Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks

Abstract Background Ticks puncture the skin of their hosts and secrete saliva, containing antiplatelet proteins, into the blood. Here, we studied disagregin, a potent platelet‐inhibiting protein derived from the salivary glands of Ornithodoros moubata, an African soft tick. Whereas conventional αIIbβ3 antagonists contain an Arg‐Gly‐Asp (RGD) sequence for platelet integrin binding, disagregin contains an Arg‐Glu‐Asp (RED) sequence, hypothesizing a different mode of inhibitory action. Objectives We aimed to compare the inhibitory effects of disagregin and its RGD variant (RGD‐disagregin) on platelet activation and to unravel the molecular basis of disagregin‐αIIbβ3 integrin interactions. Methods Disagregin and RGD‐disagregin were synthesized by tert‐butyloxycarbonyl –based solid‐phase peptide synthesis. Effects of both disagregins on platelet aggregation were assessed by light transmission aggregometry in human platelet‐rich plasma. Whole‐blood thrombus formation was investigated by perfusing blood over collagen I with and without tissue factor at a high wall‐shear rate (1000 s−1) in the presence of disagregin, RGD‐disagregin, or eptifibatide. Results Disagregin showed inhibition of collagen‐ and ADP‐induced platelet aggregation with half maximal inhibitory concentration values of 64 and 99 nM, respectively. This resembled the complete antiaggregatory effect of eptifibatide. Multiparameter assessment of thrombus formation showed highly suppressed platelet adhesion and aggregate formation with both disagregins, in contrast to eptifibatide treatment, which incompletely blocked aggregation under flow. Fibrin formation under flow was delayed by both disagregin and RGD‐disagregin (P < .01) and eptifibatide (P < .05). Conclusions Both αIIbβ3‐blocking disagregins have a strong potential to suppress collagen‐tissue factor–mediated platelet adhesion, thrombus formation, and fibrin formation. Both disagregins can be seen as potential new αIIbβ3 inhibitors.


| INTRODUCTION
Ticks are hematophagous parasites that collect blood by puncturing the skin of their hosts. This vascular puncturing leads to blood exposure to vascular collagens, which induces primary hemostasis by platelet adhesion and aggregation to produce a platelet plug or thrombus, initially arresting the bleeding. 1 Interaction of integrin receptor αIIbβ3 with fibrinogen accomplishes the formation of platelet aggregates and thrombi. In addition, vascular puncturing also leads to blood contact with subendothelial tissue factor (TF), initiating secondary hemostasis, which via the extrinsic coagulation pathway triggers thrombin generation and fibrin clot formation to stabilize the platelet plug. 1 To suppress primary and secondary hemostatic pathways of their hosts to enable continuation of feeding, ticks secrete saliva that contains a rich mixture of antiplatelet and anticoagulant proteins. 2 Ticks of the Argasidae family are so-called soft ticks equipped with a soft, leathery shield. A prominent member of this family, Ornithodoros moubata, produces the protein disagregin in its salivary glands. 3 This 6.9 kDa protein acts as an antihemostatic agent by binding to the αIIbβ3 integrin on platelets and preventing platelet aggregation. 3 Upon activation (inside-out signaling), integrin αIIbβ3 is known to change from a closed to an open conformation, making its binding sites for fibrinogen accessible. Disagregin belongs to the disintegrins, which are nonenzymatic, 6-10 kDa, cysteine-rich proteins, originally described in viper venom. 4 Disintegrins generally expose an Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) sequence, allowing specific binding to the activated integrins, thereby inhibiting binding of fibrinogen to activated platelets. 3,[5][6][7][8][9][10][11][12][13][14] Interestingly, disagregin lacks an RGD sequence but instead contains an Arg-Glu-Asp (RED) sequence, suggesting a different mode of action. One of the clinically approved αIIbβ3 inhibitors is eptifibatide, a cyclic heptapeptide found in snake venom. 15 It contains a KGD sequence in which the lysine is modified into a homo-arginine that increases specificity for the receptor. 16 In this study, we investigated whether (i) a chemically synthesized peptide, based on the tick-derived integrin antagonist disagregin, interferes with platelet activation, and (ii) the action mechanism is unique due to the absence of an RGD sequence. Therefore, both native disagregin (referred to as disagregin) and an E15G variant (referred to as RGD-disagregin) were synthesized by solid-phase peptide synthesis and native chemical ligation. Subsequently, we revealed the effects of the synthesized proteins on human platelet adhesion, aggregation, and ensuing fibrin formation.

| MATERIALS AND METHODS
Full methods are described in the supplemental information.

