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Keywords:

  • platelet;
  • ADP;
  • refractoriness;
  • adhesion;
  • flow;
  • extracellular matrix

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Exposure of whole blood (WB) to subendothelial extracellular matrix (ECM) under shear stress in the cone and plate(let) analyser (CPA) results in platelet adhesion, followed by release reaction and aggregation of circulating platelets on the adherent platelets. The properties of circulating non-adhered platelets in the CPA was studied by exposure of WB to ECM at a high shear rate (1300/s) for 2 min (1st run), followed by transfer of the suspension to a new ECM-coated well for a second run (2nd run) under similar conditions. The results of the 2nd run demonstrated transient adhesion refractoriness associated with platelet microaggregate formation in the suspension. The adhesion refractoriness was dependent on platelet activation during the 1st run and was prevented by addition of apyrase (ADP scavenger) or ADP receptor inhibitor, suggesting a role for ADP in mediating this response. Furthermore, exposure of WB samples to suboptimal concentrations of ADP (0·4–1 μmol/l) or a thrombin receptor activating peptide (TRAP) (5 μmol/l) for 2 min resulted in a similar transient platelet adhesion refractoriness to ECM under flow conditions. The transient platelet refractoriness and microaggregate formation induced by ADP was associated with a transient reduction in glycoprotein (GP)Ib, increased P-selectin expression and increased fibrinogen binding by circulating platelets. These data suggest a role for platelet agonists at suboptimal concentrations in modulating platelet function and limiting the expansion of the thrombus.

Circulating resting platelets can adhere to the exposed subendothelium at the site of vascular injury (Baumgartner & Haudenschild, 1972). The adhesion process occurs under flow conditions in which shear stress plays a major role in mediating mechanical and biochemical responses (Weiss et al, 1978; Ikeda et al, 1991). The role of platelet receptors and extracellular matrix (ECM)-specific ligands in this complex interaction has been established (George et al, 1984; Sakariassen et al, 1986; Ruggeri, 1997; Sixma et al, 1997). Upon adhesion under flow conditions, platelets undergo a series of changes, including spreading and release of their granular contents (Baumgartner et al, 1976). These granular contents include agonists that can activate circulating platelets and thus induce changes which modulate their functional state (Eldor et al, 1985). The integrin receptor αIIbβ3[glycoprotein (GP)IIb-IIIa] undergoes conformational changes on the activated circulating platelet and mediates binding of fibrinogen, von Willebrand factor and other ligands, allowing platelet aggregation to occur (Nurden, 1994; Frojmovic, 1998; Liu et al, 1998).

In spite of numerous reports on each of these steps (adhesion, release reaction and aggregation), there is little information in the literature regarding the inter-relationship of these processes under physiological conditions. This deficiency stems primarily from the lack of useful methods for the simultaneous study of different platelet properties. Thus, investigating platelet aggregation response using a routine aggregometer or flow cytometric analysis of platelet activation does not allow simultaneous testing of adhesion and aggregation properties. We recently described a method and a device, the cone and plate(let) analyser (CPA), in which whole blood (WB) platelet deposition on ECM is tested under close to physiological flow conditions (Varon et al, 1997, 1998).

In the CPA system, a small volume (0·2 ml) of WB is tested and investigation of circulating platelets during the course of platelet deposition on ECM can be conducted. In the present study, the application of the CPA method in studying the fate of circulating platelets near the site of adherent activated platelets, is described.

