Human platelets contain and release TWEAK

The multifunctional cytokine, TWEAK (TNF-like weak inducer of apoptosis), is a member of the TNFα superfamily. TWEAK is found in a broad range of cell types and has been linked to cell growth and survival, angiogenesis and other inflammatory processes. These functions and their importance in inflammatory diseases have made TWEAK an attractive pharmaceutical target, particularly for immunotherapy with monoclonal antibodies (mAbs). Immunotherapy targeting another TNFα family member, CD154, was associated with thrombosis in clinical trials. Subsequent studies identified platelets, which contain CD154, as a possible contributing factor to thrombosis in these trials. Since clinical trials with anti-TWEAK mAbs have already begun, we considered it important to determine whether platelets contain TWEAK. Using a variety of immunologic methods we found that, upon activation, human platelets expose TWEAK antigen and release it in soluble form (sTWEAK). By flow cytometry we determined that human platelets activated by TRAP (Thrombin Receptor Agonist Peptide) and other agonists expose TWEAK antigen (22% median positivity) and release TWEAK positive microparticles. The presence of TWEAK on platelets was confirmed by confocal microscopy. By ELISA, we found that sTWEAK is released by activated platelets. Finally, western blot analysis revealed TWEAK protein (～34 kDa) in washed platelet lysates. The finding that human platelets contain TWEAK raises important questions about its possible functions in normal physiology, as well as in inflammatory diseases and their treatment.     

滤白手工血小板的临床应用分析

目的：观察滤白手工血小板的临床输注效果,并就其与机采血小板进行比较,为临床如何选择滤白手工血小板和机采血小板提供依据。方法：统计我院2005—2008年临床血小板用量,了解手工血小板和机采血小板临床应用情况;通过对40例患者输用62次滤白手工血小板和67次机采血小板的比较,用血细胞计数仪分别计数血小板输注前后20～24h的数,根据输注后血小板校正增加指数（CCI）和血小板回收率（PPR）,及临床出血症状有无改善进行疗效判断。统计2007年全年滤白手工血小板和机采血小板的输血反应率。结果：滤白手工血小板和机采血小板输注后的CCI和PPR值差异无统计学意义,滤白手工血小板和机采血小板的输注效果差异无统计学意义,滤白手工血小板和机采血小板输血反应率比较差异无统计学意义。结论：滤白手工血小板的应用能大量节约血资源,值得临床推广,滤白手工血小板和机采血小板在临床应用具同等价值。     

Progress in bio-manufacture of platelets for transfusion

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.     

Anthrax lethal and edema toxins fail to directly impair human platelet function

Hemorrhage is a prominent clinical manifestation of systemic anthrax. Therefore, we have examined the effects of anthrax lethal and edema toxins on human platelets. We find that anthrax lethal toxin fails to cleave its target, mitogen-activated protein kinase 1, and anthrax edema toxin fails to increase intracellular cyclic adenosine monophosphate. Surface expression of toxin receptors tumor endothelial marker 8 and capillary morphogenesis gene 2, as well as coreceptor low density lipoprotein receptor-related protein 6 (LRP6), are markedly reduced, preventing toxin binding to platelets. Our studies suggest that the hemorrhagic clinical manifestations of systemic anthrax are unlikely to be caused by the direct binding and entry of anthrax toxins into human platelets.     

Comparison of the measurement of surface or total platelet-associated IgG in the diagnosis of immune thrombocytopenia

Platelet-associated IgG (PAIgG) can be measured on intact platelets or following platelet lysis. Measurement of PAIgG following platelet lysis may provide different or additional information compared to PAIgG measured on intact platelets. The PAIgG of lysed platelets represents "total" PAIgG, ie, IgG on the surface of platelets plus any IgG that was inside the platelet. To investigate the clinical relevance of the two types of PAIgG assay we performed a prospective study on washed platelets collected from 47 patients with idiopathic thrombocytopenic purpura (ITP). The PAIgG was measured on intact and lysed platelets using an immunoradiometric assay. Platelet-associated IgG was 2-3 times higher when measured on lysed platelets from healthy controls or patients with ITP compared to PAIgG measured on the same intact platelets. The higher level of PAIgG observed following platelet lysis was not due to the reactions not achieving equilibrium. Using lysed platelets, PAIgG was elevated on 29 of 47 samples from different ITP patients and elevated in 31 samples when measured on intact platelets. The PAIgG is invariably higher when measured following platelet lysis compared measurements made on intact platelets. Neither technique offers a diagnostic advantage over the other.     

