Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Abstract

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.

Fig. 1. Characterization of mice with β1-null platelets. (A) Western blot analysis of β1 integrin and GPVI in control and β1-null platelets. (B) Platelet counts are expressed as the mean count ± SD for groups of six mice. (C) Tail bleeding times were determined in groups of nine control and 10 β1-null mice. Each point represents one individual.

Fig. 2. Aggregation of β1-null platelets in response to fibrillar collagen. (A) Heparinized platelet-rich plasma (prp) from control and β1-null mice was stimulated with the indicated concentrations of fibrillar collagen, and light transmission was recorded in a standard aggregometer. (B) The delay in platelet aggregation is expressed as time (s) between addition of collagen and maximum shape change. Results are given as mean ± SD (n = 6). (C) Washed platelets were stimulated with fibrillar collagen (5 µg/ml) and samples were lysed at the indicated time points. Protein tyrosine phosphorylation was detected by immunoblotting.

Fig. 3. Normal aggregation of β1-null platelets in response to various agonists. Platelets from control (filled circles) and β1-null (open circles) mice were stimulated with different concentrations of ADP (A), thrombin (B), CRP (C) and convulxin (D). Results are given as percent aggregation and are expressed as mean ± SD for groups of six mice.

Fig. 4. GPVI mediates two activation pathways, one of which involves α2β1. (A) Heparinized prp from control and β1-null mice was pre-incubated with 20 µg/ml JAQ1 Fab fragments and then stimulated with fibrillar collagen (5 or 50 µg/ml). As a control, prp from GPVI-depleted and FcRγ chain-deficient mice was stimulated with collagen in the absence of JAQ1. (B) Prp from the indicated mice was stimulated with soluble collagen (50 µg/ml). Where indicated, the prp was pre-treated with JAQ1 Fab fragments (20 µg/ml) for 5 min. (C) Washed platelets from control and β1-null mice were stimulated with soluble collagen (50 µg/ml) and samples were lysed at different time points. Protein tyrosine phosphorylation was detected by immunoblotting.

Fig. 5. Platelet adhesion to fibrillar and soluble collagen under static conditions. Washed platelets from the indicated mice were allowed to adhere under static conditions to fibrillar (A) or soluble (B) collagen immobilized in microtiter plates. The experiments were performed in the presence (black bars) or absence (gray bars) of Mg²⁺/Ca²⁺ (1 mM each). Where indicated, platelets were pre-incubated with JAQ1 Fab fragments (20 µg/ml). Adherent platelets were quantitated fluorimetrically. The data shown are from a single experiment, representative of four identical experiments and expressed as the mean of triplicate readings ± SD for the indicated times. (*) To exclude any residual GPVI activity, the platelets were used in the presence of (10 µg/ml) JAQ1.

Fig. 6. GPVI-mediated integrin activation is a prerequisite for platelet adhesion to collagen. (A) Washed FcRγ chain-deficient platelets were allowed to adhere to fibrillar collagen immobilized in microtiter plates in the presence of Mn²⁺ with or without addition of blocking antibodies against α2β1 or αIIbβ3. Where indicated, the collagen substrate had been pre-exposed to vWF (20 µg/ml) or plasma for 30 min. Adherent platelets were quantitated fluorimetrically. The data shown are from a single experiment, representative of three identical experiments, and are expressed as the mean of triplicate readings ± SD for the indicated times. (B) Flow cytometric analysis of collagen-induced integrin activation on control, FcRγ chain-deficient or GPVI-depleted platelets. Diluted prp was stimulated with 30 µg/ml fibrillar collagen for 5 min at room temperature. Activated β1 integrin was detected directly with 9EG7–FITC, and activated αIIbβ3 indirectly by quantitation of surface-bound fibrinogen. Platelets were gated by FSC/SSC characteristics and Fl2 positivity (anti-CD9^PE). The results shown are representative of six individual experiments. Note that only a subpopulation of the control platelets is activated. This is based on the fact that fibrillar collagen is insoluble and, therefore, not all platelets come in contact with this agonist. The percentage of activated control platelets varied between 10.3 and 19.6 in these experiments.

Fig. 7. GPVI, but not α2β1, is essential for platelet adhesion to collagen under flow. Whole blood from the indicated mice was loaded with calcein and perfused at wall shear rates of 150 s^–1 (10 min) or 1000 s^–1 (4 min) over a collagen-coated surface. Blood from control mice was assessed in the absence or presence of 20 µg/ml JAQ1 Fab fragments. Upper panels: representative phase-contrast microscope images after perfusion. Lower panels: surface area coverage of calcein fluorescence at the end of the perfusion period (mean ± SEM, n = 3–6). Similar results were obtained when analyzing surface area coverage of platelets from phase-contrast images.

Fig. 8. Model for platelet adhesion to collagen. 1. The initial tethering to the reactive surface is mediated predominantly by GPIbα–vWF interactions and is important at high shear rates (>500 s^–1), but may not be relevant at lower shear rates (Savage et al., 1998). 2. GPVI–collagen interactions lead to cellular activation followed by shifting of integrins to a high affinity state. This activation step is proposed to be a strict prerequisite for adhesion. 3. Firm adhesion of platelets to collagen through activated α2β1 (directly) and αIIbβ3 (indirectly via vWF or other ligands). Integrin α2β1–collagen interactions are not essential for this process. This scheme only includes interactions examined in the current study (with the exception of GPIbα– vWF) and does not exclude the involvement of other receptor–ligand interactions.