SEARCH

SEARCH BY CITATION

Keywords:

  • fibrinogen;
  • heterotypic aggregates;
  • ischemia;
  • P-selectin;
  • platelets;
  • thrombosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Summary. Background: Previous studies have shown that ischemic preconditioning (PC) not only limits infarct size, but also improves arterial patency in models of recurrent thrombosis. We hypothesize that this enhanced patency is presumably because of a PC-induced attenuation of platelet-mediated thrombosis. However, there is, at present, no direct evidence that PC acts on the platelets per se and favorably down-regulates platelet reactivity. Objectives: Our goal was to test the concept that PC ischemia attenuates molecular indices of platelet activation-aggregation. Methods: Anesthetized dogs were randomly assigned to receive 10 min of PC ischemia followed by 10 min of reperfusion or a time-matched control period. Spontaneous recurrent coronary thrombosis was then initiated in all dogs by injury + stenosis of the left anterior descending coronary artery. Coronary flow was monitored for 3 h poststenosis, and molecular indices of platelet activation-aggregation were quantified by whole blood flow cytometry. Results: Coronary patency was, as expected, better-maintained following injury + stenosis in the PC group vs. controls (53% ± 5%* vs. 23% ± 5% of baseline flow, respectively; *P < 0.05). Moreover, PC was accompanied by: (i) a significant down-regulation of platelet-fibrinogen binding and formation of neutrophil-platelet aggregates (112% ± 14%* vs. 177% ± 21% and 107% ± 8%* vs. 155% ± 19% of baseline values in PC vs. control groups); and (ii) a trend towards a reduction in platelet P-selectin expression (148% ± 12%† vs. 190% ± 21% of baseline; *P < 0.05 and †P = 0.09 vs. control). Conclusion: These data provide novel, direct evidence in support of the concept that ischemic PC attenuates molecular indices of platelet activation-aggregation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Ischemic preconditioning (PC) is the well-described phenomenon whereby brief episodes of myocardial ischemia paradoxically render cardiomyocytes resistant to a later, sustained ischemic insult. While the hallmark of PC is reduction of infarct size [1,2], emerging evidence suggests that antecedent PC ischemia may have an ancillary, favorable effect on the maintenance of vessel patency in models of coronary thrombosis [3–6]. For example, we have found, using a classic canine model of spontaneous recurrent thrombosis mimicking unstable angina (i.e. the Folts preparation [7–11]), that vessel patency was better preserved in animals that received PC ischemia vs. controls [3].

The enhanced coronary patency seen with PC is presumably because of a PC-induced attenuation of platelet-mediated thrombosis. However, there is no direct evidence that PC attenuates platelet activation-aggregation. Elucidation of the mechanisms by which PC improves coronary patency is further confounded by the fact that, surprisingly, molecular markers of platelet activation-aggregation have not been fully characterized in the Folts model. Accordingly, our aims were to: (i) identify, using whole blood flow cytometry, the specific molecular indices of platelet activation-aggregation that are augmented in the canine model of recurrent thrombosis; and (ii) test our hypothesis that the improved coronary patency achieved with PC ischemia is accompanied by a favorable down-regulation in one or more molecular markers of platelet reactivity.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

This study was approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (1996).

Surgical preparation

Twenty-two adult mongrel dogs of either gender (weight 12–14 kg) were anesthetized with sodium pentobarbital (30 mg kg−1 i.v.), intubated and mechanically ventilated. Catheters were inserted in the left jugular vein for administration of fluids and supplemental anesthesia, and in the left carotid artery for measurement of heart rate and arterial pressure and collection of blood samples. After exposing the heart through a left lateral thoracotomy, two adjacent segments of the LAD were isolated, usually midway along its course. The distal LAD segment was instrumented with a Doppler flow probe (Transonic Systems, Ithaca, NY, USA) for continuous measurement of mean coronary blood flow (CBF), while the proximal segment served as the site of later injury + stenosis. Arterial pressure and CBF were recorded throughout each experiment using a MicroMed data acquisition system (Louisville, KY, USA).

Study design

After collection of baseline hemodynamic data, dogs were randomly assigned to receive 10 min of antecedent PC ischemia (achieved by placing atraumatic vascular clamps on the proximal, isolated LAD segment) followed by 10 min of reperfusion (n = 10), or a matched 20-min control period (n = 12; Fig. 1).

image

Figure 1.  Study design. PC, preconditioned; CFVs, cyclic variations in coronary flow.

