Submit Manuscripts
Author Information
Subscribe/Renew
Email Alerts
Feedback
RSS Feed

About the Journal
Editorial Board & Staff

Interventions Homepage Imaging Homepage Interventions Homepage Imaging Homepage

ACC.org
Cardiosmart
Cardiosource
CVN
Imaging Library
JACC Imaging
JACC Interventions


ACC/AHA 2008 Performance Measures for Adults With ST-Elevation and Non-ST-Elevation Myocardial Infarction

ACC/AHA 2008 Guidelines for the Management of Adults With Congenital Heart Disease
cardiology careers collections past issues search home
     

J Am Coll Cardiol, 2006; 47:155-162, doi:10.1016/j.jacc.2005.08.055 (Published online 12 December 2005).
© 2006 by the American College of Cardiology Foundation
This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow View Online Videos
Right arrow All Versions of this Article:
j.jacc.2005.08.055v1
47/1/155    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goto, S.
Right arrow Articles by Ruggeri, Z. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goto, S.
Right arrow Articles by Ruggeri, Z. M.

PRECLINICAL STUDY

Dependence of Platelet Thrombus Stability on Sustained Glycoprotein IIb/IIIa Activation Through Adenosine 5'-Diphosphate Receptor Stimulation and Cyclic Calcium Signaling

Shinya Goto, MD*,*, Noriko Tamura, MS*, Hideyuki Ishida, MD{dagger} and Zaverio M. Ruggeri, MD{ddagger}

* Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan
{dagger} Department of Basic Medicine, Tokai University School of Medicine, Kanagawa, Japan
{ddagger} Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California

Manuscript received February 13, 2005; revised manuscript received July 24, 2005, accepted August 1, 2005.

* Reprint requests and correspondence: Dr. Shinya Goto, Department of Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan (Email: shinichi{at}is.icc.u-tokai.ac.jp).


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 Appendix
 References
 
OBJECTIVES: We sought to evaluate the mechanisms that support the stability of platelet aggregates on a thrombogenic surface exposed to flowing blood.

BACKGROUND: Activation of the membrane glycoprotein (GP) IIb/IIIa—mediated in part through the P2Y1 and P2Y12 adenosine 5'-diphosphate (ADP) receptors—is necessary for platelet aggregation. Platelets in growing thrombi exhibit cyclic calcium signal, suggesting that sustained activation may be required for thrombus stability.

METHODS: Blood was perfused over type I collagen fibrils at the wall shear rate of 1,500 s–1. Three-dimensional visualization of platelet thrombi was obtained in real time with confocal microscopy. The intracytoplasmic Ca2+ concentration ([Ca2+]i) was measured in fluo-3AM–loaded platelets.

RESULTS: The height of platelet thrombi in control blood was 13.5 ± 3.3 µm after 6 min, and increased to 16.3 ± 4.5 µm (n = 8) after an additional 6 min. In contrast, the height was reduced to 5.4 ± 2.2 and 3.3 ± 1.3 µm, respectively (p < 0.01, n = 8), when the blood used in the second 6-min perfusion contained a P2Y1 (MRS2179) or P2Y12 (AR-C69931MX) inhibitor. The [Ca2+]i of platelets within forming thrombi oscillated between 212 ± 38 nmol/l and 924 ± 458 nmol/l, with cycles lasting 4.2 ± 2.8 s that were inhibited completely by AR-C69931MX and partially by MRS2179. Accordingly, thrombi became unstable upon perfusion of blood containing the Ca2+ channel blocker, lanthanum chloride. Flow cytometric studies demonstrated that AR-C69931MX, MRS2179, and lanthanum chloride reduced monoclonal antibody PAC-1 binding to platelets, indicating a decrease of membrane-expressed activated GP IIb/IIIa.

CONCLUSIONS: Continuous P2Y1 and P2Y12 stimulation resulting in cyclic [Ca2+]i oscillations is required for maintaining the activation of GP IIb/IIIa needed for thrombus stability in flowing blood.

Abbreviations and Acronyms
  ADP = adenosine diphosphate
  [Ca2+]i = intracytoplasmic Ca2+ concentration
  FITC = fluorescein isothiocyanate
  GP = glycoprotein
  PPP = platelet-poor plasma
  PRP = platelet-rich plasma
  VWF = von Willebrand factor

