Protease nexin-1 deficiency increases mouse hindlimb neovascularisation following ischemia and accelerates femoral artery perfusion

We previously identified the inhibitory serpin protease nexin-1 (PN-1) as an important player of the angiogenic balance with anti-angiogenic activity in physiological conditions. In the present study, we aimed to determine the role of PN-1 on pathological angiogenesis and particularly in response to ischemia, in the mouse model induced by femoral artery ligation. In wild-type (WT) muscle, we observed an upregulation of PN-1 mRNA and protein after ischemia. Angiography analysis showed that femoral artery perfusion was more rapidly restored in PN-1−/− mice than in WT mice. Moreover, immunohistochemistry showed that capillary density increased following ischemia to a greater extent in PN-1−/− than in WT muscles. Moreover, leukocyte recruitment and IL-6 and MCP-1 levels were also increased in PN-1−/− mice compared to WT after ischemia. This increase was accompanied by a higher overexpression of the growth factor midkine, known to promote leukocyte trafficking and to modulate expression of proinflammatory cytokines. Our results thus suggest that the higher expression of midkine observed in PN-1- deficient mice can increase leukocyte recruitment in response to higher levels of MCP-1, finally driving neoangiogenesis. Thus, PN-1 can limit neovascularisation in pathological conditions, including post-ischemic reperfusion of the lower limbs.

Protease Nexin-1 in the Cardiovascular System: Wherefore Art Thou?

The balance between proteases and protease inhibitors plays a critical role in tissue remodeling during cardiovascular diseases. Different serine protease inhibitors termed serpins, which are expressed in the cardiovascular system, can exert a fine-tuned regulation of protease activities. Among them, protease nexin-1 (PN-1, encoded by SERPINE2) is a very powerful thrombin inhibitor and can also inactivate plasminogen activators and plasmin. Studies have shown that this serpin is expressed by all cell subpopulations in the vascular wall and by circulating cells but is barely detectable in plasma in the free form. PN-1 present in platelet granules and released upon activation has been shown to present strong antithrombotic and antifibrinolytic properties. PN-1 has a broad spectrum of action related to both hemostatic and blood vessel wall protease activities. Different studies showed that PN-1 is not only an important protector of vascular cells against protease activities but also a significant actor in the clearance of the complexes it forms with its targets. In this context, PN-1 overexpression has been observed in the pathophysiology of thoracic aortic aneurysms (TAA) and during the development of atherosclerosis in humans. Similarly, in the heart, PN-1 has been shown to be overexpressed in a mouse model of heart failure and to be involved in cardiac fibrosis. Overall, PN-1 appears to serve as a “hand brake” for protease activities during cardiovascular remodeling. This review will thus highlight the role of PN-1 in the cardiovascular system and deliver a comprehensive assessment of its position among serpins.

Role of platelets in regulating activated coagulation factor XI activity

Factor (F)XI has been shown to bind platelets, but the functional significance of this observation remains unknown. Platelets are essential for hemostasis and play a critical role in thrombosis, while FXI is not essential for hemostasis, but promotes thrombosis. An apparent functional contradiction, platelets are known to support thrombin generation, yet, platelet granules release protease inhibitors, including those of activated FXI (FXIa). We aim to investigate the secretory and binding mechanisms by which platelets could support or inhibit FXIa activity. The presence of platelets enhanced FXIa activity in purified system and increased FIX activation by FXIa and fibrin generation in human plasma. In contrast, platelets reduced the activation of FXI by FXIIa and the activation of FXII by kallikrein. Incubation of FXIa with the platelet secretome, which contains FXIa inhibitors, such as protease nexin-II, abolished FXIa activity, yet in the presence of activated platelets the secretome was not able to block the activity of FXIa. FXIa variants lacking the anion-binding sites did not alter the effect of platelets on FXIa activity or interaction. Western blot analysis of bound FXIa (by FXI(a)-platelet membrane immunoprecipitation) showed that the interaction with platelets is zinc-dependent and, unlike FXI binding to platelets, not dependent on glycoprotein Ib (GPIb). FXIa binding to the platelet membrane increases its capacity to activate FIX in plasma likely by protecting it from inhibition by inhibitors secreted by activated platelets. Our findings suggest that an interaction of FXIa with the platelet surface may induce an allosteric modulation of FXIa.

