Non-parametric analysis of platelet lifespan

A model-independent and elementary method of analysis of platelet survival is proposed. The method is based on the finding that the mean and standard deviation of the platelet lifespan can be expressed in the terms of the slope at time 0 and the area under the empirical platelet survival curve. The method is tested using Monte-Carlo simulations and then applied to a set of clinical data.     

Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.     

The Effect of Sulfinpyrazone (Anturan) on Platelet Economy and Blood Coagulation in Man

Sulfinpyrazone (Anturan) administered in therapeutic doses over a period of several weeks produced prolongation of platelet survival and reduced turnover but with little change in blood coagulation. The changes in platelet survival and turnover were associated with reduced platelet adhesiveness. It is therefore possible to affect platelet economy in man significantly, without producing corresponding effects on blood coagulation.     

HLA compatibility and survival of 51 chromium-labeled allogeneic thrombocytes

In 37 patients with thrombocytopenia (mostly with ITP) the survival time of 51Cr-labeled allogeneic platelets was investigated. The HLA antigens were typed in donors and recipients and the presence of HLA cytotoxins and specific thrombocyte antibodies in sera of patients were examined. In 7 cases the identity of 2 HLA antigens, in 15 cases that of 1 HLA antigen and in 15 cases the HLA incompatibility between donor and patient were found. The survival of platelets did not depend on the degree of HLA compatibility, unless the HLA cytotoxins in sera of patients appeared. The HLA, as well the specific platelet antibodies brought about the shortened platelet survival to 1 day and less. The importance of these observations for platelet kinetics is discussed.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Bcl-2 family proteins are essential for platelet survival

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.     

Programmed anuclear cell death delimits platelet life span

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.     

Platelet kinetics--1975.

Studies of platelet kinetics have provided useful information regarding the role of platelets in the pathogenesis of thrombosis and related disorders. Additionally, platelet survival studies have been useful in evaluating the efficacy of drugs that may be useful as platelet inhibitors. Many questions remain, however, and answers are needed before much of the kinetic data can be confidently applied to clinical problems in thrombosis.     

Technetium-99m-labeled platelets: Comparison of labeling with a new lipid-soluble Sn(II)-mercaptopyridine-N-oxide and 99mTc-HMPAO

Platelets pretinned with a neutral Sn(II)-2-mercaptopyridme-N-oxide (SN-MPO) were labeled with ^99mTc and compared to those labeled with ^99mTc-HMPAO. The conditions of labeling platelets, e.g. concentrations of platelets and Sn(II)-MPO, ^99mTc in ACD-saline or ACD-plasma media, pH and incubation time, were optimized using canine platelets. Moderate labeling efficiency was obtained with 20 μg of tin(II) chloride and 30 min incubation with Sn-MPO and pertechnetate. The viability of labeled platelets was determined by platelet recovery and platelet survival times in Beagle dogs. The labeling efficiency with platelets from 43 mL of blood was 62.8 ± 7.6%. The platelet recovery was 35.7 ± 5.0% and exponential survival time was 34.6 ± 3.1 h compared to 43.3 ± 12.0% and 29.5 ± 3.3 h for ^99mTc-HMPAO-labeled platelets. These values were significantly (P < 0.01) lower than ¹¹¹In-labeled platelets. Biodistribution in dogs indicates lower retention in blood, spleen and liver after some initial ^99mTc excretion in urine. The platelet deposition with ^99mTc platelets (Sn-MPO method) on polyurethane angio-catheters was similar to ^99mTc-HMPAO-labeled platelets. This study indicates that the platelets could be successfully labeled with pertechnetate in a cost-effective manner for the evaluation of thromboembolic complications.     

Gray platelet syndrome: selective alpha-granule deficiency and thrombocytopenia due to increased platelet turnover

Clinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and alpha-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of beta-thromboglobulin were significantly reduced (3%-7% of normal). Platelet count was decreased (33-150 X 10(9)/1) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.     

Effect of dietary alpha-linolenate/linoleate balance on mean survival time, incidence of stroke and blood pressure of spontaneously hypertensive rats

Following the suckling period, stroke-prone spontaneously hypertensive rats (SHR-SP) were fed semi-purified diets supplemented either with safflower seed oil (rich in linoleic acid) or with perilla seed oil (rich in alpha-linolenic acid). The mean survival time of male SHR-SP fed the perilla diet was longer than that fed the safflower diet by 17% (p less than 0.001) while the difference was 15% in female SHR-SP (p less than 0.05). The mean survival times of female SHR-SP were more than 40% longer than those of male SHR-SP in both dietary groups. Post-mortem examinations of brains revealed apoplexy-related symptoms as the major cause of the death in both dietary groups. The systolic blood pressure was lower by ca. 10% (21 mmHg) in the perilla group than in both the safflower group and conventional diet group. The eicosapentaenoate (20:5 n-3)/arachidonate (20:4 n-6) ratio of platelet phospholipids in spontaneously hypertensive rat (SHR), a measure of platelet aggregability, was much higher in the perilla group than in the safflower group. Thus, increasing the dietary alpha-linolenate/linoleate ratio resulted in an increased mean survival time of SHR-SP rats, possibly by lowering blood pressure and platelet aggregability.     