Download figure to PowerPoint

Following the 20-min intervention phase, all dogs underwent coronary artery injury + stenosis, that is, the isolated LAD segment was squeezed with forceps, and a micromanometer constrictor was positioned around the site of injury and tightened with the objective of reducing mean CBF to 35–40% of its baseline value. This procedure rapidly (within 5 min) triggers the development of cyclic variations in CBF (CFVs; Fig. 2A) associated with the repeated, spontaneous accumulation/dislodgment of platelet-rich thrombi at the site of injury + stenosis (Fig. 2B). CBF was then monitored for 3 h without further intervention.

image

Figure 2.  Examples of cyclic variations in coronary blood flow seen following coronary artery injury + stenosis (Panel A) and medial tearing and remnants of platelet-rich thrombus at the site of arterial injury (Panel B).

Download figure to PowerPoint

At the end of the 3 h observation period, cardiac arrest was produced under deep anesthesia by intracardiac injection of KCl. As the severity of arterial injury is recognized to be a crucial determinant of patency in this model [3,6,8,12], the damaged LAD segment was collected from all dogs and stored in 10% neutral buffered formalin for later histological evaluation.

Endpoints and analysis

Heart rate, mean arterial pressure and CBF were tabulated at baseline (before randomization), throughout the intervention period, immediately before LAD injury + stenosis, and 10 s after injury + stenosis (before the development of CFVs). In addition, heart rate and arterial pressure were compiled at 1 and 3 h after the onset of CFVs.

Histologic analysis of all damaged and stenotic LAD segments was performed by one investigator (PW) without knowledge of the treatment group. Cross-sections (5 μm thickness) were cut from each artery and stained with picrosirius red (to facilitate specific visualization of collagen fibers). For each vessel, the severity of injury was assigned a semi-quantitative score according to the following criteria: 1 =endothelial denudation with little or no damage to the tunica media; 2 = tears and dissections in the media, without exposure of tunica adventitia; 3 = loss of media and/or deep tears, with focal points of adventitial exposure totaling < 10% of the arterial circumference; and 4 = loss of media with confluent and extensive (> 10%) adventitial exposure [3,6,12].

Vessel patency during the 3 h following injury + stenosis was assessed by quantifying two variables: the duration (in min) of total thrombotic occlusion (CBF = 0); and % flow-time area, defined as the area of the flow-time tracing (quantified by computerized planimetry) normalized for each dog to the baseline flow × 180 min [3,6,12–14].

Molecular indices of platelet activation-aggregation (i.e. presence of monocyte- and neutrophil-platelet aggregates, as well as platelet-fibrinogen binding and platelet expression of PECAM-1, GPIb, P-selectin and VWF) were evaluated by flow cytometry using variations of established methods employed for the quantitative assessment of platelet status in patients with acute ischemic syndromes [15–17]. In brief, 5 mL of citrated arterial blood was obtained from all dogs at baseline (post-thoracotomy, but before randomization) and at 2 h after the onset of CFVs. For assessment of heterotypic aggregates, monocytes and neutrophils were identified by their characteristic light scattering properties (Fig. 3A) and differential expression of the lipopolysaccharide receptor CD14 [phycoerythrin (PE)-conjugated TK4; DAKO Cytomation, Carpinteria, CA, USA; Fig. 3B] [16]. Heterotypic aggregates were then discerned by the expression of the platelet-specific receptor CD61 [fluorescein isothiocyanate (FITC)-conjugated Y2/51; DAKO Cytomation] on the monocyte or neutrophil populations, indicating aggregation to platelets (Fig. 3C,D) [18]. In experiments examining platelet surface antigens, platelets were identified by their characteristic light scattering properties (Figs 4A and 5A) and expression of the platelet-specific receptors CD61 (Y2/51-FITC; DAKO Cytomation) (Fig. 4B) or CD41 (5B12-PE) (Fig. 5B) [15,18,19]. Expression of PECAM-1 was measured by binding of a FITC-labeled canine cross-reactive antibody against CD31 (PECAM1.3 IgG1; supplied by P. J. Newman) [20]. Platelet GPIb, P-selectin and VWF surface expression were similarly measured using anticanine CD42b monoclonal antibody (Ab142; supplied by Dermot Kenny) [21], cross-reactive CD62P-specific antibody (1E3-PE), and FITC-labeled sheep-anticanine VWF polyclonal antibody (SACWF-IG; Affinity Biologicals, Ancaster, Canada), respectively. Fibrinogen binding was measured using FITC-labeled canine fibrinogen (F7128; Sigma, St Louis, MO, USA) against a control sample blocked with the GPIIb/IIIa antagonist eptifibatide (Integrilin; Millennium Pharmaceuticals, Cambridge, MA, USA). Phorbol myristate acetate (PMA) activation of samples served as a positive control for platelet activation-aggregation assays. All flow cytometric analyses were begun within 15 min of sample collection and performed by a single operator (MDL), who was blinded with regard to the treatment group, using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Data obtained following injury + stenosis were normalized, for each animal, to respective baseline values.

image

Figure 3.  Flow cytometric detection of heterotypic aggregates. Monocytes and neutrophils are identified by characteristic forward and side light scatter (Panel A) and differential expression of the lipopolysaccharide receptor CD14 (Panel B). Expression of platelet-specific antigen GPIIIa (CD61) denotes formation of a monocyte-platelet aggregate (Panel C) or neutrophil-platelet aggregate (Panel D). A typical result for paired samples obtained at baseline (solid profile) and following injury + stenosis (open profile) is displayed in Panels C and D.

