Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo

Activated platelets release polyP

Platelet dense granules contain polyP (Ruiz et al., 2004). We analyzed whether activated/procoagulant platelets release polyP. Washed human platelets were stimulated with ADP, thrombin receptor-activating peptide 6 (Trap6), collagen, or thrombin, after which polyP was isolated from stimulated platelet supernatants using an ion-exchange method previously used to purify polyP from cell lysates (Kumble and Kornberg, 1995). It was then separated by agarose-gel electrophoresis and probed with toluidine blue (Figure 1A) and a more sensitive DAPI-based fluorescent stain (Figure 1B) (Smith and Morrissey, 2007). Incubation of the extracted material with proteinase, RNase, DNase, or glycosaminoglycan-cleaving enzymes did not alter the staining patterns (not shown). In contrast, treatment with phosphatase (Psp), which hydrolyzes polyP, completely abolished the signal (Figures 1A and B, “thrombin+Psp”). Platelet polyP migrated in a similar manner to synthetic polyP with mean chain lengths of 70 ± 16 (polyP₇₀) phosphate units and at higher mass than polymers of 20 ± 6 phosphates (polyP₂₀). We determined the chain length of platelet polyP using ³¹P-NMR. The relative ratio of signal intensities of the phosphate end groups (α-P, -10.2 ppm) versus central phosphates (β-P, -22.1 ppm) yielded a mean chain length of platelet polyP of 80, consistent with its migration in the agarose matrix (Figure 1C). Our NMR analysis indicated that the mean chain length of “polyP₇₅” used in previous studies (Ruiz et al., 2004; Smith et al., 2006) is 125 (Figure S1). This polyP (termed polyP₁₂₅, Figures 1A and B) migrated with higher molecular weight than platelet polyP. Integration of all ³¹P signals revealed that >69 % of total platelet released inorganic phosphate is polyP.

PolyP activates the contact system in vitro

We analyzed whether platelet polyP could act as a surface for FXII activation using the chromogenic substrate S-2302, which is hydrolyzed by active FXII (FXIIa) (Figure 2A). Addition of polyP (100 μg/ml) to human (or to murine, Figure S2A) platelet-free plasma (PFP) generated FXIIa with similar kinetics and potency as an identical concentration of kaolin. Treatment of polyP with Psp before addition to plasma reduced polyP-driven FXIIa generation in human and murine plasma in a time-dependent manner. Psp digestion for >60 min completely abolished the FXIIa-generating activity of polyP (Figures 2A and S2A), suggesting that a critical polymer length is required to induce FXII activation. Indeed, synthetic polyP of mean chain length ≤45 phosphate units failed to trigger FXII activation in plasma (Figure S2B). To discriminate between polyP-mediated FXII autoactivation and FXII activation by PK, we incubated purified FXII and PK with polyP. PolyP was as efficient as dextran sulfate (a strong contact activator; Colman, 2006) in supporting PK-driven activation of FXII (Figure 2B). When autoactivation of FXII was investigated without the addition of PK to the reaction, polyP generated significant amounts of FXIIa compared to the no surface control but was not as efficient as dextran sulfate (Figure 2C). The ability of a molecule to trigger contact activation relies on interactions of contact factors with the negatively charged surface (Colman, 2006). A gel shift assay demonstrated direct binding of polyP to contact system factors (Figure 2D). Electrophoretic mobility shifts indicated strong interaction of polyP with both FXII and HK, whereas binding to PK and FXI was minor. PolyP did not bind to antithrombin or albumin. Zirconia beads coated with either polyP or albumin confirmed specific and direct polyP binding to FXII, PK, HK, and FXI, with FXII being the strongest polyP-interacting protein (Figure 2E).

PolyP triggers bradykinin generation

We examined whether polyP stimulate BK release in human plasma and observed complete cleavage of HK at concentrations >1 μg/ml (Figure 3A). Consistently, BK concentration in these samples was high (>750 ng/ml), but relatively low in samples treated with buffer (31 ± 4 ng/ml) or ≤1 μg/ml polyP, which was not sufficient to initiate HK processing (Figure 3B). To confirm that the FXII/PK cascade mediated BK generation from HK, we probed for the zymogens by Western blotting and found that polyP >1 μg/ml triggered complete PK and FXII activation with a slightly higher concentration (>2 μg/ml) resulting in complete activation of plasma FXII (Figure 3A).

