To test the biological significance of platelet–collagen interactions in the processes of adhesion and aggregation in vivo, we assessed platelet–vessel wall interactions after vascular injury of the mouse carotid artery. Vigorous ligation of the carotid artery consistently caused complete loss of the endothelial cell layer and initiated platelet adhesion at the site of injury, as assessed by scanning electron microscopy ( a). Next, we used in vivo fluorescence microscopy (18
) to directly visualize and quantify the dynamic process of platelet accumulation after vascular injury. Numerous platelets were tethered to the vascular wall within the first minutes after endothelial denudation (4725 ± 239 platelets/mm2
). Virtually all platelets establishing contact with the subendothelium initially exhibited a slow surface translocation of the “stop-start” type (23
). As we observed transition from initial slow surface translocation to irreversible platelet adhesion in 88% of all platelets (4.182 ± 253 platelets/mm2
; b), platelet arrest remained transient in only 12% (543 ± 32 platelets/mm2
). Once firm arrest was established, adherent platelets recruited additional platelets from the circulation, resulting in aggregate formation ( c). Similar characteristics of platelet recruitment have been obtained earlier with immobilized collagen in vitro (5
). In contrast, only few platelets were tethered to the intact vascular wall under physiological conditions (P < 0.05 vs. vascular injury) and ~100% of these platelets were displaced from the vascular wall without firm arrest (P < 0.05 vs. vascular injury; , a–c).
Figure 1. Platelet adhesion and aggregation after vascular injury of the common carotid artery in C57BL/6J mice in vivo. (a) Scanning electron micrographs of carotid arteries before (left) and 2 h after vascular injury (right). Endothelial denudation induces platelet (more ...)
Subendothelial fibrillar collagen has been proposed to be of major importance for platelet adhesion and aggregation at sites of vascular injury (2
) as in vitro it strongly supports platelet activation and adhesion. However, this hypothesis has not been tested in vivo where various other agonists and adhesion molecules might be involved in thrombus formation. To directly test the in vivo relevance of platelet–collagen interactions in arterial thrombus formation, we inhibited or deleted GPVI in vivo. The mAb JAQ1 (10
) blocks the major collagen-binding site on mouse GPVI and almost completely inhibits firm platelet adhesion to immobilized fibrillar collagen under high shear flow conditions in vitro (12
). To study the effect of GPVI inhibition in arterial thrombus formation, mice received syngeneic, fluorescence-tagged platelets preincubated with JAQ1 Fab fragments or isotype-matched control IgG and carotid injury was induced as described above. Very unexpectedly, platelet tethering/slow surface translocation at sites of endothelial denudation, a process thought to be mediated solely by GPIbα interaction with immobilized vWf (1
), was reduced by 89% (P < 0.05 vs. control IgG; a) in the presence of JAQ1 Fab fragments. In addition, stable platelet arrest was reduced by 93% in the presence of JAQ1 ( a). We observed transition from initial tethering/slow surface translocation to irreversible platelet adhesion in only 58% of those platelets establishing initial contact with the subendothelial surface compared with 89% with control IgG–pretreated platelets (P < 0.05; b). Aggregation of adherent platelets was virtually absent after pretreatment of platelets with JAQ1 Fab fragments but not in the controls (P < 0.05 vs. control; ). The unanticipated inhibitory effect of GPVI blockade on tethering/slow surface translocation prompted us to examine the role of GPIbα in this process. Mice received fluorescence-tagged platelets preincubated with Fab fragments of a function blocking antibody against GPIbα (p0p/B) and carotid injury was induced as described above. As shown in e, this treatment resulted in a similarly profound reduction in platelet tethering and firm adhesion (and consequently also in aggregate formation) as anti-GPVI treatment (see above) confirming the crucial role of GPIbα for platelet attachment to the damaged vascular wall under conditions of arterial shear. This finding strongly suggested that both GPVI and GPIbα are required to recruit platelets to the injured arterial wall in vivo.
Figure 2. Inhibition of GPVI abrogates platelet adhesion and aggregation after vascular injury. (a) Platelet adhesion after vascular injury was determined by intravital video fluorescence microscopy. Fluorescent platelets were preincubated with 50 μg/ml (more ...)
Together, the results described above demonstrated for the first time that direct platelet–collagen interactions are essential for initial platelet tethering and subsequent stable platelet adhesion and aggregation at sites of arterial injury. In addition, these data identify GPVI as a key regulator in this process whereas other surface receptors, most importantly GPIb-V-IX and α2β1, are not sufficient to initiate platelet adhesion and aggregation on the subendothelium in vivo.