| Molecular docking and molecular dynamics simulations
A homology model of disagregin was constructed by using the SWISS-MODEL WebServer 17 with bovine pancreatic trypsin inhibitor (BPTI K15R/R17D variant) in complex with human mesotrypsin serving as template (chain B of the PDB code 3P95). Molecular docking of disagregin and αIIbβ3 was conducted by using the protein-protein docking module implemented in the HADDOCK WebServer. 18 As in our previous work, 19 the binding pose of disagregin with αIIbβ3, which gives the lowest binding free energy (indicating the most thermodynamically favorable conformation) was selected for further investigation of binding and interaction between different disagregin variants (RED-, RGD-, [RAD], and AAA-disagregin) with the αIIbβ3 integrin. Then, these complexes were subjected to molecular dynamics (MD) simulations for 20 ns by using standard parameters and protocols as previously reported. 19,20 The binding free energy (BFE) between αIIbβ3 and the different disagregin variants was computed by applying the molecular mechanics/generalized Born surface area (GB model = 5) method. MD simulations and BFE calculations were performed by using the AMBER16 program, University of California, San Francisco.

| Light transmission aggregometry
Platelet-rich plasma (PRP) was collected by centrifuging whole blood at 240 g for 15 minutes. Platelet-poor plasma (PPP) was collected by centrifuging whole blood for 10 minutes at 2200 g twice.

| Shear-induced platelet adhesion and thrombus formation on a collagen-coated surface
Glass coverslips (24 × 60 mm, Thermo Fisher, Breda, The Netherlands) were degreased with 2M HCl in 50% ethanol and washed with dH 2 O.
The coverslips were then coated with a collagen I microspot (1 µL each, 50 µg/mL; Takeda Austria GmbH, Austria). 0.1% bovine serum albumin, 2 mM CaCl 2 and 1 U/mL heparin). After 2 minutes of stasis, a perfusion with rinse buffer was started to remove unbound label. Subsequently, representative bright-field and tricolor fluorescence images were taken. After the experiment, the bright-field and fluorescence images were blindly analyzed for parameters using FIJI software.

| Collagen/TF-induced formation of plateletfibrin thrombi in flowed whole blood
Clean and degreased coverslips were coated with 2 microspots During the flow, the blood was continuously recalcified with a coagulation mix consisting of 63 mM CaCl 2 , 32 mM MgCl 2 in modified HEPES buffer pH 7.45 via a y-shaped dual-inlet tube at a volume ratio of 10:1, as described. 27 Blood was perfused at a wall-shear rate of 1000 s -1 for 14 minutes. To evaluate the kinetics of thrombus and fibrin formation, bright-field and fluorescent microscopic images were taken, and bright-field images were taken from each microspot at 2-minute intervals. One representative image per time point was taken from both collagen I and collagen I/TF microspot to analyze parameters. Images were blindly analyzed for the parameters in Table S3.
Times to fibrin formation were measured in minutes and subtracted To compare all parameters between conditions, average values were uniformly scaled over a range from 0 to 10 and heat maps were generated, as appropriate. Using the scaled values, subtraction was conducted to visualize the difference between control and treatment. In heat maps with fibrin formation, times to first fibrin formation were subtracted from 14 minutes. 27

| Cell toxicity
Cell proliferation was measured in real time using the xCELLigence System (ACEA Biosciences, San Diego, CA, USA). EA.hy926 cells (15 000 cells per well) were seeded in a 96-xCELLigence-well plate.
After 24 hours, different compounds were added in the well and proliferation was followed for 6 days (144 hours).

| Statistical analysis
Data are presented as medians ± interquartile ranges. Statistical significance between groups was determined using a Kruskal-Wallis test with Dunn's correction for multiple comparisons.  Figure 1D).
The interaction between the Asp residue and the Ca atom was still observed in the simulation with RGD-disagregin.
To prove the importance of RED/RGD motif of disagregin for binding to αIIbβ3, we also generated complexes between RADand AAA-disagregin with αIIbβ3. For RAD-disagregin ( Figure 1E), only interactions between the Arg residue and Tyr190 and Asp232 of the αIIb domain were found, whereas the Ala residue could not establish any interactions with any residues of αIIbβ3.
The RAD motif was quite flexible during the simulation, resulting in losing the interactions between the Asp residue and the Ca atom. In the case of AAA-disagregin, the derived results demonstrated that the AAA motif cannot form any interactions with αIIbβ3 ( Figure 1F).
The BFEs of these disagregin variants (RED-, RGD-, RAD, and AAA-disagregin) with αIIbβ3 are in agreement with experimental

