S 2765

The following coagulation and coagulation-related proteases were purchased from Enzyme Research Labs (USA): kallikrein and thrombin [also called factor (F) IIa, FXa, FXIa, and FXIIa]. The following chromogenic substrates were purchased from Diapharma (USA): for thrombin, S-2238; for FXa, S-2765; for FXIa and APC, S-2366; and for kallikrein and FXIIa, S-2302. Custom synthetic double-stranded DNA fragments (gBlocks) were bought from Integrated DNA Technologies (Canada). Restriction endonucleases and glutathione agarose were purchased from Thermo Fisher Scientific (Canada). Nickel chelate affinity resin Ni-NTA agarose was bought from Qiagen (Canada). PreScission Protease [a glutathione sulfotransferase (GST)–human rhinovirus (HRV) 3C protease fusion protein] was purchased from GE Healthcare (Canada). Normal human pooled plasma (NHPP) was produced in-house. FXI-deficient plasma was purchased from Affinity Biologicals (Canada). STA PTTA reagent, STA Neoplastine CI Plus reagent, and Owren-Koller buffer were bought from Diagnostica Stago (Canada).

Assays were based on a previously published method. Briefly, human FXa (Enzyme Research Laboratories) was diluted to 50 U ml⁻¹ with PBS. The chromogenic substrate S-2765 (Diapharma) was diluted to 1 mg ml⁻¹ in water. For in vitro studies, fondaparinux and 12-mer oligosaccharides were dissolved in PBS at various concentrations (0–131 nM). 16 μl of sample was incubated with 60 μl of 35 μl ml⁻¹ antithrombin (Cutter Biologics) for 2 min at room temperature. Next, 100 μl of FXa was added and incubated for 4 min at room temperature. 30 μl of S-2765 substrate was added and the absorbance of the reaction mixture was measured at 405 nm continuously for 5 min. PBS serves as a control sample. The maximum slope for each sample was convert to percent FXa activity by dividing by the maximum slope for the control sample.
For ex vivo studies, mouse plasma collected after the 6 h reperfusion period and assayed the same as described above.

Endothelial cell medium was purchased from AllCells (Emeryville, CA, USA). Bovine serum albumin (BSA), poly-d-lysine, ethylenediaminetetraacetic acid (EDTA), lipopolysaccharide (LPS), Triton X-100, 4′,6-diamidino-2-phenylindole (DAPI), 3,3-diamino-benzidine tetrahydrochloride (DAB), gadolinium chloride (GdCl₃) and annexin V were obtained from Sigma-Aldrich (St Louis, MO, USA). 0.25% Trypsin-EDTA was from Gibco (Grand Island, NY, USA). CellTracker Green CMFDA and CellTracker Red CMTPX were from Invitrogen (Carlsbad, CA, USA). Lactadherin was prepared in our laboratory. Human factors Va, VIIa, VIII, IXa, X, Xa, prothrombin and thrombin were all from Haematologic Technologies (Burlington, VT, USA). The Chromogenix substrates S-2238 and S-2765 were from DiaPharma Group (West Chester, OH, USA). Monoclonal antibodies against CD42b (glycoprotein Ib, clone HIP1), CD62P (P-selectin, clone AK-4), CD41a (clone HIP8), CD45 (clone H130), CD36 (glycoprotein IV, clone CB38) were from Becton Dickinson Biosciences (San Jose, CA, USA). Monoclonal antibody against αvβ3 integrin was from Abcam (ab7166), Labome (clone LM609), and polyclonal antibody from Bioss (bs1310R). Alexa Fluro 488 or 647-conjugated CD41a and Alexa Fluro 647-labeled CD45 were prepared in our laboratory. FITC-labeled monoclonal antibody against CD62P was from Abcam (ab6632, Cambridge, MA, USA).