Arterial thrombosis may initiate after the rupture of an unstable atherosclerotic plaque, and it involves multiple platelet adhesion and agonist receptors (1) as well as activation of clotting with fibrin deposition (2,3). Two adenosine 5'-diphosphate (ADP) receptors, P2Y1 and P2Y12, mediate platelet stimulation induced not only by exogenous ADP (4–6), but also by shear forces or interactions with extracellular matrixes that cause ADP release from storage granules (7–9). In particular, P2Y12 concurs to the stability of platelet aggregates (10) and may exert a similar effect in developing arterial thrombi (11). In experimental studies, platelets that adhere and aggregate onto immobilized von Willebrand factor (VWF) exhibit cyclic oscillations in intracytoplasmic Ca++ concentration ([Ca2+]i) (12). These have been shown to depend on the concurrent function of P2Y12 and glycoprotein (GP) IIb/IIIa (integrin {alpha}IIbß3), and are linked to the recruitment and activation of flowing platelets into growing thrombi (12). Such findings, and the results obtained with a thrombosis model in P2Y12-deficient mice (13), suggest that continuing stimulation of ADP receptors may be required to initiate as well as propagate thrombus growth on damaged vascular surfaces. Experiments with selective inhibitors have confirmed the importance of P2Y12 in this regard (12), while P2Y1 may be key for the initial activation of ADP-stimulated platelets (14) but have no role in thrombus propagation. In the present study, we have used a fibrillar type I collagen surface exposed to flowing blood and specific antagonists of the ADP receptors and GP IIb/IIIa to evaluate how aggregating platelets are incorporated irreversibly into a thrombus. Our findings may contribute to clarifying the mechanism of action of antithrombotic agents.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 Appendix
 References
 
Blood samples.   Venous blood was obtained from medication-free volunteers (6 men, 2 women; age 28 to 43 years) with their informed consent, and transferred into plastic tubes containing 1/10 volume of the thrombin inhibitor Argatroban (Mitsubishi Kagaku, Tokyo, Japan) to yield a final concentration of 100 µmol/l (15,16), which does not decrease the plasma divalent cation concentration. Platelet-rich plasma (PRP) was separated by centrifugation at 100 g for 15 min and platelet-poor plasma (PPP) by further centrifugation at 800 g for 10 min. The platelet count in PRP was adjusted to 3 x 105/µl.

Reagents.   AR-C69931MX (17) was from AstraZeneca (Loughborough, Leicestershire, United Kingdom). MRS2179 (18) was obtained from Dr. Savi (Sanofi-Synthelabo Recherche, Toulouse, France). Lanthanum chloride (19), acid insoluble fibrillar collagen type I from bovine Achilles tendon, mepacrine (quinacrine hydrochloride), acetyl salicylic acid, ADP, and epinephrine were from Sigma Chemical Co. (St. Louis, Missouri). Tirofiban (Aggrastate) was from Merck & Co. (Allentown, Pennsylvania). Fluo-3 acetoxymethyl ester (Fluo-3AM) was from Molecular Probe (Eugene, Oregon).

Measurement of thrombus volume.   Platelets were rendered fluorescent by adding 10 µmol/l mepacrine (16,20) or 1 µg/ml of the fluorescein isothiocyanate (FITC)-labeled Fab fragment of the anti-GP IIb/IIIa monoclonal antibody, YM337 (Yamanouchi Pharmaceutical Co. Ltd., Tokyo, Japan). Thrombi of similar size were obtained in either case (not shown). A rectangular flow chamber with type I collagen fibrils coated on the glass bottom (15,16,20) was assembled onto the stage of an inverted epifluorescence microscope (Leica, Germany). Blood was aspirated through the chamber with a syringe pump (Harvard Apparatus, Holliston, Massachusetts) at a constant flow rate to yield a wall shear rate of 1,500 s–1. Images were digitized on-line with a color CCD video camera (L-600, Leica, Germany). Thrombus growth was evaluated in two dimensions by measuring the surface area covered by platelets (16) and in three dimensions by confocal microscopy (Fig. 1) as previously reported (15). The effect of inhibitors of platelet function on the stability of platelet thrombi was tested in two-stage experiments, consisting in the perfusion of untreated blood for 6 min followed by blood containing or not a test substance for an additional 6 min.


Figure 1
View larger version (41K):
[in this window]
[in a new window]
  		Figure 1 Three-dimensional projection imaging of platelet thrombi. A piezo-electric motor (a) moved the objective lens at a constant speed of 0.4 µmol/l/s to provide scanning images of the platelet thrombi (a’). The sum of the confocal images in a bottom to top stack (z-axis) was projected on planes at 10° intervals relative to the x axis to obtain the three-dimensional projection images shown on the right, including projections at 0 degrees (top view; A), 60° (B), and 90° (front view; C). The maximum height (h) of the platelet thrombi was calculated from the front view projection, as shown in C.

