The factor VIII C1 domain contributes to platelet binding

Abstract

Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrin-labeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrin-labeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluorescein-conjugated proteins. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7,000 C1C2-binding sites per platelet, approximately 1,400 for C2, and approximately 3,000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet-binding affinity.

Figure 1

Expression and purification of the FVIII C1C2 domain. (A) Lanes 1 and 2 show the insoluble and soluble fractions of an E coli cell lysate (15% SDS-PAGE, Coomassie Blue staining; marker proteins on far left). (B) Refolded C1C2 run on a 15% acrylamide gel, with 5 μg protein in lane 1 and 10 μg in lane 2; the left panel shows Coomassie blue staining (marker proteins on far left), and the right panel shows a Western blot using ESH8. (C) Removal of the amino-terminal peptide: lanes 1 and 2 show C1C2 before and after thrombin cleavage (10% SDS-PAGE, Coomassie blue staining; marker proteins on far left). The image was cropped below the C1C2 band because the lower percentage of acrylamide resulted in the loading dye running much closer to the C1C2 band than in the 15% gels; additional Coomassie-stained gels and Western blots showed no evidence of proteolytic degradation or lower molecular weight contaminants (not shown). The thrombin-cleaved C1C2 (containing a 16–amino acid amino-terminal extension) was used for all binding and inhibition studies.

Figure 2

Platelet activation by SFLLRN amide. Binding of FITC-labeled anti–P-selectin (FL1 on the Y-axis is relative fluorescence using the channel for fluorescein) to platelets was near background prior to activation with the thrombin receptor peptide but increased to 90% of cells stained following activation of fresh platelets.

Figure 3

Binding of biotinylated C1C2–2296C (C1C2^b) and fluorescein-labeled C1C2-2296C (C1C2*) to platelets. (A) Binding of C1C2^b to platelets was detected by PE-streptavidin (FL2 on the y-axis is relative fluorescence using the channel for phycoerythrin, PE). The left panel shows platelets with PE-streptavidin added as a control. The right panel shows platelets after addition of 1.2 μM C1C2^b followed by PE-streptavidin. (B) Binding of fluorescein-labeled C1C2-2296C. The left panel shows the platelet-only control. The right panel shows platelets after addition of 0.25 μM C1C2* (FL1, y-axis). Note the reduced scatter and higher percentage of platelets binding compared with panel A, although only one fifth as much labeled C1C2 was used as in panel A. Shown are percentages of platelets binding C1C2^b-PE-streptavidin or C1C2* above the background fluorescence signal from platelets without the fluorescent probe.

Figure 4

Binding of fluorescein-labeled C1C2-2296C (C1C2*) to platelets before (center panel) and after (bottom panel) activation. The results are consistent with a smaller population of lower-affinity C1C2-binding sites on platelets prior to activation, whereas more than 90% of activated platelets became labeled after addition of 0.25 μM C1C2*. The upper-left panel shows the platelet control without added C1C2. Percentages are as in previous figures.

Figure 5

Binding of fluorescein-labeled FVIIIa (FVIIIa*) to activated platelets. From 0.2 to 20 nM FVIIIa* was added to activated platelets. Maximal binding occurred at approximately 10 nM FVIIIa, and half-maximal binding gave an estimated K_d of approximately 2.5 nM. Similar results were obtained for binding of fluorescein-labeled FVIII to activated platelets (not shown). Error bars represent SD.

Figure 6

Competition assays. Fluorescein-labeled C1C2 (C1C2*), C2 (C2*), or FVIIIa (FVIIIa*) were added to activated platelets. The bound, labeled proteins were competed by adding increasing amounts of unlabeled C2, C1C2, FVIII, or FVIIIa; alternatively, platelet binding was examined after preincubation with a monoclonal antibody, ESH4. (A) C1C2* competition by C1C2 or C2: 0.125 μM C1C2* binding was reduced from 88% (left) to 5% after a 10-fold molar excess of unlabeled C1C2 (center), but adding a 20-fold molar excess of unlabeled C2 reduced the percentage of labeled platelets to only 34% (right). (B) C1C2* competition by ESH4: 0.125 μM C1C2* binding was reduced from 92% to 21% by adding equimolar ESH4 and to 16% with a 5-fold molar excess of ESH4. (C) C1C2* or C2* competition by FVIII or FVIIIa: 0.25 μM C1C2* (left) or C2* (right) was competed by adding unlabeled FVIII () or FVIIIa (); controls are shown as (). Before competition, binding was near saturation for C1C2* and for C2*; however, note the different scales for the left and right panels, which indicate that binding of C2* to activated platelets was lower than for C1C2*. Data represent the mean and standard deviations (upper error bars shown) for measurements using 6 different platelet preparations. Representative data for one of the C1C2* competition experiments plotted in histogram and scattergram format are available in Figure S1A,B, available on the Blood website; see the Supplemental Figure link at the top of the online article. (D) FVIIIa* competition by FVIIIa, C1C2, or C2: 10 nM FVIIIa* (near-saturation binding, Figure 5) was competed by FVIIIa (left panel, ), C1C2 (center panel, ), or C2 (right panel, ) at molar ratios as indicated. Data represent the mean and standard deviations (upper error bars shown) for measurements using 5 (for right and center panels) and 4 (for left panel) different platelet preparations. Representative data from one of the FVIIIa*-C1C2 competition experiments plotted in histogram and scattergram format are available in Figure S1C,D.

Figure 7

Estimated number of fluorescein-labeled C1C2 (C1C2*)–binding sites per platelet. The top histogram shows fluorescent signals from a mixture of 7.6 μm beads, where 1 is the peak for unlabeled beads and 2 through 5 are signals from the same beads containing increasing, defined amounts of FITC label. The bottom panel shows a histogram representation of near-saturation binding of 0.25 μM C1C2* to activated platelets. From 3 sets of similar measurements using different platelet preparations, the estimate ranged from 6100 to 7600 C1C2 molecules bound per activated platelet.