Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha-granule membrane protein 140 (GMP-140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium-labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP-140, progressing from a mean of 4 +/- 2 percent (SD) on the day of collection to a mean of 25 +/- 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = -0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/- 15 percent of the number predicted by the absolute platelet increment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Large platelets continue to circulate in an activated state after myocardial infarction

Abstract This study was intended to investigate the actual platelet activation status after an acute coronary event. The activation status of circulating platelets was assayed directly by measuring the membrane activation markers CD62 and CD63 with the Düssel-dorf III flow cytometry test in 22 patients with the diagnosis of acute myocardial infarction during the 48-h observation period following the acute event. The number of activated, marker-positive sample platelets was significantly increased in the post-MI patients: CD62: 5·8%× 2·25^±1 vs. 3·5%× 2·32^±1, P≤ 0·05; CD63: 18·7%× 1·77^±1 vs. 4·6%× 2·16^±1, P≤ 0·00·1. The platelet volume and count were concomitantly increased (12·1 ± 2·4 fl/ 236 ± 90 times 10³μl^-1 compared to 8·3 ± 1·6 fl/187 ± 42 times 10³μl^-1) in the control group. Particularly large platelets were identified as being activated documented by the exponential increase in the difference in CD63-binding sites per sample platelet above the 90%-percentile and below the 10%-percentile of the volume distribution: Δ+ 1341 ± 903 (MI patients) vs. Δ+ 276 ± 126 (controls), P≤ 0·00·1. Significant creatine kinase elevation and decrease in platelet count was found in the non-survivor subset (n= 5). We conclude that predominantly large platelets continue to circulate in an activated state after MI. This study provides direct evidence that the assumption of an increased thrombotic potential becomes operative in vivo in MI patients. Besides CK elevation and decrease in platelet count this might possibly constitute a prognostic factor for the short-term outcome of the patients.     

两种血小板制剂应用于儿科血液病的临床观察

目的观察并比较机器单采法及手工法分离制备血小板制剂用于儿科血液病输注的效果。方法输注机器单采血小板患儿463例次为机采制剂组,输注手工分离血小板制剂患儿155例次为手工制剂组,分别在输注后24、48及72h作外周血血小板计数,观察临床止血效果、有无输血反应发生,计算血小板计数增加校正指数(CCI)、血小板回升率(PPR)、输注无效率、输血反应发生率等指标。结果输注后24、48、72h,机采制剂组:CCI分别为18.9、15.4、14.1,PPR分别为33.4%、27.8%、25.0%;手工制剂组:CCI分别为11.3、9.4、2.9,PPR分别为20.3%、10.3%、3.8%;机采法制剂组均明显高于手工制剂组(P<0.01)。机采制剂组输注无效率10.58%、输血反应发生率3.02%,手工制剂组相应为32.90%及11.61%,机采组虽都明显低于手工组,但两组均达到较好的临床止血目的,组间无差异。结论输注机器单采血小板制剂能更有效地提高血液病患儿的血小板值,减少其血小板输注无效及输血反应的发生。     

Shiga toxin binds to activated platelets

Summary. Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)‐producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid‐citrate‐dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA‐washed preparations relative to ACD‐derived platelets. Binding of Stx was also increased with ACD‐derived platelets when activated with thrombin (1 U mL^?1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA‐exposed platelets lost their normal discoid shape and were larger. P‐selectin (CD62P) exposure was significantly increased in EDTA‐washed preparations relative to ACD‐derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on ‘activated’ platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.     

Atopic predisposition of recipients in allergic transfusion reactions to apheresis platelets