 
Measurement of [Ca2+]i.   Platelets in PRP were incubated for 30 min at 37°C with fluo-3AM (8 µmol/l), then mixed with erythrocytes separated from the same blood and washed three times by centrifugation and resuspension in a buffer composed of 10 mmol/l HEPES, 140 mmol/l NaCl, pH 7.4 (HEPES buffer). The washed cells were resuspended in homologous PPP containing Argatroban (100 µmol/l) at a 40% hematocrit. Unlike previously suggested (21) but in agreement with recent reports (22), we did not use probenecid to prevent the leakage of fluo-3 because of its effects on platelet function (23). The [Ca2+]i of 10 randomly selected platelets incorporated at different positions within a thrombus was measured using confocal microscopy. Variations in the fluorescence intensity of fluo-3AM were converted into [Ca2+]i using the equation:

Formula Formula
where Kd (495 nmol/l) is the dissociation constant of fluo-3AM for the interaction with Ca2+ (21); F is the measured fluorescent intensity of single platelets; Fmax is the fluorescence intensity of single platelets treated with the Ca2+ ionophore A23187 [GenBank] (10 µmol/l; Sigma) in the presence of 2 mmol/l Ca++; and Fmin is the fluorescent intensity of unstimulated single platelets.

Flow cytometry.   The platelet binding of FITC-conjugated PAC-1, a monoclonal antibody that selectively interacts with activated GP IIb/IIIa, was measured by flow cytometry (FACScan, Becton-Dickinson, San Jose, California). Platelets in PRP were activated with the combination of ADP and epinephrine (25 µmol/l each) or with the thrombin receptor activation peptide (1 mmol/l). Then, FITC-conjugated PAC-1 was added at a final concentration of 2.77 µg/ml, followed by HEPES buffer containing or not AR-C69931MX (100 nmol/l), MRS2179 (100 µmol/l), or lanthanum chloride (1 mmol/l). PAC-1 binding was measured 5, 10, 30, 45, and 60 min after addition of the last solution. All these experiments were performed under static conditions. The median fluorescence of 10,000 single platelets was calculated using the CellQuest software (Becton Dickinson Biosciences).

Statistical analysis.   All numerical data are expressed as mean values ± SD unless otherwise specified. The effect of various concentrations of AR-C69931MX and MRS2179 on the surface coverage by platelets was tested by one-way analysis of variance. Differences between two groups of data were compared by Newman-Keuls test. A p value of <0.05 was considered to denote statistical significance.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 Appendix
 References
 
P2Y1 and P2Y12 antagonists inhibit platelet thrombus growth.   In agreement with previous results (7,8), ADP receptor antagonists inhibited thrombus growth on type I collagen fibrils exposed to blood flowing with a wall shear rate of 1,500 s–1. Platelet surface coverage decreased from 38.0 ± 6.6% to 13.6 ± 3.9% after blocking P2Y12 with 100 nmol/l AR-C69931MX, and to 19.4 ± 5.4% after blocking P2Y1 with 100 µmol/l MRS2179 (p < 0.01). Both antagonists also inhibited thrombus volume (Fig. 2). Untreated blood perfused for 6 min formed multilayered thrombi with a height of 13.2 ± 2.3 µm (n = 8), which was reduced to a single layer of platelets with a height of 3.2 ± 1.1 µm by 100 nmol/l AR-C69931MX (n = 8). With blood containing 100 µmol/l MRS2974, the thrombus height was 6.1 ± 3.5 µm (n = 8), less than with untreated blood (p < 0.01) but more than with the P2Y12 antagonist (p < 0.01).


Figure 2
View larger version (55K):
[in this window]
[in a new window]
  		Figure 2 Three-dimensional projection images of thrombi formed in the presence or absence of adenosine diphosphate receptor antagonists. Blood with fluoresceinated platelets was perfused over immobilized collagen type I fibrils for 6 min at the wall shear rate of 1,500 s–1 in the absence (Control) or presence of MRS2179 (100 µmol/l) or AR-C69931MX (100 nmol/l), as indicated. The platelet thrombi were scanned in the z-axis by confocal microscopy, and the resulting images were projected on planes rotated around the x-axis at 10° intervals (please see the online version of this article for supplemental videos). The figure shows projection images from the top (A), 60° (B), and the front (C). These images are representative of the results obtained in eight separate experiments.

 
Inhibitors of P2Y1, P2Y12, and Ca2+ channels reduce platelet thrombus size.   Blood perfused over collagen type I fibrils for 6 min at the wall shear rate of 1,500 s–1 formed platelet thrombi with a height of 13.5 ± 3.3 µm (n = 8; Fig. 3), increasing to 16.3 ± 4.5 µm after an additional 6 min. In contrast, when blood perfused in the second 6-min period contained 100 nmol/l AR-C69931MX, thrombus size progressively decreased until a single layer of adherent platelets with a height of 3.3 ± 1.3 µm remained. A similar effect, resulting in a 3.4 ± 1.2 µm high single layer of adherent platelets, was observed with the Ca2+ channel blocker, lanthanum chloride (1 mmol/l), whereas 100 µmol/l MRS2974 reduced thrombus height to 5.4 ± 2.2 µm. The latter value was less than with untreated blood but more than with the anti-P2Y12 antagonist (n = 8; p < 0.01 for both comparisons; Fig. 3). Addition of 0.5 µmol/l tirofiban, a GP II/IIIa antagonist (15), to the blood used in the second perfusion also reduced thrombi to a single layer of adherent platelets with a height of 3.1 ± 1.1 µm (n = 8; Fig. 4), whereas 100 µmol/l aspirin allowed continued growth to a height of 15.8 ± 5.5 µm (n = 8; Fig. 4).


