Kinetics and distribution in vivo of 111In-labelled autologous platelets in idiopathic thrombocytopenic purpura

The kinetics of autologous 111In-labelled platelets were studied in 26 patients with ITP. The platelet mean life time (MLT) was considerably shortened, the platelet in vivo recovery slightly lowered and the platelet turnover normal. Comparative studies of the kinetics of simultaneously injected 111In- and 51Cr-labelled platelets in 10 patients showed the MLT and turnover of 51Cr-platelets to be shorter and higher, respectively, than those of 111In-platelets, suggesting that 51Cr-labelling in ITP may underestimate platelet MLT and overestimate platelet turnover. Our results confirm that accelerated platelet destruction is an important pathogenetic factor in ITP, and that the platelet concentration may be influenced by increased splenic platelet pooling and by inability of the bone marrow to respond adequately to the low platelet count. Our scintigraphic studies showed that the spleen played an important role for platelet destruction in most patients, with the liver contributing in some patients.     

Reevaluation of the prognostic factors for splenectomy in chronic idiopathic thrombocytopenic purpura (ITP): a report on 181 cases

From 1973 to 1986 we splenectomized 181 patients with chronic ITP after platelet kinetic studies with 51Cr or 111In. Mean age at diagnosis was 34 (range 4-79 yr). Follow-up of at least 1 yr after splenectomy was available in every patient. 141 patients (78%) achieved remission (platelets greater than 100 x 10(9)/l by 3 months after splenectomy), of whom 9 subsequently relapsed. Among the 40 non-responders at 3 months, 3 achieved a later remission spontaneously. Factors associated with response to splenectomy included a high post-operative platelet count (p = 0.0001), younger age at the time of surgery (p = 0.0077) and predominantly splenic sequestration of platelets (p = 0.0002), the two latter factors being partially correlated. In a multivariate analysis, however, only post-operative platelet count and age retained an independent prognostic significance, whereas the sequestration site of platelets had only borderline value. These results are discussed in the context of indications of platelet kinetic studies in chronic ITP, before splenectomy is considered.     

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.     

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.     

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.     

Kinetics, in vivo redistribution and sites of sequestration of indium-111-labelled platelets in giant platelet syndromes

Six patients with giant platelet syndrome were examined: four with Bernard-Soulier syndrome (two were asplenic); one with hereditary thrombopathic thrombocytopenia; and one with May-Hegglin anomaly. Autologous platelets were labelled with In-111-oxine and in vivo redistribution and sites of sequestration measured with quantitative imaging. In Bernard-Soulier syndrome platelet survival was normal or moderately shortened; platelet turnover was decreased only in the two patients with thrombocytopenia. In the patients with thrombopathia or May-Hegglin anomaly, platelet survival and turnover was moderately decreased. In those patients with normal-sized spleens, the mean splenic platelet pool consisted of 35.5% of the platelet mass, i.e. normal. The intrasplenic transmit time of the megathrombocytes was prolonged. Splenic blood flow was within normal limits. There was a marked accumulation of platelets in the liver at equilibrium: 15.5-58.8% of whole body radioactivity (normal 9.6 +/- 1.2%). This finding is unexplained. The final sites of sequestration of platelets were mainly in the liver and spleen, similar to that seen in normal subjects. We conclude that there is no inverse relationship between cell size and splenic platelet transit time. Platelet size therefore does not determine the size of the splenic platelet pool. The size of the platelets also does not seem to affect the sites of sequestration at the end of their life span.     

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.     

Platelet survival and turnover: important factors in predicting response to splenectomy in immune thrombocytopenic purpura

Autologous indium-111 platelet sequestration and survival studies were performed on 59 immune thrombocytopenic purpura (ITP) patients, 21 of whom underwent splenectomy shortly thereafter. Sequestration patterns were primarily splenic in 46 patients, primarily hepatic in 6 patients, and both splenic and hepatic in 8 patients. The mean platelet survival ranged from 15 to 211 hr (normal, 180-220 hr), and mean platelet turnover (a measure of platelet production rate) varied from 99 platelets/microliters/hr to 7,585 platelets/microliters/hr (normal 1,200-1,600 platelets/microliters/hr). Among splenectomy patients, 13 had an excellent response, and 8 had a fair or poor response. Neither the pattern of platelet sequestration nor the quantity of platelet-associated IgG was useful in predicting response to splenectomy. There was, however, a striking correlation between platelet studies showing short survival/high turnover and subsequent excellent response to splenectomy. Conversely, patients with only moderately decreased survival and low turnover had an unpredictable response to splenectomy. This investigation demonstrates that ITP patients are a heterogeneous population and include a significant subset whose thrombocytopenia results primarily from decreased turnover. Platelet kinetic studies appear useful in predicting beneficial response to splenectomy.     