Download figure to PowerPoint

image

Figure 4.  Flow cytometric detection of platelet surface P-selectin expression. Platelets were identified by characteristic forward and side light scatter (Panel A) and expression of the platelet-specific glycoprotein IIb (CD41) via double gating and threshold on CD41 (Panel B). Platelet surface P-selectin expression was determined by CD62P fluorescence (Panel C). A typical result for paired samples obtained at baseline (solid profile) and following injury + stenosis (open profile) is displayed in Panel C.

Download figure to PowerPoint

image

Figure 5.  Flow cytometric detection of platelet surface fibrinogen binding. Platelets were identified by characteristic forward and side light scatter (Panel A) and expression of the platelet-specific glycoprotein IIIa (CD61) via double gating and threshold on CD61 (Panel B). Platelet surface fibrinogen binding was determined by fluorescent detection of labeled fibrinogen on the platelets (Panel C). A typical result for paired samples obtained at baseline (solid profile) and following injury + stenosis (open profile) is displayed in Panel C.

Download figure to PowerPoint

Statistics

All variables measured repeatedly throughout the experiment (hemodynamics, CBF, indices of platelet activation-aggregation) were compared by two-factor anova (for group and time) with replication, and, if significant F-values were obtained, post hoc pair-wise comparisons were made using the Newman-Keuls test. Discrete variables (arterial injury scores, zero flow duration, % flow-time area) were compared by t-tests. All data are reported as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Hemodynamics and CBF

There were no group differences in heart rate or mean arterial pressure at any time during the protocol (Table 1).

Table 1.   Hemodynamics
 BaselineEnd-treat§Prestenosis10 sec poststenosisDuring CFVs
1 h3 h
  1. PC, preconditioned; CFVs, cyclic variations in coronary flow. §End-treat: data obtained upon relief of 10 min PC ischemia or matched time-point in controls. na: discrete measurements of CBF not applicable after the onset of CFVs. *P < 0.05 vs. control; P < 0.05 and P < 0.01 vs. baseline.

Heart rate (beats min−1)
 Control151 ± 7151 ± 7149 ± 8150 ± 6161 ± 5175 ± 4
 PC151 ± 8142 ± 11155 ± 11158 ± 11163 ± 9179 ± 11
Mean arterial pressure (mmHg)
 Control128 ± 7129 ± 7125 ± 8124 ± 7134 ± 7132 ± 6
 PC130 ± 4124 ± 5129 ± 4130 ± 4128 ± 4123 ± 7
Coronary blood flow (CBF) (% of baseline)
 Control100%104 ± 4%103 ± 7%34 ± 3%nana
 PC100%455 ± 49%*98 ± 7%37 ± 2%nana

CBF averaged 11.3 and 10.9 mL min−1 at baseline in the control and PC groups (P = ns). As expected, dogs that received PC ischemia displayed myocardial cyanosis and dyskinesis during the 10-min episode of antecedent LAD occlusion, and were hyperemic (with CBF increasing to 455% of baseline) upon reflow. This hyperemic response was, however, transient: coronary flow in the PC group returned to 98% of baseline during the 10-min reperfusion period. Moreover (and as further expected), there was no evidence of CFVs or recurrent thrombosis in the 10 min following relief of PC ischemia.

Immediately following application of the stenosis, CBF was similar in both cohorts, averaging 34–37% of baseline (Table 1).

Arterial damage

Arterial damage in all groups was characterized by medial tearing and dissection with minimal adventitial exposure (Fig. 2B): mean injury score was 2.7 ± 0.3 in both control and PC groups (P = ns).

Vessel patency

All dogs developed spontaneous recurrent thrombosis following coronary injury + stenosis and displayed persistent CFVs throughout the 3 h observation period. Vessel patency was, however, better maintained in dogs that received antecedent PC ischemia: zero flow duration averaged 13 ± 5* vs. 59 ± 14 min, while % flow-time area was 53% ± 5%* vs. 23% ± 5% in the PC-treated group vs. controls (*P < 0.05 vs. controls; Fig. 6).

image

Figure 6.  Indices of coronary patency (zero flow duration and flow-time area) following injury + stenosis. PC, preconditioned. *P < 0.05 vs. control.

