Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression by Chang-jun Hu, Frank A. Baglia, David C.B. Mills, Barbara A. Konkle, and Peter N. Walsh Blood Volume 91(10):3800-3807 May 15, 1998 ©1998 by American Society of Hematology

Flow cytometric assay for the detection of platelet factor XI on the surface of normal platelets. Flow cytometric assay for the detection of platelet factor XI on the surface of normal platelets. The experiment was performed as described in the Materials and Methods. The Y axis displays the number of platelets at any specific fluorescence intensity noted on the X axis as a log scale. The C gate was set at the edge of the background of platelet fluorescence intensity without adding any primary antibody or preimmune goat IgG. A representative result is shown, in which affinity-purified anti-factor XI antibody was bound to unactivated platelets (A) and to platelets activated by 10 μmol/L thrombin receptor peptide (B). Results in (C) depict preimmune goat IgG nonspecifically bound to unactivated and (D) to activated platelets. The mean fluorescence intensity in (A) minus that in (C) or (B) minus (D), respectively, represent the net mean fluorescence intensity due to anti-factor XI antibody binding to platelet factor XI. Chang-jun Hu et al. Blood 1998;91:3800-3807 ©1998 by American Society of Hematology

Comparison of the exposure of platelet factor XI, GPIIb/IIIa, and P-selectin on platelets. Comparison of the exposure of platelet factor XI, GPIIb/IIIa, and P-selectin on platelets. One hundred percent binding of anti-factor XI antibody, FITC-PAC1, or FITC-S12 to platelets was defined as the mean fluorescence intensity of platelets activated by 10 μmol/L thrombin receptor peptide (SFLLRN amide) affinity-purified factor XI antibody and incubated with PAC1 antibody as described in the Materials and Methods. The results shown are the means (±SEM) of data obtained with platelets from four normal donors probed for platelet factor XI (•), GPIIb/IIIa (□), or P-selectin (○) . Chang-jun Hu et al. Blood 1998;91:3800-3807 ©1998 by American Society of Hematology

Flow cytometric assay for the detection of GPIIb/IIIa or P-selectin in activated platelets using PAC1 and S12 antibodies. Flow cytometric assay for the detection of GPIIb/IIIa or P-selectin in activated platelets using PAC1 and S12 antibodies. Two FITC-labeled activation-dependent monoclonal antibodies, PAC1 (A and B) or S12 (C and D), were incubated with unactivated platelets (A and C) or to platelets activated by 10 μmol/L thrombin receptor peptide (B and C), and flow cytometry was performed as described in the Materials and Methods. The Y axis displays the number of platelets at any specific fluorescence intensity noted on the X axis as a log scale. The C gate was set at the edge of the background of platelet fluorescence intensity without adding any antibody. The maximal binding of PAC1 or S12 to platelets was defined as the mean fluorescence intensity of platelets activated by 10 μmol/L thrombin receptor peptide and incubated with FITC-PAC1 (80 μg/mL) or FITC-S12 (16 μg/mL). The mean fluorescence intensity obtained in the absence of antibody was similar to that shown in (A) and (C) and was subtracted from that obtained in (B) and (D) to obtain the net mean fluorescence intensity due to PAC1 binding to GPIIb/IIIa or S12 binding to P-selectin on activated platelets. Chang-jun Hu et al. Blood 1998;91:3800-3807 ©1998 by American Society of Hematology

Effects of platelet agonists on the exposure of platelet factor XI in normal and factor XI-deficient donors. Effects of platelet agonists on the exposure of platelet factor XI in normal and factor XI-deficient donors. Four normal donors and four patients with plasma factor XI deficiency were tested for the exposure of platelet factor XI on unactivated platelets and on platelets activated by ADP, thrombin, collagen, or TRP as described in the Materials and Methods. The percentage of exposure of platelet factor XI was calculated as described in the text and in the legends to Figs 1 and 3. The total exposure (100%) of platelet factor XI was defined as the net mean fluorescence intensity of normal platelets activated with 10 μmol/L TRP. (A) depicts the mean percentage of platelet factor XI exposure in four normal platelets, whereas (B) depicts results obtained with platelets from four patients with plasma factor XI deficiency. Chang-jun Hu et al. Blood 1998;91:3800-3807 ©1998 by American Society of Hematology

Effects of platelets and factor XI in thrombin-activated coagulation assays. Effects of platelets and factor XI in thrombin-activated coagulation assays. Coagulation assays were performed as described in the Materials and Methods. Either factor XI-deficient plasma (A and C) or normal plasma (B and D) was incubated with phospholipid vesicles (PS:PC 1:3 ratio, 10 μmol/L; A and B) or gel-filtered platelets (200,000 platelets/μL; C and D) activated with the thrombin receptor peptide, SFLLRN-amide (5 μmol/L), and CaCl2 (5 mmol/L). In some assays, affinity-purified goat antihuman factor XI (14.6 μmol/L) was added, followed by thrombin at various concentrations (0.05, 0.1, 0.25, 0.5, and 1.0 U/mL) to initiate clot formation. Data shown are the means (±SEM) for four experiments, each performed in triplicate. In the absence of added thrombin, the clotting times of all samples were greater than 5 minutes. (A) Factor XI-deficient plasma and phospholipid vesicles in the absence (○) or presence (•) of anti-factor XI antibody. (B) Normal plasma and phospholipid vesicles in the absence (○) or presence (•) of anti-factor XI antibody. (C) Factor XI-deficient plasma and normal activated platelets (□) or platelets from factor XI-deficient patients (▵) or normal platelets in the presence of anti-factor antibody (▪). (D) Normal plasma and normal activated platelets (□) or platelets from factor XI-deficient patients (▵) or normal platelets in the presence of anti-factor XI antibody (▪). Chang-jun Hu et al. Blood 1998;91:3800-3807 ©1998 by American Society of Hematology