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(Circulation. 2004;110:2946-2951.)
© 2004 American Heart Association, Inc.

Vascular Medicine

Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced ₂ß₁ Levels or Concomitant Aspirin Therapy

Sabine Grüner, MD^*; Miroslava Prostredna, MSc^*; Barsom Aktas, PhD; Alexandra Moers, PhD; Valerie Schulte, PhD; Thomas Krieg, MD; Stefan Offermanns, MD; Beate Eckes, PhD; Bernhard Nieswandt, PhD

From the Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Würzburg (S.G., M.P., B.A., V.S., B.N.); the Institute of Pharmacology, University of Heidelberg, Heidelberg (A.M., S.O.); and the Department of Dermatology, University of Cologne, Cologne (T.K., B.E.), Germany.

Correspondence to Bernhard Nieswandt, PhD, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany. E-mail bernhard.nieswandt{at}virchow.uni-wuerzburg.de

Received March 5, 2004; revision received May 13, 2004; accepted May 21, 2004.

	Abstract

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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 ₂ß₁, 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 ₂^–/– or 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 ₂^–/– or ₂^+/– platelets to collagen. This process required the simultaneous activation of the G_q 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 -chain–deficient but not control mice.

Conclusions— Anti–GP VI therapy may result in defective hemostasis in patients with reduced ₂ß₁ 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.

Key Words: collagen • glycoproteins • receptors • platelets • thrombosis

	Introduction

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Platelet adhesion and aggregation on the extracellular matrix is a key mechanism for normal hemostasis limiting blood loss after tissue trauma but may also lead to occlusion and irreversible tissue damage or infarction in diseased vessels.^1,2 Therefore, platelet inhibition has become an important strategy for the control of cardiovascular diseases.^1,2 Collagen is one of the major constituents of the subendothelial extracellular matrix and initiates adhesion and aggregation of platelets. In addition to glycoprotein (GP) Ib-V-IX and _IIbß₃ integrin, which indirectly interact with collagen via von Willebrand factor,³ several direct collagen receptors have been identified on platelets, most importantly ₂ß₁ integrin⁴ and GP VI,^5,6 which noncovalently associates with the signal transducing Fc receptor (FcR) -chain in the platelet membrane.⁷ In vitro, GP VI is essential for platelet-collagen interaction because it mediates the activation of ß₁- and ß₃-integrins, thereby allowing firm adhesion and thrombus growth.⁸ In vivo, GP VI/FcR deficiency results in profound protection in different mouse models of arterial thrombosis,^9,10 but GP VI–deficient humans^5,11 and mice^12,13 display no major bleeding defect, suggesting that other receptors can substitute for GP VI during normal hemostasis. Thus, GP VI may serve as an attractive antithrombotic target, particularly because the receptor can be irreversibly depleted from circulating platelets in vivo, resulting in a GP VI–knockout-like phenotype and prolonged antithrombotic protection.^12,14

The role of ₂ß₁ in platelet-collagen interactions has been controversial. Initially, ₂ß₁ was thought to be essential for shear-resistant adhesion on collagen but not to be involved in the activation process.¹⁵ However, it is now recognized that the integrin plays a significant but not essential role for the adhesion process⁸ and that it contributes to signaling directly¹⁶ and indirectly by reinforcing GP VI–collagen interactions.¹⁷ Despite this clear function of ₂ß₁ in vitro, ₂-deficient mice display no hemostatic defect,^18,19 and they form occlusive thrombi in the injured carotid artery,²⁰ although in one study, this process was found to be delayed.²¹ In healthy humans, ₂ß₁ expression levels on platelets vary up to 10-fold,²² demonstrating that even individuals with very low platelet ₂ß₁ levels suffer no bleeding problem.

Although ₂ß₁ plays a supportive rather than an essential role in normal platelets, recent in vitro studies have revealed a more prominent function of the integrin when GP VI levels are reduced or the function of the receptor is inhibited.^8,23 Signaling through ₂ß₁ is similar to that induced by GP VI, because both receptors regulate a similar set of intracellular signaling molecules, including Syk, SLP-76, and phospholipase C2.¹⁶ Thus, ₂ß₁ and GP VI appear to have partially redundant functions, but the in vivo significance of this is unclear.