Download figure to PowerPoint

Indices of platelet activation-aggregation

In the control cohort, LAD injury + stenosis was associated with significant increases in platelet surface P-selectin expression, platelet-fibrinogen binding and formation of heterotypic aggregates, and a trend (P = 0.3) towards an increase in expression of VWF. There were no differences in PECAM or GPIb in blood samples obtained following injury + stenosis when compared with baseline (Fig. 7). Most notably, however, the improved coronary patency seen with PC ischemia was accompanied by a significant decline in platelet-fibrinogen binding, a decrease in the formation of neutrophil-platelet aggregates, and a trend towards a reduction in platelet P-selectin expression when compared with controls (Fig. 8).

image

Figure 7.  Indices of platelet activation-aggregation in control animals, measured at 2 h following injury + stenosis and expressed as a percentage of baseline values. MPA, monocyte-platelet aggregates; NPA, neutrophil-platelet aggregates. †P < 0.05 vs. baseline.

Download figure to PowerPoint

image

Figure 8.  Monocyte-platelet aggregates, neutrophil-platelet aggregates, P-selectin expression and platelet-fibrinogen binding, measured at 2 h following injury + stenosis and expressed as percentage of baseline values. PC, preconditioned. †P < 0.05 vs. baseline; *P < 0.05 and §P = 0.09 (ns) vs. control.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

In this study, we report novel evidence that PC-induced attenuation of recurrent arterial thrombosis is accompanied by a favorable down-regulation in multiple markers of platelet activation-aggregation, including platelet-fibrinogen binding, formation of neutrophil-platelet aggregates, and a trend towards a decline in platelet P-selectin expression.

Characterization of platelet activation-aggregation in the setting of coronary injury + stenosis

The hallmark of the Folts model is the development of CFVs, an observation that has been attributed to recurrent spontaneous formation/dislodgement of platelet-rich thrombi at the site of injury + stenosis [7]. Indirect evidence to support this concept has been provided by studies showing that in vivo administration of inhibitors and antibodies targeting selectins, VWF, GPIb, and GPIIb/IIIa, suppresses the frequency (number per hour) of CFVs [22–27]. Nonetheless, with the exception of previous investigations that focused exclusively on P-selectin [27], no studies have utilized flow cytometry in order to provide a comprehensive assessment of the specific molecular indices of platelet activation-aggregation that are up-regulated in this model. Accordingly, we quantified multiple markers of platelet reactivity, including platelet surface expression of P-selectin, PECAM, GPIb, VWF and platelet-fibrinogen binding (achieved via activation-dependent conformational changes in the GPIIb/IIIa receptor on platelets) [28]. In addition, we probed for the presence of heterotypic (monocyte-platelet and neutrophil-platelet) aggregates, formed largely by activation-dependent expression of P-selectin on the platelet binding to its ligand, PSGL-1, constitutively expressed on leukocytes [29].

Our results corroborate previous reports that the Folts model is characterized by an increase in platelet P-selectin expression [27]. Moreover, we expand on previous pharmacologic data implicating (but not documenting) an increase in platelet-leukocyte interactions [26,27] and provide direct, quantitative evidence that initiation of CFVs is associated with robust increases in the formation of monocyte- and neutrophil-platelet aggregates, platelet-fibrinogen binding, and a trend towards augmented expression of platelet VWF. In contrast, PECAM and GPIb expression remained unchanged against baseline. These observations are consistent with previous findings that expression of PECAM, GPIb and VWF is less responsive to platelet activation than P-selectin, the GPIIb/IIIa receptor (as demonstrated by fibrinogen binding) and leukocyte-platelet aggregation [15,30,31], and provide the first flow cytometric characterization of platelet reactivity in this classic model mimicking unstable angina.

PC ischemia attenuates indices of platelet activation-aggregation

Results of the current study confirm our previous report that PC ischemia improves subsequent vessel patency in damaged and stenotic coronary arteries [3]. In addition, we make the novel observation that the improved patency seen with PC ischemia is accompanied by significant reductions in platelet-fibrinogen binding and formation of neutrophil-platelet aggregates and a trend towards a decrease in platelet P-selectin expression when compared with controls. These data provide the first direct evidence that PC ischemia has a favorable, inhibitory effect on molecular indices of platelet activation-aggregation.

