THROMBOSIS RESEARCH Thrombosis
SUPPLEMENT
Research 92 (1998) S47-S52
ISSUE
Platelet Adhesion and Aggregation in Pulsatile Shear Flow: Effects of Red Blood Cells J. Heinrich Joist’, Joan E. Baumanl and Salvatore P. Sutera” ‘Departments of Pathology and Internal Medicine, Saint Louis University School of Medicine, and ‘Department of Biomedical Engineering, Washington University, St. Louis, Missouri, USA.
Abstract An in vitro test system was developed to examine the effects of red blood cells (RBC) on shearinduced platelet adhesion (SIPAD) and platelet aggregation (SIPAG). Suspensions of human platelets labeled with Mepacrine and suspended in titrated plasma were exposed to single, continuous or repetitive (120-300X) one second shear stress pulses of varying amplitude (15-100 dyn/cm2) in a cone-plate viscometer in the presence or absence of fresh, untreated (intact) RBC or glutaraldehyde (GLA)-fixed, rigid, adenosine diphosphate (ADP)depleted (GLA)-RBC. SIPAG was expressed as percent loss of single platelets. SIPAD was assessed by measuring the amount of Mepacrine-related fluorescent material remaining on glass disks in the plate of the viscometer after washing with EDTAsaline to remove platelet aggregates. Intact RBC were twice as effective as GLA-RBC in potentiating SIPAG at all shear stress levels. Potentiation of SIPAD by intact RBC was markedly less than that observed with GLA-RBC at stresses below 50 dyn/cm2. These findings are consistent with the concept that while both physical and chemical (ADP) mechanisms are substantially involved in potentiation by RBC of SIPAG, RBC support RBC, red blood cells; SIPAD, shear-induced platelet adhesion; SIPAG, platelet aggregation; GLA, glutaraldehyde; vWF, von Willebrand factor: GPIb. glycoprotein Ib-IX-V: GPIIbIIIa, cq&,, glycoprotein IIb-IIIa; C-PPP, titrated platelet-poor plasma; C-PRP. titrated platelet-rich plasma: ADC, acid citrate dextrose solution. Corresponding author: J. Heinrich Joist, Departments of Pathology and Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO 63110, USA.
Abbreviations:
SIPAD largely by enhancement of platelet transport from the bulk flow to the bounding surfaces. The findings also indicate that it is feasible to assess SIPAD and SIPAG in the same flow system simultaneously. A less complicated version of the method described here should prove useful in the evaluation of patients with platelet functional disorders, and in the evaluation and monitoring of antiplatelet agents. 0 1998 Elsevier Science Ltd. Key Words: Shear; Pulsatile; Platelet; Adhesion; Aggrega-
tion; Hematocrit; Viscometer
T
he initial arrest of bleeding from small blood vessels in response to vascular injury is accomplished by platelet adhesion to subendothelial connective tissue structures and subsequent platelet aggregation leading to the formation of a hemostatic platelet plug [l]. Much insight into the complex mechanisms involved in hemostatic platelet plug formation in vivo has been gained from in vitro experiments. In these, anticoagulated or native blood or suspensions of isolated platelets, and other blood cells in artificial media were exposed to de-endothelialized vascular [2-61 or artificial foreign [6-91 surfaces under defined, biologically relevant shear flow conditions. These studies identified major factors essential for or affecting shear-induced platelet adhesion (SIPAD) and aggregation (SIPAG), such as von Willebrand factor (vWF), fibrinogen, platelet receptors (integrins), i.e., glycoprotein Ib-IX-V (GPIb) and glycoprotein IIb-IIIa (GPIIb-IIIa, (Y&&), and thromboxane AZ. Furthermore, it has been shown that red blood cells (RBC) increase substantially the efficiency of
0049-3848/98 $-see front matter 0 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII SoO49-3848(98)00160-l
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J.H. Joist et al./Thrombosis
platelet deposition on subendothelium [lo-121 and platelet interaction with artificial surfaces [13-H] in different in vitro flow systems, apparently involving: 1) hematocrit and shear rate dependent platelet-surface transport, also called platelet diffusivity [16,17], augmented by the increased concentration of RBC in the center of the blood stream (reverse Fahraeus effect, physical mechanisms) [18]; and 2) liberation from RBC of adenosine diphosphate (ADP) [13-151 and other factors [19] (chemical mechanism) [20]. SIPAG when studied in these in vitro flow systems involves both platelet adhesion to the foreign surfaces and platelet aggregation and there is little information on the relative contribution of RBC-physical and chemical effects on SIPAD. We developed an in vitro flow system using a conventional, rotational cone-plate viscometer in which platelet suspensions can be exposed to repetitive shear pulses of biologically relevant amplitudes to examine SIPAG [21]. We adapted this system to allow quantitative determination of SIPAD. Using this system, we tested the hypothesis that RBC can potentiate SIPAD and that this effect is mediated by both physical and humoral factors.