Figure 3
View larger version (53K):
[in this window]
[in a new window]
  		Figure 3 Reduction in the size of platelet thrombi exposed to blood containing different antagonists of platelet function. These experiments were performed as described in the caption of Figure 2, with the difference that the surface was exposed to two subsequent aliquots of blood each perfused for 6 min. (A) Representative images (0-degree and 90° projections) of platelet thrombi after perfusion of the first (before) or second (after) control blood aliquot (please see the online version of this article for supplemental videos). (B) Representative images of platelet thrombi after perfusion of the first control blood aliquot (before) or a second blood aliquot containing the P2Y12 inhibitor AR-C69931MX (100 nmol/l) (please see the online version of this article for supplemental videos). (C) Cross-sectional area occupied by fluorescent platelets in horizontal planes passing through the thrombi at the indicated distance from the collagen surface, calculated as percentage of the area in the plane closest to collagen surface. The bars labeled "Before" show the area of thrombi formed after perfusion of untreated blood for 6 min. The bars labeled "No Addition," AR-C, MRS, and LAN show the area of thrombi remaining on the surface after an additional 6 min perfusion of untreated blood, or blood treated with the P2Y12 inhibitor AR-C69931MX (100 nmol/l), or the P2Y1 inhibitor MRS2179 (100 µmol/l), or the putative Ca2+ channel inhibitor lanthanum chloride (LAN) (1 mmol/l), respectively. Mean and SEM of eight experiments are shown.

 

Figure 4
View larger version (46K):
[in this window]
[in a new window]
  		Figure 4 Changes in the two-dimensional and three-dimensional structure of platelet thrombi exposed to blood containing antiplatelet agents. These experiments were performed essentially as described in the caption of Figure 3. (A) Representative two-dimensional fluorescence microscopy images of platelet thrombi immediately after perfusion of the first control blood aliquot (0 min) or at different times after beginning the second perfusion with either untreated blood (No Addition) or blood containing tirofiban (0.5 µmol/l), as indicated (please see the online version of this article for supplemental videos). (B) Cross-sectional area of thrombi at the indicated distances from the collagen surface after perfusion of untreated blood for 6 min (No Addition), or after perfusion for an additional 6 min of blood containing aspirin (100 µmol/l) or tirofiban (0.5 µmol/l), as indicated. See Figure 3C for additional details.

 
Platelets within thrombi exhibited cyclic [Ca2+]i oscillations (Fig. 5). The lowest average [Ca2+]i was 212 ± 38 (SEM) nmol/l and the highest 924 ± 458 nmol/l, with a cycle length from peak to peak of 4.2 ± 2.8 s (Fig. 5). These [Ca2+]i variations appeared to depend on specific ion channels because they were blocked by the Ca2+ channel antagonist lanthanum chloride (not shown). Addition of the P2Y12 antagonist to the perfused blood rapidly decreased the platelet [Ca2+]i within formed thrombi; after 2 min the value ranged between 182 ± 22 nmol/l and 244 ± 96 nmol/l without detectable cycles in most platelets (Fig. 5). After addition of the P2Y1 antagonist, the low and high [Ca2+]i values ranged between 192 ± 34 nmol/l and 558 ± 211 nmol/l, respectively, and some but not all platelets showed measurable cycles of 5.8 ± 2.5 s (Fig. 5). Neither tirofiban, in spite of the pronounced effect on platelet thrombus size, nor aspirin had any influence on the cyclic [Ca2+]i of aggregated platelets (not shown).


Figure 5
View larger version (59K):
[in this window]
[in a new window]
  		Figure 5 Changes in the intracytoplasmic Ca2+ concentration ([Ca2+]i) of platelet thrombi caused by adenosine diphosphate receptor antagonists. These experiments were conducted as described in the caption of Figure 3, except that blood was replaced with a cell suspension containing fluo-3AM–loaded platelets, washed erythrocytes, and homologous platelet-poor plasma with the specific thrombin inhibitor argatroban (100 µmol/l) as the anticoagulant. The cell suspension was perfused over type I collagen fibrils at the shear rate of 1,500 s–1 for 4 min to form platelet thrombi. Then, the same cell suspension without or with the addition of the P2Y12 antagonist AR-C69931MX (100 nmol/l), or the P2Y1 antagonist MRS2179 (100 µmol/l), was perfused for an additional 4 min. The [Ca2+]i of platelets incorporated into thrombi was measured during the second perfusion. (Left panels) Intracytoplasmic Ca2+ concentration of five randomly selected platelets recorded for 10 s beginning 2 min after the start of the second perfusion. (Right panels) Images reflecting the concentration of Ca2+ ions in platelets within thrombi formed during perfusion of untreated blood (Control) or blood containing 100 nmol/l AR-C69931MX (ARC).