The reasons for the apparently selective effect of PC ischemia on the formation of neutrophil-platelet (but not monocyte-platelet) aggregates are unknown. This may reflect the observations made in human and primate blood samples that monocyte-platelet aggregates are highly sensitive to small changes in platelet activation status [32], or a differential effect of PC ischemia on neutrophils and platelets vs. monocytes. For example, although data obtained in patients has shown that formation of heterotypic aggregates is largely initiated by the interaction between P-selectin and PSGL-1 [29], aggregates are stabilized by multiple mechanisms, including fibrinogen binding to GPIIb/IIIa on platelets and CD11b/CD18 on leukocytes [33]. Accordingly, one intriguing possibility is that PC ischemia may not only act on platelets, but may also favorably attenuate neutrophil activation and adhesion, that is, in particular, may evoke a decrease in the expression of activated CD11b/CD18 and a resultant destabilization of neutrophil-platelet aggregates. This concept is, however, speculative: although adenosine (a purine known to be liberated from myocytes during brief PC ischemia-reperfusion) reportedly initiates a down-regulation of CD11b/CD18 [34–36], the effect of PC per se on neutrophil adhesion molecule expression has, to date, not been quantified.

Potential mechanisms

Our observation of a significant down-regulation in molecular indices of platelet activation-aggregation with PC ischemia raises three obvious questions. Firstly, what is the time-course of the favorable ‘antiplatelet’ effect of PC? Measurement of flow cytometric indices of platelet activation-aggregation at multiple time points throughout the protocol would potentially have provided meaningful information. However, the number of samples analyzed was limited by the comprehensive nature of the analysis performed on each sample: we were concerned that interrogation of additional samples would have subjected the blood aliquots to an unacceptably long waiting period before beginning the analyses. Accordingly, the temporal profile of this aspect of PC is, at present, unresolved.

A second, related, question is: were platelets activated during the PC stimulus and, as a result, rendered refractory to subsequent activation? We cannot provide a definitive answer to this question, as we did not, as discussed above, assess platelet reactivity during brief ischemia-reperfusion. Nonetheless, it is well-recognized that ischemia-reperfusion achieved by the placement of atraumatic vascular clamps on an intact coronary artery does not initiate CFVs or recurrent thrombosis [37–39] (an observation corroborated in the current protocol), thereby implying that the PC stimulus did not cause appreciable platelet activation. We can, however, provide preliminary insight into whether platelets in PC-treated animals may have been rendered refractory to aggregation. Specifically, as an ancillary component of our flow cytometric analysis, we assessed platelet activation-aggregation in response to exogenous addition of IBOP (thromboxane mimetic, 0.25 μm). Blood samples obtained at 2 h following injury + stenosis showed similar activation in both PC and control groups as judged by formation of neutrophil-platelet aggregates (43% vs. 32%; P = ns), platelet P-selectin expression (86% vs. 87%; P = ns) and platelet-fibrinogen binding (69% vs. 65%; P = ns), data that argue against the concept that platelets were rendered unresponsive by antecedent PC ischemia.

Finally, what are the cellular mechanism(s) responsible for the attenuation in platelet activation-aggregation seen in the PC group? Our working hypothesis is that the favorable effects of PC are due, at least in part, to release of one or more mediators in the setting of brief PC ischemia-reperfusion that may modulate platelet function. Potential candidates theoretically include adenosine, epinephrine, endogenous opioids, bradykinin, nitric oxide (NO) and prostacyclin (PGI2) [1,2]. Epinephrine is not a plausible option, as it is well recognized to potentiate (not attenuate) platelet aggregation [8,40,41]. With regard to opioids, there is no consensus on their effect on platelet reactivity or, indeed, whether opioid receptors are present on platelets [42–44]. Moreover, while both NO and PGI2 inhibit platelet aggregation, temporal issues argue against their contribution as trigger(s) for the prolonged (3 h) improvement in arterial patency seen with PC ischemia: the antiplatelet effects of NO and PGI2 are transient and rapidly reversed [12,45–49]. Continuous exposure to NO and PGI2 is needed to achieve a sustained antiplatelet effect [12,45,47], a requirement that is incongruous with the short-term release of these candidates following PC ischemia.

In contrast, adenosine, despite its highly labile nature (plasma half-life of c. seconds to minutes), reportedly elicits a sustained (c. hours) inhibition of platelet reactivity initiated via stimulation of adenosine A2 receptors on the platelets’ surface [41,50–53]. Indeed, our group has postulated that release of adenosine from ischemic-reperfused cardiomyocytes during the PC stimulus may serve as the trigger for the down-regulation in platelet activation-aggregation (and, thus, the improvement in vessel patency [3,6,13]) seen with PC. Recent pharmacologic data have, however, failed to support this hypothesis: we found that the potent adenosine A2 receptor agonist CGS 21680, given in lieu of brief PC ischemia, failed to mimic the favorable effects of PC on markers of platelet reactivity in the canine model [54,55].