Serpins, New Therapeutic Targets for Hemophilia

Hemostasis is a tightly regulated process characterized by a finely tuned balance between procoagulant and anticoagulant systems. Among inherited hemostatic conditions, hemophilia is one of the most well-known bleeding disorders. Hemophilia A (HA) and B (HB) are due to deficiencies in coagulation factor VIII (FVIII) or FIX, respectively, leading to unwanted bleeding. Until recently, hemophilia treatment has consisted of prophylactic replacement therapy using plasma-derived or recombinant FVIII in cases of HA or FIX in cases of HB. Because FVIII and FIX deficiencies lead to an imbalance between procoagulant and anticoagulant systems, a recent upcoming strategy implies blocking of endogenous anticoagulant proteins to compensate for the procoagulant factor deficit, thus restoring hemostatic equilibrium. Important physiological proteins of the anticoagulant pathways belong to the serpin (serine protease inhibitor) family and, recently, different experimental and clinical studies have demonstrated that targeting natural serpins could decrease bleeding in hemophilia. Here, we aim to review the different, recent studies demonstrating that blocking serpins such as antithrombin, protein Z-dependent protease inhibitor, and protease nexin-1 or modifying a serpin like α1-antitrypsin could rebalance coagulation in hemophilia. Furthermore, we underline the potential therapeutic use of serpins for the treatment of hemophilia.

Correction: Protease Nexin I is a feedback regulator of EGF/ PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of Actr2 gene Variants on Mouse Viability, Gene Expression, and Cytoskeletal Dynamics

Venous thromboembolism (VTE) is a prevalent human disease that exhibits significant heritability. Factor V Leiden (F5L) is the most common known VTE risk factor, but modifier genes significantly influence VTE development. We recently identified a p.R258G missense mutation in the Actr2 gene (Actr2+/G, ARP2 protein) as a genetic suppressor of lethal thrombosis in mice homozygous for F5L (F5L/L) and hemizygous for tissue factor pathway inhibitor (Tfpi+/-). However, the antithrombotic mechanism of Actr2+/G is unknown. We used genetic and genomic techniques to investigate the phenotypic effects of the Actr2G variant on cells and mice. We previously analyzed progeny from Actr2+/G x Actr2+/G (N7 backcross generations to C57BL6/J) to investigate the effects of Actr2G on mouse survival. The progeny deviated from Mendelian frequencies, as only ~12% were Actr2G/G mice (N=303; p<9x10-7) and these mice have a significantly reduced lifespan of ~six months (N=131; p<0.0001). The Actr2+/G mice, when compared to wildtype mice, were morphologically indistinguishable and were born at the anticipated frequency. In the context of F5L, a F5L/LActr2+/G x F5L/LActr2+/G cross yielded only 3 F5L/LActr2G/G, which also significantly departed from expected frequencies (N=66; p<0.01). The F5L/LActr2+/G mice were born at the anticipated frequency. To determine if the mouse embryos were produced at normal frequencies, we examined pups at embryonic day 14.5 of this F5L/LActr2+/G x F5L/LActr2+/G cross. We observed normal frequencies at embryonic day 14.5, with 5 F5L/LActr2G/G embryos out of 18 total. Taken together, our results suggest that Actr2G exhibits gene dosage lethality, with Actr2G/G homozygosity detrimental for late stage embryonic development/early adulthood. We next studied the mechanism(s) of Actr2G thrombosis suppression by gene expression studies. RT-qPCR for multiple coagulation genes was performed in liver samples from Actr2+/+ and Actr2+/G mice. Serpine2 mRNA (Protease Nexin-1 (PN-1)) was significantly increased in Actr2+/G mice (N=3; p<0.0001). We also observed an increase in plasma PN-1 by quantitative Western Blot analysis. To investigate the Actr2G regulation of PN-1 expression, mouse N2a cells containing Actr2+/G or V5-tagged PN-1 were generated using CRISPR/Cas9. Genomic analysis revealed homology directed repair (HDR)-mediated knock-in frequencies of 24.3% (9/37) for Actr2+/G and 5.6% (1/18) for PN-1-V5. We then attempted to generate PN-1-V5 cells carrying mutant and/or Myc-tagged ARP2 (GMyc2). For the GMyc2 lines, the HDR efficiency for double knock-in was 15.6% (7/45). Analysis of all of our cell lines revealed however, that indels were also present within both the endogenous Actr2 and Serpine2 loci. Subsequent analysis of our GMyc2 cells revealed 5 Actr2 alleles. This suggests that cells avoid the loss of Actr2 function and highlights the difficulty with creating an Actr2 mutant cell model. To further investigate the effects of the Actr2G mutation, we isolated mouse embryonic fibroblasts (MEFs) from Actr2+/+ and Actr2G/G mice. The Actr2G/G MEFs grew poorly in culture, and displayed a significant reduction in cell surface area 20 minutes after plating on fibronectin when compared to Actr2+/+ (q=0.0003). Further analysis revealed F-actin aggregation in the root of cellular protrusions of Actr2G/G MEFs. These results indicate cytoskeletal remodeling defects in Actr2G/G cells. In summary, our studies indicate that mutations affecting ARP2 function can have profound effects on mouse and cell survival, as well as pronounced effects on specific physiological processes like blood coagulation and the transcriptional regulation of coagulation related genes. Disclosures No relevant conflicts of interest to declare.