 
Inhibition of P2Y1 and P2Y12, and blockade of Ca2+ entry reduce activated GP IIb/IIIa on platelets.   Fluorescein isothiocyanate-conjugated PAC-1 binding to platelets, measured as the median fluorescence intensity of 10,000 platelets, increased when platelets were activated by the combination of ADP and epinephrine or the thrombin receptor activating peptide (the results were similar and only the former are shown). Bound PAC-1 slowly but significantly decreased in time when no additional exogenous agonist was added after the initial activation (Fig. 6). The reduction was more marked after adding the P2Y12 inhibitor (Fig. 6), suggesting that continuous stimulation is necessary to maintain the active state of GP IIb/IIIa. A similar effect was observed with the P2Y1 inhibitor or the Ca2+ channel blocker (Fig. 7), but not with aspirin (data not shown).


Figure 6
View larger version (31K):
[in this window]
[in a new window]
  		Figure 6 Changes in PAC-1 binding to activated platelets caused by adenosine diphosphate (ADP) receptor antagonists. To activate platelets, 25 µl of HEPES buffer containing ADP and epinephrine (400 µmol/l each) was mixed with 375 µl of platelet-rich plasma and incubated for 20 min. Fifty µl of fluorescein isothiocyanate-conjugated PAC-1 (25 µg/ml) was then added, and the fluorescence of individual platelets was measured 5, 15, 30, 45, and 60 min after the addition of 50 µl of HEPES buffer containing or not the P2Y12 antagonist AR-C69931MX (100 nmol/l). (Upper panel) Mean and SEM of the median fluorescence of 10,000 platelets in eight experiments. (Lower panels) Representative flow cytometric results at selected time points in one experiment without (solid lines) or with (dotted lines) the addition of AR-C69931MX.

 

Figure 7
View larger version (29K):
[in this window]
[in a new window]
  		Figure 7 Effect of different platelet inhibitors on PAC-1 binding to activated platelets. These experiments were performed as described in the caption for Figure 6, with the only difference that the P2Y1 inhibitor, MRS2179, or the putative Ca2+ channel blocker, lanthanum chloride, was also added to platelets after activation by adenosine diphosphate and epinephrine. The upper panel shows the mean and SEM of the median fluorescence of 10,000 platelets measured 30 min after addition of AR-C69931MX (final concentration: 100 nmol/l), MRS2179 (final concentration: 100 µmol/l), or lanthanum chloride (final concentration: 1 mmol/l) as compared to control in which only buffer was added (n = 8). The lower panels show the actual flow cytometric results of one representative experiment. Solid lines represent the results in the presence of the inhibitor shown in each panel, while dotted lines represent the results in the absence of inhibition.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
Appendix
 References
 
Our findings show that platelet thrombi growing on collagen may disperse several minutes after the initial aggregation when exposed to blood containing an ADP receptor antagonist or a putative Ca2+ channel blocker or a GP IIb/IIIa inhibitor. Thrombus stability, therefore, may depend on sustained ligand binding to activated GP IIb/IIIa, which in turn may be mediated by [Ca2+]i elevations induced by the continuous stimulation of specific signaling pathways (Fig. 8). Our findings add to the concept of intercellular calcium communication, suggesting that it operates bidirectionally from platelets at the growing edge of a thrombus not only to activate newly recruited platelets (12) but also to ensure stability throughout the entire aggregate. Such a conclusion is based on the observation that perfusion of blood containing ADP receptor antagonists reduced the cyclic [Ca2+]i increases within platelet thrombi even several minutes after the initial adhesive contacts had been established. Our experiments, however, could not define whether platelets at the center of thrombi differed in their Ca2+ responses from those at the outer edges. It has been proposed that cooperation between P2Y12-mediated activation and GP IIb/IIIa engagement by ligand promotes the activation of newly recruited platelets in a thrombus (12). We observed, however, that blocking GP IIb/IIIa on the perfused platelets had no effect on the [Ca2+]i oscillations within the thrombus even though it caused progressive disaggregation. Thus, the effect of GP IIb/IIIa inhibition on Ca2+ signals may reflect the need of maintaining newly recruited platelets in contact with adherent ones until activation is induced. In the case of platelets already aggregated into a thrombus, the replacement of an adhesive ligand by a GP IIb/IIIa antagonist may result in the detachment of platelets at the edge without effects on the activation of those still in the thrombus.


