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Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP).

ABSTRACT: FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K(D) estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.

SUBMITTER: Nakazawa F

PROVIDER: S-EPMC1134760 | biostudies-other | 2005 Feb

REPOSITORIES: biostudies-other

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Project description:Essentials Knowledge of genetic regulators of plasma factor VII activating protease (FSAP) levels is limited. We performed a genome-wide analysis of variants influencing FSAP activity in Scandinavian cohorts. We replicated an association for Marburg-1 and identified an association for a HABP2 stop variant. We identified a novel locus near ADCY2 as a potential additional regulator of FSAP activity.SummaryBackground Factor VII-activating protease (FSAP) has roles in both coagulation and fibrinolysis. Recent data indicate its involvement in several other processes, such as vascular remodeling and inflammation. Plasma FSAP activity is highly variable among healthy individuals and, apart from the low-frequency missense variant Marburg-I (rs7080536) in the FSAP-encoding gene HABP2, determinants of this variation are unclear. Objectives To identify novel genetic variants within and outside of the HABP2 locus that influence circulating FSAP activity. Patients/Methods We performed an exploratory genome-wide association study (GWAS) on plasma FSAP activity amongst 3230 Swedish subjects. Directly genotyped rare variants were also analyzed with gene-based tests. Using GWAS, we confirmed the strong association between the Marburg-I variant and FSAP activity. HABP2 was also significant in the gene-based analysis, and remained significant after exclusion of Marburg-I carriers. This was attributable to a rare HABP2 stop variant (rs41292628). Carriers of this stop variant showed a similar reduction in FSAP activity as Marburg-I carriers, and this finding was replicated. A secondary genome-wide significant locus was identified at a 5p15 locus (rs35510613), and this finding requires future replication. This common variant is located upstream of ADCY2, which encodes a protein catalyzing the formation of cAMP. Results and Conclusions This study verified the Marburg-I variant to be a strong regulator of FSAP activity, and identified an HABP2 stop variant with a similar impact on FSAP activity. A novel locus near ADCY2 was identified as a potential additional regulator of FSAP activity.

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Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP).

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