PolyP initiates bradykinin-dependent mechanism in vivo

To analyze whether polyP initiates contact system-mediated capillary leakage via BK formation, we employed a Miles edema model in genetically altered mice (Figures 4A-F). In WT animals, platelet polyP or synthetic polyP₁₂₅ (10 μg each) initiated leakage was increased 7.9 ± 0.8 and 7.4 ± 1.0 fold, respectively, higher than vehicle alone (Figures 4A and G). BK stimulation in WT mice increased leakage to 11.3 ± 1.5 fold over vehicle (Figure 4A). To confirm that polyP induces vascular leakage by releasing BK, we employed B2R-deficient (B2R^-/-) mice, which are protected from BK-driven edema formation (Han et al., 2002). B2R^-/-mice were mostly resistant to polyP-induced increase in permeability (polyP 1.1 ± 0.2 fold and polyP₁₂₅ 1.1 ± 0.1 fold, Figures 4B and G). Similarly, BK failed to induce a response in B2R^-/- mice (1.1 ± 0.2 fold, not depicted) whereas injection of histamine (100 μM) resulted in considerable leakage (7.7 ± 1.3 fold compared to vehicle), confirming that B2R^-/- mice are susceptible to edema formation via contact system independent pathways. Because FXII-independent mechanisms for PK activation exist (Muller and Renne, 2008), we analyzed polyP-triggered leakage in FXII-deficient mice (FXII^-/-), which are defective in contact system-driven BK formation (Pauer et al., 2004). FXII^-/- mice were almost completely resistant to polyP-induced leakage, and the degree of edema was not significantly different from the buffer-treated group (polyP 1.3 ± 0.2 fold and polyP₁₂₅ 1.2 ± 0.2 fold, n=10, p>0.05, Figures 4C and G). BK-stimulated edema formation in FXII^-/- mice was similar to that observed in WT animals (10.4 ± 1.3 fold).

Hereditary angioedema is characterized by recurrent attacks of swelling owing to a deficiency of a functional C1 esterase inhibitor (C1INH), which is the major plasma inhibitor for several complement proteases, FXIIa and PK (Zuraw, 2008). PolyP initiated edema in C1INH null (C1INH^-/-) animals that exceeded levels in WT by >40% (polyP 11.8 ± 0.9 fold and polyP₁₂₅ 10.9 ± 0.8 fold, Figures 4D and G), whereas BK-induced (11.0 ± 0.8 fold) tracer extravasation was similar to WT mice levels (11.3 ± 1.5 fold).

Since congenital deficiency in B2R or FXII protects mice from polyP-driven edema formation, pharmacological targeting of BK-signaling or -formation should provide similar protection. To address this hypothesis, we treated WT mice with the FXIIa inhibitor H-D-Pro-Phe-Arg-chloromethylketone (PCK) (Kleinschnitz et al., 2006), or with the B2R antagonist icatibant (Icat), 5 min prior to polyP application. PolyP was unable to induce leakage in WT mice pretreated with PCK (polyP 1.9 ± 0.4 fold and polyP₁₂₅ 1.2 ± 0.2 fold, Figures 4E and G) or Icat (polyP 1.6 ± 0.2 fold, polyP₁₂₅ 1.1 ± 0.2 fold, Figures 4F and G). BK function was blocked by Icat (1.2 ± 0.1 fold) but was independent of FXII activity (9.6 ± 2.3 fold, Figure 4F).