While our current thinking has focused on the release of mediators that may attenuate platelet activation-aggregation, there is a second intriguing and as-yet unexplored possibility: that the better maintenance of coronary patency seen with PC ischemia may be explained by a decrease in one or more well-established pro-thrombotic factor(s). Among these, ADP is an unlikely candidate, as: (i) the well-documented catabolism of ATP in ischemic cardiomyocytes and resultant release of adenosine would favor a transient increase (rather than decrease) in intracellular ADP concentrations in the PC group [56]; and (ii) it is questionable whether these intracellular alterations would appreciably change the intracoronary (blood) concentration of ADP. Differences between groups in exposure to collagen is, in all likelihood, also not the explanation, as blinded histologic analysis demonstrated that the severity of arterial injury was comparable in both PC and control groups. With regard to other pro-thrombotic triggers, while there is no evidence to suggest that PC ischemia is accompanied by a reduction in blood concentrations of thrombin, thromboxane, serotonin, etc., this concept cannot, at present, be excluded.

Future prospective investigations will be required to discern the specific mechanisms that contribute to the favorable down-regulation in platelet status seen with PC ischemia. Nonetheless, our current results provide novel and direct evidence that brief PC ischemia attenuates molecular indices of platelet activation-aggregation.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References

Supported by R01-HL72684 from the National Institutes of Health (to KP).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of Conflict of Interests
  8. References
  • 1
    Przyklenk K, Kloner RA. Ischemic preconditioning: exploring the paradox. Prog Cardiovasc Dis 1998; 40: 51747.
  • 2
    Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev 2003; 83: 111351.
  • 3
    Hata K, Whittaker P, Kloner RA, Przyklenk K. Brief antecedent ischemia attenuates platelet-mediated thrombosis in damaged and stenotic canine coronary arteries: role of adenosine. Circulation 1998; 97: 692702.
  • 4
    Andreotti F, Pasceri V, Hackett DR, Davies GJ, Haider AW, Maseri A. Preinfarction angina as a predictor of more rapid coronary thrombolysis in patients with acute myocardial infarction. N Engl J Med 1996; 334: 712.
  • 5
    Muller DW, Topol EJ, Califf RM, Sigmon KN, Gorman L, George BS, Kereiakes DJ, Lee KL, Ellis SG. Relationship between antecedent angina pectoris and short-term prognosis after thrombolytic therapy for acute myocardial infarction. Thrombolysis and Angioplasty in Myocardial Infarction (TAMI) Study Group. Am Heart J 1990; 119: 22431.
  • 6
    Przyklenk K, Whittaker P. Brief antecedent ischemia enhances recombinant tissue plasminogen activator-induced coronary thrombolysis by adenosine-mediated mechanism. Circulation 2000; 102: 8895.
  • 7
    Folts JD, Crowell Jr EB, Rowe GG. Platelet aggregation in partially obstructed vessels and its elimination with aspirin. Circulation 1976; 54: 36570.
  • 8
    Folts J. An in vivo model of experimental arterial stenosis, intimal damage, and periodic thrombosis. Circulation 1991; 83: IV314.
  • 9
    Ikeda H, Koga Y, Kuwano K, Nakayama H, Ueno T, Yoshida N, Adachi K, Park IS, Toshima H. Cyclic flow variations in a conscious dog model of coronary artery stenosis and endothelial injury correlate with acute ischemic heart disease syndromes in humans. J Am Coll Cardiol 1993; 21: 100817.
  • 10
    Eichhorn EJ, Grayburn PA, Willard JE, Anderson HV, Bedotto JB, Carry M, Kahn JK, Willerson JT. Spontaneous alterations in coronary blood flow velocity before and after coronary angioplasty in patients with severe angina. J Am Coll Cardiol 1991; 17: 4352.
  • 11
    Willerson JT, Golino P, Eidt J, Campbell WB, Buja LM. Specific platelet mediators and unstable coronary artery lesions. Experimental evidence and potential clinical implications. Circulation 1989; 80: 198205.
  • 12