Kinetics of 111In-labelled platelets in healthy subjects

25 healthy subjects underwent platelet kinetic and scintigraphic studies following injection of 111In-labelled platelets. Using the multiple hit model, platelet mean lifetime averaged 185.3 hours (range 141.2-225.6), and platelet in vivo recovery averaged 56% (range 42-89). The platelet disappearance pattern was almost linear. However, in comparison with simultaneously injected 51Cr-labelled platelets (7 subjects studied), the 111In-platelet survival curves were slightly more curved. Our results indicate that this may be ascribed to a slight degree of elution of 111In from the circulating platelets. The plateau or even slight increase in circulating activity following the initial steep decline in activity appears mainly to reflect reversible sequestration of platelets in the spleen and maybe in the liver. Using a semiquantitative scintigraphic approach, we estimated 45%, 25% and 30% platelet destruction to occur in the spleen, liver and bone marrow, respectively. Our results indicate that the discrepancy between the course of circulating platelet-bound activity and of chest wall activity reflects platelet destruction in the bone marrow.     

The site of platelet destruction in thrombocytopenic purpura as a predictive index of the efficacy of splenectomy

The significance of the site of platelet sequestration in determining the indication for splenectomy in idiopathic thrombocytopenic purpura (ITP) is a controversial subject. However, most of the negative conclusions are based on 51chromium labelling of homologous platelets. We report here the results of an analysis of 222 cases in which the kinetic study of 111indium-oxinate-labelled autologous platelets was performed under homogeneous technical conditions. 103 of these patients subsequently underwent splenectomy. This study demonstrates that the site of platelet sequestration in active ITP constitutes a variable independent of the patient's age, history of the disease and its severity (platelet count, lifespan). The sequestration site is a good predictive element of the short-term efficacy of splenectomy (71/76 cases with splenic sequestration obtained a platelet count exceeding 100 x 10(9)/l versus 7/13 cases with mixed sequestration and 1/14 cases with hepatic sequestration), and the long-term results (6 months to 5 years after splenectomy) do confirm the clinical value of this study.     

Kinetics of indium-111-labelled platelets in HIV-infected patients with and without associated thrombocytopaenia.

Seven to 12% of HIV-infected patients have thrombocytopaenia. The pathophysiology of the thrombocytopaenia is not clear. It has been variously suggested that it may be caused by an increased peripheral platelet destruction, a defect in platelet production, or by a combination of these. The aim of the study was to elucidate the pathogenesis of HIV-associated thrombocytopaenia. We determined the mean platelet life span (MPLS) and calculated the turnover of autologous indium-111-labelled platelets in 17 HIV-positive patients, seven with thrombocytopaenia. The sites of sequestration of labelled platelets were quantified. The thrombocytopaenic patients had a very short MPLS (3.0+/-3.8 h) and a marked increase in platelet production (18.2+/-12.6x10(9)/l/h). The majority of these patients (5 of 7) had excessive sequestration of platelets in the spleen. Five of the patients with a normal blood platelet count had a shortened MPLS (109+/-23 h) and increased platelet turnover (3.8+/-1.2x10(9)/l/h), i.e. the increased peripheral platelet destruction was compensated for by increased platelet production. The other five patients with a normal platelet count had normal MPLS (195+/-11 h) and slightly increased platelet production (2.5+/-0.6x10(9)/l/h). We conclude that patients with HIV-associated thrombocytopaenia have increased peripheral platelet destruction. Platelet production is elevated but is insufficient to maintain a normal peripheral platelet count. In these patients platelets are predominantly sequestrated in the spleen. Patients with HIV infection and a normal blood platelet count may also have increased platelet production. This may be an early subclinical phase in the development of full-blown HIV-associated thrombocytopaenia.     