BK is an important mediator in bacterial infections. Escherichia coli that locally assemble contact system proteins on their surface generate BK (Herwald et al., 1998). We analyzed bacterial polyP for BK formation. PolyP was purified from E. coli, separated by agarose-gel electrophoresis, and stained with toluidine blue. Consistent with chain lengths of 100 - 1000 in E. coli (Rao et al., 2009) the polymer migrated with an apparent molecular weight of 10 -100 kDa (Figure 4H). As little as 1 μg/ml E. coli-derived polyP initiated contact activation with complete cleavage of FXII, FXI, PK, and HK, but not of low molecular weight kininogen (LK) (Figure 4I); E. coli polyP potently triggered BK generation (Figure 4J). Psp treatment of E.coli polyP abrogated activation of the contact system, as detected by persistence of zymogen forms (right lanes, Figure 4I) and low levels of BK (28 ± 3 ng/ml, Figure 4J). Intraperitoneal application of polyP (750 μg/g body weight) in WT mice induced strong writhing reactions (a pain reaction) and decreased systemic arterial blood pressure (109 ± 17 to 57 ± 39 mmHg, n=10, survivors and non-survivors); 9/10 mice died within 15 min of challenge (Figure 4K). In contrast, FXII^-/- and B2R^-/- mice were protected from polyP-induced hypotonic shock, writhing reaction, edema formation, with 8/10 and 7/10 animals (**p<0.01 FXII^-/- or B2R^-/- vs. WT), respectively, surviving.

PolyP initiates the intrinsic pathway of coagulation in plasma

To analyze the procoagulant activity of polyP, we incubated human plasma with increasing concentrations of the polymer (0.5 - 1000 μg/ml) and followed generation of FXIIa and activation of its principal substrate in the intrinsic pathway of coagulation, FXI, by Western blotting (Figure 5A); we also quantified thrombin generation (Figure 5B). PolyP ≥2 μg/ml triggered the conversion of FXII and FXI to active proteases (as indicated by the disappearance of the zymogen forms at 80 kDa, Figure 5A), which correlated with polyP-triggered thrombin generation (Figure 5B). Digestion of polyP with Psp (0.05 U/μg polyP) impeded the procoagulant activity of the polymer. Furthermore, we compared polyP with kaolin for initiating thrombin formation in plasma in real time using a fluorogenic thrombin substrate. PolyP- and kaolin-driven thrombin generation had almost identical kinetics, potency, and effects on total thrombin generation (Figure S2C). Pre-incubation of polyP with Psp largely abolished thrombin formation (ETP <100 nM*min). Addition of polyP to normal plasma but not FXII-depleted plasma shortened the clotting time in a dose-dependent manner (Figure 5C). Clotting times in recalcified plasma triggered by synthetic polyP₁₂₅ and polyP₁₀₀₀ were similar to kaolin, whereas polyP₃ stimulated clotting was similar to buffer control (Figure S2D).

We analyzed tissue factor (TF)- and polyP-initiated clotting in normal and FXII deficient human plasma (Figure 5D). Application of TF or polyP shortened the clotting time to 80 ± 6 and 78 ± 5 sec, respectively (untreated plasma >450 sec). PolyP addition to TF-stimulated plasma further accelerated clotting (29 ± 5 sec). The ability of polyP to induce clotting in FXII deficient plasma was impaired (590 ± 35 sec) compared to normal plasma, whereas TF-stimulated clotting was unaffected (82 ± 2 sec). Co-application of polyP and TF to FXII-deficient plasma further shortened clotting times slightly to 66 ± 3 sec. Similar to human plasma, polyP-induced clotting of WT mouse plasma (74 ± 5 sec) and accelerated TF-initiated clotting (TF alone: 69 ± 5, TF and polyP: 54 ± 6 sec). In FXII^-/- mouse plasma, polyP had minor procoagulant activity (380 ± 42 sec) and slightly increased TF-initiated clotting (76 ± 5 vs. 65 ± 4 sec) (Figure S3A).