Figure 8
View larger version (34K):
[in this window]
[in a new window]
  		Figure 8 Schematic representation of the mechanism that stabilizes platelets at the edge of a growing thrombus exposed to elevated shear rates. (A) Circulating platelets adhere and become activated onto collagen through multiple adhesive interactions, initiated by glycoprotein (GP) Ib{alpha} binding to von Willebrand factor (VWF) under high shear rate conditions. Full activation depends on released adenosine diphosphate (ADP) and leads to the binding of soluble adhesive ligands such as VWF and fibrinogen. These form the new substrate for the recruitment of circulating platelets, again initiated under high shear rate conditions by GP Ib{alpha}-VWF binding. Adhesive interactions and soluble agonists present in the environment of the growing thrombus lead to activation of the newly recruited platelets and further ADP release. (B) Cyclic Ca2+ signaling induced by released ADP and mediated by P2Y1 and P2Y12 maintains GP IIb/IIIa activation necessary for the sustained binding of adhesive molecules and stability of the aggregate. The first layer of platelets interacting with the collagen surface may not require sustained ADP stimulation for stable adhesion.

 
We propose that cyclic [Ca2+]i increases within platelet thrombi reflect a mechanism that maintains GP IIb/IIIa activation and, thus, thrombus stability. The experiments with lanthanum, a putative Ca2+ channel blocker, support such a concept, but in this study we could not identify the ion channel involved or the signaling pathway regulating its function. Lanthanum, therefore, could have other effects, such as altering the ion binding sites of adhesive receptors and/or ligands (24) resulting in thrombus dissolution independently of Ca2+ channel blocking. Moreover, the cyclic [Ca2+]i increases of aggregated platelets within thrombi could be a consequence, not the cause of, maintaining GP IIb/IIIa activation. In the alternative, the observation that [Ca2+]i oscillations are linked to ADP receptor function may suggest an involvement of store-dependent Ca2+ entry (25–27) mediated by P2Y12 simulation. From a technical viewpoint, changes in platelet volume could have affected the results obtained with fluo-3AM, a single-wave Ca2+ indicator, thus influencing our conclusions. It should be noted that we have attempted to minimize such possible effects by using ultra-fast confocal microscopy, and no cyclic platelet volume changes have been reported during thrombus growth. In the end, sustained platelet activation necessary for thrombus stability may involve various signaling molecules, including ephrin/eph kinases (28), regulated by platelet-platelet contacts at the upper edge of the growing thrombus.

Although there is agreement that P2Y12 is involved in the calcium signaling that sustains activation, conclusions on the role of P2Y1 are not univocal, perhaps because previous studies were focused on the activation of newly recruited platelets (12) while we analyzed the activation of platelets already incorporated within thrombi. The two processes may be distinct, or the discrepant results may be caused by methodological differences. For example, continuous rather than periodic monitoring of [Ca2+]i cycles may better reveal the relatively weak effect of blocking P2Y1. Moreover, others have reported that only the combined blockade of P2Y1 and P2Y12 can inhibit platelet thrombus formation on the surface of collagen (7), whereas we have shown previously (8) and confirm here that inhibition of either receptor is almost equally effective in doing so. This discrepancy may result from differences in experimental observation times, because we measured thrombus volume after several minutes when the stability may be more influenced by interplatelet communication and less by the effects of the platelet-collagen interactions at the base of the thrombus.

Perhaps the main limitation of an ex vivo blood perfusion model is the need to use an anticoagulant to preserve blood fluidity, which in turn prevents the generation of thrombin and, consequently, of fibrin, both likely to have a role in providing thrombus stability (3,29). Nonetheless, ADP-induced signaling pathways may contribute to the overall effects of other agonists, including thrombin, that lead to nucleotide release from storage granules after primary platelet stimulation. Moreover, the mechanism highlighted here may provide initial stability to aggregated platelets exposed to flowing blood and, through the procoagulant function of activated platelets (30), contribute to fibrin formation. Our results, therefore, provide new insights into the mechanism of action of antiplatelet agents used in clinical practice and currently targeted against the P2Y12 receptor. In situations such as acute coronary syndromes or coronary interventions with elevated thrombotic risk, P2Y12 inhibitors may be used in combination with anticoagulants such as heparin, suggesting that an experimental model in which thrombin activity and fibrin formation are inhibited may be representative of at least some clinical conditions.