1. Materials and Methods 1.2. Blood Collection and Preparation of Platelet-Rich-Plasma Blood was obtained from healthy male and female volunteers who had given informed written consent and had not been taking aspirin or other plateletinhibitory medications or estrogen. Blood was collected into 0.1 volume of 3.8% sodium citrate solution for preparation of titrated platelet-poor plasma (C-PPP) and isolation of RBC, and l/7 volume of acid citrate dextrose solution (ADC) for preparation of Mepacrine-labeled platelets. Citrated blood was centrifuged at 180g for 10 minutes at room temperature
to yield titrated platelet-rich plasma (C-PRP) w hic h was again centrifuged at 25OOg for 10 minutes at room temperature to yield C-PPP.
was centrifuged at 200g for 10 minutes at room temperature to yield ACD-PRP which was
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Removable Disk
Fig. 1. Cut away depiction of flow chamber of cone and plate viscometer (not to scale) with removable disks for measuring shear-induced platelet adhesion simultaneously with shear-induced platelet aggregation.
resuspended in C-PPP to yield platelet concentrations of about 1.0X106/pL. 2.3. Preparation of Intact and ADP-Depleted
RBC
Following centrifugation of C-blood and removal of supernatant C-PPP the remainder was centrifuged at 2500g for 10 min at room temperature, the supernatant C-PPP and buffy coat were carefully removed and this procedure repeated once. RBC were depleted of ADP by exposure to glutaraldehyde (GLA) according to the method previously described [14] and resuspended in C-PPP and the mixture centrifuged at 2500g for 10 min at room temperature. 1.4. Shear Experiments Aliquots of packed intact and GLA-RBC were added to aliquots of Mepacrine labeled C-PRP to yield
different
packed
RBC
concentrations
(he-
matocrit). Aliquots (1.3 ml) of C-PRP/RBC mix-
ACD-blood
tures were applied
incubated with Mepacrine for 30 minutes at 37°C to yield a final concentration of 20 PM. The mixture was centrifuged at 3500 g for 5 min, the supernatant PPP was removed and the platelets immediately
into which four removable glass disks had been inserted (Figure 1). The cone was then lowered to point contact with the plate and, following a two-minute warm-up period, the desired shear stress amplitude, appar-
to the plate of the viscometer
J.H. Joist et al./Thrombosis
ent viscosity, shear pulse ramp, and shear pulse duration, between shear pulse pause and numbers of shear pulses were entered into a microcomputer and the shear run was initiated. Upon completion of the shear run the cone was raised and the sheared mixture carefully transferred by plastic pipette to a plastic tube containing a fixative solution (0.1% formalin/l% GLA) for quantitation of SIPAG which was expressed as percent loss of single platelets as determined by phase contrast microscopy. The shear chamber was carefully rinsed with 1% EDTA-saline to remove the remaining non-adherent platelets and platelet aggregates. The four glass disks were removed and immersed in 1 mL of 0.1 N NaOH and Mepacrine-related fluorescence was measured. The results were expressed as number of platelets using a standard curve prepared with dilutions of intact Mepacrine-labeled C-PRP. 2. Results Figure 2 shows the effects of intact RBC and rigid, ADP-depleted GLA-RBC at different hematocrits on SIPAG under continuous, sustained (5 min) shear flow at an intermediate shear stress of 50 dyn/cm2. SIPAG increased progressively with increasing hematocrit. Mean SIPAG in C-PRP in the absence of RBC at 50 dyn/cm2 was 9%. Figure 3 shows the effects of intact RBC and GLA-RBC at a fixed hematocrit of 30% on SIPAD at different shear amplitudes using repetitive, short duration (1 set) shear pulses with a 0.7 set shear ramp and 1 set pauses between shear pulses. GLARBC potentiated SIPAD considerably at shear stresses up to 50 dyn/cm2. Mean SIPAD in the absence of RBC did not exceed 0.6~10~ platelets at any shear stress amplitude. At the higher shear stress of 100 dyn/cm2, the SIPAD-potentiating effect of GLA-RBC declined markedly. 3. Discussion There is substantial evidence that RBC can potentiate SIPAG in different experimental in vitro flow systems [lo-151. These findings are consistent with and seem to provide a basis for observations in patients with anemia of uremia [22] and other types of anemia [23] indicating that RBC play an important role in hemostasis in vivo.