PolyP also acts on proteins downstream of FXII in the coagulation cascade such as tissue factor pathway inhibitor (TFPI), factor V (FV), and thrombin-activatable fibrinolysis inhibitor (TAFI) (Smith et al., 2006). We analyzed the relative importance of coagulation initiation via FXII versus acceleration of fibrin formation via FV, TFPI, and TAFI for procoagulant polyP-activity (Figure 5E). Plasma was incubated with increasing polyP concentrations (0 - 2 μg/ml, not sufficient to completely activate FXII) in the presence or absence of active FV (FVa), PCI (a TAFI inhibitor), or TFPI function-inhibiting antibodies. Adding exogenous FVa to plasma accelerated clotting somewhat in the absence of polyP. The procoagulant activity initiated by as little as 0.5 μg/ml polyP, however, greatly exceeded polyP-effects on FV (Figure 5E). PolyP-driven clotting was largely independent of anti-TFPI antibodies or PCI in this assay. PolyP completely failed to trigger clotting in FXII deficient plasma, indicating that polyP effects on downstream coagulation proteins are not sufficient to induce clotting without FXII activation. To specifically analyze polyP-stimulated thrombin generation via the intrinsic pathway, we blocked TF/FVII activity with 30 pM FVIIai. In a dose dependent fashion, polyP (0 - 4.8 μg/ml) increased total and maximal thrombin (ETP, 569-971 nM*min, peak: 58-163 nM), and shortened lag time (19.5-10.5 min) and time to peak (24-14 min) (Figure 5F). In the absence of TF/FVII activity there was no effect of polyP on thrombin generation in FXII deficient plasma (FXII-def., Figure 5F). In contrast, polyP accelerated thrombin generation in FXII-deficient plasma stimulated with TF (0.5 pM), although not by a large amount. PolyP did not increase thrombin formation stimulated by TF in FXI-deficient plasma (Figure 5G). Neither inhibition of TAFI or TFPI activity affected polyP–driven thrombin formation in the presence or absence of TF/FVII activity (not depicted), arguing against direct effects of the polymer on these proteins.

The originally described patient with FXII-deficiency, John Hageman, died from pulmonary embolism, which apparently originated from a hemipelvis fracture-associated deep vein thrombosis (Ratnoff et al., 1968). Does platelet polyP-initiated FXII activation have implications for the embolic disease of Mr. Hageman? We used thromboelastography in whole blood to analyze clot firmness dependent on polyP and FXII activity (Figure 5H). This method measures mechanical clot stability (represented by the maximal amplitude of the curves in Figure S3B). Addition of polyP (5 μg/ml) increased clot firmness to 128 ± 4 % vs. untreated blood (100%). Pharmacological targeting of FXII activity abolished polyP-mediated clot stabilization (85 ± 3 vs. 83 ± 3 % in CTI treated blood, p>0.05). As polyP does not affect factor XIII (FXIII) activity or activation (Figure S3C), increased clot firmness conferred by the polymer appears to be mediated by enhanced fibrin structures (Smith and Morrissey, 2008a) and fibrin production (Figure 5F).

Deficiencies in intrinsic pathway proteases protect mice from polyP-induced thrombosis

To analyze polyP for fibrin formation in vivo, we challenged WT and FXII^-/- mice in a model of lethal pulmonary thromboembolism (PE) by intravenous infusion of polyP (300 μg/g body weight). All WT mice with the exception of a single animal (14/15) died within 5 min of polyP application (Figure 6A). In contrast, FXII^-/- mice were significantly protected from polyP-induced PE, with 12 out of 15 FXII^-/- mice surviving the challenge for >30 min (**p<0.01 FXII^-/- vs. WT). To show that the high level of survivors observed in FXII^-/- mice was a direct result of defective polyP-driven FXII activation, we reconstituted FXII null mice with human FXII (hFXII). Intravenous infusion of human protein corrected the prolonged aPTT clotting time of FXII-deficient murine plasma to normal values of WT mice (28 ± 4 sec) and restored susceptibility to lethal PE after polyP infusion, with 12 out of 15 reconstituted animals dying (Figure 6A). Pretreatment of polyP with Psp abolished its procoagulant activity, with 14/15 mice surviving infusion of degraded polyP (Figure 6B). The infestin4-based recombinant FXIIa-inhibitor CSL829 (Schmidtbauer et al., 2009) and PCK irreversibly inhibit the amidolytic activity of FXIIa and PK-mediated activation of FXII. CSL829 blocked polyP-driven FXII and consecutive FXI, PK, and HK activation steps in plasma (Figure 6C). To test the protective potential of FXIIa-inhibitors for polyP-driven thrombosis, WT mice were intravenously injected with CSL829 10 min prior to polyP infusion, which prolonged the aPTT (48 ± 9 sec) and significantly protected mice from lethal PE (12 out of 15 survived; **p<0.01 vs. WT, Figure 6A). PCK (8 μg/g body weight) provided similar protection from polyP-triggered thromboembolism (12/15 survived, not shown). Histological sections of lung tissue from polyP treated mice and counting of formed thrombi are shown in Figures 6D and E. While the majority of vessels was obstructed in WT and hFXII-reconstituted FXII^-/- mice (dead and survivors), virtually no thrombi were found in FXII^-/- mice and in WT animals treated with FXIIa-inhibitors.