The profound inhibition of platelet tethering by GPVI blockade was surprising and suggested a previously unrecognized function of this receptor in the very initial phase of thrombus formation. To exclude the possibility that this effect was based on steric impairment of other receptors, e.g. GPIb-V-IX, by surface-bound JAQ1, we generated GPVI-deficient mice by injection of JAQ1 5 d before vascular injury. As reported previously, such treatment induces virtually complete internalization and proteolytic degradation of GPVI in circulating platelets, resulting in a GPVI knockout–like phenotype for at least 2 wk (11
). As illustrated in a, GPVI was undetectable in platelets from JAQ1-treated mice on day 5 after injection of 100 μg/mouse JAQ1 but not control IgG, whereas surface expression and function of all other tested receptors, including GPIb-V-IX, αIIb
, and α2
was unchanged in both groups of mice, confirming earlier results ( a and ; reference 11
Figure 3. Platelet adhesion after endothelial denudation in GPVI-deficient mice. (a) JAQ1-treated mice lack GPVI. On the top, platelets from mice pretreated with irrelevant control IgG or anti-GPVI (JAQ1) were stained for GPVI and GPIIb/IIIa (top) or GPIa and GPIbα (more ...)
Surface Expression of GPs on Platelets from JAQ1-treated Mice
As shown by scanning electron microscopy, platelet adhesion and aggregation after endothelial denudation of the common carotid artery were virtually absent in GPVI-deficient, but not in IgG-pretreated, mice ( b). Next, in vivo video fluorescence microscopy was used to define platelet adhesion dynamics after vascular injury in GPVI-deficient mice (). The loss of GPVI profoundly reduced tethering/slow surface translocation of platelets at the site of vascular injury by 83% compared with IgG-pretreated mice (P < 0.05). This GPVI-independent slow surface translocation required vWf-GPIbα–interaction as it was abrogated by preincubation of the platelets with Fab fragments of p0p/B (anti-GPIbα), confirming the critical role of GPIbα in this process (not depicted). In the absence of GPVI, stable platelet adhesion was reduced by ~90% compared with the (IgG-treated) control, whereas aggregation of adherent platelets was virtually absent (). We saw transition from platelet tethering to stable platelet adhesion in only 58% of all platelets initially tethered to the site of injury compared with 89% with control mAb–pretreated platelets (P < 0.05; d), indicating that GPIbα-dependent surface translocation is not sufficient to promote stable platelet adhesion and subsequent aggregation.
To further substantiate the role of GPVI in the process of platelet recruitment after endothelial disruption, we next examined platelet adhesion/aggregation using two additional models of arterial thrombosis. First, arterial injury was induced in control or GPVI-depleted mice by local administration of ferric chloride to the adventitial surface of the carotid artery as previously described (21
). Time to arterial occlusion was monitored by in vivo fluorescence microscopy. As shown in , FeCl3
exposure resulted in a rapid thrombotic response in control animals. 9 out of 10 carotid arteries showed complete occlusion after 235 ± 33 s. In contrast, arterial thrombus formation was dramatically retarded in GPVI-deficient mice (P < 0.05 vs. control mice). In fact, 6 out of 10 GPVI-deficient mice did not show arterial occlusion until 600 s after removal of the FeCl3
-saturated filter paper. In the remaining vessels, occlusion was markedly delayed (356 ± 55 s). These results further support a crucial role of GPVI in the process of arterial thrombus formation.
Figure 4. Role of GPVI in arterial thrombosis after ferric chloride exposure. Vascular injury of the carotid artery was induced by local application of ferric chloride on the carotid artery in GPVI-deficient or control mice. The time to thrombotic occlusion of (more ...)
Next, we assessed platelet recruitment in the carotid artery after wire-induced endothelial disruption (22
). As reported earlier by Zhu et al. (28
) and Lindner et al. (22
), vascular injury with a flexible wire consistently caused complete endothelial denudation (unpublished data). In untreated control animals and mice pretreated with irrelevant control IgG, disruption of the endothelial surface initiated platelet tethering and adhesion as assessed in vivo by video fluorescence microscopy () . Numerous platelets were tethered to the vascular wall within the first minute after endothelial denudation (11.495 ± 1.283 tethered platelets/mm2
). ~46% of all platelets establishing contact with the subendothelium showed transition from initial slow surface translocation to irreversible platelet adhesion (5.266 ± 915 firmly adherent platelets/mm2
). Platelet adhesion at the site of injury was associated with the formation of platelet aggregates attached to the site of injury. Platelet adhesion dynamics in mice pretreated with irrelevant IgG did not differ significantly from untreated control animals (13.521 ± 2.519 and 5.474 ± 1.575 tethered and firmly adherent platelets/mm2
, respectively). In contrast to control animals, platelet tethering/slow surface translocation and firm adhesion at sites of wire-induced endothelial denudation were reduced by 90 and 95% in GPVI-depleted mice (P < 0.05 vs. control mice; ). We observed transition from initial tethering/slow surface translocation to irreversible platelet adhesion in only 24% of those platelets establishing initial contact with the subendothelial surface compared with 46% with control animals (P < 0.05). Aggregation of adherent platelets was virtually absent in GPVI-deficient mice (P < 0.05 vs. control; ). Together, these data add additional strong evidence to the concept that GPVI-mediated direct platelet–collagen interactions are essential for initial platelet tethering and subsequent stable platelet adhesion and aggregation at sites of arterial injury.
