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

Danique L. van den Kerkhof; Magdolna Nagy; Kanin Wichapong; Sanne L. N. Brouns; Johan W. M. Heemskerk; Tilman M. Hackeng; Ingrid Dijkgraaf

doi:10.1002/rth2.12466

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

Danique L. van den Kerkhof, Magdolna Nagy, Kanin Wichapong, Sanne L. N. Brouns, Johan W. M. Heemskerk, Tilman M. Hackeng, Ingrid Dijkgraaf^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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 alpha IIb beta 3 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-alpha IIb beta 3 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 alpha IIb beta 3-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 alpha IIb beta 3 inhibitors.

Original language	English
Pages (from-to)	231-242
Number of pages	12
Journal	Research and practice in thrombosis and haemostasis
Volume	5
Issue number	1
Early online date	18 Dec 2020
DOIs	https://doi.org/10.1002/rth2.12466
Publication status	Published - Jan 2021

Keywords

blood coagulation
synthetic chemistry techniques
platelet activation
platelet aggregation inhibitors
ticks
ORNITHODOROS SAVIGNYI ACARI
GLYCOPROTEIN-IIB-IIIA
SOFT TICK
AGGREGATION
ANTAGONIST
APYRASE
DISAGREGIN
KNOWLEDGE
PROTEINS
RECEPTOR

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Cite this

@article{7f01c39dc7ce45dbb427dbafc6f63721,

title = "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 alpha IIb beta 3 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-alpha IIb beta 3 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 alpha IIb beta 3-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 alpha IIb beta 3 inhibitors.",

keywords = "blood coagulation, synthetic chemistry techniques, platelet activation, platelet aggregation inhibitors, ticks, ORNITHODOROS SAVIGNYI ACARI, GLYCOPROTEIN-IIB-IIIA, SOFT TICK, AGGREGATION, ANTAGONIST, APYRASE, DISAGREGIN, KNOWLEDGE, PROTEINS, RECEPTOR",

author = "{van den Kerkhof}, {Danique L.} and Magdolna Nagy and Kanin Wichapong and Brouns, {Sanne L. N.} and Heemskerk, {Johan W. M.} and Hackeng, {Tilman M.} and Ingrid Dijkgraaf",

note = "Funding Information: We gratefully acknowledge Wouter Aarts for support with peptide synthesis. This work was supported by Netherlands Organization for Scientific Research (NWO) Grants VIDI 723.013.009 and Aspasia 0.15.010.005 (to ID) and in part by the Van de Laar Foundation on Biochemistry of Cardiovascular Disease (to DLK). Publisher Copyright: {\textcopyright} 2020 Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).",

year = "2021",

month = jan,

doi = "10.1002/rth2.12466",

language = "English",

volume = "5",

pages = "231--242",

journal = "Research and practice in thrombosis and haemostasis",

issn = "2475-0379",

publisher = "Wiley",

number = "1",

}

Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks. / van den Kerkhof, Danique L.; Nagy, Magdolna ; Wichapong, Kanin et al.
In: Research and practice in thrombosis and haemostasis, Vol. 5, No. 1, 01.2021, p. 231-242.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks

AU - van den Kerkhof, Danique L.

AU - Nagy, Magdolna

AU - Wichapong, Kanin

AU - Brouns, Sanne L. N.

AU - Heemskerk, Johan W. M.

AU - Hackeng, Tilman M.

AU - Dijkgraaf, Ingrid

N1 - Funding Information: We gratefully acknowledge Wouter Aarts for support with peptide synthesis. This work was supported by Netherlands Organization for Scientific Research (NWO) Grants VIDI 723.013.009 and Aspasia 0.15.010.005 (to ID) and in part by the Van de Laar Foundation on Biochemistry of Cardiovascular Disease (to DLK). Publisher Copyright: © 2020 Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).

PY - 2021/1

Y1 - 2021/1

N2 - 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 alpha IIb beta 3 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-alpha IIb beta 3 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 alpha IIb beta 3-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 alpha IIb beta 3 inhibitors.

AB - 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 alpha IIb beta 3 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-alpha IIb beta 3 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 alpha IIb beta 3-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 alpha IIb beta 3 inhibitors.

KW - blood coagulation

KW - synthetic chemistry techniques

KW - platelet activation

KW - platelet aggregation inhibitors

KW - ticks

KW - ORNITHODOROS SAVIGNYI ACARI

KW - GLYCOPROTEIN-IIB-IIIA

KW - SOFT TICK

KW - AGGREGATION

KW - ANTAGONIST

KW - APYRASE

KW - DISAGREGIN

KW - KNOWLEDGE

KW - PROTEINS

KW - RECEPTOR

U2 - 10.1002/rth2.12466

DO - 10.1002/rth2.12466

M3 - Article

C2 - 33537548

SN - 2475-0379

VL - 5

SP - 231

EP - 242

JO - Research and practice in thrombosis and haemostasis

JF - Research and practice in thrombosis and haemostasis

IS - 1

ER -