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(Circulation. 2004;110:2946-2951.)
© 2004 American Heart Association, Inc.
Vascular Medicine |
2ß1 Levels or Concomitant Aspirin Therapy
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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Methods and Results Mice lacking (/) or expressing half-levels (+/) of the other major platelet collagen receptor, integrin
2ß1, were injected with the antiGP VI antibody JAQ1 and analyzed on day 5. AntiGP 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 antiGP VItreated wild-type but not
2/ or
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 antiGP VItreated or GP VI/Fc receptor
-chaindeficient but not control mice.
Conclusions AntiGP 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 antiGP VIbased therapeutics in the prevention of cardiovascular disease.
Key Words: collagen glycoproteins receptors platelets thrombosis
| Introduction |
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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 VIdeficient humans5,11 and mice12,13 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 VIknockout-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 VIcollagen 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 C
2.16 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 VIdepleted wild-type but not
2/ or
2+/ platelets to collagen in vitro and was necessary to arrest bleeding in antiGP VItreated or FcR
chaindeficient but not wild-type mice. These results provide the first evidence that platelet collagen receptors are essential for normal hemostasis and strongly indicate that antiGP VI therapy may induce a bleeding defect in patients with reduced
2ß1 levels or concomitant aspirin therapy.
| Methods |
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2 integrin- (
2/) and G
q-subunits (G
q/) as well as the conditional G
13-deficient mouse line (G
13/) 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 antiGP Ib-FITC were from Emfret Analytics. The antiGP 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-Tyrodes 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 Tyrodes 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 s1). 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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2ß1 in hemostasis, we depleted GP VI in
2/ mice by injection of the antiGP 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 VIdeficient, as shown by analysis with the antiGP 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.
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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 antiGP 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 VIdeficient (not shown). Very unexpectedly, the antiGP 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 antiGP VItreated mice. Although the data obtained in mice cannot be directly extrapolated to the situation in humans, these results strongly suggest that antiGP VI treatment would severely impair primary hemostasis in humans with low
2ß1 levels, thereby potentially increasing the risk of uncontrolled bleeding.
|
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 VIdeficient or blocked platelets fail to adhere to collagen because of defective activation of integrins
2ß1 and
IIbß3.8 To test whether adhesion of GP VIdeficient 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 s1) 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 VIdepleted 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 VIdepleted 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 VIdepleted 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
IIbß3von 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
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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 antiGP VItreated 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 VIdeficient 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 antiGP VItreated mice, with 10 of 18 animals (55.5%) bleeding longer than 20 minutes (Figure 4a). To further substantiate this surprising finding, we used FcR
-chaindeficient 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 antiGP VI treatment might be blunted in patients concomitantly taking aspirin at antithrombotic, analgetic, or antiphlogistic dosages.
|
In platelets, the TxA2 receptor couples to the Gq 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 VIdeficient platelets, we injected wild-type, G
q-, and G
13-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 G
13/ platelets, demonstrating that both signaling pathways are essential for this process to occur (Figure 4b).
| Discussion |
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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 VIcompromised individuals is important to develop effective antiGP 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-proteinmediated 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 antiGP VI treatment. These observations may have important implications for the development and potential use of antiGP VIbased therapeutics for the prevention of ischemic cardiovascular disease.
| Acknowledgments |
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| Footnotes |
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