Figure 5. Role of GPVI in the regulation of platelet recruitment after wire injury of the carotid artery. Wire-induced endothelial denudation of the carotid artery was induced in GPVI-deficient mice. Untreated animals served as controls. The left shows representative (more ...)
Fibrillar collagen is a major constituent of the normal vessel wall but also of atherosclerotic lesions (29
). In the process of atherogenesis, enhanced collagen synthesis by intimal smooth muscle cells and fibroblasts has been shown to significantly contribute to luminal narrowing (30
). Plaque rupture or fissuring, either spontaneously or after balloon angioplasty, results in exposure of collagen fibrils to the flowing blood but their contribution to arterial thrombus formation has been elusive. Platelets express a large number of different collagen receptors, which made it very difficult to identify the role of each of these receptors in the processes of adhesion and activation in vitro. In addition, reagents suitable for specific inhibition of individual collagen receptors in vivo have not been available. Only recently has GPVI been identified as the central platelet receptor that is essential for both adhesion and activation of platelets on collagen in vitro (12
In contrast, the absence of other major collagen receptors such as integrin α2
or GPV only results in more subtle defects in the platelet–collagen interaction (7
), suggesting that inhibition or deletion of GPVI, but no other collagen receptor, is required to abrogate platelet collagen–interactions in vivo.
The results of this study provide the first definitive evidence that subendothelial collagens are the major trigger of arterial thrombus formation and reveal an unexpected function of GPVI in platelet recruitment to the injured vessel wall. The processes of platelet tethering and slow surface translocation under conditions of elevated shear are known to largely depend on GPIbα interaction with immobilized vWf (1
). In addition, a number of studies have shown that GPIbα or even its NH2
-terminal 45-kD domain, which carries the binding site for vWf, mediates tethering of cells or coated beads, respectively, to a vWf-coated surface under high shear flow conditions (32
). Together, these findings suggested that GPIbα–vWf interactions might be sufficient to establish the initial contact and slow surface translocation of platelets at sites of vascular injury. However, the results presented here demonstrate that tethering/slow surface translocation of platelets at sites of arterial injury in vivo is largely inhibited in the absence of functional GPVI although expression and function of GPIb-V-IX is not altered under these experimental conditions ( and ; reference 11
). On the other hand, inhibition of the vWf binding site on GPIbα by Fab fragments of the p0p/B mAb also virtually abrogated platelet adhesion to the injured vessel wall, confirming the strict requirement for this interaction under conditions of high shear in mice ( e). Thus, it appears that GPIbα and GPVI act in concert to recruit platelets to the subendothelium in vivo by yet undefined mechanisms. This strongly suggests that presentation of vWf on the extracellular matrix of the damaged vessel wall may differ significantly from the conditions found in vitro when it is homogeneously coated to a glass surface. At sites of vascular injury, vWf is thought to become immobilized mostly on fibrillar collagen (1
). Based on our results, one may speculate that the vWf layer on collagen fibrills might be inhomogeneous and frequently interrupted making efficient interactions between GPIbα and vWf impossible unless a second receptor interacts with the “gaps,” i.e., collagen not covered with vWf. GPVI is known to be a low affinity collagen receptor mediating loose, but not firm adhesion that may support this hypothesis (14
). Another point in favor of the idea that GPIb and GPVI act in concert is the recent identification of different snake venom–derived proteins that interact with platelets specifically through both GPIb and GPVI, indicating that these two receptors might be physically and functionally linked (34
During platelet tethering, ligation of GPVI can shift αIIb
integrins from a low to a high affinity state (12
). Both αIIb
then act in concert to promote subsequent stable arrest of platelets on collagen (5
) whereas αIIb
is essential for subsequent aggregation of adherent platelets. Thus, ligation of GPVI during the initial contact between platelets and subendothelial collagen provides an activation signal that is essential for subsequent stable platelet adhesion and aggregation. Our results suggest that occupation or lateral clustering of GPIbα (during GPIbα-dependent surface translocation), which has been shown to induce low levels of αIIb
integrin activation in vitro (32
), may not be sufficient to promote platelet adhesion in vivo.
This revised model of platelet attachment to the subendothelium highlights a central role of GPVI–collagen interactions in all major phases of thrombus formation, i.e., platelet tethering, firm adhesion, and aggregation at sites of arterial injury (e.g., during acute coronary syndromes). Although the data obtained in mice cannot be directly extrapolated to the situation in humans, the profound antithrombotic protection that was achieved by inhibition or depletion of GPVI strongly indicates that a selective pharmacological modulation of GPVI–collagen interactions may become a promising strategy to control the onset and progression of pathological arterial thrombosis.