Anticoagulant and Antithrombotic Effect of Platelet Protease Nexin-1

Protease nexin-1 (PN-1) is a serpin which inhibits plasminogen activators, plasmin and thrombin. PN-1 is barely detectable in plasma, but is expressed by platelets. Here we have characterized platelet PN-1 localization and function in human and mouse platelets. PN-1 is expressed at the platelet surface and stored within granules from which it is released after activation by thrombin receptor activating peptide (TRAP) or collagen, but not by ADP. The granules storing PN-1 are alpha-granules, as indicated by the total absence of PN-1 in platelets from patients with the Gray platelet syndrome. Using a PN-1 blocking antibody, we observed that PN-1, released after stimulation of human washed platelets (5 × 10⁸/ml) by TRAP, inhibited thrombin (0.25 nM) by 94% ± 3% and urokinase (uPA, 1nM) by 65% ± 3.2%. Consistent with these findings, the secreted products of platelets from PN-1-deficient mice no longer inhibited thrombin or uPA, whereas the secreted products of platelets from PAI-1-deficient mice still inhibited these proteases. These data point out the supremacy of platelet PN-1 compared to platelet PAI-1 in the regulation of thrombin activity. The effect of platelet PN-1 on tissue factor-induced thrombin generation was measured by thrombinography. A significant increase in the rate and the peak of thrombin generation was observed in human platelet-rich plasma (PRP) preincubated with a blocking anti-PN-1 antibody and in the PRP from PN-1-deficient mice whereas no modification of the thrombogram parameters were observed in PPP. This indicates that platelet PN-1 limits the rate and the extent of thrombin generation. The effect of platelet PN-1 on thrombin-induced platelet activation was analysed by measuring P-selectin exposure by flow cytometry and aggregation of washed platelets. Platelets from PN-1-deficient mice showed a higher sensitivity to thrombin. Half maximal P-selectin exposure was obtained with 0.08 ± 0.008 nM thrombin in wild-type (WT) versus 0.04 ± 0.008 nM thrombin in PN-1-deficient platelets (p=0.0186), indicating that PN-1-deficient platelets were twice as sensitive to thrombin than WT platelets. Furthermore, the lag period preceding platelet aggregation was shorter, the rate and the extent of aggregation were higher when PN-1 deficient platelets were stimulated with up to 0.3 nM thrombin as compared to WT platelets, but increasing concentrations of thrombin overcame these defects. The effect of platelet PN-1 on thrombus formation was analysed ex vivo by flowing whole blood over collagen-coated-flow chambers. Platelets adhesion and aggregation were increased for PN-1-deficient mice. After 5 min of perfusion at 1500 s^-1, PN-1 -/- thrombi were larger than WT thrombi, with a surface coverage which increased from 2314 µm² ± 89 µm² for WT mice to 3607 µm² ± 130 µm² for PN-1-deficient mice (p< 0.001). The role of PN-1 on thrombus formation was further analysed in vivo by intravital microscopy in the FeCl₃-induced thrombosis. Complete occlusion of the mesenteric veins was twice as rapid in PN-1 deficient mice compared to WT mice, with occlusion times being respectively 22.1 min ± 1.7 min and 42.4 min ± 2.3 min post-injury (p<0.0001). Mesenteric arterioles of PN-1-deficient mice were also occluded more rapidly than arterioles of WT mice with occlusion times after injury being 20 min ± 2 min and 35 min ± 1 min respectively (p<0.0001). These data demonstrate that platelet PN-1 raises the threshold above which platelet activation, thrombin generation and thrombus formation occurs. Our results establish for the first time that platelet PN-1 possesses efficient anticoagulant and antithrombotic properties, and may be an important regulator in the initiation of thrombosis.