Effects of pathogen reduction systems on platelet microRNAs,mRNAs, activation,and function

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen?+?ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin?+?UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p?<?0.05) and an impaired platelet aggregation response to ADP (p?<?0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.     

Galactosylation does not prevent the rapid clearance of long-term, 4 degrees C-stored platelets

Cold storage of platelets for transfusion is desirable to extend platelet storage times and to prevent bacterial growth. However, the rapid clearance of cold-stored platelets prevents their use. A novel method for preventing the rapid clearance of cold-stored platelets has previously been developed in a murine model. Cold storage induces the clustering and recognition of exposed beta-N-acetylglucosamine (betaGlcNAc) on platelet surfaces. Glycosylation of betaGlcNAc residues with uridine 5'-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0 degrees C-stored murine platelets. Based on this finding, we developed a similar glycosylation process by adding UDP-galactose to human apheresis platelets. A phase 1 clinical trial was conducted transfusing radiolabeled autologous apheresis platelets stored for 48 hours at 4 degrees C with or without pretreatment with UDP-galactose. In contrast to the murine study, galactosylation of human platelets did not prevent the accelerated platelet clearance routinely observed after 4 degrees C storage. We next developed a murine model of platelet storage for 48 hours at 4 degrees C and showed that UDP-galactose treatment of murine platelets also did not prevent their rapid clearance, in agreement with the human platelet study. We conclude that different mechanisms of clearance may exist for short- and long-term cold-stored platelets.     

Role of the retention test Homburg in evaluating platelet hyperactivity and in monitoring therapy with antiplatelet drugs

Decreased retention in the retention test Homburg (RTH) indicates a loss of platelet function; increase is associated with an increased activation of platelets, for example, in patients with vascular diseases. Compared with other materials (e.g., collagen, glass pearls, etc.) the filter surface in the retention tubes is nonthrombogenic. Therefore, the RTH seems to be well suited for measuring an in vivo over-reactivity of platelets. In a pilot study using the RTH we evaluated the postoperative over-reactivity of platelets in 14 patients and observed a significant heterogeneity of the platelet population concerning size and stickiness. Reliable platelet function tests are also necessary for "drug monitoring," since they deliver important clinical laboratory parameters for efficient control of a therapy with antiplatelet drugs. Therefore, we evaluated in vitro how, after administration of platelet aggregation-inhibiting medications (such as acetylsalicylic acid, Prostaglandin and ReoPro in various concentrations, the adenosine diphosphate (ADP), collagen, ristocetin, or suprarenin increased retention can be reduced. The reaction of the platelets in platelet-rich plasma of different patients or donors to the addition of ADP is variable. The platelet function inhibitor effect is dose dependent. In a clinical pilot study, a significant platelet-inhibiting effect of clopidogrel using the RTH has been shown.     

The use of electron microscopy in the investigation of the ultrastructural morphology of immune thrombocytopenic purpura platelets

Ultrastructural studies have proven useful for the accurate identification and classification of certain lymphoid and hematopoietic disorders; however, the value of electron microscopy in the diagnosis and clinical management of platelet pathology is less well defined. Electron microscopy has been used to evaluate inherited platelet disorders. In these disorders, certain platelet structural defects can be characteristic. Recently, we investigated the ultrastructural morphology and immunogold localization of IgG in platelets from patients with idiopathic thrombocytopenic purpura (ITP). The accelerated platelet destruction of ITP is mediated by antiplatelet autoantibodies directed against platelet surface glycoproteins and by the functional capacity of the reticuloendothelial system. The basic dysregulation that occurs in these patients remains unexplained. Traditionally, laboratory investigation of ITP has focused on the development of serologic assays to measure the autoantibodies. As an alternative investigative approach, determination of the immunomorphologic characteristics of platelet-associated IgG (PAIgG) in ITP platelets may prove useful in the diagnosis or clinical management of patients with ITP. Our results showed that the ultrastructural morphology of ITP platelets is similar to that observed for normal platelets. No structural abnormalities are observed in ITP platelets. Immunogold labeling of IgG within alpha-granules of ITP platelets is significantly higher than that of normal platelets. Additionally, in some ITP platelets, immunogold labeling is also observed on the platelet surface and within channels of the open-cannalicular system. In comparison, immunogold labeling of these structures in normal platelets, is rare, or absent. In conclusion, electron microscopic studies should contribute to furthering our understanding of this common autoimmune disorder, and may provide possible biological explanations for the increased levels of PAIgG in platelets from patients with ITP.     