We report here that the induction of a GP VI deficiency in mice lacking or expressing half-levels of ₂ß₁ results in a severe hemostatic defect, as shown by markedly increased bleeding times. Thromboxane (Tx) A₂ restored defective adhesion of GP VI–depleted wild-type but not ₂^–/– or ₂^+/– platelets to collagen in vitro and was necessary to arrest bleeding in anti–GP VI–treated or FcR chain–deficient but not wild-type mice. These results provide the first evidence that platelet collagen receptors are essential for normal hemostasis and strongly indicate that anti–GP VI therapy may induce a bleeding defect in patients with reduced ₂ß₁ levels or concomitant aspirin therapy.

	Methods

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Animals
All animal experiments and care were approved by the local Animal Care and Use Committee. Classic mouse mutants deficient in the ₂ integrin- (₂^–/–) and G_q-subunits (G_q^–/–) as well as the conditional G₁₃-deficient mouse line (G₁₃^–/–) were produced as described.^18,24,25 Both mutant and wild-type control animals were of 129/Sv x C57BL/6 genetic background and were used at the age of 10 to 16 weeks. To induce GP VI depletion, mice were injected with 100 µg JAQ1 intraperitoneally, and platelets were used for analysis on day 5.

Antibodies and Chemicals
JON/A-PE, which preferentially binds to activated integrin _IIbß₃,²⁶ and anti–GP Ib-FITC were from Emfret Analytics. The anti–GP VI monoclonal antibody JAQ2¹⁴ and all other antibodies and chemicals were from previously described sources.¹²

Platelet Preparation
Platelet preparation was performed as described previously.¹² Briefly, whole blood was drawn from the retro-orbital plexus of anesthetized mice and collected in TBS containing 20 U/mL heparin. After washing the platelets twice with HEPES-Tyrode’s buffer (137 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaHCO₃, 0.3 mmol/L NaH₂PO₄, 5.5 mmol/L glucose, 5 mmol/L HEPES, 0.35% BSA), platelets were resuspended in Tyrode’s buffer containing 1 mmol/L CaCl₂ and 1 mmol/L MgCl₂ to 2x10⁶ platelets/µL.

Flow Cytometry
Platelets (2x10⁶) were left untreated or stimulated with ADP (5 µmol/L), U46619 (1 µmol/L), or both and then stained with fluorophore-conjugated monoclonal antibodies at saturating concentrations for 15 minutes at room temperature and analyzed directly on a FACScalibur (Becton Dickinson). Platelets were identified by FSC/SSC characteristics.

Aggregometry
To determine platelet aggregation, light transmission was recorded by use of platelet-rich plasma (200 µL with 0.5x10⁶ platelets/µL) in a Fibrintimer 4 channel aggregometer (APACT Laborgeräte und Analysensysteme).

Adhesion Under Flow Conditions
Mouse blood (1 vol) was collected into 0.5 vol of HEPES buffer, pH 7.45, containing 137 mmol/L NaCl, 5.6 mmol/L glucose, 5 mmol/L HEPES, 2.7 mmol/L KCl, 2 mmol/L MgCl₂, 0.42 mmol/L NaH₂PO₄, and 0.1% BSA, 120 µmol/L PPACK, and 15 U/mL heparin. Rectangular coverslips (24x60 mm) were coated with 0.25 mg/mL "Horm type" collagen (Nycomed) for 1 hour at 37°C and blocked with 1% BSA. Perfusion of whole blood was performed as described.⁸ Briefly, transparent flow chambers with a slit depth of 50 µm, equipped with the collagen-coated coverslips, were rinsed with HEPES buffer supplemented with 2 mmol/L CaCl₂ and 1 U/mL heparin and connected to a syringe filled with the anticoagulated blood. Perfusion was performed at room temperature with a pulse-free pump at high shear stress (4 minutes, flow rate of 7.53 mL/h, equivalent to a wall shear rate of 1000 s^–1). When indicated, ADP (5 µmol/L), U46619 (1 µmol/L), or a combination of the 2 agonists was coinfused to the blood directly before it entered the flow chamber. During perfusion, microscopic phase-contrast images were recorded in real time. Thereafter, the chambers were rinsed by a 10-minute perfusion with HEPES buffer, pH 7.45 (2 mmol/L CaCl₂ and 1 U/mL heparin), at the same shear stress. Phase-contrast images were recorded from at least 5 different microscopic fields (x63 objectives). Image analysis was performed offline with Metamorph software (Visitron). The platelet adhesion results are expressed as the mean percentage of total area covered or single platelets per microscopic field.

