Concurrent label method with 111In and 51Cr allows accurate evaluation of platelet viability of stored platelet concentrates

Summary. The precision and reproducibility of ¹¹¹In and ⁵¹Cr platelet radiolabel agents for in vivo kinetic studies of stored platelet concentrates (PC) were investigated. The objective was to develop a precise method with concurrent labelling of two platelet populations using different isotopes, which would allow identification of small differences in in vivo platelet quality. Identical labelling procedures were used to investigate the effects of PC storage age, different methods of red cell (RBC) and white cell (WBC) contamination correction, and label elution correction on the results of ¹¹¹In and ⁵¹Cr kinetic studies. ¹¹¹In and ⁵¹Cr platelet survival curves from the same PC, even when uncorrected for elution and RBC contamination, exhibited excellent correlation, irrespective of the age of the concentrate and its viability. However, slightly higher, but statistically significant, post-infusion per cent recoveries with ⁵¹Cr labelled platelets were found. Two factors were identified as the cause for this difference. There was a higher affinity of contaminating RBC/WBC in PC for ⁵¹Cr than for ¹¹¹In. With determination of RBC/WBC activity by centrifugation/density separation, RBC/WBC fractions from the injectate were found to contain 12.6 ± 3·8%v 7·1 ± 3·6% of total ⁵¹Cr and ¹¹¹In activity, respectively, in 20 studies. In addition, there was a significantly higher ¹¹¹In activity in plasma immediately post-infusion than with ⁵¹Cr, 5·2 ± 1·3%v 2·8 ± 1·6%, respectively, suggesting more label elution or carryover. After correction for the activity of RBC/WBC and for elution or carryover, essentially identical ⁵¹Cr/¹¹¹In platelet survival curves were found. In 31 stored PC studies, the absolute average difference between ⁵¹Cr and ¹¹¹In per cent recoveries was only 4 ± 3% in a group of donors whose platelet recoveries ranged from 10% to 80%. Similarly, the average difference between ⁵¹Cr and ¹¹¹In survival was only 8±4 h within a range of survivals from 40 to 220 h. In conclusion, after correction for elution and contaminating RBC/WBC binding, these studies show that ⁵¹Cr and ¹¹¹In may be used interchangeably for labelling of stored PC, and that small differences between test and control platelets could be reliably detected using concurrent labelling with simultaneous infusion.     

Radionuclide Distribution folowing Injection of 111Indium-labelled Platelets

Platelets labelled with 111In displayed similar survival curves after incubation with 111In-oxine in plasma, plasma-saline and dextrose saline media. The use of autologous red cells to cushion platelets during high-speed centrifugation facilitated platelet resuspension without greatly affecting the duration of the labelling procedure. Quantitative scanning after reinjection of labelled platelets in haematologically normal subjects showed that, initially, splenic indium amounted to about 35% of the injected dose and hepatic indium about 12%; these levels rose only slightly over the subsequent duration of the platelet life span. Subtraction of the signal from indium in platelets thought to be normally pooled within the spleen from the total indium signal gave splenic indium uptake curves which reflected splenic platelet destruction. Initially, the sum of indium levels in spleen, liver and blood equalled 100% of the dose. Thereafter, the sum fell progressively at a rate thought to be approximately equal to the rate of bone marrow uptake.     

The kinetics of unmatched and HLA-matched 111In-labelled homologous platelets in recipients with chronic marrow hypoplasia and anti-platelet immunity

The kinetics of homologous platelets, labelled in plasma with 111In-tropolonate, have been studied in five recipients with chronic marrow hypoplasia and severe thrombocytopenia, who were refractory to platelet transfusions as a result of alloimmunization. Mean platelet life span (MPLS), recovery, plasma 111In level and splenic and hepatic uptake kinetics were studied on two occasions, one using HLA-matched platelets and the other unmatched platelets. In each case, recovery of labelled platelets at 1 h post-injection and MPLS improved with HLA matching, although this improvement was highly variable. Only two of the five subjects would have derived any significant benefit from HLA-matched as compared with unmatched platelet transfusions. It was concluded that the need exists for additional cross-matching procedures, possibly related to platelet specific antigens, in patients who remain refractory to platelet transfusion.     

Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr- labeled platelets differ

Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr- labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.     