A profound adhesion refractoriness of circulating platelets was found, which is probably as a result of partial activation by released agonists from adherent platelets. This adhesion refractoriness is transient and may serve as a negative feedback control for limiting thrombus expansion.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Materials Tissue culture and virgin polystyrene four-well plates were purchased from Nunc (Roskilde, Denmark). Dextran T-40, Triton X-100, rat tail collagen type I, glutaraldehyde, fibrinogen, bovine serum albumin (BSA) fraction V, May–Grünwald stain, Prostaglandin E1 (PGE1), apyrase, thrombin receptor activating peptide (TRAP; ser-phe-leu-leu-arg-asn-pro-asn-asp-lys-tyr-glu-pro-phe), adenosine 2′,5′-diphosphate and tetrapeptide RGDS (arg-gly-asp-ser) were purchased from Sigma (St. Louis, MO, USA). 3-aminopropyltrimethoxysilane was obtained from Fluka (Buchs, Switzerland). Tissue culture media, sera and antibiotics, as well as fibronectin, were purchased from Biological Industries (Beit Haemek, Israel). Purified von Willebrand factor (VWF) was obtained from Alexis (Läufelfingen, Switzerland). An anti-GPIIb-IIIa monoclonal antibody (mAb) fragment (ReoPro) was obtained from Centocor (Malvern, PA, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-GPIb mAb AN51, FITC-conjugated anti-P-selectin, FITC-conjugated anti-human fibrinogen mAb and FITC-conjugated normal mouse IgG were purchased from Dako A/S (Glostrup, Denmark).

Preparation of ECM-coated and protein-coated plates Tissue culture plates coated with ECM were prepared as previously described (Gospodarowicz et al, 1980). Briefly, bovine corneal endothelial cells grown to confluence for 12–14 d in the presence of 4% Dextran T-40 were washed with phosphate-buffered saline (PBS) and dissolved by exposure to 0·5% Triton X-100 and 0·1 mol/l NH4OH solution followed by extensive washing with distilled water. The wet ECM-coated plates were sealed in plastic bags and maintained at 4°C for up to 12 months prior to use. This ECM is composed of collagen, adhesive glycoproteins and proteoglycans, however, unlike vascular ECM it does not contain VWF, which is apparently acquired from the tested blood during the course of the test.

Collagen type I (rat tail)-coated plates were prepared by covalent cross-linking of soluble collagen (1 mg/ml) to polystyrene four-well plates. The plastic surface was activated by incubation with 3-aminopropyltrimethoxysilane (4%) in 0·1 mol/l sodium acetate (pH 5·5) for 3 h at 90°C (Wikstrom et al, 1988). The plates were washed and further incubated with glutaraldehyde (3%) for 18 h, rewashed and exposed to collagen (1 mg/ml in 10 mmol/l HCl) for 2 h at 22°C, followed by an extensive wash and maintenance at 4°C for up to 1 week prior to use. Fibrinogen, fibronectin and VWF-coated plates were prepared by exposure of polystyrene plates to these proteins at concentrations of 50 μg/ml, 50 μg/ml and 1 U/ml, respectively, followed by blocking of excess binding sites with 1% BSA solution.

Cone and Plate(let) analyser (CPA) procedure Blood samples were taken from healthy volunteers or patients with Glanzmann's thrombasthenia and placed in a sodium citrate solution (final concentration of 0·38%). Samples containing 0·2 ml of the anticoagulated WB were placed on ECM, purified plasma and matrix protein-coated four-well plates. Shear force was applied using a rotating Teflon cone at a shear rate of 1300/s for ECM-coated plates and 1800/s for protein-coated plates, for 2 min at room temperature (Shenkman et al, 2000). Plates were then washed with PBS, stained with May–Grünwald stain and air dried. Platelet adhesion and aggregation on the ECM- and protein-coated plates were evaluated using an image analyser (Varon et al, 1997). Performing the CPA assay at 37°C resulted in similar surface coverage (SC) and average size (AS) values to the values obtained at room temperature for both normal and refractory blood samples. The use of heparin (2·5 U/ml) instead of citrate did not change the pattern of results with normal and refractory blood samples. Results were expressed as mean ± SD of surface coverage (SC, %) and average size (AS, μm2) of adhered particles, the n-value representing the number of blood donors.

