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.     

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.     

The in vivo kinetics of 111In- and 51Cr-labelled platelets: a comparative study using both stored and fresh platelets

The in vivo kinetics of simultaneously injected 51Cr- and 111In-labelled platelets was investigated in 20 healthy male volunteers. The studies were carried out using both fresh platelets and platelets stored for 5 d at 22 degrees C; the disappearance of platelet-bound radioactivity was measured on whole blood samples as well as platelet pellets. Compared to 111In, the labelling efficiency was notably lower for 51Cr, and a higher amount of 51Cr was bound to contaminating red cells. As regards the fresh platelets, only small dissimilarities were observed in the in vivo kinetics obtained with the two labels. 51Cr yielded a slightly higher platelet recovery and longer T1/2 than 111In, when whole blood samples were used for calculations; no differences were seen when using platelet pellets. When stored platelets were studied, 51Cr gave significantly longer T1/2 and mean life-span (MLS) than did 111In. This difference was present for all mathematical models used for the calculation of MLS, and when whole blood samples as well as platelet pellets were employed. It was demonstrated that the estimation of MLS was also highly dependent upon techniques of blood sampling and curve fitting. Calculation, in which the initial data points were excluded, gave consistently longer MLS (P less than 0.0001), as compared to when all data points were included. Furthermore, when all survival studies were grouped together, calculations using platelet pellets gave a significantly (P less than 0.001) shorter platelet MLS than calculations using whole blood samples. It is concluded that both 51Cr and 111In are acceptable as radiolabels for both fresh and stored platelets. However, it appears that the viability of stored platelets may be influenced by the choice of label, and caution must be taken when these isotopes are used together in dual tracer experiments. Also, our results show that a meticulously standardized processing of blood samples and experimental data is required to enable interlaboratory comparisons of the results.     

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.     

A simple,fluorescent method to internally label platelets suitable for physiological measurements

Current methods for studying platelet survival in vivo are limited by the use of radioisotopes, with their inherent safety and regulatory concerns, systemic drug administrations that produce biochemical modifications of platelet functions, or external labeling techniques, which may produce artifacts due to surface modifications. For these reasons, we sought to develop a simple, nonisotopic method for labeling platelets internally, thereby producing platelets more likely to have in vivo properties equivalent to native cells. Murine platelets in protein-free buffer were fluorescently labeled internally by incubation with 2.5 μM 5-chloromethyl fluorescein diacetate (CMFDA), and without washing, were injected into mice for platelet survival studies. CMFDA-labeled platelets were unactivated, as shown by minimal P-selectin expression. When tested in vitro for function by aggregometry, the response of CMFDA-labeled platelets to collagen and thrombin was identical to that of unlabeled platelets. Flow cytometric analysis demonstrated that CMFDA platelets were an intensely stained, unimodal population that was completely separated from unlabeled platelets. The mean half-life of labeled platelets in the murine circulation was 37.5 ± 4.5 hr (±1 SD), and the mean survival time was 3.1–3.3 days (n = 24), similar to results reported using ⁵¹Cr and ¹¹¹In. No evidence of in vivo transfer of dye from labeled platelets to unlabeled cells was observed. CMFDA produces a population of platelets that are nonradioactively, internally labeled with a highly fluorescent, stable product. The labeled platelets function equivalently to native platelets, as demonstrated by immunocytometry and aggregometry, and importantly, in vivo, by normal platelet survival. Am. J. Hematol. 56:17–25, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Morphological Changes Associated with pH Changes during Storage of Platelet Concentrates in First-Generation 3-Day Container

The platelet injury and loss of viability that has been shown to occur with storage of platelet concentrates (PC) under conditions with increasing or falling pH were examined using scanning and transmission electron microscopy. After storage, samples were taken for measurement of pH value, platelet count and size distribution, release of lactate dehydrogenase (LDH) into plasma, and for SEM and TEM. Increased levels of LDH were observed in PC with pH above 7.3 and below 6.1. In PC with pH above 7.3 this was related to an increased number (23%) of platelets that were lysed or had a swollen disintegrated internal structure (balloons) as seen with TEM. SEM and Coulter counter studies also showed that platelet fragmentation and formation of microvesicles were prominent in PC with pH above 7.3. The electron microscopic pictures confirmed previous suggestions that platelet disc-to-sphere transformation and cytoplasmic swelling occur when pH falls below 6.7-6.8 during storage. SEM studies showed that concomitant with this change, folds and bulky projections appeared on the platelet surface. In PC with pH below 6.1 the morphological change was irreversible with the appearance of more than 90% lysed and balloon platelets. In conclusion, these studies suggest that the loss of viability observed with PC with pH above 7.3 or below 6.1 after storage is related to an increased percentage of lysed and balloon platelets.     

