Prostasomes, PC cells, and PC tissue expose procoagulant polyP. Isolation of polyP from prostasomes and PC cells: (A-B) PolyP was extracted from PC3 (A) prostasomes and (B) cells by an anion exchanger chromatography, separated by electrophoresis on polyacrylamide/urea gel and visualized by DAPI-negative staining. Synthetic polyP with mean chain lengths of 134, 383, and 637 serves as molecular size standard. Purified polyP and synthetic LC polyP were loaded before and after incubation with phosphatase (Psp, 10 U/mL for 1 hour). (C,E) Epifluorescence images of polyP on PC3 prostasomes using Alexa 594-labeled PPBD. The polymer colocalizes with (C) prostasome surface membrane marker CD63 (anti-CD63) and (E) FXIIa (3F7). (D,F) Confocal laser scanning microscopy of polyP in (D) nonpermeabilized and (F) permeabilized PC3 cells. PolyP is stained with Alexa 488-conjugated PPBD, and DNA was visualized with DAPI. Bar is 500 nm in C and E and 10 µm in D and F. (G-H) PC polyP activates FXII. Platelet-free plasma was incubated with 1.5 µg/mL PC3 (G) prostasome or (H) cell polyP in the absence or presence of PPBD (1 mg/mL), generation 1.0 dendrimer (25 µg/mL), or Psp (1 U/mL). PolyP-treated FXII-deficient plasma (FXII def.) and buffer-stimulated normal plasma (buffer) is shown as control. Hydrolysis of the chromogenic substrate S-2302 measures formed FXIIa and PK. Mean ± SEM, n = 6. (I-J) Real-time thrombin formation generated in platelet-free plasma stimulated with 1.5 µg/mL (I) prostasome or (J) PC3 cell polyP in the absence or presence of PPBD (0.5 and 2 mg/mL) or generation 1.0 dendrimer (25 µg/mL). Thrombin formation in polyP-activated FXII-deficient plasma (FXII def.) and buffer-stimulated normal plasma is blotted for comparison. A representative thrombin generation curve of a series of n = 6 is shown. (K) Mortality associated with intravenous injection of PC3 prostasomes (0.8 µg/g bw) in WT mice pretreated with saline or PPBD (150 µg/g bw) was assessed in each group (n = 5); animals alive at 30 minutes after challenge were considered survivors. *P < .05 vs saline, unpaired Student t test. (L) PolyP detection on seminal, PC3 cell, and patient prostasomes using PPBD binding in an ELISA. Bars represent polyP content relative to seminal prostasomes, mean ± SEM, n = 3. ***P < .001 vs seminal prostasomes, 1-way ANOVA. (M-P′) Immunohistochemistry of FXII, polyP, and fibrin in human PC tissue sections. (M-M′) Hematoxylin/eosin-staining of the adenocarcinoma (arrows denote occluded vessels). Immunohistochemical localization of FXII using (N-N′) anti-FXII antibodies (arrows indicate FXII-positive occluded vessels and cancer tissue, respectively), (O-O′) polyP using recombinant PPBD as a probe (arrows), and (P-P′) fibrin deposits using 59D8 antibody (arrows) in (M-N′) paraffin and (O-P′) cryo sections. Black and yellow asterisks denote tissue areas containing cancer cells and nonmalignant tissue, respectively. Sections were counterstained with Mayer’s hematoxylin. Scale bars, 100 µm.