Integrin αIIbβ3 mediates platelet adhesion and aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbβ3 is expressed on the surface membrane and in intracellular compartments (1, 2). Inside-out signaling triggered by agonists such as thrombin, collagen, collagen-related peptide (CRP), or adenosine diphosphate (ADP) induces a cation-dependent transformation of receptors from a low affinity to a high affinity state (3). Additionally, the number of surface-expressed receptors is increased by the translocation of internal pools to the plasma membrane (1, 4). Soluble, multivalent ligands, most importantly fibrinogen (Fg) and von Willebrand factor (vWF), bind to activated αIIbβ3 and crosslink adjacent platelets leading to thrombus formation (5). The activation of integrin αIIbβ3 is the final common pathway of platelet activation. Therefore, the direct assessment of integrin αIIbβ3 activation is critical for accurate studies on platelet function.
On human platelets, the high-affinity conformation of integrin αIIbβ3 is detected specifically by the monoclonal antibody (mAb), PAC-1. PAC-1 is a ligand-mimetic antibody, i.e., it competes with the ligand for binding to the receptor. The binding of PAC-1 is cation dependent and is blocked by RGD-containing peptides. In flow cytometry, PAC-1 discriminated between the resting and the activated form of the receptor as platelet activation is a prerequisite for its binding (6, 7). PAC-1 has become a widely used tool for studies on αIIbβ3 activation on human platelets.
Recent advances in the manipulation of the mouse genome have contributed enormously to a better understanding of platelet function in hemostasis and thrombosis. Mouse integrin αIIbβ3 shares high homology with its human counterpart and also plays a crucial role in platelet adhesion and aggregation (8, 9). However, there are no reagents that allow direct assessment of the activated mouse αIIbβ3 integrin. Therefore, the amount of surface-bound fluorophore-labeled Fg is used most frequently as a measure of αIIbβ3 activation. This method, however, has some limitations. First, the labeled Fg competes with plasma and platelet-derived Fg for binding to αIIbβ3. Second, activation of αIIbβ3 occurs transiently (as in the case of ADP-induced platelet activation), leading to the loss of bound Fg before analysis. Third, fibrin(ogen) may polymerize on the surface of activated platelets, particularly in the presence of thrombin, yielding inaccurate results.
We describe the development of a fluorescent derivative of a novel mAb against mouse αIIbβ3 (JON/APE) that allows the detection of the activated integrin in flow cytometry. Binding of JON/APE to activated mouse αIIbβ3 is irreversible and the conjugate can be used for studies on whole blood. The use of JON/APE in flow cytometry provides a rapid and easy-to-handle method for studies on activation of this major platelet receptor in mice.
- Top of page
- Materials and Methods
- LITERATURE CITED
In the current study, we describe a fluorescent derivative of a novel mAb directed against mouse αIIbβ3 (JON/APE) that specifically binds to the activated receptor and therefore resembles PAC-1 binding to the human receptor. However, in contrast to PAC-1, which is a ligand-mimetic antibody that competes with Fg for binding to αIIbβ3 (6), binding of JON/APE is not affected by the presence of plasma Fg. When platelets are preincubated with JON/A, however, Fg binding and platelet aggregation in response to different agonists are inhibited (Fig. 1). JON/A may affect Fg binding by allosteric inhibition of the receptor as proposed by the model of dual interacting ligand binding sites on this integrin (18). However, because JON/A does not replace receptor-bound Fg, this hypothesis is not favorable. The inhibitory effect of JON/A on Fg binding may be based on steric effects resulting from binding to an epitope located in close vicinity to the Fg-binding pocket on αIIbβ3.