Flow cytometry analysis of platelet activation A WB cytometric analysis was used as previously described (Michelson et al, 1991) with some modifications. Blood samples (5 μl) were placed in tubes containing 35 μl of modified Tyrode's buffer (134 mmol/l NaCl, 2·9 mmol/l KCl, 12 mmol/l NaHCO3, 1 mmol/l MgCl2, 0·34 mmol/l Na2HPO4, 10 mmol/l HEPES, 5·5 mmol/l dextrose, 0·35% BSA, pH 7·4) and 10 μl of either FITC-conjugated anti-CD42b, anti-P-selectin or anti-fibrinogen mAb. After 5 min of incubation at room temperature, the reaction was stopped by dilution, 20-fold, with cold Tyrode's-EDTA (5 mmol/l) buffer (pH 6·5). Samples were immediately analysed in an EPICS XL Coulter Flow Cytometer (Coulter, Miami, FL, USA) that was equipped with a 500 mW argon laser, operated at 15 mW with a wavelength of 488 nm. After identification of platelets by gating of both FITC positivity and characteristic light scatter, 5000 individual platelets were analysed. Background binding obtained from parallel samples with FITC-conjugated normal IgG was subtracted from each test sample.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Transient platelet adhesion refractoriness

A sample of WB was applied to an ECM-coated well and subjected to flow for 2 min in the CPA system (1st run), then the blood was immediately transferred into a new ECM-coated well and subjected to similar flow conditions (2nd run) (Fig 1A). Staining and analysis of the 1st run plate demonstrated normal platelet interaction with the ECM (SC = 17·9% and AS = 54·2 μm2). In the 2nd run plate, platelets poorly adhered to the ECM (SC = 0·8%) and the few that did adhere appeared as single platelets (AS = 12·4 μm2).

image

Figure 1. Platelet adhesion refractoriness after exposure to ECM under flow conditions. (A) WB sample was circulated on ECM for 2 min (1st run) and then the blood in the suspension was immediately transferred into a new ECM well and re-circulated under the same conditions (2nd run). Both plates were washed, stained and analysed. The SC, AS and representative pictures of platelet adhesion are presented. (B) WB samples were circulated on ECM for 2 min (CPA 1st run) followed by incubation of the blood in the suspension at room temperature for the indicated time intervals. At the end of the incubation period samples were applied to ECM plates for a 2nd run under similar conditions. The SC of plates after the 1st run was considered as 100%, the SC after the 2nd run was determined as a percentage of the SC obtained on the 1st run and presented as mean ± SD (n = 8).

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To explore the nature of this platelet adhesion refractoriness, blood samples were applied to the CPA system for 2 min (1st run) and then incubated at 22°C for increasing time periods before application to the 2nd run (Fig 1B). The 2nd run demonstrated a time-dependent recovery of platelet interaction with the ECM. After 10–20 min incubation, both the SC and the AS (data not shown) were 80–90% of the values obtained at the 1st run.

Platelet adhesion refractoriness is platelet activation dependent

WB was exposed to wells coated with various substrates under flow conditions (1st run), then the blood was immediately transferred into a new ECM-coated well under flow conditions to measure the platelet adhesion refractoriness. On the 1st run, platelet adhesion and aggregation occurred on ECM-, VWF- and collagen-coated surfaces, as indicated by high SC (Fig 2A) and AS (data not shown). In the BSA-coated surface, very low SC was observed, while in the fibrinogen- and fibronectin-coated surfaces, a moderate SC was detected, representing adherence of single platelets, indicated by the low AS (data not shown). The 2nd run demonstrated complete platelet adhesion refractoriness in WB samples that were exposed (on the 1st run) to ECM-, VWF- and collagen-coated plates. WB samples circulated on the 1st run over BSA-, fibrinogen- and fibronectin-coated wells demonstrated platelet adhesion on the 2nd run, but at a lower level compared with that of ECM-coated wells on the 1st run (Fig 2A). These results may suggest that platelet adhesion refractoriness is dependent on platelet activation which is induced by the flow of blood over the appropriate substrate. Furthermore, some platelet activation may be induced by the application of shear force alone, resulting in partial refractoriness, as observed with BSA-coated plates. To further prove the importance of platelet activation in the induction of platelet adhesion refractoriness, WB samples were preincubated with PGE1, RGDS or ReoPro for 10 min before the 1st run in ECM-coated plates (Fig 2B). In all treated samples, extensive adhesion of single platelets to the ECM on the 1st run was observed, however, it was not accompanied by platelet activation and aggregation (as reflected by low AS). On the 2nd run, single platelets adhered to the ECM to the same extent as on the 1st run and no refractoriness was observed (Fig 2B). Blood samples of Glanzmann's thrombasthenia patients demonstrated extensive adhesion of single platelets to the ECM, as reflected by close to normal SC but low AS (10–14 μm2), on both runs (Fig 2B). Apparently, activation and aggregation do not follow this process and therefore no adhesion refractoriness could be demonstrated.