To analyze the mechanism of polyP-initiated PE in vivo we used FXI^-/- mice and generated FXII^-/-/FXI^-/--double gene deficient animals. If the prothrombotic effect of polyP is mediated through FXII activation and the consecutive activation of FXI by FXIIa, then FXI^-/- and FXII^-/-/FXI^-/--deficient animals should be protected from PE to the same extent as FXII^-/- mice (Figure 6A). Indeed, FXI and FXII/FXI-combined deficiency conferred resistance to polyP-induced PE and both mouse strains were largely protected from polyP challenge (11/15 and 13/15 survived; **p<0.01 FXI^-/- and FXII^-/-/FXI^-/- vs. WT; p>0.05 FXI^-/- and FXII^-/-/FXI^-/- vs. FXII^-/-). Targeting B2R was shown to confer protection from arterial occlusion induced by vascular injury (Gailani and Renne, 2007a). We analyzed B2R^-/- mice in our PE model and found that almost all (13/15) animals died, suggesting that BK is not required for polyP initiated PE. To confirm that polyP-initiated FXII activity contributes to pathological thrombosis by the intrinsic pathway of coagulation, we measured fibrin formation in lung tissues by quantitative immunoblot analysis of urea-insoluble tissue extracts with the fibrin specific antibody 59D8. Fibrin accumulation was significantly reduced in the lungs of FXII^-/- and inhibitor treated mice, compared to WT and reconstituted animals (Figure 6F).

PolyP initiates fibrin formation on activated platelets

To assess whether polyP may present the “foreign surface” that initiates FXII-driven fibrin formation on platelets we determined whether Psp, which efficiently degrades polyP (Figure 2A), inhibits procoagulant platelet activity. Platelets of human (Figure 7A) and mouse (Figure 7B) origin were stimulated with calcium ionophore A23187 prior to recalcification. Platelet activation reduced clotting times in both species up to 3.2- and 4.2-fold compared to recalcification times in unstimulated PRP. Addition of Psp prior to recalcification abrogated the procoagulant activity of stimulated platelets resulting in similar clotting times to unstimulated PRP. Psp did not alter the recalcification time in PRP without platelet activation. Consistent with our initial observation that clotting in A23187-stimulated PRP depends on FXII (Renne et al., 2005a), clotting in FXII deficient PRP in response to the platelet activator was severely impaired in the presence or absence of Psp (Figures S4A and S4B).

We analyzed whether platelet inhibitors, prostaglandin E1 (PGE1) and N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (dBcAMP), which interfere with thrombin-stimulated platelet activation, could inhibit the procoagulant activity of Trap6-stimulated human (Figure 7C) and murine (Figure S4C) platelets. PGE1, dBcAMP or Psp did not alter recalcification times in PRP without platelet activation. Trap6 reduced clotting times 3.2-fold in human and 2.6-fold in mouse PRP. Addition of Psp prior to recalcification interfered with Trap6 enhanced clotting (1.2- in human and 1.4-fold in mouse PRP compared to unstimulated control). Application of PGE1 and dBcAMP prior to Trap6 reduced procoagulant platelet activity (1.9- and 1.6-fold in human and 1.6- and 1.8-fold in murine PRP). Treatment with Psp in addition to PGE1 or dBcAMP further reduced the procoagulant activity of Trap6-treated platelets until clotting times were no longer significantly different from unstimulated PRP (p>0.05).