Conclusions.   Our studies, although directly relevant only for thrombus formation on collagen fibrils under artificial blood flow conditions, suggest a possible role for distinct ADP receptors and their operating calcium signals in sustaining the long-term GP IIb/IIIa activation required to maintain platelet aggregate stability before the occurrence of fibrin generation. Such a mechanism may provide a more comprehensive understanding of the pharmacological effects of anti-P2Y12 drugs, and suggest novel strategies to achieve the dispersion of platelet thrombi after they are formed.


   Appendix
Top
 Abstract
 Methods
 Results
 Discussion
 Appendix
References
 
For the supplemental videos, please see the online version of this article.


   Acknowledgments
 
The authors gratefully acknowledge the contribution of the staff of Tokai University Educational and Research Support Center.


   Footnotes
 
Supported by a Grant-in-Aid for Scientific Research in Japan (15590771, 17590764); by Tokai University School of Medicine, Project Research 2004 and 2005; by a grant from the Vehicle Racing Commemorative Foundation; by a grant for the leading project supported by the Ministry of Education and Science, Sports, and Culture, Japan; by a Health and Labor Sciences research grant from the Ministry of Health, Labor, and Welfare, Japan; and by grants HL31950, HL48728, and HL77436 from the U.S. National Institutes of Health.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 Appendix
 References
 

