Mouse Anti-PF4 Recombinant Antibody (clone KKO) (CAT#: PABL-299)

Figure 1 Binding of mAb to human platelets in the presence of added hPF4.

(A) The graph shows the fold increase in the mean fluorescence intensity (MFI) of antibody binding in the presence and absence of the noted concentrations of PF4. Gray squares indicate TRA isoimmune control; open diamonds, anti–human CD41 mAb; and filled circles, KKO. Each antibody was added at 50 μg/mL. (B) The fold change in antigenicity for KKO in the presence of PF4 at 12.5 μg/mL (open diamonds), 50 μg/mL (gray squares), and 200 μg/mL (filled circles), with heparin added at the concentrations shown. The y-axis indicates fold change from that at baseline without heparin. (C) Platelet activation by KKO (50 μg/mL) at the indicated PF4 concentrations as measured by annexin V binding. (D) Kinetics of KKO binding (50 μg/mL) in the presence of 50 μg/mL of PF4.

Rauova, L., Zhai, L., Kowalska, M. A., Arepally, G. M., Cines, D. B., & Poncz, M. (2006). Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood,107(6), 2346-2353.

Figure 2 KKO binding to murine platelets in the presence of hPF4.

(A) Details are the same as for Figure 1A but mPF4null platelets were studied with increasing amounts of added hPF4. Diamonds indicate total platelet PF4; squares, total surface immunogenic PF4; and circles, KKO-detectable surface PF4. (B) Details are the same as for panel A for KKO but mPF4null platelets pretreated with CS ABC were used. (C) Details are the same as for panel A for KKO but genotypes of the mice are as shown and the platelets were incubated with either the FcγRIIA blocking mAb IV.3 or an isotype control (50 μg/mL) for 30 minutes at RT prior to addition of PF4 and KKO. (D) Details are the same as for panel A for KKO with the genotypes of the mice indicated. Relative annexin binding was measured. (E) Time course of KKO and RTO binding (50 μg/mL) in the whole blood samples to transgenic murine platelets. +/+ indicates hPF4high/FcγRIIA+ double-transgenic mouse platelets; +/–, hPF4high/FcγRIIA– transgenic mouse platelets. MFI is in absolute values. For panels A-D, the mean ± 1 SD is shown. Each experiment was performed 3 times. In panel E, a study representative of 3 is shown.

Rauova, L., Zhai, L., Kowalska, M. A., Arepally, G. M., Cines, D. B., & Poncz, M. (2006). Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood,107(6), 2346-2353.

Figure 3 Characterization of hPF4 mice.

(A) Total platelet-associated hPF4 expressed per mL of blood in WT animals and the 3 hPF4 transgenic mice lines studied. Controls (Ctl) were platelets from 4 human donors. The mean ± 1 SD is shown for the experiments performed 3 times, each in triplicate. (B) Flow cytometric measurement of CD41+–platelet-bound FITC-KKO in the same animals as in panel A measured 10 minutes after intravenously injected FITC-KKO.

Rauova, L., Zhai, L., Kowalska, M. A., Arepally, G. M., Cines, D. B., & Poncz, M. (2006). Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood,107(6), 2346-2353.

Figure 4 KKO-induced thrombocytopenia in hPF4 mice.

(A) Platelet counts in mice after intraperitoneal injection of KKO. The first time point is at 3 hours after injection. Filled circles indicate hPF4high mice, 200 μg KKO; and open circles, FcγRIIA transgenic mice, 200 μg KKO. Diamonds indicate hPF4high/FcγRIIA double-transgenic mice. Open diamonds, light gray diamonds, dark gray diamonds, and filled diamonds indicate 50, 100, 200, and 400 μg KKO intraperitoneally, respectively. The mean of 3 experiments, each performed in triplicate, is shown. (B) Animals were all hPF4high/FcγRIIA double-transgenic mice. ○ indicates 200 μg TRA, intraperitoneally; ♦, 200 μg RTO, intraperitoneally; and •, 200 μg KKO, intraperitoneally. The mean ± 1 SD of 3 experiments, each in triplicate is shown. *P <.05 (C) All animals received 200 μg KKO. • indicates hPF4high mice, 200 μg KKO; ○, FcγRIIA transgenic mice, 200 μg KKO; ⋄, hPF4low/FcγRIIA; An external file that holds a picture, illustration, etc. Object name is graydiam.jpg, hPF4mid/FcγRIIA; and •, hPF4high/FcγRIIA. The mean ± 1 SD of 3 experiments, each performed in triplicate, is shown. *P <.05 from baseline value. (D) All animals received 200 μg KKO, intraperitoneally. • indicates hPF4high mice, 200 μg KKO; An external file that holds a picture, illustration, etc. Object name is graydiam.jpg, hPF4mid mice; and •, hPF4mid mice that also received 20 U heparin subcutaneously daily for 4 days as indicated by arrows. The mean ± 1 SD of 3 experiments, each performed in triplicate, is shown. *P <.05 of heparin treated from untreated hPF4mid/FcγRIIA mice.

Rauova, L., Zhai, L., Kowalska, M. A., Arepally, G. M., Cines, D. B., & Poncz, M. (2006). Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood,107(6), 2346-2353.

Figure 5 Binding of KKO to structure-based PF4 mutants.

Cai, Z., Yarovoi, S. V., Zhu, Z., Rauova, L., Hayes, V., Lebedeva, T.,... & Greene, M. I. (2015). Atomic description of the immune complex involved in heparin-induced thrombocytopenia. Nature communications, 6, 8277.

Figure 1 Anti-PF4 Recombinant Antibody (PABL-299) in SDS-PAGE

SDS-PAGE analysis of PABL-299 in non-reduced (Lane 1) and reduced (Lane 2) conditions.

Figure 2 Anti-PF4 Recombinant Antibody (PABL-299) in HPLC

The purity of PABL-299 was greater than 95% as determined by HPLC.