Platelet protease nexin-1, a serpin that strongly influences fibrinolysis and thrombolysis

Abstract

Background: Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the α-granules of platelets.

Methods and results: In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice.

Conclusions: Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator-mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

Figure 1. Inhibition of fibrin-bound tPA activity by PN-1

(A–B) tPA (2.5 nM) was bound to fibrin surfaces, and plasmin formation was recorded in absence or presence of recombinant PN-1 (10 nM), with the chromogenic substrate CBS0065. The curves represent the average of the raw data corresponding to the change in absorbance as a function of time in the absence and the presence of rPN-1. To simplify the plots, error standards are represented only every 20 or 40 min. (B) Rates of substrate hydrolysis were calculated from the initial velocity and compared to a plasmin standard curve. Data are representative of 3 different experiments, each performed in triplicate. *P < 0.05 significantly different from tPA alone. (C–D) Plasminogen activation by tPA was measured by fibrin-plasminogen-agar zymography after incubation of tPA with the supernatant from resting platelets or activated platelets by PAR1-AP or PAR4-AP as described in Methods. tPA was incubated (C) with human platelet secretion products in the presence or absence of an anti-PN-1 IgG, or (D) with platelet secretion products from WT and PN-1−/− mice. Data are representative of 5 separate experiments from different donors or mice.

Figure 2. Inhibition of fibrin-bound plasmin activity by PN-1

(A–B) Plasmin (50 nM) was bound to fibrin surfaces and its activity was measured in absence or presence of recombinant PN-1 (10 nM), with the chromogenic substrate CBS0065. The curves represent the average of the raw data corresponding to the change in absorbance as a function of time in the absence and the presence of rPN-1. To simplify the plots, error standards are represented only every 20 or 40 min. (B) Rates of substrate hydrolysis were calculated from the initial velocity and compared to a plasmin standard curve. Data are representative of 3 different experiments, each performed in triplicate. *** P < 0.001 significantly different from plasmin alone. (C–D) Plasmin activity was measured by fibrin-agar zymography after incubation of plasmin with the supernatant from resting platelets or activated platelets by PAR1-AP or PAR4-AP as described in Methods. Plasmin was incubated (C) with human platelet secretion products in the presence or absence of an anti-PN-1 IgG, or (D) with platelet secretion products from WT and PN-1−/− mice. Data are representative of 5 separate experiments from different donors or mice.

Figure 3. Effect of platelet PN-1 in PRC lysis

(A–B) PRCs from PRP of healthy donors, in the presence of an irrelevant IgG or an anti-PN1 IgG, or an anti-PAI-1 IgG or (C–D) PRCs from WT or PN-1−/− mice PRP, were incubated with FITC-fibrinogen prior to clot formation. (A, C) The percentage reduction in clot weight and (B, D) the percentage of released fluorescence were analyzed over 24 hours. Data are presented as means ± SEM of 5 independent experiments from different donors and mice. ***P < 0.001, significantly different from control IgG or WT clots. *P < 0.05, significantly different from control IgG. **P < 0.01, significantly different from WT clots.

Figure 4. Effect of platelet PN-1 on ROTEM^® ex vivo clot lysis

(A) Representative human PRP thromboelastogram (ROTEM^®) profiles. PRP was preincubated with a subthreshold concentration of tPA in presence of an irrelevant IgG, or an anti-PN-1 IgG, an anti-PAI-1 IgG or both. (B) In each condition, the rate of fibrinolysis was assessed by the reduction of the amplitude of the thrombolelastogram profile at 60 minutes. (C) Representative mice PRP thromboelastogram (ROTEM^®) profiles. WT and PN-1−/− PRPs were preincubated with a subthreshold concentration of tPA and (D) the rate of fibrinolysis was quantified at 60 minutes. Data are presented as means ± SEM of 5 independent experiments from different donors and mice. ***P < 0.001, significantly different from control IgG. **P < 0.01 significantly different from WT clots.

Figure 5. Effect of platelet PN-1 on lysis-front velocity

PRCs were labelled with Alexa 488-fibrinogen. (A) A series of confocal micrographs showing the dynamic lysis by rtPA, of human PRCs in presence of an irrelevant IgG or an anti-PN-1 IgG. (B) Confocal images of the dynamic lysis from WT or PN-1−/− PRCs. Progressive lysis-front motions are visualized and confocal micrographs are representative of 5 independent experiments from different donors and mice. Bar, 20μm.

Figure 6. Effect of PN-1 on thrombolysis

(A) Representative intravital images of vessel recanalization after tPA-treatment following an occlusion induced by FeCl₃. Bar, 200μm. (B) Quantification of the incidence of recanalized vessels within 1 hour post tPA-treatment. (C) Analysis of thrombus size after tPA-treatment in WT and PN-1 −/− mice. Data are means ± SEM for 13 vessels injured in 7 mice per group. *P< 0.05 significantly different from WT mice at equivalent time post-tPA treatment.