image

Figure 2. Role of various immobilized ligands and platelet inhibitors in platelet adhesion refractoriness. (A) WB samples were circulated on various adhesive substrates (1st run) and then the blood was immediately transferred to ECM-coated wells and re-circulated under the same conditions (2nd run). SC after the 1st run and 2nd run was evaluated and presented as mean ± SD (n = 5). (B) Normal WB samples were preincubated for 10 min at 22°C with no addition (none; n = 19), or with PGE1 (100 ng/ml; n = 8), RGDS (4 μmol/l; n = 7) or ReoPro (4 μg/ml; n = 5) and then subjected to the CPA procedure (1st run), followed by an immediate transfer to the 2nd run. WB samples of Glanzmann's thrombasthenia (GT) patients (n = 8) were subjected to the CPA procedure (1st run) followed by an immediate transfer to the 2nd run. All CPA runs were done on ECM-coated wells. The SC on both the 1st and 2nd run was determined and presented as mean ± SD.

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Role of platelet agonists in platelet refractoriness

To explore the potential involvement of ADP in the refractoriness phenomenon, apyrase (an ADP scavenger) and adenosine 2′,5′-diphosphate (an ADP receptor antagonist) were added to the blood sample before the 1st run. The addition of apyrase (10 U/ml) or adenosine 2′,5′-diphosphate (5 mmol/l) resulted in a slight but not significant increase in SC on the 1st run, however, it significantly eliminated the refractoriness phenomenon on the 2nd run (Fig 3A). These experiments indicated that ADP might mediate the shear/ECM-induced platelet adhesion refractoriness. The role of ADP was further explored by pre-exposure of blood samples to suboptimal concentrations of ADP (0·75 μmol/l) (inducing partial aggregation in the aggregometer) for various time periods, followed by the CPA procedure (Fig 3B). Immediately after exposure to ADP, platelets did not adhere to the ECM, as reflected by a SC close to zero. Further incubation of the ADP-treated blood for 20–40 min prior to the CPA procedure resulted in almost complete recovery of platelet adhesion to the ECM. Similar results were observed when TRAP was used as a platelet activator instead of ADP (Fig 3B).

image

Figure 3. Role of ADP and TRAP in platelet refractoriness. (A) Normal WB samples were subjected to the CPA procedure (1st run) followed by an immediate 2nd run in the absence (control) or presence of either apyrase (10 U/ml) or adenosine 2′,5′-diphosphate (5 mmol/l). The SC of the ECM-coated wells was determined and presented as mean ± SD (n = 5). (B) WB samples were gently mixed and incubated at room temperature in the presence of ADP (0·75 μmol/l) or TRAP (5 μmol/l) for the indicated time intervals and then subjected to the CPA procedure. The SC was determined and presented as mean ± SD (n = 6). The initial SC determined for the blood samples before the addition of ADP or TRAP (zero time) was considered as 100%.

