Background: Protease Nexin-1 (PN-1), is a strong inhibitor of serine proteases. We recently established platelet PN-1 as a negative regulator of thrombin generation and thrombosis. Altered clot architecture and retraction are known to be associated with abnormal thrombin generation.

Aims: To evaluate the role of platelet PN-1 on clot structure and retraction.

Methods: Clot retraction was assessed after recalcification in glass tubes of human platelet-rich plasma (PRP) incubated or not with a PN-1-blocking IgG, or of PRP obtained from wild-type (WT) or PN-1 knock-out (PN-1^-/-) mice. Clot architecture was evaluated with confocal microscopy of human or mice platelet-rich clots (PRC). Platelet adhesion and spreading were assessed on immobilized fibrinogen at 37°C. Clot viscoelasticity properties were analysed with ROTEM and the phosphorylation state of platelet tyrosine proteins and myosin light chain (MLC) by western blot of solubilized clots.

Results: Surprisingly, clot weight was increased by 66% in human PRC incubated with a blocking anti-PN-1 IgG and by 40% in PN-1^-/- PRC compared to their controls. Confocal microscopy images showed a fibrin network structure more porous in PRC from PN-1^-/- mice or in human PN-1 inhibited PRC. TEMograms showed that the blocking anti-PN-1 IgG induced a 26% decrease of the maximum clot elasticity. Adhesion to fibrinogen was markedly decreased (P< 0,001) in TRAP-4 or convulxin- activated platelets from PN-1^-/- mice compared to WT. Platelets PN-1^-/- display a more pronounced actin polymerization in response to TRAP-4 activation. Interestingly, platelets from PN-1^-/- mice exhibit a persistence of MLC phosphorylation during clot formation.

Conclusions: Despite its anti-thrombin effect, platelet PN-1 accounts for the formation of a tight structure of PRC. We thus identify a critical positive role of PN-1 on platelet-driven clot retraction. Platelet PN-1 has an inhibitory effect on F actin polymerization and MLC phosphorylation.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/platelet-pn-1-regulates-clot-structure-and-retraction/