Bleeding-Time Experiments
Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (ketamine 100 mg/kg, Parke-Davis; xylazine 5 mg/kg, Bayer AG), and a 3-mm segment of the tail tip was cut off with a scalpel. Tail bleeding was monitored by gently absorbing the bead of blood with a filter paper without contacting the wound site. When no blood was observed on the paper after 15-second intervals, bleeding was determined to have ceased.²⁷ The experiment was stopped after 20 minutes. In some experiments, mice were treated with 100 mg/kg acetylsalicylic acid (Aspisol, Bayer AG) in sterile PBS intravenously 3 hours before the experiments.

Statistical Evaluation
Statistical analysis was performed by use of ANOVA (Scheffé test).

	Results

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To better define the function of GP VI and ₂ß₁ in hemostasis, we depleted GP VI in ₂^–/– mice by injection of the anti–GP VI antibody JAQ1. In agreement with previous results,¹² on day 5, JAQ1-treated wild-type and ₂^–/– mice had normal platelet counts (Figure 1a) but were GP VI–deficient, as shown by analysis with the anti–GP VI monoclonal antibody JAQ2¹⁴ by flow cytometry (Figure 1b) and Western blot analysis (data not shown). In contrast, all other receptors examined, including GP Ib and _IIbß₃, were not affected by the treatment. Because of the absence of GP VI, both wild-type and ₂^–/– platelets did not aggregate in response to high doses of collagen (20 µg/mL), whereas they normally responded to other agonists such as ADP (5 µmol/L) or the stable TxA₂ analog U46619 (1 µmol/L) (Figure 1c). To test whether the ₂/GP VI double deficiency would have any effect under in vivo conditions, we determined tail bleeding times as a measure of primary hemostasis. As reported previously,¹² JAQ1-treated wild-type mice displayed only slightly increased bleeding times. In marked contrast, however, in ₂^–/– mice, the same treatment induced a severe hemostatic defect, with 20 of 33 mice (60.6%) bleeding longer than 20 minutes (Figure 1d). Furthermore, the mean bleeding time of the other 13 mice was 2.6 times increased compared with JAQ1-treated wild-type mice. This finding demonstrated that the presence of either functional GP VI or ₂ß₁ is absolutely required for normal primary hemostasis.

View larger version (28K): [in this window] [in a new window]   Figure 1. Anti–GP VI treatment severely compromises hemostasis in 2–/– mice. Wild-type (wt) or 2-deficient (2–/–) mice were injected with 100 µg JAQ1 or irrelevant IgG intraperitoneally. On day 5, mice had normal platelet counts (a) (n=8 per group). b, Flow cytometric analysis of platelet glycoprotein expression. c, Platelet-rich plasma from indicated mice was stimulated with ADP, U46619, or collagen and at 37°C under stirring conditions and light transmission was recorded. b, c, Results are representative of 8 mice per group. d, Tail bleeding times (wt, n=26; 2–/–, n=21; JAQ1-treated wt, n=31; JAQ1-treated 2–/–, n=33). Experiment was stopped manually after 20 minutes. Each symbol represents 1 individual.

It is well documented that ₂ß₁ expression levels in platelets vary greatly (up to 10-fold) among the normal population because of several linked polymorphisms within the ₂ gene.²² Although there is growing evidence that high ₂ß₁ levels may be associated with an increased risk of cardiovascular disease, low levels of the integrin do not seem to affect hemostasis in otherwise hemostatically normal individuals.²⁸ However, low ₂ß₁ levels may become clinically significant in a setting in which the overall platelet-collagen interaction is already partially compromised, as in the case of mild type I von Willebrand disease.²⁹ To test whether reduced ₂ß₁ levels would interfere with the safety of anti–GP VI treatment, we examined ₂^+/– mice, which express 50% of normal ₂ß₁ on their platelets¹⁸ (Figure 2a). Five days after injection of 100 µg JAQ1, these mice had normal platelet counts and were GP VI–deficient (not shown). Very unexpectedly, the anti–GP VI treatment resulted in a marked bleeding time prolongation in the heterozygous mice that was similar to that observed in the homozygous mutant mice, with 8 of 14 animals (57.1%) bleeding longer than 20 minutes (Figure 2b). Thus, a 50% reduction in the ₂ß₁ level is sufficient to induce a severe hemostatic defect in anti–GP VI–treated mice. Although the data obtained in mice cannot be directly extrapolated to the situation in humans, these results strongly suggest that anti–GP VI treatment would severely impair primary hemostasis in humans with low ₂ß₁ levels, thereby potentially increasing the risk of uncontrolled bleeding.