一种快速、灵敏的血小板释放功能检测方法及其在抗血小板药物研究中的应用

本文报道了一种快速、灵敏的血小板释放功能检测方法:利用荧光素-荧光素酶在有ATP、Mg~(2+)、O_2存在时产生的生物发光素测定血小板ATP的释放量,以反映血小板的释放功能;研究了ADP、AA、胶原、凝血酶等四种诱导剂对血小板释放功能的作用,发现ADP的诱导释放能力较其他三者为弱;观察在不同剂量ADP和AA的诱导下,血小板聚集强度和释放能力之间的关系,研究了血小板数等因素对ATP释放功能测定的影响。应用该方法研究了Aspirin及活血化淤药物川芎嗪,毛冬青甲素对血小板释放功能的影响,发现Aspirin对AA诱导的释放反应有强烈的抑制作用。在以ADP诱导的释放反应中,川芎嗪的抑制作用较毛冬青甲素更为强烈。     

Thrombophilic states in late gestosis

In 80 pregnant women with pre-eclampsia and in 38 healthy pregnant women malondialdehyde production by blood platelets, platelet survival and several haemostatic variables were determined. It was demonstrated that due to the enhanced platelet turnover pre-eclampsia was invariably accompanied by a thrombophilic state which in severe cases developed into chronic consumption coagulopathy.     

青少年慢性粒细胞白血病急变临床分析

目的:分析青少年慢性粒细胞白血病(CGL)急变患者临床表现及实验检查特点,以提高对青少年CGL急变临床特点的认识。方法:将CGL急变患者按年龄分组,将青少年组和中老年组患者的急变时间、确诊时和急变时的脾脏大小、白细胞和血小板数目、急变时骨髓幼稚细胞的比例,以及患者的总生存时间、急变后生存时间进行对比分析。结果:青少年组和中老年组的脾脏大小、确诊时血小板数目、急变时的白细胞数目、急变时间、以及总生存时间无显著性差异;青少年组确诊时的白细胞数目较中老年组高,青少年组的急变后生存时间较长。结论:青少年CGL患者的临床表现及实验室检查结果具有CGL的普遍特征,但其确诊时白细胞数目较高,急变后生存时间较长。     

Platelets contribute to allograft rejection through glutamate receptor signaling

Platelets recruit leukocytes and mediate interactions between leukocytes and endothelial cells. Platelets have been long described as markers of transplant rejection, but the contribution of platelets to transplant rejection has not been critically examined. We demonstrate in this study that following T cell initiation of allograft rejection, platelets contribute to T cell recruitment and increased plasma inflammatory mediators and accelerate T cell-meditated skin graft rejection. Prior work from our laboratory has shown that platelets secrete glutamate when activated, which then induces platelet thromboxane production by signaling through platelet-expressed ionotropic glutamate receptors. Glutamate receptor antagonists therefore represent, to our knowledge, novel inhibitors of platelet-accelerated inflammation. We have found that plasma glutamate is increased in mice that receive skin grafts and that mice treated with glutamate receptor antagonists have improved graft survival and decreased plasma thromboxane, platelet factor 4 (CXCL4), and IFN-γ. Taken together, our work now demonstrates that subsequent to T cell initiation of skin graft rejection, platelets contribute to further T cell recruitment and that by blunting glutamate-mediated platelet activation, graft survival is improved.     

Effect of altitude exposure on platelets.

Since decompression from depth is known to produce a fall in platelet count, the effect of altitude decompression and high-altitude exposure on platelets was investigated. Sixteen subjects decompressed without hypoxia to 20,000 ft simulated altitude for two hours showed a significant (P less than 0.01) drop in circulating platelet count of approximately 10% for three days following decompression. Four of five subjects similarly exposed had a shortened autologous platelet survival compared to that prior to exposure. Subjects exposed to 9,800 ft and then 17,600 ft in a mountain environment showed a significant mean decrease in platelet count on day 2 of 7% and 25% respectively, which had returned to control by day 5. Nonhypoxic and hypoxic decompressed rabbits which received homologous chromium-51-labeled platelets had an increase in lung radioactivity compared with sea-level controls. It is postulated that altitude decompression produces platelet reductions similar to these seen after decompression from depth, and that platelets sequester in the pulmonary vascular bed.     

Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin

BACKGROUND: To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 microg/ml; high, 10 microg/ml). METHODS: Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis. RESULTS: Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P < 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater than those of PKH-labeled platelets (P < 0.001). Survival of platelets labeled with biotin did not differ (low, 83.3 h; high, 85.2 h) and was longer than for PKH-labeled platelets (P < 0.01). Labeling methods did not activate platelets (measured by P-selectin expression), nor did they affect platelet responses to adenosine diphosphate (ADP), collagen, or thrombin. CONCLUSIONS: Labeling with two levels of biotin is superior to labeling with PKH dyes, and is useful for measuring concurrently the survival of two differing platelet populations.     

Fluid storage of thrombocytes. In vitro viability of thrombocytes at 12 degrees C storage temperature

Platelet concentrates from ACD-blood were stored with and without agitation at +12 degrees C for 3 days. pH-values, hypotonic shock response, serotonin uptake and adenine nucleotides were investigated. In addition platelet shape was morphologically differentiated in discs with and without pseudopods, in spheres and irregular platelets. In accordance with platelet storage at +22 degrees C an optimum platelet number and gentle agitation were essential to maintain in vitro vitality at +12 degrees C for 3 days. The number of discoid platelets declined from 76% in fresh platelet concentrates to 25% after 3 days at +12 degrees C although pH-values did not fall below 6.8 in the agitated concentrates. A diminished post-transfusion survival could result from this, unless the shape change reverses in the circulation.     

Treatment of chronic kidney transplant rejection with prostacyclin — reduction of platelet deposition in the transplant; Prolongation of platelet survival and improvement of transplant function

8 patients with chronic kidney transplant rejection were treated intravenously with prostacyclin during 5 days. This treatment seemed to have a beneficial effects as measured by platelet deposition in the transplant and prolongation of platelet survival. In the majority of cases the transplant function improved. A longer duration of this therapeutic effect could be a new way for the long-term treatment of chronic kidney transplant rejection.     

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.