Blood platelets and inflammation: their relationship with liver and digestive diseases

An expansion of knowledge from basic and clinical research has highlighted the critical role of platelets in inflammation and tissue repair in addition to their established contribution to hemostasis. Activated platelets are a rich source of mediators participating to inflammation and tissue regeneration. Platelet-derived microparticles recapitulate essential platelet functions and their contribution to the pathogenesis of inflammatory diseases has been emphasized. Recent findings suggest that platelets are both friends and foes for the liver. Platelets are essential to liver regeneration, platelet-derived serotonin being critical. However platelets can also exacerbate liver damage, as in immune-mediated injury. The dual role of platelets has recently been exemplified in animal models of liver fibrosis. Platelets release profibrogenic mediators, such as CXC Chemokine Ligand 4, that is instrumental in the progression of liver fibrosis. On the other hand, thrombocytopenia aggravates liver fibrosis, an outcome linked to the downregulation of hepatic stellate cell collagen production by platelet derived hepatocyte growth factor. CD154, a key molecule in inflammation, is expressed by platelets and is a pathogenic mediator in inflammatory bowel disease. Here, we summarize some of the mechanisms linking platelets with inflammation and comment few recent articles indicating why platelets may prove to be important pathogenic mediators in liver and gastrointestinal diseases.     

Detection of In Vivo Activated Platelets in Experimental Cerebral Thrombosis: Studies Using a New Monoclonal Antibody 2T60, Specific for Activated Human and Rabbit Platelets

To detect in vivo activated platelets in humans as well as in animal models of thrombosis, we developed a new murine monoclonal antibody, 2T60, specific for activated human and rabbit platelets by immunizing with human thronibin-activated platelets. 2T60 (IgG(1) subclass) showed a great difference between binding to the thrombin-activated and resting human and rabbit platelets on ELISA and flow cytometer analysis. Immunoblotting analysis revealed that 2T60 reacted with a 130 or 106 kDa protein of human or rabbit platelets, respectively, only under non-reducing conditions. (125)I-labeled 2T60 inserted into the intermediate gel of CIE of solubilized human platelets was incorporated into a immunoprecipitation line. 2T60 immunoprecipitated a protein of 130 or 115 kDa from human or rabbit platelets, respectively, which had been activated and (125)I-surface-labeled. The N-terminal sequence of the affinity purified 2T60 antigen of human platelets was identical to that of GMP 140. There were differences in the carbohydrate chain content of GMP 140 between human and rabbit platelets. In experimental cerebral thrombosis of rabbits that had been injected with 2T60, the platelets adhering to the exposed subendothelium and contained in thrombi were found to bind 2T60 prominently. These results suggest that 2T60 may be a useful tool for clinical and experimental studies of thrombotic disease.     

Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P < 0.05) percentage of P-selectin-positive and fibrinogen-positive platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P < 0.05) percentage of responsive platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P < 0.05), while the percentage of platelets responsive to in vitro agonists was decreased (P < 0.05 in autologous transfusion patients), consistent with platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.     

Role of platelets in chronic liver disease and acute liver injury

Platelets contain not only hemostatic factors but also many growth factors that play important roles in wound healing and tissue repair. Platelets have already been used for the promotion of tissue regeneration in the clinical setting, such as dental implantation and plastic surgery. Thrombocytopenia, which is frequently found in patients with chronic liver disease and cirrhosis, is due to various causes such as decreased thrombopoietin production and accelerated platelet destruction caused by hypersplenism. However, the relationship between thrombocytopenia and hepatic pathogenesis and the role of platelets in chronic liver disease are poorly understood. In acute liver injury, it is reported that platelets are recruited to the liver and contribute to liver damage by promoting the induction of chemotactic factors and the accumulation of leukocytes in the liver, whereas platelets or mediators released by platelets can have a protective effect against liver injury. In this review, we highlight the recent accumulated knowledge concerning the role of platelets in chronic liver disease and acute liver injury.     

The MYH9 syndrome: report of a new case with a new mutation of the MYH9 gene

INTRODUCTION: Familial macrothrombocytopenias are a group of rare autosomal dominant platelet disorders including many syndromes in particular the May-Hegglin anomaly. They are characterized by thrombocytopenia with giant platelets and in some cases neutrophilic inclusions in peripheral blood granulocytes. Recently these different clinical entities have been demonstrated to be linked to mutations in the same gene, MYH9. CASE REPORT: We report in a young African woman presenting as a May-Hegglin anomaly a new mutation of the MYH9 gene. In regard of this case we present a brief review of the MYH9 syndrome. CONCLUSION: The MYH9 syndrome includes now several clinical entities who share some common clinical and biological characteristics such as a thrombocytopenia with giant platelets, presence or absence of other manifestations including Dohle like bodies, nephritis, sensorineural hearing loss, cataract. We report a new case in which a new mutation of the MYH9 gene was evidenced.     