BACKGROUND: The biologic mechanisms of allergic transfusion reactions (ATRs) are largely unknown. We sought to compare the atopic predisposition of platelet (PLT) recipients who experienced an ATR to nonreactive control recipients. STUDY DESIGN AND METHODS: We identified 37 consecutive apheresis PLT recipients who experienced an ATR and 26 matched controls. Total immunoglobulin (Ig)E and aero‐ and food allergen–specific IgE were quantified in plasma by a blood test for allergies (ImmunoCAP: Phadiatop and Fx5). IgE testing of apheresis PLT supernatants was also performed. RESULTS: Pruritus and urticaria were manifest in 91.9 and 83.8% of all ATRs, with more severe respiratory symptoms and angioedema occurring in less than 15% of cases. No subject had anaphylaxis. Sex, age, and primary diagnosis were balanced between the two groups. Total and aeroallergen‐specific IgE was higher among subjects experiencing an ATR in comparison to control subjects (median total IgE, 55.5 kU/L vs. 8.3 kU/L, p = 0.002; and median aeroallergen‐specific IgE, 0.57 kUa/L vs. 0.36 kUa/L, p = 0.046). IgE antibody levels in apheresis products associated with ATRs were similar to control products (p > 0.1 for all IgE tests). CONCLUSION: Recipient atopic predisposition, as defined by IgE sensitization, is a risk factor associated with ATRs.     

HPA配型在免疫性血小板输注无效中的应用研究

目的探讨以HPA配型解决免疫性血小板输注无效的方案。方法 1)建立PCR-SSP方法检测HPA-1～5基因型检测方法,建立机采血小板供者库;2)采用微柱凝胶法和Capture-P法对32名血小板输血无效患者作血小板同种抗体筛查,并对2种方法比较;3)对血小板同种抗体筛查阳性患者采用已知HPA基因型的标准谱血小板作抗体鉴定并采取HPA基因型同型输注的原则寻找供者。结果 1)采用PCR-SSP方法成功检测出HPA-1～5基因型,并对1 000名血小板供者的HPA-1～5基因型定型;2)32例血小板输注无效病例中,微柱凝胶法检测血小板同种抗体阳性率为50%,Capture-P法血小板抗体阳性检出率为40%;3)32例血小板输血无效病例中2种方法同时血小板抗体阳性13例,其中2例鉴定为抗-HPA,分别为抗-HPA-5b(P=1/84)、抗-HPA-1a(P=1/55)。结论对抗-HPA引起的血小板输注无效患者采用HPA基因型相合的方法寻找供者是有效的。     

血小板输注无效患者的血小板抗体筛查与配合性输注

目的分析血小板输注无效（PTR）的主要原因,为患者选择适用的血小板,用以提高输注疗效。方法对多次输血患者于输血前后监测血小板计数值,并计算1h和24h血小板增高指数（CCI值）,对判断为血小板输注无效者,采用简易致敏血小板血清学技术（SEPSA）进行血小板抗体筛查,并进行血小板交叉配型,对配合性输注效果进行分析。结果278例多次输血患者发生PTR共88例（31．7％）;88例PTR患者中血小板抗体阳性67例（76．1％）;67例抗体阳性患者,血小板交叉配型成功37例,血小板输注有效32例,有效率达86．5％。结论血小板抗体的产生是导致血小板输注无效的主要免疫性因素,血小板配合性输注能显著提高血小板输注效果。     

Acute hemolytic transfusion reaction in a pediatric patient following transfusion of apheresis platelets

The practice of transfusing ABO-incompatible platelets, driven primarily by concerns about inventory management, has been considered generally safe because the accompanying plasma is usually diluted in the recipient's total blood volume. However, if the platelet product contains a large volume of plasma or a high concentration of incompatible isoagglutinin, there may be hemolysis of the recipient's red cells. Patients with a small blood volume, such as babies and children, are considered to be at particular risk for such a complication. We describe the case of a baby who suffered massive hemolysis of her group A red cells after transfusion of group O Apheresis Platelets containing a high-titered anti-A isoagglutinin. We also offer a review of the literature on this subject and recommendations to avoid acute hemolytic reactions as a result of platelet transfusion.     

Binding of collagen alpha1 chains to human platelets.

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.     

Temperature cycling improves in vivo recovery of cold‐stored human platelets in a mouse model of transfusion