To analyze polyP functions for pathological clotting driven by stimulated platelets in vivo, we established a model of lethal PE initiated by Trap6 infusion (Figure 7D). Trap6 activates platelets and initiates polyP secretion (Figure 1), but Trap6 by itself has no direct effect on the plasma coagulation cascade. Almost all (13/15) WT mice died within 5 min after intravenous infusion of Trap6 (Figure 7D). In contrast, FXII^-/- mice were largely protected from Trap6-induced lethal PE (13/15 survived). Consistent with anticoagulant activity in human and mouse plasma (Figures 7C and S4C), PGE1 and dBcAMP infusion into WT mice conferred protection from PE; 10/15 mice treated with each inhibitor survived the Trap6 challenge. Infusion of Psp prior to Trap6 application protected WT mice from lethal PE and 13 out of 15 animals survived the challenge for >30 min. Lung histology from Trap6 treated mice confirmed PE (Figure S4D). While the vast majority of vessels was obstructed in WT animals, virtually no thrombi were found in FXII^-/- mice and in WT mice treated with Psp. Endogenous Psp activity in plasma (Smith et al., 2006) and on endothelial cells (Figures S5A and S5B) degrades polyP slowly and polyP released from activated platelets coincubated with endothelial cells (Figure S5D, inset) initiated FXII-dependent thrombin and BK formation (Figures S5C-E).

We further tested the importance of this concept for human disease states. Hermansky-Pudlak Syndrome (HPS) is a rare, complex hereditary disease (Gahl et al., 1998), in which a bleeding diathesis results from platelet storage pool deficiency. HPS patients have reduced or absent platelet dense granules, the polyP storage site in platelets. We analyzed platelets isolated from HPS patients for their ability to initiate clotting (Figure 7E). Normal or HPS platelets were stimulated with Trap6 before adding them to normal PFP. Supplementing plasma with normal platelets resulted in a recalcification time of 280 sec with no additional shortening of the clotting time upon addition of exogenous polyP₁₂₅ (10 μg/ml). Time to clot formation triggered by stimulated HPS platelets was longer (425 sec) and could be shortened to a ‘normal’ time of approximately 300 sec by addition of polyP. These results indicate that the concentration of polyP found in platelets is sufficient to trigger plasma coagulation, but that a reduction in the normal range of these concentrations, such as in HPS platelets, impairs the procoagulant potential of activated platelets.

Cumulatively, these findings support the concept that inorganic polyP is a new class of platelet-derived proinflammatory and procoagulant mediator that exerts its effect by activation of the FXII-driven contact activation system. PolyP initiates fibrin formation on procoagulant platelets, linking primary to secondary hemostasis.

Extraction of polyP

PolyP was purified from platelet concentrates of healthy volunteers that had donated to the blood bank of Würzburg University Hospital, Germany. Washed platelets were adjusted to 7.5×10¹¹ platelets/ml in New Tyrode's buffer (145 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.5 mM Na₂HP04, 5.5 mM glucose, 10 mM Hepes pH 7.4, 0.35 % bovine serum albumin, pH 7.4) before activation with thrombin (2 U/ml) (Roche), Trap6 (0.2 mM) (Bachem) and collagen (0.2 mg/ml) (Nycomed) for 1 min, or ADP (0.1 mM) (Sigma-Aldrich) for 5 min at 37°C. Supernatants were immediately collected by two centrifugation steps at 3000× g for 15 min at 4°C. An anion exchanger-based procedure was employed (Kumble and Kornberg, 1995) with minor modifications to extract polyP from platelets. Briefly, supernatants were incubated with proteinase K (750 μg/ml, 37°C, 1 h) and extracted with a 1:1 phenol/chloroform mixture. The aqueous phase was combined with extracts from the phenol layer in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, and again chloroform extracted. PolyP was precipitated from the extracts with barium acetate (0.1 M) at pH 4.5 with subsequent incubation for 4 h at 4°C and centrifugation at 14000× g for 30 min. The polyP precipitates were transformed to soluble state by adding ion-exchange resin Dowex 50Wx8 in NH₄⁺ form (SERVA Electrophoresis, Heidelberg, Germany). The mixture was incubated on a shaker for 10 min and then centrifuged at 10000× g for 30 min. The supernatant was incubated with Benzonase (350 μg/ml) and chondroitinase ABC/heparitinase (5 U each) for 5 h. The reaction mixture was extracted with phenol/chloroform as described above and polyP was isolated by evaporation. Yield was 20 mg polyP from 3.7×10¹³ platelets.