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Fate of platelets in the suspension during the CPA procedure

The number of single platelets in the suspension of blood samples treated using the CPA procedure or ADP was studied (Fig 4). Prior to counting, blood samples were fixed in 1% formaldehyde. Both treatments induced platelet adhesion refractoriness associated with a significant decrease in the number of single platelets in the suspension. The platelets transiently formed microaggregates in the suspension that dissociated upon further incubation, confirmed by studying blood smears, with about 60–78% recovery of single platelets after 20 min incubation. The recovery of platelet number demonstrated a similar kinetic pattern as the recovery in platelet adhesion to ECM. To evaluate the fate and the state of activation of platelets exposed for 2 min to suboptimal concentrations of ADP (0·4 μmol/l), the treated samples were counted and analysed using either CPA or FACS, the latter measuring P-selectin and GPIb expression as well as fibrinogen binding (Fig 5). The exposure of platelets to 0·4 μmol/l ADP for 2 min did not induce the decrease in platelet count that was observed with 0·75 μmol/l ADP (Fig 5A). However, this treatment was sufficient to induce a significant increase in the cell surface expression of P-selectin (from 2% to 10%), fibrinogen binding (from 3% to 40%) and a significant decrease in GPIb expression (about 50% of control) (Fig 5B). These changes in the platelet surface receptors were accompanied by a decrease in platelet adhesion to ECM in the CPA (Fig 5C).

image

Figure 4. Platelet count in the CPA and ADP treated blood samples. (A) CPA: Normal WB samples were subjected to the CPA procedure on ECM-coated wells for 2 min and then the blood samples were collected and maintained at room temperature. After the CPA procedure, at the time points indicated, blood samples were taken, fixed with 1% buffered formaldehyde and the platelet count determined. Each value was calculated as the percentage of the initial platelet count determined before the CPA procedure and the mean ± SD (n = 6) is presented. (B) ADP: Normal WB samples were incubated in the presence of ADP (0·75 μmol/l). Then blood samples were taken at the time points indicated, fixed with 1% buffered formaldehyde and counted for platelets. Each value was calculated as the percentage of the initial platelet count determined before ADP addition and the mean ± SD (n = 6) is presented.

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image

Figure 5. Effect of suboptimal concentration of ADP on platelet properties. (A) Citrated WB was incubated for 2 min with (black bars) or without (empty bars) ADP (0·4 μmol/l). The samples were then fixed with formaldehyde (1%) and subjected to blood counting. (B) WB samples diluted 1:20 were incubated with or without ADP (0·4 μmol/l) in the presence of mAb against P-selectin, fibrinogen or GPIb (all conjugated to FITC). After 5 min, the samples were washed, diluted 1:200 and immediately subjected to FACS analysis. The results are presented as a percentage of fluorescence-positive cells (FPC). (C) WB was incubated with or without ADP (0·4 μmol/l) for 2 min. The samples were then placed on an ECM surface and subjected to flow (1300/s) for 2 min, washed, stained and analysed using an image analysis system. The results are presented as a percentage of the surface covered with platelets.(***P < 0·05).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In a recent review by Nurden (1997), it was suggested that a logical consequence of platelet activation in suspension should be a transient decrease in platelet adhesiveness. Indeed, in the present study we describe a transient adhesion refractoriness of activated platelets in suspension. These results suggest a negative feedback at the site of a growing thrombus in which released agonists from the adherent and activated platelets induce adhesion refractoriness in circulating platelets. This conclusion is based on the following observations: (i) circulating platelets in the CPA system lost their adhesion properties after the 1st run (Fig 1); (ii) this refractoriness induction was dependent on the capability of the platelet to be activated, as both metabolic (by PGE1) and receptor (by RGDS or ReoPro) inhibitors eliminated this response (Fig 2). Further support was given by the lack of refractoriness of Glanzmann's thrombasthenia platelets in this system (Fig 2) and the specificity of the matrix that can induce refractoriness. Thus, only ECM-, VWF- and collagen-coated but not BSA-, fibrinogen- or fibronectin-covered plates could induce this response (Fig 2). (iii) the refractory state of circulating platelets was mediated by agonists released from adherent activated platelets, as pretreatment of WB by apyrase (an ADP scavenger) or ADP receptor antagonist inhibits this response (Fig 3). In addition, suboptimal activation of platelets using either ADP or TRAP induced the same adhesion refractoriness (Fig 3).