| Peptide synthesis
Disagregin and RGD-disagregin were successfully synthesized with Boc-based solid-phase peptide synthesis and native chemical ligation (Figure 2A, B). Due to oxidative folding problems with other variants, we chose to continue experiments with disagregin and RGD-disagegin. Purity was verified by HPLC analysis and mass spectrometry ( Figure 2C). HPLC chromatograms demonstrated a single UV absorbance peak for both proteins ( Figure S1). The measured monoisotopic mass of disagregin (6951.1) and RGD-disagregin (6879.4) corresponded well with their calculated monoisotopic masses, 6950.9 Da and 6878.9 Da, respectively ( Figure S2). The predicted structure of disagregin is shown in Figure 2D. and collagen (P < .01). The lower dose of RGD-disagregin (P < .01), but not of disagregin, significantly reduced platelet aggregation with collagen ( Figure 3D).

| Assessment of whole-blood platelet aggregation and thrombus formation under flow
To further investigate the observed antiaggregatory effect of disagregin, a multiparameter assay of collagen-dependent thrombus Glanzmann thrombasthenia, lacking integrin αIIbβ3, 28 or with leukocyte adhesion deficiency III syndrome, defective αIIbβ3 activation. 29 For this assay, citrate-anticoagulated blood was preincubated with disagregin, RGD-disagregin or eptifibatide, and subsequently recalcified in the presence of PPACK and flowed at a high shear rate (1000 s -1 ) over collagen I microspots. After 3.5 minutes of flow, bright-field and fluorescence microscopic images were collected to measure P-selectin expression (AF647-labeled anti-P-selectin mAb), platelet procoagulant activity (AF568annexin A5) and activated integrins by fibrinogen binding (FITClabeled fibrinogen mAb). Representative stills after 3.5 minutes are shown in Figure 4A. In control condition, medium aggregates were formed of contracted platelets, exposing P-selectin as well as PS, with activated integrins. In the presence of 1-µM disagregin or RGD-disagregin, the formation of multilayered thrombi and contracted thrombi was strongly suppressed (P < .05), resulting in single platelets adhesion. In contrast, the low concentration of both disagregins and 1 µM eptifibatide still showed the presence of multilayered and contracted thrombi ( Figure 4A, B, C). Both PS

F I G U R E 4
Effect of (RGD-)disagregin on microspot-induced whole-blood thrombus formation. Citrated blood was preincubated with disagregin or RGD-disagregin, recalcified with D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK), and then flowed through a microfluidic chamber at wall-shear rate of 1000 s -1 . After 3.5 minutes, thrombi were stained with AF647-labeled anti-P-selectin monoclonal antibody (mAb), AF568-annexin A5, and fluorescein isothiocyanate-labeled fibrinogen mAb. Eptifibatide was used as a reference integrin agonist. A, Representative bright-field and fluorescence microscopic images from collagen I microspots after 3.5 minutes of flow. Bars = 20 μm. Note dose-dependent reduction of platelet adhesion, platelet aggregation, P-selectin, phosphatidylserine exposure, and integrin activation (measured by fibrinogen binding) with indicated inhibitors. B, Decreased morphological score with various integrin inhibitors. C, Decreased contraction score with various integrin inhibitors. D, Decreased integrin activation with various integrin inhibitors. Data are presented as medians ± interquartile ranges. *P < .05 exposure and P-selectin expression were partially decreased in all conditions compared to control; however, it has not reached significance ( Figure 4A). In the presence of disagregin (1 µM), fibrinogen binding was no longer observed, indicative of abolished integrin activation (P < .05) ( Figure 4D). Table S4 shows the significant decrease in morphological score, multilayer score, contraction score, and integrin activation by the addition of disagregin and RGD-disagregin (Table S4).

F I G U R E 5
Similar impairments in platelet-fibrin thrombus formation by disagregin and RGD-disagregin. Citrated blood was prelabeled with 3,3′-dihexyloxacarbocyanine iodide, AF568-annexin A5, and AF647 human fibrinogen. Samples were preincubated with disagregin or RGD-disagregin, recalcified with CaCl 2 and MgCl 2 , and then flowed through a microfluidic chamber at a wall-shear rate of 1000 s -1 .
Eptifibatide was used as a reference integrin agonist. A, Representative bright-field and fluorescence microscopic images from collagen I/ tissue factor microspots after 10 minutes of flow. Bars = 20 μm. Note dose-dependent reduction of platelet adhesion, absence of platelet aggregation, and delay of fibrin formation with indicated inhibitors. B, Delayed time to first fibrin formation under flow with various integrin inhibitors. Data are presented as medians ± interquartile ranges. *P < .05, **P < .01