To study JON/A binding to murine αIIbβ3 in flow cytometry, we used two fluorescent derivatives of the mAb that markedly differ in size: the 150-kD (approximately) JON/AFITC and the 630-kD (approximately) JON/APE. JON/AFITC normally bound to resting and activated receptors when compared with staining with the FITC derivative of the well-established anti-αIIbβ3 mAb MWReg30 (10). Unexpectedly, a profound difference in binding was observed between the two PE-coupled mAbs. Although MWReg30PE rapidly bound to resting and activated platelets, virtually no binding of JON/APE to resting platelets was detected (Fig. 2). This finding strongly suggests that the increased size of the PE derivative (compared with unlabeled/ FITC-labeled JON/A) sterically impedes its binding to αIIbβ3 on resting platelets. This hypothesis is supported by the observation that the intermediate-sized PE-labeled Fab fragments of JON/A (about 290 kD) yielded a weak but significant staining of resting platelets (data not shown). On the other hand, it is very unlikely that PE conjugation of JON/A affects the antigen recognition sites of the antibody. This is because coupling occurs between the sulfhydryl-reactive maleimide group of the chemical crosslinker SMCC (coupled to PE) and free sulfhydryl groups in the hinge region of the partially reduced antibody. Therefore, the increase in the molecular size of JON/A directly influences its binding properties to resting αIIbβ3 integrin.
Upon cellular activation, however, a strong, calcium-dependent increase in JON/APE (intact IgG) binding to platelets was detectable. This strongly suggests that the conformational changes of the receptor required for binding of Fg (5, 15, 16) also allow binding of JON/APE. Therefore, JON/AFITC and JON/APE provide the first example of fluorescent antibody derivatives with identical antigenic specificitiy that allow the discrimination between the resting and the activated state of an integrin.
One of the major advantages of mAbs like PAC-1 is that they can be used to detect activation of αIIbβ3 in whole blood flow cytometry (7). This approach requires only microliter volumes of blood and significantly lowers the risk of unintentional platelet activation induced by platelet preparation. However, whole blood flow cytometric detection of activated αIIbβ3 can only be successful if the analysis is not affected by plasma Fg. For PAC-1, which competes with Fg for binding to αIIbβ3, it has been proposed that this may be due to the higher affinity of the mAb compared with the ligand (7). In our experiments with JON/APE, no significant difference in binding to αIIbβ3 was observed when platelets were activated (CRP) in the presence or absence of plasma Fg, even when the antibody derivative was added to platelets 10 min after stimulation of the cells (Fig. 4).
Fg binding to αIIbβ3 is a reversible process. However, the extent of the reversibility depends on the agonist that activates the platelet. JON/APE bound to αIIbβ3 stabilizes the activation-dependent conformation of the receptor and does not dislocate from the receptor, even when EDTA is added (Fig. 3d). The stabilizing effect of JON/APE was confirmed in time course experiments. These experiments tested whether JON/APE can be used to detect changes in the activation state of αIIbβ3 in response to the weak agonist ADP, ADP in combination with the anti-GPVI mAb JAQ1, and in response to the strong agonist CRP (Fig. 5). For all agonists, maximum binding of JON/APE was detected already 1 min upon stimulation. However, although no significant change in JON/APE binding to CRP-activated platelets was detectable for 15 min (Fig. 5c), a rapid decrease was observed on ADP-activated platelets, reaching almost basal values at 6 min (Fig. 5a). For platelets activated with ADP in combination with JAQ1, αIIbβ3 activation was prolonged but partly reversible (Fig. 5b). The observed time-dependent changes in the activation state of αIIbβ3 in response to agonists of different strength correlate well with the stability of aggregates under stirring conditions. These findings demonstrate that JON/APE firmly binds to activated αIIbβ3 and strongly suggest that it stabilizes the high-affinity conformation of the receptor.
In summary, we report the generation of a fluorescent derivative of a mAb against mouse αIIbβ3 (JON/APE) that specifically binds to the activated form of the receptor. JON/APE provides an easy-to-handle reagent for direct assessment of αIIbβ3 function in mice and may become a very important tool for future studies on the regulation of this dominant platelet integrin.