Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function

Abstract

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6^tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

Keywords: JAQ1; glycoprotein VI; platelet activation; platelet inhibition; platelet receptors.

Figure 1

Anti-mouse GPVI monoclonal antibody JAQ1 binds to human GPVI and modulates its function. (A) Washed human or mouse blood was pre-incubated with JAQ1 IgG-FITC and Mean Fluorescence Intensity (MFI) was measured by flow cytometry; where indicated, human blood was pre-incubated with 20 µg/mL IV.3. Irrelevant rat-IgG-FITC was used as control; (B) Human blood was pre-incubated with 20 µg/mL EMF1, EMF-2 or control IgG for 10 min and subsequently incubated with JAQ1-FITC for 10 min, irrelevant IgG-FITC was used as control; (C) Mouse (WT) and human platelet lysates were separated by 10% SDS-PAGE under non-reducing condition and blotted onto PVDF membrane. JAQ1-HRP was used to detect GPVI on the membrane. GAPDH served as loading control; (D) Murine or human washed platelets were pre-incubated with either JAQ1 or a control IgG and aggregometry was performed; crosslinking of bound antibody was induced by adding an anti-Rat IgG antibody and light transmission was recorded for 15 min. When indicated, human platelets were incubated with IV.3 prior to JAQ1 addition; (E) Human washed platelets were pre-incubated with either JAQ1 or a control IgG and aggregometry was performed; aggregation was induced using the indicated agonists and for 10 min; (F,G) Human-washed platelets were pre-incubated with IV.3 plus JAQ1 IgG or a control IgG and let spread on a 100 µg/mL fibrinogen-coated surface for 45 min at 37 °C. Experiments shown are representative of n = 4. Flow cytometry and spreading data are expressed as mean ± SD, significance is expressed as * p < 0.05, ** p < 0.01, vs. indicated group (t-test).

Figure 2

hGP6^tg/tg mice confirm that JAQ1 binds to native human GPVI on the platelet surface, but not in Western blot analysis. (A) WT, hGP6^wt/tg, hGP6^tg/tg and Gp6^−/−-washed blood was incubated JAQ1-FITC and MFI was measured by flow cytometry; (B) hGP6^tg/tg-washed blood was pre-incubated with either EMF-1, EMF-2 or control IgG and subsequently incubated with JAQ1-FITC; (C) WT, hGP6^wt/tg and hGP6^tg/tg platelet lysates were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. JAQ1 or EMF-1 were used to detect GPVI on the membrane. GAPDH served as loading control; (D,E) Western blot quantitative analysis relative to loading control; (F) hGP6^tg/tg washed platelets were pre-incubated with JAQ1 or control IgG and aggregate formation was induced using anti-rat IgG antibodies (20 µg/mL). Experiments shown are representative of n = 4, Western blot of n = 3. Flow cytometry and Western blot data are expressed as means ± SD.

Figure 3

Differential effect of JAQ1 on huGPVI and mGPVI. (A–D) WT (A,B) or hGP6^tg/tg (C,D) diluted heparinized blood was pre-incubated with 20 µg/mL JAQ1 or control-IgG. Treated platelets were incubated with WUG 1.9-FITC (A,C), JON/A-PE (B,D) and stimulated with CRP (10 µg/mL), convulxin (1.25 µg/mL) or vehicle; (E,F) Washed platelets from WT (E) or hGP6^tg/tg (F) were pre-incubated with 20 µg/mL JAQ1 or control IgG and aggregation was induced with the indicated agonists; aggregation was measured for 10 min; (G–I) Heparinized WT or hGP6^tg/tg blood was pre-incubated with 20 µg/mL JAQ1 or control IgG and tested in flow adhesion assay on a collagen-coated surface. Percentage of the covered surface (H) and relative volume of thrombi (I) were analyzed based on fluorescence intensity of anti-GPIX-AF488 derivative. Experiments shown are representative of n = 4. Flow cytometry data are expressed as means ± SD, significance is expressed as * p < 0.05, ** p < 0.01, *** p < 0.001 vs. indicated group (t-test).