BACKGROUND: Platelet (PLT) storage at room temperature (RT) is limited to 5 days to prevent growth of bacteria, if present, to high levels. Storage in cold temperatures would reduce bacterial proliferation, but cold‐exposed PLTs are rapidly cleared from circulation by the hepatic Ashwell‐Morell (AM) receptor, which recognizes PLT surface carbohydrates terminated by β‐galactose. We cycled storage temperature between 4 and 37°C to preserve PLT function and reduce bacterial growth. STUDY DESIGN AND METHODS: Temperature‐cycled (TC) human PLTs were stored at 4°C for 12 hours and then incubated at 37°C for 30 minutes before returning back to cold storage. PLTs stored at RT or at 4°C (COLD) or TC for 2, 5, and 7 days were infused into SCID mice and the in vivo recovery was determined at 5, 20, and 60 minutes after transfusion. RESULTS: PLTs stored for 2 days in COLD had significantly lower in vivo recoveries than RT PLTs. TC PLTs had improved recoveries over COLD and comparable to RT PLTs. After 5‐ and 7‐day storage, TC PLTs had better recoveries than RT and COLD PLTs. PLT surface β‐galactose was increased significantly for both COLD and TC PLTs compared to RT. Blocking of the AM receptor by asialofetuin increased COLD but not TC PLT recovery. CONCLUSION: TC cold storage may be an effective method to store PLTs without loss of in vivo recovery. The increased β‐galactose exposure in TC PLTs suggests that mechanisms in addition to AM receptors may mediate clearance of cold‐stored PLTs.     

Binding of human von Willebrand factor to collagen and to collagen-stimulated platelets

Binding of von Willebrand factor (vWF) to collagen and to "collagen-stimulated platelets" was studied. Purified human iodine 125-labeled vWF binds to collagen. Binding was rapid, dependent on collagen and vWF concentration, independent of divalent ion concentration, and inhibited by unlabeled vWF as well as by high concentrations of fibronectin and fibrinogen. It was blocked by six monoclonal antibodies (MAbs) to human vWF (B200 through B205). Conversely, binding of 125I-vWF to platelets in the presence of collagen (collagen-treated platelets) was time dependent, saturable, and dependent on collagen, vWF, and divalent ion concentration. It was also inhibited by unlabeled vWF, fibronectin, or fibrinogen as well as by MAbs to vWF B200 through B205. In addition, binding of vWF to collagen-treated platelets was blocked by MAb to vWF 9 previously shown to inhibit binding of vWF to glycoprotein (GP) IIb-IIIa as well as by a MAb to GPIIb-IIIa. This binding was also strikingly decreased with use of patient platelets deficient in either GPIIb-IIIa or GPIa, whereas it was normal in the presence of a MAb to GPIb or when testing patient platelets deficient in GPIb. In conclusion, binding of vWF to platelets in the presence of collagen appeared to be a three-step phenomenon, including: binding of vWF to collagen; interaction of collagen with its receptor on the platelet membrane (GPIa ?), leading to activation of platelets and exposure of GPIIb-IIIa; and binding of vWF to GPIIb-IIIa.     

Reversibility of severe metabolic stress in stored platelets after in vitro plasma rescue or in vivo transfusion: restoration of secretory function and maintenance of platelet survival

BACKGROUND: Determining reversible aspects of the platelet storage lesion may result in improved function and survival of transfused platelets. STUDY DESIGN AND METHODS: Using a model of high-dose (apheresis-derived) platelet concentrates (PC), functional changes imposed by transient adverse metabolic conditions (pH < 6.0 for 1-2 hr) that could be reversed by autologous plasma rescue followed by standard platelet storage were investigated. Whole-blood-derived PCs were transfused into a small number of normal volunteers to determine platelet recovery and survival. RESULTS: Without rescue, high-dose PCs developed severe in vitro functional derangements at the time of the pH nadir including 1) loss of resting morphology; 2) complete abrogation of osmotic recovery and platelet aggregation and glycoprotein IIb/IIIa up-regulation to agonist; and 3) decreased alpha-granule release. By contrast, spontaneous and agonist-induced binding of annexin V were unaffected by adverse metabolic conditions. Plasma rescue to an optimal pH improved morphology scores, stabilized osmotic recovery, and completely restored platelet secretory responses, as measured by aggregation, glycoprotein IIb/IIIa up-regulation, and alpha-granule release. In a limited number of studies, plasma rescue was accompanied by preserved in vivo platelet recovery and survival after autologous transfusion after 5 days of storage. CONCLUSION: Transient derangement of platelet metabolism, which does not increase membrane phosphatidylserine exposure, causes in vitro functional abnormalities that are fully reversed or stabilized by metabolic rescue. Preliminary data suggest that such rescued platelets may have normal posttransfusion recovery and survival.