# An Update on Polyphosphate In Vivo Activities
## 7 Extracellular PolyP
Injection of polyP into the skin of normal mice induced BK-mediated oedema. In contrast, mice with inherited deficiency in or pharmacologically inhibited expression of FXII and kinin B2 receptors were protected from polyP-induced vascular leakage, indicating that polyP activates FXIIa that drives BK-stimulated vascular leakage [98; 104]. Recently, targeting FXIIa has proven to be an efficient therapy for patients with hereditary angioedema (HAE, a life-threatening swelling disease) [103]. An engineered fully human antibody, Garadacimabtm, specifically neutralised FXIIa (originally named 3F7) and potently blocks BK-driven swelling attacks in a prophylactic setting [105]. The exact trigger for FXII activation and kallikrein-kinin-system-mediated BK formation in HAE is not entirely clear; however, clearly, mast cell-derived heparin [106] or polyP (which is a component of mast cell granules [77]) are promising candidates. Activated platelets and platelet microparticles stimulate thrombin generation in an FXII-contact activation-dependent manner in vivo and human plasma with critical implications for thrombosis in vivo [98; 107]. Vice versa, FXII-dependent plasma clotting and thrombus formation are defective in mice and humans with a genetic deficiency in polyP (inositol hexakisphosphate kinase 1 null [\([lp6k1^{-/-}]\) mice [108] and Hermansky-Pudlak syndrome patient platelets [98], respectively). Natural polyP is largely insoluble Ca\({}^{2+}\), and Zn\({}^{2+}\)-rich polyP nanoparticles potently initiate FXII contact activation similarly to kaolin (a silicate commonly used to trigger FXII activation in diagnostic aPTT clotting assays) [94]. Based on the original discovery that FXII-deficient mice are protected from arterial and venous thrombosis but do not bleed excessively [107; 109], the FXIIa-triggered contact system has emerged as a promising target for safe anticoagulant drugs. Mimicking FXII/FXIIa inhibitors targeting extracellular polyP have emerged as a novel treatment strategy to dampen thrombosis with preserved haemostasis [103]. Various polycation agents have been used to target extracellular polyP, including spermidine, histone H1, polymyxin B, and cationic polymers [30]. Neutralising the polyP charge interferes with the ability of the polyanion to initiate FXII contact activation and ablates the prothrombotic activities of the polymer while sparing haemostasis [30; 103]. Another strategy for interference with polyP orthorhombic activity is based on polymer backbone degradation. Originally, alkaline phosphatase has been shown to interfere with polyP coagulation activity and to block activated platelet-driven thrombosis in mouse models [110; 111]. Alkaline phosphatase cleaves the polymer chain; however, it also removes phosphate groups from lipids and proteins, suggesting the use of polyP-specific phosphatases. _E. coli_ exopolynophatase (PPX), a member of the PPX-GppA phosphatases family, specifically cleaves polyP of chain length \(>\) 35 P\({}_{\rm l}\) but no other polyanions such as heparin, DNA/RNA, or synthetic dextran sulphate. PPX efficiently degrades synthetic and natural polyP and ablates the procoagulant activity of the polymer in human plasma. Infusion of PPX into mice confers protection against polyP procoagulant activity and blunts activated platelet-stimulated vascular occlusions in mouse thrombosis models [112; 113; 114]. Similarly to full-size PPX, the recombinant polyP-binding domains of the enzyme (domains 3 and 4) bind with high affinity to polyP, interfere with procoagulant activities of the polymer, and block polyP-mediated FXII contact activation. Both polyP-degrading PPX and non-degrading PPX_A12 inhibit thrombus formation in lethal pulmonary embolism without interfering with haemostasis in vivo, reproducingthe effects of FXII inhibitors [68; 115]. The data show that targeting polyP mimics the selective importance of FXIIa in thrombosis, sparing hemostasis [103; 116].