tiff (16.8MB, tiff)

RESOURCES