The term ‘platelet refractoriness’ has been used to describe more than one phenomenon. Grant et al (1976) and Hagen et al (1977) were the first to describe a refractory state for ristocetin of disaggregating platelets following exposure to an aggregating agent. It was further characterized by Mills et al (1990), who showed that membrane alterations, accompanying shape change caused by an aggregating agent, were responsible for inhibiting subsequent agglutination by VWF. Other studies suggest that platelet activation decreases the binding of VWF to GPIb/IX on platelet membranes (Michelson & Barnard, 1987; George & Torres, 1988). However, in a more recent study, White & Rao (1996) described the refractory state of activated platelets in the presence of calcium ions and demonstrated that the refractory platelets remained sensitive to agglutination by ristocetin, indicating that GPIb/IX receptors are still present on agonist-activated platelets. ADP-induced platelet refractoriness of turbidimetrically measured aggregation has also been reported (Rozenberg & Holmsen, 1968; Holme et al, 1977; Peerschke, 1985). In gel-filtered platelets, the refractoriness was accompanied by shape change induced by low or optimal ADP concentrations. Partial recovery of the aggregability and a return of discoid morphology of the platelets following addition of apyrase were observed. Further studies with ADP demonstrated that, at low concentrations, it initiates platelet shape change without aggregation but reduces platelet adhesion to collagen (Meyer et al, 1981). In all the above studies, except for Meyer et al (1981), platelet refractoriness was induced by optimal concentrations of agonists. This resulted in full platelet activation accompanied by release reaction and formation of irreversible aggregates. The dissociation of these aggregates by PGE1 treatment (White & Rao, 1996) or any other means released platelets that were refractory to a second stimulation by any platelet agonist. In contrast to previous studies, in this study, platelet refractoriness was apparently associated with microaggregate formation, which may occur during a physiological haemostatic process, presumably owing to release of a suboptimal level of agonists. The microaggregates formed under these conditions were transient, as well as the adhesion refractoriness to ECM. The refractory platelets described in this study were probably different from those in most of the previous studies, but similar to those described by Meyer et al (1981), deriving from partial activation and microaggregate formation that spontaneously disaggregate within a period of 10–30 min. During the period that platelets are in microaggregates, they do not adhere to the ECM (or immobilized VWF) under flow conditions. As soon as the microaggregates dissociate, platelets regain their normal properties of adhesion to ECM under flow conditions. The microaggregate formation described in this study is similar to the previously described low ADP concentration-dependent aggregation (Frojmovic et al, 1983) that was distinct from high ADP concentration-dependent aggregations. These two types of aggregates are probably two successive steps in platelet aggregation with shape change and reversible aggregation in the first step, followed by stable aggregation associated with release from dense granules (Holmsen, 1982). It is noteworthy that, in our system, the adhesion refractory state was associated with changes in cell surface receptors that preceded the transient microaggregate formation (Fig 5).

This study suggests a role for platelet agonists at suboptimal concentrations in modulating platelet function and limiting the expansion of the thrombus. The results of this study should, however, be considered cautiously. The CPA system consists of a very small volume of WB that continuously circulates over the ECM for about 120 s in order to achieve a full refractory response. As a consequence of these conditions, the concentration of the released agonists may be increased above physiological levels. Therefore, more studies using flow-through chambers are necessary to elucidate the potential role of such a mechanism in vivo. However, it may be speculated that, in the microcirculation and especially at a bleeding site where a thrombus is being formed, static (or close to static) conditions may be present that allow an accumulation of the released agonists to levels similar to those operating in our assay system (Goldsmith & Karino, 1987). Furthermore, the role of ADP (at optimal concentrations) released by activated platelets in the recruitment and activation of platelets at the site of thrombus formation is well established. It is reasonable to assume that suboptimal concentrations similar to those achieved in our in vitro system may exist downstream of a growing thrombus and, thus, limit its expansion.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This research was supported by a grant from the National Council for Research and Development, Israel, and Deutsche Forschungsanstalt für Luft und Raumfahrt.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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