PolyP were isolated from E.coli strain BL21, which were grown to an OD₆₀₀ of 2.5 in LB-medium within 8 h. Cells were pelleted, lysed in buffer (50 mM Tris, 1 M Urea, 0.1 % SDS, 10 mM EDTA), and suspension was sonicated on ice for 3×10 sec bursts and polyP was purified as described above. 1 mg polyP was purified from a 100 ml culture.

Activation of the intrinsic pathway in vitro

Human citrate-anticoagulated plasma was incubated with Ca²⁺-saturated polyP (0.5 -1000 μg/ml) for 30 min at 37°C. The reaction was stopped by the addition of reducing Läemmli buffer containing 4% (w/v) SDS. 0.2 μl plasma per lane were analyzed by Western blotting as described (Renne et al., 2005b). BK plasma concentrations were quantified with MARKIT-M-Bradykinin ELISA (Dainippon Pharmaceutical, Osaka, Japan). Activation of purified FXII (200 nM) by kallikrein (0.5 nM) was analyzed by incubating at 37°C in the presence of either dextran sulfate (1 μg/ml), polyP₁₂₅ (5 μg/ml) or buffer alone (20 mM Hepes pH 7.4, 100 mM NaCl; HBS). Reactions were stopped at various times (0 - 60 min) in HBS buffer containing polybrene (5.5 μg/ml) and soy trypsin inhibitor (100 μg/ml). Activity was quantified using a chromogenic substrate for FXIIa (200 nM, Bachem) and read against a standard curve of purified FXIIa for 60 min at 405 nm in a VERSAmax microplate reader (Molecular Devices). Autoactivation of FXII (200 nM) was analyzed in a similar manner with addition of surfaces but in the absence of kallikrein.

PolyP-induced pulmonary thromboembolism

Mice were anesthetized by i.p. injection of 2,2,2-tribromoethanol and 2-methyl-2-butanol (0.15 ml/10 g of body weight from a 2.5 % solution), and polyP (300 μg/g body weight in NaCl) was slowly injected into the inferior vena cava. Alternatively, Trap6 (0.7 μg/g body weight) was infused. In some experiments, mice were injected i.v. with Psp (15 U/g body weight) before the challenge. Animals still alive after 30 min were considered survivors.

Skin vascular leakage assay

Stimulus-triggered leakage of skin microvessels was analyzed with a Miles assay. Briefly, anesthetized mice were retro-orbitally injected with 10 μl/g body weight 0.25 % Evans Blue solution; 5 min later, 50 μl saline (negative control), BK (positive control, 100 μM), or platelet-derived or synthetic polyP₁₂₅ were intradermally injected in the dorsal skin of mice. After 30 min, the animals were sacrificed and the skin was removed and photographed. Skin samples were excised and the Evans Blue dye was extracted by incubation in N,N-dimethyl formamide overnight at 55°C. After centrifugation at 20000× g for 60 min, the supernatant was collected and the concentration of the extracted dye was determined by fluorescence spectroscopy.

NMR-Analysis

A sample of commercial polyP₁₂₅ (25 mg) (Sigma-Aldrich) and a sample of lyophylized lysate (10 mg) were dissolved in heavy water (D₂O) and the ³¹P NMR spectra recorded at 202.4 MHz and 25°C using a Bruker Avance 500 NMR spectrometer. Pulse delay and repetiton time were set at 0.03 and 100 sec, respectively, to ensure correct signal integration. The content of the various species was then calculated directly from the signal intensities.

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