View larger version (19K): [in this window] [in a new window]   Figure 2. Defective hemostasis in JAQ1-treated 2+/– mice. a, Flow cytometric analysis of integrin 2-levels in wild-type (+/+) and heterozygous 2-knock out (+/–) mice. b, Integrin 2+/– mice were injected with 100 µg JAQ1 (n=15) or irrelevant IgG (n=12) intraperitoneally, and bleeding times were determined on day 5. Experiment was stopped manually after 20 minutes. Each symbol represents 1 individual.

To examine the molecular determinants of hemostasis, and in particular the role of ₂ß₁, in the absence of functional GP VI, we assessed platelet adhesion in ex vivo whole-blood perfusion studies. GP VI–deficient or –blocked platelets fail to adhere to collagen because of defective activation of integrins ₂ß₁ and _IIbß₃.⁸ To test whether adhesion of GP VI–deficient platelets can be restored by integrin activation through other signaling pathways, we coinfused ADP, U46619, or a combination of the 2 agonists into the flowing blood (1000 s^–1) directly before it entered the flow chamber.⁸ In the case of ADP (5 µmol/L), which induces reversible integrin activation at intermediate levels (Figure 3a), both GP VI–depleted wild-type and ₂^–/– platelets formed large thrombi on the collagen surface within 2 minutes. These thrombi were, however, very unstable and completely washed out when the flow chamber was rinsed (data not shown). In contrast, coinfusion of U46619 (1 µmol/L), which on its own induces weak but sustained integrin activation³⁰ (Figure 3a), consistently resulted in firm adhesion of single GP VI–depleted wild-type but not ₂^–/– or ₂^+/– platelets (Figure 3b). When strong and sustained integrin activation was induced by the coinfusion of ADP and U46619 in combination³⁰ (Figure 3a), GP VI–depleted wild-type, ₂^–/–, and ₂^+/– platelets formed large thrombi on the collagen, the size and stability of which was comparable in all groups (Figure 3b). This ₂ß₁-independent adhesion is mediated by _IIbß₃–von Willebrand factor interaction, because it was completely inhibited in the presence of Fab' fragments of the anti-_IIbß₃ antibody JON/A (30 µg/mL, Figure 3c). These findings suggest that ₂ß₁ plays a major role for shear-resistant platelet adhesion on collagen under conditions of weak but not strong integrin activation, because under the latter conditions, _IIbß₃ is sufficient to arrest the cells. This is in line with the well-documented requirement for ₂ß₁ for adhesion to monomeric or degraded collagen, which is a weak agonist at GP VI and therefore induces only low-level integrin activation.³¹

View larger version (47K): [in this window] [in a new window]   Figure 3. Adhesion of GP VI–depleted platelets. a, Integrin activation in platelets in response to different agonists. Washed wild-type platelets were stimulated with ADP, U46619, or combination of both agonists, and IIbß3 activation was determined with JON/A-PE after 1 minute (black line) and 15 minutes (gray line). Nonactivated platelets are indicated as gray histogram. Data shown are from wild-type platelets, but similar results were obtained with 2-deficient platelets. b, Whole blood was perfused over a "Horm type" collagen-coated coverslip at high shear (1000 s–1).8 Where indicated, agonists were coinfused to blood. Top, Representative phase-contrast images taken at end of experiment. Bottom, Firmly adherent platelets or surface area covered by thrombi at end of experiment of JAQ1-treated wt (white bars), 2–/– (black bars), and 2+/– mice (gray bars) (mean±SD, n=8). c, Whole blood from JAQ1-treated wild-type (white bar) or 2–/– (black bar) mice was preincubated with Fab' fragments of blocking anti-IIbß3 antibody JON/A and perfused as above together with U46619 and ADP. Representative phase-contrast images (left) and numbers of firmly adherent platelets (mean±SD, n=8 per group) at end of experiment are shown.