Kinetics and in vivo distribution of 111-In-labelled autologous platelets in chronic hepatic disease: mechanisms of thrombocytopenia

The kinetics and distribution in vivo of autologous 111-In-labelled platelets were studied in 20 patients with chronic hepatic disease. The patients, 16 of whom were thrombocytopenic, exhibited a shortened platelet mean life time, a reduced platelet recovery and a normal platelet turnover, the latter 2 of which were positively correlated to the platelet count. Platelet in vivo recovery was negatively correlated to the spleen volume. In accordance with this, scintigraphic studies revealed that the spleen was the major organ of platelet sequestration and destruction, the role of the liver being almost negligible. Signs of platelet destruction in the bone marrow were also found. Our results indicate that splenic platelet pooling and accelerated platelet destruction, accompanied by inability of the bone marrow to compensate for the thrombocytopenia are the main causes of the thrombocytopenia accompanying chronic hepatic disease.     

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.     

The Platelet Destruction Site in Thrombocytopenic Purpuras

The site of sequestration of ⁵¹Cr-labelled platelets has been studied in 465 subjects, of whom 317 suffered from idiopathic thrombocytopenic purpura. The validity of the method was demonstrated in several ways: a given subject usually showed the same site of platelet sequestration when investigated more than once even after a long interval; there were characteristic and very different platelet sequestration curves in the thrombocytopenias due to bone marrow hypoplasia, hypersplenism or ITP; and there was a correlation between the preoperative in vivo results and the radioactivity found in the spleen after splenectomy.
In ITP the destruction of labelled platelets was more often splenic in children and in patients whose thrombocytopenia responded to steroid therapy. There was a perfect correlation between the site of platelet destruction and the platelet rise immediately after splenectomy. There was a good correlation between the site of platelet destruction and the long-term effectiveness of splenectomy.     

Platelet Survival and Platelet Production in Idiopathic Thrombocytopenic Purpura (ITP)

S ummary . Platelet mean life span (MLS) and platelet production were measured in 3–5 patients with idiopathic thrombocytopenic purpura (ITP) and in 21 healthy subjects.
The mean platelet production in ITP was 2.3 times normal: the highest values were 3–5 times normal. There was a highly significant negative correlation between platelet production and peripheral platelet count. With platelet counts above 50000μl, platelet turnover was within the upper part of the normal range, but with lower platelet counts, turnover progressively increased. It is concluded that the bone marrow in ITP increases platelet production in response to a low platelet count and that this response does not differ from that hitherto known to occur in man and animals rendered thrombocytopenic by thrombopheresis.
The disappearance curve of ⁵¹Cr labelled platelets in ITP consists of two components, a rapid initial one covering the first 15 min after infusion and then a slower one. The pattern was the same whether autologous or homologous platelets were used for labelling. It is suggested that the initial part of the curve does not represent in vivo survival but is due to slight damage to the platelets during the labelling procedure. These slightly damaged cells can resume normal viability when infused into a normal recipient but are rendered less viable when further damaged by platelet antibodies in patients with ITP. This explains the low recovery of infused labelled platelets in ITP recipients, despite the fact that the size of their splenic platelet pool is normal.     

A comparative study in volunteers of apheresis and buffy coat derived platelets

It is important that, at the time of transfusion, platelets prepared by different techniques are effective in vivo and meet acceptable criteria. A paired study was undertaken in 8 volunteer donors to compare apheresis platelets collected on the COBE Spectra with platelets derived from the buffy coat of whole blood donations after 5 days storage. In vivo recovery, survival and biodistribution were determined following indium-111 labelling of the platelets and autologous infusion into volunteers together with the in vitro evaluation of platelet function and biochemistry. The in vivo and in vitro characteristics of both types of platelet concentrate were not significantly different. Both products were equally viable after 5 d storage and both were of an acceptable quality as judged by currently used platelet products. Mean platelet survival (multiple hit) was 5.4 d for the apheresis platelets and 6.9 d for the buffy coat derived platelets and maximum recovery in circulation was 28.1% and 34.8%, respectively. There was a significantly higher platelet yield, as expected, from apheresis and a 1.5 log lower level of white cell contamination. This would be advantageous for patients in need of prolonged or recurrent transfusions as the number of donor exposures and the risk of alloimmunisation or viral transmission would be reduced.     

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.     

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.     

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.     

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.