Indium-III: a New Radionuclide Label for Studying Human Platelet Kinetics

Indium-III. when complexed with 8-hydroxyquinoline (oxine), has been employed as a radioactive platelet label for thrombus imaging in animals and man. The short half-life (2.8 d) and high yield of gamma photons of 111In make it ideal for in vitro counting and external imaging. To evaluate its suitability for studies of platelet turnover in man, platelet kinetic studies were carried out on 10 healthy volunteers using 111In-and 51Cr-platelets concurrently. For 111In labelling, platelets were harvested by differential centrifugation from 43 ml of whole blood drawn into acid-citrate dextrose (ACD) solution. The platelets were washed and suspended in a mixture of ACD and isotonic saline and then incubated with 111In-oxine, rewashed, and suspended in plasma for reinfusion. 51Cr labelling was performed using standard methods. Mean labelling efficiency was 73% with 111In and 6.5% with 51Cr. In vitro studies demonstrated minimal release, elution, and reutilization of the 111In label. There was no significant difference in the aggregation response of 111In- and 51Cr-platelets to ADP and collagen. The in vivo recovery of 111In-platelets was approximately 50% greater than that of 51Cr-platelets whereas the platelet life spans were similar. These results indicate that 111In labelled platelets may be useful for thromobokinetic studies in man. The new method offers the advantages of reduced blood requirements, higher labelling efficiency, and the ability to perform external imaging of platelet distribution in vivo.     

Survival of baboon biotin-X-N-hydroxysuccinimide and (111)In-oxine-labelled autologous fresh and lyophilized reconstituted platelets

BACKGROUND AND OBJECTIVES: In accordance with Food and Drug Administration (FDA) regulations, platelets can be stored in the liquid state at 22 degrees C for only 5 days. Platelets frozen with 6% dimethylsulphoxide (DMSO) can be stored at -80 degrees C for 2 years, and platelets frozen with 5% DMSO can be stored at -150 degrees C for 3 years. Studies are being conducted to determine the effects of lyophilization of platelets. In the present study, we assessed the survival of autologous lyophilized-reconstituted platelets in the baboon. MATERIALS AND METHODS: We studied fresh baboon platelets and baboon platelets that had been treated with paraformaldehyde, frozen, lyophilized, thawed and reconstituted. Aliquots of these platelets were labelled with (111)In-oxine or biotin-X-N-hydroxysuccinimide (biotin-X-NHS) before autotransfusion, and measurements were made of the in vivo recovery and lifespan. We also evaluated the response of fresh and lyophilized platelets to in vitro agonists by measuring the level of platelet surface markers and heterotypic aggregates in the peripheral blood following the autotransfusions. RESULTS: The (111)In-oxine- or biotin-X-NHS-labelled lyophilized, reconstituted platelets exhibited survival times of less than 15 min. These platelets did not respond to stimulation with agonists to decrease platelet GPIb and increase platelet P-selectin and platelet GPIIb-IIIa levels 1 min post-transfusion and they accumulated more procoagulant factor V than did the fresh platelets. CONCLUSIONS: Lyophilized reconstituted baboon platelets labelled with (111)In-oxine or biotin-X-NHS before autotransfusion exhibited an in vivo circulation time of less than 15 min. Further study of the lyophilized, reconstituted platelets is required to evaluate their haemostatic function.     

Kinetic Studies of 51Cr and DF32P Labelled Granulocytes

In vivo kinetic studies of granulocytes labelled in vitro with 51Cr and DF32P were carried out in nine haematologically normal subjects by isolation of the cells in the blood samples by the Ficoll-Isopaque flotation method. 51Cr and 32P specific activity of blood samples made of 93-98% granulocytes was studied. Distribution between marginated and circulating granulocyte pools was identical for both labelled cells and the marginated pool was similar to the circulating pool, except that it was lower in one subject who had a previous splenectomy. The half-disappearance time (T 1/2) was 16.1+/-2.2 h for 51Cr-labelled and 5.4+/-2.1 hr for DF32P-labelled granulocytes. In one case of a normal subject who previously received multiple transfusion homologous 51Cr-labelled granulocytes had a T 1/2 of less than 1 h.     