  1. Ruggeri ZM. Platelets in atherothrombosis Nat Med 2002;8:1227-1234.[CrossRef][ISI][Medline]
  2. Goto S. Propagation of arterial thrombilocal and remote contributory factors. Arterioscler Thromb Vasc Biol 2004;24:2207-2208.[Free Full Text]
  3. Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse Nat Med 2002;8:1175-1181.[CrossRef][ISI][Medline]
  4. Hollopeter G, Jantzen HM, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs Nature 2001;409:202-207.[CrossRef][Medline]
  5. Savi P, Combalbert J, Gaich C, et al. The antiaggregating activity of clopidogrel is due to a metabolic activation by the hepatic cytochrome P450-1A Thromb Haemost 1994;72:313-317.[ISI][Medline]
  6. Jin J, Quinton TM, Zhang J, Rittenhouse SE, Kunapuli SP. Adenosine diphosphate (ADP)-induced thromboxane A(2) generation in human platelets requires coordinated signaling through integrin alpha(IIb)beta(3) and ADP receptors Blood 2002;99:193-198.[Abstract/Free Full Text]
  7. Turner NA, Moake JL, McIntire LV. Blockade of adenosine diphosphate receptors P2Y(12) and P2Y(1) is required to inhibit platelet aggregation in whole blood under flow Blood 2001;98:3340-3345.[Abstract/Free Full Text]
  8. Goto S, Tamura N, Handa S. Effects of adenosine 5'-diphosphate (ADP) receptor blockade on platelet aggregation under flow Blood 2002;99:4644-4646.[Free Full Text]
  9. Remijn JA, Wu YP, Jeninga EH, et al. Role of ADP receptor P2Y(12) in platelet adhesion and thrombus formation in flowing blood Arterioscler Thromb Vasc Biol 2002;22:686-691.[Abstract/Free Full Text]
  10. Trumel C, Payrastre B, Plantavid M, et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase Blood 1999;94:4156-4165.[Abstract/Free Full Text]
  11. Bhatt DL, Topol EJ. Scientific and therapeutic advances in antiplatelet therapy Nat Rev Drug Discov 2003;2:15-28.[CrossRef][ISI][Medline]
  12. Nesbitt WS, Giuliano S, Kulkarni S, Dopheide SM, Harper IS, Jackson SP. Intercellular calcium communication regulates platelet aggregation and thrombus growth J Cell Biol 2003;160:1151-1161.[Abstract/Free Full Text]
  13. Andre P, Delaney SM, LaRocca T, et al. P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries J Clin Invest 2003;112:398-406.[CrossRef][ISI][Medline]
  14. Gachet C. ADP receptors of platelets and their inhibition Thromb Haemost 2001;86:222-232.[ISI][Medline]
  15. Goto S, Tamura N, Ishida H. Ability of anti-glycoprotein IIb/IIIa agents to dissolve platelet thrombi formed on a collagen surface under blood flow conditions J Am Coll Cardiol 2004;44:316-323.[Abstract/Free Full Text]
  16. Goto S, Tamura N, Handa S, Arai M, Kodama K, Takayama H. Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions Circulation 2002;106:266-272.[Abstract/Free Full Text]
  17. Ingall AH, Dixon J, Bailey A, et al. Antagonists of the platelet P2T receptora novel approach to antithrombotic therapy. J Med Chem 1999;42:213-220.[CrossRef][ISI][Medline]
  18. Boyer JL, Mohanram A, Camaioni E, Jacobson KA, Harden TK. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate Br J Pharmacol 1998;124:1-3.[CrossRef][ISI][Medline]
  19. Friedman H, Meir S, Rosenberger I, Halevy AH, Kaufman PB, Philosoph-Hadas S. Inhibition of the gravitropic response of snapdragon spikes by the calcium-channel blocker lanthanum chloride Plant Physiol 1998;118:483-492.[Abstract/Free Full Text]
  20. Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor Cell 1996;84:289-297.[CrossRef][ISI][Medline]
  21. Merritt JE, McCarthy SA, Davies MP, Moores KE. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+ Biochem J 1990;269:513-519.[ISI][Medline]
  22. Heemskerk JW, Willems GM, Rook MB, Sage SO. Ragged spiking of free calcium in ADP-stimulated human plateletsregulation of puff-like calcium signals in vitro and ex vivo. J Physiol 2001;535:625-635.[Abstract/Free Full Text]
  23. Packham MA, Rand ML, Perry DW, Ruben DH, Kinlough-Rathbone RL. Probenecid inhibits platelet responses to aggregating agents in vitro and has a synergistic inhibitory effect with penicillin G Thromb Haemost 1996;76:239-244.[Medline]
  24. Achyuthan KE, Mary A, Greenberg CS. Tb(III)-ion-binding-induced conformational changes in platelet factor XIII Biochem J 1989;257:331-338.[Medline]
  25. Redondo PC, Harper AG, Salido GM, Pariente JA, Sage SO, Rosado JA. A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets J Physiol 2004;558:99-109.[Abstract/Free Full Text]
  26. Rosado JA, Lopez JJ, Harper AG, et al. Two pathways for store-mediated calcium entry differentially dependent on the actin cytoskeleton in human platelets J Biol Chem 2004;279:29231-29235.[Abstract/Free Full Text]
  27. Rosado JA, Sage SO. The ERK cascade, a new pathway involved in the activation of store-mediated calcium entry in human platelets Trends Cardiovasc Med 2002;12:229-234.[CrossRef][ISI][Medline]
  28. Prevost N, Woulfe DS, Jiang H, et al. Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets Proc Natl Acad Sci U S A 2005;102:9820-9825.[Abstract/Free Full Text]
  29. Gast A, Tschopp TB, Baumgartner HR. Thrombin plays a key role in late platelet thrombus growth and/or stability. Effect of a specific thrombin inhibitor on thrombogenesis induced by aortic subendothelium exposed to flowing rabbit blood Arterioscler Thromb 1994;14:1466-1474.[Abstract/Free Full Text]
  30. Goto S, Tamura N, Li M, et al. Different effects of various anti-GP IIb-IIIa agents on shear-induced platelet activation and expression of procoagulant activity J Thromb Haemost 2003;1:2022-2030.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 JEMHome page
H. Nishikii, K. Eto, N. Tamura, K. Hattori, B. Heissig, T. Kanaji, A. Sawaguchi, S. Goto, J. Ware, and H. Nakauchi
Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells
J. Exp. Med., August 4, 2008; 205(8): 1917 - 1927.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
J. M. Auger and S. P. Watson
Dynamic Tyrosine Kinase-Regulated Signaling and Actin Polymerisation Mediate Aggregate Stability Under Shear
Arterioscler. Thromb. Vasc. Biol., August 1, 2008; 28(8): 1499 - 1504.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
D. J. Angiolillo, E. Bernardo, M. Sabate, P. Jimenez-Quevedo, M. A. Costa, J. Palazuelos, R. Hernandez-Antolin, R. Moreno, J. Escaned, F. Alfonso, et al.
Impact of Platelet Reactivity on Cardiovascular Outcomes in Patients With Type 2 Diabetes Mellitus and Coronary Artery Disease
J. Am. Coll. Cardiol., October 16, 2007; 50(16): 1541 - 1547.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Jirouskova, J. K. Jaiswal, and B. S. Coller
Ligand density dramatically affects integrin {alpha}IIb{beta}3-mediated platelet signaling and spreading
Blood, June 15, 2007; 109(12): 5260 - 5269.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
L. F. Brass
Thrombus formation: stability matters
Blood, November 1, 2006; 108(9): 2883 - 2884.
[Full Text] [PDF]

Home page
 BloodHome page
J. M. E. M. Cosemans, I. C. A. Munnix, R. Wetzker, R. Heller, S. P. Jackson, and J. W. M. Heemskerk
Continuous signaling via PI3K isoforms beta and {gamma} is required for platelet ADP receptor function in dynamic thrombus stabilization
Blood, November 1, 2006; 108(9): 3045 - 3052.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow View Online Videos
Right arrow All Versions of this Article:
j.jacc.2005.08.055v1
47/1/155    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goto, S.
Right arrow Articles by Ruggeri, Z. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goto, S.
Right arrow Articles by Ruggeri, Z. M.

 
  cardiology careers collections past issues search home