    Przyklenk K, Hata K, Whittaker P, Elliott GT. Monophosphoryl lipid A: a novel nitric oxide-mediated therapy to attenuate platelet thrombosis? J Cardiovasc Pharmacol 2000; 35: 36675.
  • 13
    Hata K, Whittaker P, Kloner RA, Przyklenk K. Brief myocardial ischemia attenuates platelet thrombosis in remote, damaged, and stenotic carotid arteries. Circulation 1999; 100: 8438.
  • 14
    Przyklenk K, Kloner RA. Sildenafil citrate (Viagra) does not exacerbate myocardial ischemia in canine models of coronary artery stenosis. J Am Coll Cardiol 2001; 37: 28692.
  • 15
    Linden MD, Frelinger III AL, Barnard MR, Przyklenk K, Furman MI, Michelson AD. Application of flow cytometry to platelet disorders. Semin Thromb Hemost 2004; 30: 50111.
  • 16
    Weiss DJ. Flow cytometric evaluation of hemophagocytic disorders in canine. Vet Clin Pathol 2002; 31: 3641.
  • 17
    Moritz A, Walcheck BK, Deye J, Weiss DJ. Effects of short-term racing activity on platelet and neutrophil activation in dogs. Am J Vet Res 2003; 64: 8559.
  • 18
    Barnard MR, Krueger LA, Frelinger III AL, Furman MI, Michelson AD. Whole blood analysis of leukocyte-platelet aggregates. In: RobinsonJP, DarzynkiewiczZ, DeanPN, HibbsAR, OrfaoA, RabinovitchPS, eds. Current Protocols in Cytometry. New York: John Wiley & Sons, 2003: 06.15.016.15.08.
  • 19
    Krueger LA, Barnard MR, Frelinger III AL, Furman MI, Michelson AD. Immunophenotypic analysis of platelets. In: RobinsonJP, DarzynkiewiczZ, DeanPN, HibbsAR, OrfaoA, RabinovitchPS, eds. Current Protocols in Cytometry. New York: John Wiley & Sons, 2003: 06.10.016.10.7.
  • 20
    Newman PJ, Berndt MC, Gorski J, White GC II, Lyman S, Paddock C, Muller WA. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 1990; 247: 121922.
  • 21
    Kenny D, Morateck PA, Fahs SA, Warltier DC, Montgomery RR. Cloning and expression of canine glycoprotein Ibalpha. Thromb Haemost 1999; 82: 132733.
  • 22
    Zoldhelyi P, Beck PJ, Bjercke RJ, Ober JC, Hu X, McNatt JM, Akhtar S, Ahmed M, Clubb Jr FJ, Chen Z-Q, Dixon RAF, Yeh ETH, Willerson JT. Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor. Am J Physiol 2000; 279: H306575.
  • 23
    Wu D, Vanhoorelbeke K, Cauwenberghs N, Meiring M, Depraetere H, Kotze HF, Deckmyn H. Inhibition of the von Willebrand (VWF)-collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons. Blood 2002; 99: 36238.
  • 24
    Wu D, Meiring M, Kotze HF, Deckmyn H, Cauwenberghs N. Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons. Arterioscler Thromb Vasc Biol 2002; 22: 3238.
  • 25
    Kageyama S, Yamamoto H, Nakazawa H, Yoshimoto R. Anti-human vWF monoclonal antibody, AJvW-2 Fab, inhibits repetitive coronary artery thrombosis without bleeding time prolongation in dogs. Thromb Res 2001; 101: 395404.
  • 26
    Ueyama T, Ikeda H, Haramaki N, Kuwano K, Imaizumi T. Effects of monoclonal antibody to P-selectin and analogue of sialyl Lewis X on cyclic flow variations in stenosed and endothelium-injured canine coronary arteries. Circulation 1997; 95: 15549.
  • 27
    Ikeda H, Ueyama T, Murohara T, Yasukawa H, Haramaki N, Eguchi H, Katoh A, Takajo Y, Onitsuka I, Ueno T, Tojo SJ, Imaizumi T. Adhesive interaction between P-selectin and sialyl Lewis(x) plays an important role in recurrent coronary arterial thrombosis in dogs. Arterioscler Thromb Vasc Biol 1999; 19: 108390.
  • 28
    Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem 1985; 260: 1110714.
  • 29
    Sarma J, Laan CA, Alam S, Jha A, Fox KA, Dransfield I. Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation 2002; 105: 216671.
  • 30
    Michelson AD. Flow cytometry: a clinical test of platelet function. Blood 1996; 87: 492536.
  • 31
    Michelson AD. Platelet function testing in cardiovascular diseases. Circulation 2004; 110: e48993.
  • 32
    Michelson AD, Barnard MR, Krueger LA, Valeri CR, Furman MI. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation 2001; 104: 15337.
  • 33
    Rinder HM, Bonan JL, Rinder CS, Ault KA, Smith BR. Activated and unactivated platelet adhesion to monocytes and neutrophils. Blood 1991; 78: 17609.
  • 34
    Wollner A, Wollner S, Smith JB. Acting via A2 receptors, adenosine inhibits the upregulation of Mac-1 (Cd11b/CD18) expression on FMLP-stimulated neutrophils. Am J Respir Cell Mol Biol 1993; 9: 17985.