The value of a 51Cr platelet lysis assay as crossmatch test in patients with leukaemia on platelet transfusion therapy

Current platelet crossmatch testing still results in a significant percentage of false positive or false negative results in oncological patients during platelet support. A 51Cr platelet lysis assay was used for the detection of platelet alloantibodies. We determined the predictive value of this assay as crossmatch procedure on 28 occasions in 14 patients who received random single donor platelet transfusions. To deal with the problem of spontaneous lysis we included a panel of 10 control sera from normal individuals and applied several methods of statistical analysis to these data. It appeared that the use of only one control serum was sufficient when the percentage relative counts was used as the criterion variable, which is of advantage in the practical application of the test. The test values were retrospectively compared with the clinical transfusion response, determined as the 1 h post-transfusion platelet recovery. The 51Cr platelet lysis crossmatch showed false negative results in 1/21 cases and false positive results in 1/7 cases. These data indicate that the 51Cr platelet lysis assay adds a useful dimension to the solution of the problem of selecting compatible platelet donors. Spontaneous lysis of target platelets appeared not to be a problem in the interpretation of test results.     

Loss of 111Indium as indicator of platelet injury

We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.     

Survival of density subpopulations of rabbit platelets: use of 51Cr-or 111In-labeled platelets to measure survival of least dense and most dense platelets concurrently

The origin of the density heterogeneity of platelets was studied by measuring the survival of density subpopulations of rabbit platelets separated by discontinuous Stractan density gradient centrifugation. When a total population of 51Cr-labeled platelets was injected into recipient rabbits, the relative specific radioactivity of the most dense platelets decreased rapidly. In contrast, that of the least dense platelets had not changed 24 hr after injection, and then decreased slowly. To distinguish between the possibilities that most dense platelets are cleared from the circulation more quickly than least dense platelets or that platelets decrease in density as they age in the circulation, the concurrent survival of least dense and most dense platelets, labeled with either 51Cr or 111In-labeled total platelet populations, determined concurrently in the same rabbits, were identical, calculated from 1 hr values as 100%. However, the 1-hr recovery of 111In-labeled platelets was slightly but significantly less than that of 51Cr-labeled platelets. Therefore, we studied the survival of 51Cr-labeled least dense and 111In-labeled most dense platelets as well as that of 111In-labeled least dense and 51Cr-labeled most dense platelets. Mean 1-hr recovery of least dense platelets, labeled with either isotope (78% +/- 7%, SD) was similar to that of most dense platelets, labeled with either isotope (77% +/- 8%; SD). Mean survival of least dense platelets was 47.3 +/- 18.7 hr (SD), which was significantly less than that of most dense platelets (76.1 +/- 21.6 hr; SD) (p less than 0.0025). These results indicate that platelets decrease in buoyant density as they age in the circulation and that most dense platelets are enriched in young platelets, and least dense in old. Thus, the events that affect platelets as they age in the circulation contribute to platelet density heterogeneity, although they may not be the sole cause of it.     

Post-transfusion recovery of function of 5-day stored platelet concentrates

Summary Platelets show a rapid reduction in their responsiveness to aggregating agents during storage for transfusion, but little is known about reversal of this defect in vivo after transfusion. In this study, fresh and stored platelets from the same donor ( n =12) were labelled with ¹¹¹In or ⁵¹Cr, respectively, mixed, and simultaneously infused. Blood samples were taken for up to 5 d post-infusion, and the functional behaviour of the labelled platelets ex vivo was measured by retention on glass bead columns, and by whole blood aggregability to ADP, epinephrine and ristocetin. Aggregation was determined by filtering aggregated samples through a column of cotton wool to remove the aggregates, and quantitated as per cent decrease in radioactive counts. The study showed that, although infused radiolabelled 5 d stored platelets had a significantly lower aggregability towards ADP and epinephrine immediately (1 h) after infusion (32% and 29%, respectively, of fresh platelet values), a complete restoration to fresh platelet levels was found 24–72 h post-infusion, with no further change observed over the ensuing 5 d with either fresh or stored labelled platelets. A slightly (6–9%) lower adhesion to both uncoated and collagen-coated beads was found for the stored platelets throughout the 5 d period of study post-infusion.
In conclusion, these studies show that, with ex vivo testing, platelets stored for 5 d quickly recovered adhesion and aggregability capabilities similar to that of fresh platelets, suggesting that the functional lesion developed during storage is quickly and completely reversed after infusion.     