| Time-dependent assessment of platelet-fibrin thrombus formation under flow
Subsequently, we established how the disagregins affected flowdependent thrombus formation in the presence of coagulation triggered by TF, that is, when thrombin and fibrin are generated.
Therefore, a previously validated multiparameter test was used, in which whole blood is perfused at a high shear rate over collagen I and collagen I/TF microspots. Series of microscopic images were collected in real time to measure platelet deposition (DiOC 6 label), platelet procoagulant activation (AF568-annexin A5) and fibrin formation (AF647-fibrinogen label, with threshold setting above fibrinogen fluorescence). 27 In the control samples, at the 10-minute time point, large aggregates were formed of contracted platelets, showing massive PS exposure and fibrin formation ( Figure 5A). In the presence of 1 µM disagregin, only single platelets adhered, which did neither aggregate nor displayed PS exposure or fibrin formation formation than disagregin at the same dose (not significant). This was also observed by measurement of the times to first fibrin formation.
To quantitatively compare the effects of integrin inhibitors on all scaled parameters over time (Table S3), subtraction heat maps were constructed for the spots containing collagen I and collagen I/TF ( Figure 6A, B). As before, we distinguished between platelet adhesion parameters (P1-2), thrombus parameters (P3-6), and fibrin parameters (P7-9). 27 Remarkably, the lower dose of 100 nM disagregins only moderately reduced and delayed all parameters, with RGD-disagregin being slightly more effective than disagregin.
On the other hand, the higher dose of 1 µM disagregin or RGDdisagregin effectively suppressed all thrombus (and platelet aggregation) parameters, and inhibited fibrin formation on the TF microspots. Eptifibatide at 1 µM showed a similar inhibitory pattern but overall was less effective. Note that on collagen I only microspots, the amounts of fibrin formed were already low in F I G U R E 6 Time-dependent sustained effects of platelet-aggregation inhibitors on platelet, thrombus, and fibrin parameters under coagulating conditions. Citrated blood was preincubated with indicated concentrations of disagregin, RGD-disagregin, or eptifibatide. Blood perfusion was performed under continuous recalcification over collagen I microspots with or without tissue factor (TF) at a shear rate of 1000 s -1 . Data of thrombus parameters (P1-9) were obtained from real-time microscopic images taken at 2, 4, 6, 8, 10, 12, 14 minutes (t arrows). For coding, see Table S3. Mean values were univariate scaled per parameter (scale 0-10). Data of P1-9 without addition (control) were set at 0 for reference. A, Subtraction heat map after scaling representing effects of inhibitors on thrombus and fibrin formation without TF. B, Subtraction heat map after scaling representing effects of inhibitors on thrombus and fibrin formation with TF. Color scale from green to black to red: light green represents lower than control, black represents same as control. Time to fibrin formation (P9) subtracted. Data are means of n = 5 blood samples the absence of inhibitors. Taken together, these results indicated that RGD-disagregin is similarly active as disagregin in abrogating platelet adhesion, aggregation, and fibrin formation under flow conditions.

| Cell proliferation with the addition of different compounds
Cell proliferation studies were performed to determine if the disagregins have toxic effects on endothelial cells (EA.hy926) ( Figure S3).
In this cell line used, the disagregins did not show obvious toxic effects, in that proliferation of cells was maintained by the addition of disagregin, RGD-disagregin, or eptifibatide.

| DISCUSSION
In this study, the inhibitory effects of disagregin and RGD-disagregin were investigated by light transmission aggregometry and whole- Our in silico molecular docking and molecular dynamics simulations revealed that disagregin binds to αIIbβ3 in a similar way as eptifibatide. The RED/RGD sequence of disagregin and RGD-disagregin, respectively, interacts with residues from both the αIIb and β3 do- Cell proliferation studies pointed to the absence of strong toxic effects ( Figure S3). However, more toxicity research in other cell lines is necessary to conclude that in vivo studies can be performed without expected toxicity.
Research is necessary to further modify the reversible binding ability of disagregin to the αIIbβ3 integrin receptor and to design the best selective protein sequence for a dose-controllable antagonist.
A limitation of this study might be the use of labeled fibrinogen to detect fibrin in our experiments. However, we decreased the contribution of fibrinogen to the fibrin signal by threshold settings and bright-field images were used to confirm fluorescent fibers. 36,37 With labeled fibrinogen we prevent the underestimation of fibrin because of poor permeation of an antibody in the thrombus. 38 Taken together, we conclude that disagregin and RGD-disagregin are potential αIIbβ3 integrin inhibitors by inhibiting platelet adhesion, aggregation, and fibrin formation.

RELATIONSHIP DISCLOSURE
The authors declare no conflicts of interest.