Tumor interaction with vascular endothelium

Clinical and experimental observations suggest that tumor-induced endothelial cell (EC) injury may be one of several initial events in the establishment of tumor metastases. This work investigates tumor-induced EC injury and the interaction between tumor-damaged EC and platelets. We used cultured bovine EC and extracts of four cultured human malignancies. EC injury was assessed by 51Cr and lactic dehydrogenase (LDH) release. Incubation of EC with melanoma, breast carcinoma or lung carcinoma caused significant LDH and 51Cr release, whereas colon cancer seemed ineffective. Increased adhesion of platelets to tumor-injured EC was noted. These observations indicate that certain varieties of tumor cause EC injury. Adhesion of platelets to tumor-injured EC results in the formation of platelet-tumor thrombi at the endothelial surface, an event that may initiate tumor invasion of the vessel wall.     

Subcellular Distribution of 51Cr and Characterization of its Binding Sites in Human Platelets

Subcellular fractionation of human platelets labeled with radioactive sodiumchromate was utilized to study distribution and characterization of the ⁵¹Crbinding sites in these cells. The cytoplasmic fraction of the platelets containedthe major portion of the radioactivity while microsomal and mitochondrialfractions played a minor role in the binding of chromate. The stromal sediment had approximately one third of the total radioactivity. Only about 20per cent of the ⁵¹Cr taken up by the platelets was in a free ionic form andwas in the cytoplasmic fraction. The major portion of the ⁵¹Cr in the plateletswas bound to nucleotides in the platelet cytoplasm. Nucleotides of the-granules had no radioactive chromate. Hypertonic media and platelet antiserum produced marked release of ⁵¹Cr from the platelets, whereas aggregatingagents produced none.

In physiologic conditions, elution of ⁵¹Cr from labeled platelets is verylimited because there is only little ⁵¹Cr in free ionic form in the cells. Releaseof considerable amount of ⁵¹Cr from the platelets can occur only in case ofcell damage with release of nucleotides from the cytoplasmic pool. This wasseen with hypertonic solutions and with platelet antibody. Platelet degranulation, as it occurs with aggregating agents, is not associated with release of ⁵¹Crsince the granular pool of nucleotides is not labeled by this compound.

     

Comparative Studies of the Function and Morphology of 111In-and 51Cr-Labelled Human Platelets

Comparative studies of the aggregability in vitro and ex vivo, and of the surface/volume ratio of ¹¹¹In- and ⁵¹Cr-labelled human platelets were carried out. The ADP-induced aggregation in vitro of ¹¹¹In-platelets was superior to that of ⁵¹Cr-platelets, as was that of ⁵¹Cr-platelets labelled in plasma as compared to ⁵¹Cr-platelets labelled in buffer. These differences seemed to be reversed in vivo, as identical collagen-induced aggregation responses were observed ex vivo when comparisons were made between ¹¹¹In- and ⁵¹Cr-platelets, and between labelled and unmanipulated platelets. Morphometric determination of the surface/volume ratios of the labelled platelets indicated a higher degree of platelet activation of ⁵¹Cr-platelets labelled in buffer as compared to those labelled in plasma. In this respect, no difference seemed to be present between ¹¹¹In- and ⁵¹Cr-platelets. The results of the ex vivo aggregation studies were unaffected by the time spent by the platelets in the circulation within 24 h post-injection, by platelet isolation yield, and by the medium used in ⁵¹Cr-labelling. Our results indicate that it will be possible to conduct comparative studies of simultaneously induced aggregation ex vivo of different platelet populations labelled with ¹¹¹In and ⁵¹Cr.     

Platelet size does not correlate with platelet age

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense- body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.     

Studies of the Fate of Platelets in Rats and Man

The tissue distribution of ⁵¹Cr-labeled platelets and erythrocytes was determined in normal and asplenic rats. Two hours after injection, the distributions in tissues of RBC and platelet ⁵¹Cr were not significantly differentexcept in the spleen which contained platelet ⁵¹Cr (12 per cent), significantlyin excess of red cell ⁵¹Cr (1.4 per cent). As labeled platelets were removedfrom the bloodstream, 80.2 per cent of the injected ⁵¹Cr was deposited in thespleen, liver and marrow of normal animals. During the same interval, radioactivity in all other sites examined diminished to less than 10 per cent ofinitial values. In splenectomized animals, hepatic uptake of radioactivity wastwice that observed in normal animals.

Studies in normal human subjects using external scintillation scanningsuggest that similar quantitative considerations apply to man in that, normally, one-third of total platelets are sequestered in the liver and one-thirdin the spleen while in asplenic individuals two-thirds are destroyed in the liver.