  • 35
    Thiel M, Chambers JD, Chouker A, Fischer S, Zourelidis C, Bardenheuer HJ, Arfors KE, Peter K. Effect of adenosine on the expression of beta(2) integrins and L-selectin of human polymorphonuclear leukocytes in vitro. J Leukoc Biol 1996; 59: 67182.
  • 36
    Vinten-Johansen J, Thourani VH, Ronson RS, Jordan JE, Zhao ZQ, Nakamura M, Velez D, Guyton RA. Broad-spectrum cardioprotection with adenosine. Ann Thorac Surg 1999; 68: 19428.
  • 37
    Bauer B, Simkhovich BZ, Kloner RA, Przyklenk K. Does preconditioning protect the coronary vasculature from subsequent ischemia/reperfusion injury? Circulation 1993; 88: 65972.
  • 38
    Przyklenk K. Nicotine exacerbates postischemic contractile dysfunction of ‘stunned’ myocardium in the canine model. Possible role of free radicals. Circulation 1994; 89: 127281.
  • 39
    Ovize M, Kloner RA, Hale SL, Przyklenk K. Coronary cyclic flow variations ‘precondition’ ischemic myocardium. Circulation 1992; 85: 77989.
  • 40
    Folts JD, Rowe GG. Epinephrine potentiation of in vivo stimuli reverses aspirin inhibition of platelet thrombus formation in stenosed canine coronary arteries. Thromb Res 1988; 50: 50716.
  • 41
    Hjemdahl P, Larsson PT, Wallen NH. Effects of stress and beta-blockade on platelet function. Circulation 1991; 84: VI4461.
  • 42
    Gryglewski RJ, Szczeklik A, Bieron K. Morphine antagonises prostaglandin E1- mediated inhibition of human platelet aggregation. Nature 1975; 256: 567.
  • 43
    Hsiao G, Shen MY, Fang CL, Chou DS, Lin CH, Chen TF, Sheu JR. Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments. J Biomed Sci 2003; 10: 292301.
  • 44
    Reches A, Eldor A, Vogel Z, Salomon Y. Do human platelets have opiate receptors? Nature 1980; 288: 3823.
  • 45
    Bertha BG, Folts JD. Inhibition of epinephrine-exacerbated coronary thrombus formation by prostacyclin in the dog. J Lab Clin Med 1984; 103: 20414.
  • 46
    Machleidt C, Rose P, Mittmann U. Prevention of coronary platelet aggregation with a phosphodiesterase inhibitor RX-RA 69. Thromb Res 1985; 37: 595604.
  • 47
    Folts JD, Stamler J, Loscalzo J. Intravenous nitroglycerin infusion inhibits cyclic blood flow responses caused by periodic platelet thrombus formation in stenosed canine coronary arteries. Circulation 1991; 83: 21227.
  • 48
    Yao SK, Ober JC, Krishnaswami A, Ferguson JJ, Anderson HV, Golino P, Buja LM, Willerson JT. Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries. Circulation 1992; 86: 13029.
  • 49
    Brune B, Hanstein K. Rapid reversibility of nitric oxide induced platelet inhibition. Thromb Res 1998; 90: 8391.
  • 50
    Bullough DA, Zhang C, Montag A, Mullane KM, Young MA. Adenosine-mediated inhibition of platelet aggregation by acadesine. A novel antithrombotic mechanism in vitro and in vivo. J Clin Invest 1994; 94: 152432.
  • 51
    Kitakaze M, Hori M, Sato H, Takashima S, Inoue M, Kitabatake A, Kamada T. Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs. Circ Res 1991; 69: 14028.
  • 52
    Minamino T, Kitakaze M, Asanuma H, Tomiyama Y, Shiraga M, Sato H, Ueda Y, Funaya H, Kuzuya T, Matsuzawa Y, Hori M. Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs. J Clin Invest 1998; 101: 164353.
  • 53
    Sandoli D, Chiu PJ, Chintala M, Dionisotti S, Ongini E. In vivo and ex vivo effects of adenosine A1 and A2 receptor agonists on platelet aggregation in the rabbit. Eur J Pharmacol 1994; 259: 439.
  • 54
    Linden MD, Michelson AD, Barnard MR, Frelinger III AL, Furman MI, Przyklenk K. Does adenosine A2 receptor stimulation trigger the favorable ‘anti-platelet’ effect of preconditioning ischemia? Arterioscler Thromb Vasc Biol 2006; 25: e-46.
  • 55
    Linden MD, Michelson AD, Barnard MR, Frelinger III AL, Furman MI, Przyklenk K. In vitro adenosine A2 receptor stimulation inhibits platelet activation in humans. Arterioscler Thromb Vasc Biol 2006; 26: e-58 (abstract).
  • 56
    Harrison GJ, Willis RJ, Headrick JP. Extracellular adenosine levels and cellular energy metabolism in ischemically preconditioned rat heart. Cardiovasc Res 1998; 40: 7487.