On fibrillar collagens as found in the extracellular matrix, GP VI mediates strong and sustained integrin activation,³¹ which may explain why ₂ß₁ is not required for hemostasis under normal conditions.^18,19 In anti–GP VI–treated mice, however, ₂ß₁ is strictly required to arrest bleeding, suggesting that the stimuli leading to integrin activation in the absence of functional GP VI must be rather weak, ie, insufficient to induce _IIbß₃-dependent adhesion. We suspected TxA₂ of playing a role in this process, because in vitro, this activation pathway induces ₂ß₁-dependent adhesion of GP VI–deficient platelets to collagen (Figure 3b). To test this hypothesis directly, we inhibited TxA₂ synthesis in control and JAQ1-treated wild-type mice (day 5) with aspirin (100 mg/kg) and determined the tail bleeding times 3 hours after aspirin treatment. Strikingly, although aspirin had only a minor effect on bleeding times in control mice, it severely compromised hemostasis in anti–GP VI–treated mice, with 10 of 18 animals (55.5%) bleeding longer than 20 minutes (Figure 4a). To further substantiate this surprising finding, we used FcR-chain–deficient mice, which fail to express GP VI on their platelets.³¹ These mice are largely protected from arterial thrombus formation and subsequent neointimal hyperplasia⁹ as well as collagen-induced thromboembolism,²¹ but they display no increased bleeding tendency.³¹ Treatment of these mice with aspirin, however, almost completely blocked hemostasis, as shown by bleeding times >20 minutes in 7 of 10 mice (Figure 4a). These results demonstrate that the TxA₂-mediated activation pathway becomes crucial for primary hemostasis in the absence of functional GP VI, suggesting that the safety of anti–GP VI treatment might be blunted in patients concomitantly taking aspirin at antithrombotic, analgetic, or antiphlogistic dosages.

View larger version (24K): [in this window] [in a new window]   Figure 4. TxA2 is required for hemostasis in GP VI–deficient mice. a, Control (n=12), JAQ1-treated (day 5, n=18), or FcR–/– (n=9) mice received 100 mg/kg acetylsalicylic acid (ASA), and bleeding times were determined after 3 hours. Experiment was stopped manually after 20 minutes. b, Wild-type (wt, white bar), G13–/– (black bar), and Gq–/– (gray bar) were treated with JAQ1. On day 5, whole blood was perfused over a Horm-type collagen-coated coverslip (1000 s–1). Platelets were stimulated by coinfusion of 1 µmol/L U46619. Top, Representative phase-contrast images taken at end of experiment. Bottom, Firmly adherent platelets (mean±SD, n=4 per group).

In platelets, the TxA₂ receptor couples to the G_q and G₁₂/G₁₃ signaling pathways, both of which are crucial for the formation of stable thrombi under flow conditions in vitro and in vivo.^24,25 To determine which of these signaling pathways is required for U46619-induced adhesion of GP VI–deficient platelets, we injected wild-type, G_q-, and G₁₃-deficient mice with JAQ1 and tested the adhesion of their platelets to collagen under flow conditions ex vivo on day 5. Interestingly, U46619 was unable to induce adhesion of G_q^–/– or G₁₃^–/– platelets, demonstrating that both signaling pathways are essential for this process to occur (Figure 4b).

	Discussion

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Thus, platelet adhesion in the absence of functional GP VI requires the concerted action of different G-protein–mediated signaling pathways. This indicates that a reduced activity of one of these pathways, which by itself may not be sufficient to affect hemostasis under normal conditions, could cause a severe bleeding defect in individuals receiving anti–GP VI treatment. Although there is only a weak correlation between the bleeding time and bleeding risk, it is tempting to speculate that the combination of anti–GP VI agents with other antiplatelet compounds to improve the antithrombotic effectiveness of the therapy, as currently discussed for clopidogrel and aspirin,³² would have to be carefully evaluated to avoid uncontrolled bleeding in humans.

GP VI mediates platelet activation at sites of vascular injury and plays a central role in pathological thrombus formation.^9,10 Therefore, this receptor has been recognized as a promising antithrombotic target particularly because its inhibition or absence does not cause major bleeding in humans or mice. Identifying the mechanisms underlying primary hemostasis in GP VI–compromised individuals is important to develop effective anti–GP VI agents and to predict their safety. Our data clearly indicate that the loss of GP VI signaling is compensated for by a very sensitive network of different G-protein–mediated signaling pathways that converge in the activation of ₂ß₁, allowing it to arrest the cells and to reinforce activation through "outside-in" signals.¹⁶ Any impairment of this alternative pathway, which may not be of any significance under normal conditions, might lead to an intolerable bleeding risk under anti–GP VI treatment. These observations may have important implications for the development and potential use of anti–GP VI–based therapeutics for the prevention of ischemic cardiovascular disease.

	Acknowledgments

 
This work was supported by grant Ni556/4-1 (to Dr Nieswandt) and SFB 589 (to Dr Eckes and Dr Krieg) from the Deutsche Forschungsgemeinschaft (DFG). We thank M. Koch and S. Hartmann for excellent technical assistance.

	Footnotes

 
*The first 2 authors contributed equally to this work.

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