Kinetics, Distribution and Sites of Destruction of 111Indium-labelled Human Platelets

The survival, tissue distribution and fate of ¹¹¹In-oxine labelled autologous platelets in six normal humans were studied with serial blood sampling, scintillation camera and computer-assisted imaging, whole body profile scanning, and rectilinear scanning. ¹¹¹In-platelets recovery in the circulation was 72±16% and survival was 216±17 h. Platelet survival curves fitted a linear function best. Initially platelets pooled rapidly in the spleen as a single exponential function, and at 90 min 26% of the injected ¹¹¹In was located in this organ. Early hepatic uptake was also significant and at 90 min constituted 16% of total body ¹¹¹In-activity. As labelled platelets disappeared from the circulation there was a threefold increase of radioactivity in the liver to reach 39% of whole body activity at 216 h. Radioactivity also increased significantly in the spleen (33±3% at 216 h). There was significant residual radioactivity in the thoracic and lower abdominal regions at 216 h, suggesting that platelets are also sequestered in the bone marrow. Radioactivity in the lower limbs almost disappeared with time (0±7% at 216 h), indicating that utilization of platelets in the peripheral vasculature is not marked in normal subjects.     

Serotonin uptake and release by platelets adhering to polyethylene

Heparinized human blood containing platelets labelled with 14C-serotonin and 51Cr was exposed to a polyethylene surface by rotation in Chandler loops. Both uptake of platelets on the surface and platelet aggregation in the blood occurred. More 14C- than 51Cr activity accumulated on the surface. It was demonstrated that this was due to an active uptake of 14C-serotonin by the surface-adherent platelets, which also retained their capacity to exert a release reaction.     

Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets

The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of "abnormal" platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy.     

In vitro and in vivo behavior of 111In-labelled platelets: An experimental study of healthy male volunteers

The aim of this study was to obtain a critical evaluation of a simple method for labelling platelets with ¹¹¹In-oxine. All experiments were carried out on healthy volunteers. 65 ± 7 (SD) % of the platelets in collected blood were labelled and reinjected. As compared to control experiments, only in response to a low final ADP concentration (1.0 μmol/l) did ¹¹¹In-labelled platelets show reduced in vitro aggregability. The mean platelet volume for ¹¹¹In-labelled platelets was slightly lower than the mean platelet volume in whole blood. The results for initial platelet recovery and platelet mean lifespan closely agreed with those of other studies in which considerably higher platelet extraction from whole blood was obtained. After injection, the splenic uptake and blood disappearance of ¹¹¹In-labelled platelets followed a monoexponential function with almost identical rate constants. By compartmental analysis of the equilibration of platelets between blood and spleen, the splenic blood flow was estimated to be 4.8 ± 1.9 (SD) % of the total blood volume/min; the intrasplenic platelet transit time was 9.7 ± 1.6 (SD) min, and the exchangeable splenic platelet pool 31 ± 8 (SD) %. Highly significant relationships were present between the splenic blood flow and the splenic platelet pool size, as well as between the splenic blood flow and the initial platelet recovery. It is concluded that the requirements for adequate interpretation of platelet kinetics are well met with the present method for harvesting and labelling of platelets.     

Biotinylated platelets: a new approach to the measurement of platelet life span

Summary The in vivo life span of dog platelets was determined by derivatizing whole blood with N-hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 ± 1.1 d as determined by curve-fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either ¹¹¹Indium-oxine or ⁵¹Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.     

