Valerie Schulte

Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen

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 α2β1 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 β1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of β1‐null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, β1‐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 αIIbβ3 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 α2β1 integrin, which strengthens adhesion without being essential.

A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet–vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by ∼89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet–collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.

Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen.

The integrin α2-subunit was ablated in mice by targeted deletion of the ITGA2 gene. α2-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin α2β1 has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, α2-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of α2-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, α2-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of α2-deficient but not wild type platelets is abrogated. These results demonstrate that integrin α2β1 significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of α2-deficient platelets to soluble collagen. Thus, α2β1 plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that α2β1-deficient animals suffer no bleeding anomalies.

Costimulation of Gi- and G12/G13-mediated Signaling Pathways Induces Integrin αIIbβ3 Activation in Platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Gαq-deficient platelets that do not respond with activation of integrin αIIbβ3 to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Gαq-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin αIIbβ3-mediated aggregation of Gαq-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin αIIbβ3 activation. Although Gq-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin αIIbβ3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving Gq and Gi as well as G12/G13.

Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced α2β1 Levels or Concomitant Aspirin Therapy

Background—Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. Methods and Results—Mice lacking (−/−) or expressing half-levels (+/−) of the other major platelet collagen receptor, integrin &agr;2&bgr;1, were injected with the anti–GP VI antibody JAQ1 and analyzed on day 5. Anti–GP VI treatment resulted in a marked hemostatic defect in &agr;2−/− or &agr;2+/− mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti–GP VI–treated wild-type but not &agr;2−/− or &agr;2+/− platelets to collagen. This process required the simultaneous activation of the Gq and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti–GP VI–treated or GP VI/Fc receptor &ggr;-chain–deficient but not control mice. Conclusions—Anti–GP VI therapy may result in defective hemostasis in patients with reduced &agr;2&bgr;1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti–GP VI–based therapeutics in the prevention of cardiovascular disease.

Rhodocytin (Aggretin) Activates Platelets Lacking α2β1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibα

Although α2β1integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom ofCalloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against α2β1 integrin. This finding suggested that clustering of α2β1 integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to α2β1 integrin. Here we show that the Cre/loxP-mediated loss of β1 integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of α2β1integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibα either on normal or on β1-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both α2β1 integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbα rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.

Complementary roles of glycoprotein VI and α2β1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen‐bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso‐occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin α2β1, is essential in priming platelet‐collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen‐dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin β1 reduces the GPVI‐evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in Gαq or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen‐induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin α2β1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

Rhodocytin (aggretin) activates platelets lacking a2b1 integrin, GPVI, and the ligand binding domain of GPIba

Although α2β1integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom ofCalloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against α2β1 integrin. This finding suggested that clustering of α2β1 integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to α2β1 integrin. Here we show that the Cre/loxP-mediated loss of β1 integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of α2β1integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibα either on normal or on β1-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both α2β1 integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbα rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.

Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands

We have investigated the effects of decreased levels of the complex between glycoprotein VI (GPVI) and the Fc receptor gamma-chain (FcRgamma) on responses to collagen and GPVI-specific ligands in murine platelets. We show that levels of GPVI-FcRgamma of the order of 50% and 20% of wild-type levels caused 2- and 5-fold shifts to the right respectively in the dose-response curve for aggregation in response to collagen, the snake toxin convulxin and the monoclonal antibody JAQ1. In addition, there is a delay in the onset of aggregation in response to collagen. In contrast, the stimulation of protein tyrosine phosphorylation by collagen (as measured after 150 s) and adhesion to a collagen-coated surface under static conditions were unaffected in platelets with 50% and 20% of wild-type levels of GPVI. In contrast, responses to a collagen-related peptide (CRP), made up of repeat glycine-proline-hydroxyproline motifs, were markedly inhibited and abolished in platelets expressing 50% and 20% of wild-type levels of GPVI respectively. We suggest that the marked effect of a reduction in GPVI levels on the CRP-induced activation of platelets is due to the multivalent nature of CRP and the fact that GPVI is its sole receptor on platelets. Thus it appears that the interaction of CRP with GPVI is determined by a combination of affinity and avidity. The observation that collagen does not behave like CRP in platelets expressing reduced levels of GPVI, even in the combined presence of blocking antibodies against integrin alpha2beta1 and GPV, suggests that collagen has a greater affinity than CRP for GPVI, and/or that other receptors are involved in its binding to platelets. The clinical significance of these results is discussed.

Evidence for Two Distinct Epitopes within Collagen for Activation of Murine Platelets

It has recently been shown that the monoclonal antibody JAQ1 to murine glycoprotein VI (GPVI) can cause aggregation of mouse platelets upon antibody cross-linking and that collagen-induced platelet aggregation can be inhibited by preincubation of platelets with JAQ1 in the absence of cross-linking (Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E., and Zirngibl, H. (2000) J. Biol. Chem.275, 23998–24002). In the present study, we have shown that cross-linking of GPVI by JAQ1 results in tyrosine phosphorylation of the same profile of proteins as that induced by collagen, including the Fc receptor (FcR) γ-chain, Syk, LAT, SLP-76, and phospholipase Cγ2. In contrast, platelet aggregation and tyrosine phosphorylation of these proteins were inhibited when mouse platelets were preincubated with JAQ1 in the absence of cross-linking and were subsequently stimulated with a collagen-related peptide (CRP) that is specific for GPVI and low concentrations of collagen. However, at higher concentrations of collagen, but not CRP, aggregation of platelets and tyrosine phosphorylation of the above proteins (except for the adapter LAT) is re-established despite the presence of JAQ1. These observations suggest that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelets, is occupied in the presence of high concentrations of collagen. Although this could be a second site on GPVI that is activated by a novel motif within the collagen molecule, the absence of LAT phosphorylation in response to collagen in the presence of JAQ1 suggests that this is more likely to be caused by activation of a second receptor that is also coupled to the FcR γ-chain. The possibility that this response is mediated by a receptor that is not coupled to FcR γ-chain is excluded on the grounds that aggregation is absent in platelets from FcR γ-chain-deficient mice.