These observations suggest that the majority of platelets are cleared fromthe circulation by the reticuloendothelial system after becoming "senescent"but they do not elucidate the nature of "senescence" or the circumstancesleading to it.

Submitted on September 6, 1968 Accepted on March 27, 1969     

The effect of intravascular neutrophil chemotactic factors on blood neutrophil and platelet kinetics

Intravenous infusion of an analogue (f-met-leu-phe [FMLP]) of a bacterial-derived polymorphonuclear leukocyte (PMNL) chemotactic factor, or of the complement-derived chemotactic stimulus, zymosan-activated plasma (ZAP, containing C5ades Arg) into rabbits induces acute PMNL margination in the pulmonary vasculature. This process also occurs during hemodialysis and the adult respiratory distress syndrome. The pulmonary PMNL sequestration is accompanied by thrombocytopenia. Because of the role platelets and PMNLs play in hemostasis and defense against infection, we studied the fate of these blood elements following sequestration induced by chemotactic factors. By employing 111In-labelled platelets and external radioisotope scanning, platelets were found to sequester in the pulmonary vasculature during FMLP infusion. Simultaneous 51Cr PMNL and 111In-platelet studies showed that following sequestration, PMNLs returned to the circulation and disappeared with a normal half-life (T1/2) whereas the T1/2 of the platelets was markedly shortened (T1/2 of control = 49 +/- 3.0 hr; FMLP or ZAP infused T1/2 = 27 +/- 2.7 hr). Infusion of platelet-activating factor (PAF) induced PMN and platelet sequestration with similar abnormalities in platelet kinetics. Studies with 51Cr- and 14C-serotonin-labelled platelets showed that platelets did not release serotonin during FMLP, ZAP, or low dose PAF-induced sequestration. In contrast to platelet survival, platelet size, platelet aggregation responses, and platelet glycoproteins were not affected by transient sequestration. These results indicate that during PMNL margination induced by relatively "pure" PMNL stimuli such as FMLP, platelets may reversibly marginate and subsequently be cleared at an accelerated rate. The reason for accelerated platelet clearance is not a result of circulating platelet aggregates or detectable proteolytic modification of membrane glycoproteins. Such altered platelet kinetics may contribute to thrombocytopenia during sepsis, the adult respiratory distress syndrome, and other states in which excess PMNL margination occurs.     

Evolution of antithrombin III during the postoperative period

With a view to throwing some light on the age characteristics of platelets released into the blood in response to short-term physical exercise, we conducted exercise experiments on 23 healthy subjects previously injected with different platelet populations labelled with ¹¹¹In and ⁵¹Cr. Based on comparative platelet kinetic studies of pre- and post-exercise platelets and on exercise experiments carried out while labelled-platelet populations of different age composition were circulating, we cannot support the concept that the platelets released into the blood in response to exercise are enriched with young platelets. In addition, we found that simultaneously-injected large and small platelets were released to the same extent in response to exercise, but that the sequestration patterns of large and small platelets seem to be different.     

The reversible binding of vinblastine to platelets: implications for therapy

The ability of platelets to adsorb vinblastine has been used to treat patients with immune thrombocytopenia. It is hypothesized that the drug- platelet complex is coated with antibody, taken up by macrophages which are then destroyed by the drug. We gave 16 courses of vinblastine- platelets to six patients with immune thrombocytopenia. Only one patient responded, and therefore we examined possible reasons for the lack of benefit. Using 3H-vinblastine, the kinetics of vinblastine binding to platelets was studied in vitro. The binding of vinblastine to both human and rabbit platelets was identical with maximal binding occurring within 10 min at 600 microgram/ml vinblastine. Similarly, the plasma half-life of vinblastine in rabbits was close to that reported for man, and therefore, in vivo binding of vinblastine to platelets in rabbits was considered a suitable model for man. Homologous donor rabbit platelets were labeled with 51Cr alone, 51Cr plus vinblastine, or 3H-vinblastine and infused into recipient rabbits. Vinblastine had no effect on 51Cr survival, but all measureable vinblastine had left the platelets within 2 hr of the infusion. These observations suggest that delivery of the vinblastine to the macrophages depends on the platelets being phagtocytized before the drug leaves the platelets. This would be likely to occur only in those patients with severe immune thrombocytopenia. Further investigations into this treatment should be directed at methods to maintain the drug within the platelet.     