Comparison of posttransfusion recoveries achieved with either fresh or stored platelet concentrates

Summary The effectiveness of platelet concentrate transfusion depends on such variables as blood bag material, donor — recipient compatibility, and time elapsed between donation and transfusion. To study the latter a corrected thrombocyte increment for recovery in the recipients was evaluated with 108 platelet transfusions in 31 patients. In 83 treatment programs, the mean recovery at the one-hour post-transfusion time point was 8.6×10⁹ platelets/l with fresh platelets and 5.9×10⁹ platelets/l with stored platelets. Significantly better recovery was achieved with freshly prepared platelet over the total of platelet concentrates stored for up to 96 hours; however, if the recoveries in different patient groups given stored platelets were considered separately in terms of storage times of up to 48 h or 48–96 h, the good recovery with fresh platelets was significantly better only when compared to the older (p=0.034) but not to the younger group of stored platelets. In patients with signs indicating enhanced platelet destruction (fever, splenomegaly, disseminated intravascular coagulation) the transfusion with fresh platelet concentrates gave a significantly better recovery compared to stored platelet concentrates (p=0.028), whereas in the absence of such signs the recovery produced by fresh concentrates was not significantly higher than with stored concentrates. These findings may be relevant for the logistics in blood banking.     

Kinetics and distribution of platelets in man

⁵¹Cr sodium-chromate, though having been widely used in the last two decades for labeling platelets, suffers from several serious drawbacks, ie, low labeling efficiency, long physical half-life, and low gamma photon yields. ¹¹¹In-oxine and ¹¹¹In-tropolone overcome these shortcomings and have the potential of precise determination of platelet kinetics as well as visualization and in vivo quantification of the temporal and spatial distribution of platelets in man. Computer analysis of platelet kinetics reveals that the multiple-hit model fits the survival curve better than the linear or the exponential model. The multiple-hit model provides not only the mean platelet survival time but also information on the initial recovery and the shape of the survival curve. The application of these techniques in normal and disease states should greatly enhance our understanding of the physiology and pathophysiology of platelets.     

Human blood platelet serotonin studied in vitro: endogenous serotonin may stimulate thrombin-induced serotonin release in stored platelets

Human platelets with a high content of endogenous serotonin took up more serotonin when incubated with exogenous serotonin, than platelets with a low endogenous content of serotonin. Also, thrombin-stimulated serotonin secretion (%) was high when endogenous serotonin was high. This was not found with fresh platelets, but was found when platelets were stored as platelet concentrates for 5 and 7 days. With platelets stored for 5 or 7 days, both uptake and secretion were increased after preincubation of the platelets with an amount of exogenous serotonin that was completely taken up. Inhibition of the reuptake of serotonin by imipramine during thrombin-induced secretion increased the secretion in stored platelets. Agonists like collagen, ADP, and a prostaglandin analogue (U46619) gave only 3–7% secretion. Imipramine increased the secretion induced by U46619, but not the secretion induced by ADP or collagen. The specific 5-HT₂ receptor inhibitor, ketanserin, had no effect on agonist stimulated secretion, or secretion stimulated by a calcium ionophore (A23187).

Both the uptake and the thrombin-induced secretion of serotonin correlated significantly with endogenous serotonin in stored platelets. In fresh platelets the uptake of serotonin correlated positively, although not significantly, with endogenous serotonin. It is speculated that endogenous serotonin may affect secretion through stimulation of the thrombin receptor, at least in stored platelets.     

Human blood platelet serotonin studied in vitro: endogenous serotonin may stimulate thrombin-induced serotonin release in stored platelets

Human platelets with a high content of endogenous serotonin took up more serotonin when incubated with exogenous serotonin, than platelets with a low endogenous content of serotonin. Also, thrombin-stimulated serotonin secretion (%) was high when endogenous serotonin was high. This was not found with fresh platelets, but was found when platelets were stored as platelet concentrates for 5 and 7 days. With platelets stored for 5 or 7 days, both uptake and secretion were increased after preincubation of the platelets with an amount of exogenous serotonin that was completely taken up. Inhibition of the reuptake of serotonin by imipramine during thrombin-induced secretion increased the secretion in stored platelets. Agonists like collagen, ADP, and a prostaglandin analogue (U46619) gave only 3-7% secretion. Imipramine increased the secretion induced by U46619, but not the secretion induced by ADP or collagen. The specific 5-HT(2) receptor inhibitor, ketanserin, had no effect on agonist stimulated secretion, or secretion stimulated by a calcium ionophore (A23187). Both the uptake and the thrombin-induced secretion of serotonin correlated significantly with endogenous serotonin in stored platelets. In fresh platelets the uptake of serotonin correlated positively, although not significantly, with endogenous serotonin. It is speculated that endogenous serotonin may affect secretion through stimulation of the thrombin receptor, at least in stored platelets.