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Experimental data using tube flow model systems indicate that RBC, by concentrating in the center of the blood stream [18] and colliding with platelets [16,17], may facilitate transport of platelets to the periphery of the blood stream near the bounding surfaces, also called the reverse Fahraeus effect [18] (physical mechanism). This would increase the platelet concentration near the wall thus increasing the likelihood of collisions of platelets with the vessel wall or artificial surfaces of the in vitro or ex vivo flow system. However, it has also been postulated that RBC may enhance sheardependent platelet deposition on subendothelial vessel wall structures such as collagen and microfibrils (SIPAG) by a chemical mechanism, i.e., liberation of ADP which could induce a conformational change in platelet GP IIb-IIIa, a prerequisite for binding of fibrinogen and vWF to this platelet receptor and thus, platelet cohesion, as well as platelet adhesion [20]. In previous experiments designed to determine the relative contribution of physical and chemical mechanisms in RBC-potentiation of SIPAG, we compared the effects of fresh, untreated (intact) human RBC capable of liberating ADP with those of GLA-fixed, rigid, ADP-depleted RBC [14]. Using a Couette rotational viscometer and continuous, five-minute shear stresses we observed that both intact RBC and GLA-RBC enhanced SIPAG and intact RBC were substantially more effective in potentiating SIPAG than GLARBC. We also demonstrated that the amounts of ADP liberated from RBC could yield concentrations well within the range sufficient to initiate and potentiate platelet aggregation in vitro. The findings presented here using a cone and plate viscometer and single, five-minute shear pulses of intermediate amplitude confirmed our earlier data indicating that both intact RBC and GLARBC can potentiate SIPAG and that the effect of intact RBC is much greater than that of GLARBC. Thus, it is apparent that physical and chemical mechanisms contribute roughly equally to potentiation by RBC of SIPAG. Similar findings were reported by others using RBC ghosts instead of GLA-RBC [19]. In the present report we also present new findings indicating that intact RBC and GLA-RBC also potentiate SIPAD. However, in contrast to our findings with SIPAG, the effect of GLA-RBC on SIPAD was much more pronounced than that of intact RBC. This may be explained by
J.H. Joist et al./Thrombosis
1 •j
Fresh REC
Research 92 (1998) S47-S52
1
P < 02 1
50 dynlcm’ 1 x 300 set N=5
Fig. 2. Effects of different concentrations of fresh, intact and GLA-fixed, rigid, ADPdepleted RBC on shearinduced platelet aggregation using a single, continuous fiveminute shear stress of intermediate amplitude. The means (iSEM) for fresh and GLA-RBC were compared by analysis of variance (ANOVA).
pulse
20%
Hematocrit
(%)
the rigid GLA-RBC having a greater potential to facilitate transport of platelets to the bounding surfaces of the viscometer. These data seem to indicate that RBC exert their potentiating effect on SIPAD predominantly via enhancement of platelet diffusivity/transport, i.e., a physical mechanism, whereas the RBC effect on SIPAG is substantially mediated by chemical mechanisms, e.g., liberation of ADP from RBC [14] or other mechanisms [19] (Figure 4).
The findings must be interpreted with caution because GLA-RBC (like RBC-ghosts) are not ideal models of ADP-depleted RBC because they are rigid and may have GLA-induced membrane surface alterations with unknown effects on interactions with platelets in shear flow, which could affect both SIPAD and SIPAG. Experiments currently in progress to examine the effects of GPIb-, GPIIb-IIIa- and ADP receptor blocking agents and
4.5-
=
4.0 -
120x1 secpulses N=3-12 30% Hematocrit
3.5-
1.5NS l.O-
Fig. 3. Effects of fresh, intact RBC and GLA-fixed, rigid, ADP-depleted RBC on shearinduced platelet adhesion in pulsatile shear flow. The means (%SEM) observed at different shear stresses were compared with that observed at 0 shear by analysis of variance (ANOVA).
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Fig. 4. Mechanisms involved in potentiation by RBC of high shear-induced platelet adhesion and aggregation. The initial platelet contact with the surface, mediated by binding of platelet glycoprotein Ib (GPIb) to surface-bound, structurally altered von Willebrand factor (vWF) is facilitated by RBC-mediated platelet transport to the surface (physical mechanism). Initial platelet-surface contact under the influence of high shear and release from platelets of adenosine diphosphate (ADP) lead to platelet activation and conformational changes in GPIIb-IIIa which allows: 1) binding of the receptor to surface-bound vWF, facilitating firm platelet attachment to and spreading on the surface, release of ADP, activation of prostaglandin metabolism and formation of thromboxane A1 (TxA& and, 2) binding of plasma vWF and fibrinogen to altered GPIIb-IIIa on activated platelets in close proximity leading to platelet cohesion/aggregation. Shear-induced platelet aggregation is enhanced by both RBC-mediated platelet diffusivity and ADP liberated from RBC and platelets.
ADP removal systems may provide further insight into the complex mechanisms by which RBC enhance SIPAD and SIPAG. A number of methods for routine in vitro evaluation of platelet function in the clinical laboratory are available. However, these methods measure predominantly platelet aggregation or a combination of platelet adhesion and aggregation under poorly defined flow conditions. The method described here allows simultaneous evaluation of platelet adhesion and aggregation without the addition of a plateletactivating agent under well defined, laminar, pulsatile shear flow. A technically less complicated and time consuming modification of this method may be useful in the evaluation of patients with suspected
impaired or increased platelet function, of the effects of antiplatelet agents and for monitoring patients treated with antiplatelet drugs.
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