Labelling autologous platelets with 111In tropolonate for platelet kinetic studies: limitations imposed by thrombocytopenia

An in vitro method of radiolabelling platelets with 111In tropolonate in plasma has been devised enabling imaging and cell kinetic studies to be performed in patients with thrombocytopenia (TP) using autologous, rather than donor, platelets. Platelets from 10 TP patients, with platelet counts ranging from 4-91 x 10(9)/l, were labelled in 50% plasma with 111In tropolonate, containing the optimum tropolone concentration of 2 x 10(-4) mol/l, at platelet concentrations ranging from 0.08-4.5 x 10(9)/ml resulting in labelling efficiencies (LE) between 51 and 86%. In 4 patients, red cells contaminated the platelets but this was corrected for by measuring the proportion of 111In on the platelets prior to injection and in the post-injection blood samples. Platelet recovery (PR), mean platelet life-span (MPLS) and platelet production rate (PPR) were calculated and splenic and hepatic images were taken. The results clearly show that useful clinical data can be obtained by this method even in patients with severe TP.     

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.     

Porcine model of stent thrombosis: Platelets are the primary component of acute stent closure

Acute stent thrombosis remains a major concern of coronary stent implantation. Animal studies using stents do not adequately mimic this clinical problem, since stent placement is rarely associated with acute closure. The purpose of this study was to develop and characterize a porcine model of stent thrombosis. Improved understanding through such a model may be useful toward preventing and treating acute stent closure. Whole blood was drawn from domestic crossbred swine one day before study. Platelets were isolated, labeled with 111-In tropolone, and reinjected within 18 hr of the study. Bilateral carotid arteries were exposed, and severe injury induced by a series of mechanical crushes. This method produced histologic injury similar to human coronary angioplasty, with medial disruption and large dissections protruding into the lumen. Stenting was performed in standard fashion with 3.5-mm JJIS stents. Local platelet deposition was measured and recorded as 111-In radioactivity using a miniaturized scintillation detector (Dosimeter Corp.) mounted directly at the artery injury site. This measurement was made in real time at 1-min intervals. Similarly, volumetric blood flow was measured in real time by Doppler flowmeter. Eighteen arteries of nine pigs were studied. In nine arteries from nine pigs, crush injury only was performed and monitored. In the contralateral artery, crush injury was followed immediately by placement of a 3.5-mm Palmaz-Schatz (coronary) stent. Blood flow decreased rapidly following injury in both groups and followed a cyclic pattern. Eight arteries of the crush alone and two arteries of the crush plus stent groups were totally occluded 1 hr after crush. 111-In counts normalized to baseline were significantly higher at 1 hr in both groups compared to baseline; in the stented group, counts were higher than in the unstented group. Blood flow was higher in the stented group than in unstented group for 1 hr. Histopathologic observation of the thrombi forming in both crush-only and crush-stent injuries showed severe medial dissections with obstructing medial flap formation. The thrombi forming in both groups were highly platelet rich. This model of stent and arterial thrombosis showed rapid formation of platelet-rich thrombus, cyclic blood flow variations, and acute occlusion in 20% of cases. Stent placement at arterial injury sites is associated with thrombus that is predominantly platelet rich. Stent placement at injury sites enhances platelet deposition over crush injury alone. Despite greater numbers of platelets, as shown by increased 111-In counts, stenting improved vessel patency. These were likely due to higher volumetric blood flow, continuous deposition, and embolization of labeled platelets. © 1996 Wiley-Liss, Inc.     

Radioactive Diisopropyl Fluorophosphate as a Platelet Label: An Evaluation of in Vitro and in Vivo Technics

A technic for the in vitro labeling of human platelets with DFP³² is presented, critically evaluated, and compared to in vivo methods employing DFP³² and to in vitro methods using Cr⁵¹. The initial recovery of platelets labeled in vitro with DFP³² averaged 79 per cent, but the survival curve was characterized by an irreversible initial loss of platelet radioactivity. Experiments in which platelets were simultaneously labeled in vitro with both DFP³² and Cr⁵¹ suggest that this is not due to elution of DFP³². The survival curve of platelets labeled in vivo with DFP³² shows an initial transient reduction in platelet radioactivity. It is suggested that both of these aberrations in initial survival are the result of platelet injury by DFP³². Significant "tailing" was observed in the survival curves obtained with DFP³², and possible explanations of this phenomenon are discussed. DFP³²-labeled platelets circulating after 5 hours apparently survive normally and disappear from the circulation as a rectilinear function over the next 6-8 days. Although both in vitro and in vivo labeling methods employing DFP³² provide a meaningful approximation of platelet lifespan, the initial and terminal aberrations of the survival curves greatly complicate further interpretation. Dextran had no detectable effect on platelet survival, and epinephrine, Mecholyl, and cutaneous vasodilatation did not alter the platelet count or the specific activity of circulating labeling platelets in human subjects. The problem of initial platelet survival and the question of an extravascular or marginal platelet pool is discussed in the light of these data.

Submitted on May 24, 1966 Accepted on November 8, 1966