Current viewpoints on platelet contribution to inflammation

Inflammation is an underlying feature of a variety of human diseases. Because inflammatory diseases are a major cause of morbidity and mortality in developed countries, understanding the interaction of the most important factors involved is an important challenge. Although platelets are widely recognized as having a critical role in primary hemostasis and thrombosis, basic and clinical evidence increasingly identifies these enucleated cells as relevant modulators, as both effector and target cells, of the inflammatory response. The cross-talk between platelets, endothelial cells and leukocytes in the inflammatory milieu mat be seen as a double-edged sword which functions not only as an effective first-line defense mechanism but may also lead to organ failure and death in the absence of counter-regulation systems. The molecular mechanisms involved in the reciprocal activation of platelets, endothelial cells and leukocytes are beginning to be elucidated. In the light of the existing data from experimental and clinical studies it is conceivable that platelet adhesion molecules and platelet mediators provide promising targets for novel therapeutic strategies in inflammatory diseases. The potentially adverse effects of these approaches need to be carefully addressed and monitored, including alterations in hemostasis and coagulation and particularly the impairment of host defense mechanisms, given the recently identified pivotal role of platelets in pathogen recognition and bacterial trapping. In this review we discuss the most important recent advances in research into the cross-talk between platelets and vascular cells during inflammation and the clinical consequences of these interactions.     

Platelet concentrates for bone regeneration: Current evidence and future challenges

Activated platelet concentrates are autologous blood preparations containing supraphysiological concentration of platelets. Platelet concentrates are commonly used for bone regeneration purposes based on the fact that growth factors released from activated platelets alpha granules have osteoinductive effects on bone cells. Although most preclinical and clinical studies show that platelet concentrates improve the outcomes of bone regeneration procedures, some studies reported conflicting results and even negative effects on bone healing. Several confounding parameters have been suggested as possible reasons for such inconsistencies (i.e. preparation and activation methods). However, heterogeneity in clinical studies makes drawing evidence-based conclusions difficult. On the other hand, recent findings show that the constituents of platelets dense granules (i.e. serotonin, ATP, Ca²⁺) have potential inhibitory effects on bone metabolism. Accordingly, we suggest that a partial explanation for the conflicting results could be the potential negative effects that dense granules may have on bone healing.     

Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet lifespan in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from patients with immune thrombocytopenia, which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleavage of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of an ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment of the production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.     

Platelets and HIV-1 infection: old and new aspects

In this review we summarize the data on interaction of platelets with HIV-1 infection. Thrombocytopenia is a common finding among HIV-1 infected patients; several combined factors contribute to low peripheral platelet counts, which are present during all the stages of the disease. In addition, a relationship between platelet count, plasma viral load and disease progression has been reported, and this shows the potential influence platelets may have on the natural history of HIV-1 disease. Several lines of evidence have shown that platelets are an integral part of inflammation, and can be also potent effector cells of innate immune response as well as of adaptive immunity. Thus, we rewieved the role of inflammatory cytokines, and chemokines as activators of platelets during HIV-1 infection. Moreover, platelets show a direct interaction with HIV-1 itself, through different pathogenic mechanisms as binding, engulfment, internalisation of HIV-1, playing a role in host defence during HIV-1 infection, by limiting viral spread and probably by inactivating viral particles. Platelets may also play an intriguing role on endothelial dysfunction present in HIV-1 infection, and this topic begins to receive systematic study, inasmuch as interaction between platelets and endothelial cells is important in the pathogenesis of atherosclerosis in HIV-1 infected patients, especially in those patients treated with antiretroviral drugs. Finally, this review attempts to better define the state of this emerging issue, to focus areas of potential clinical relevance, and to suggest several directions for future research.     

Aspirin can stimulate luminol-enhanced chemiluminescence of activated platelets

A preliminary investigation was conducted into the influence of aspirin on the luminol-enhanced chemiluminiscence of platelets stimulated with platelet-activating factor (PAF). Ten coronary artery disease patients and six volunteers without coronary artery disease were included in the study. All the patients received aspirin (daily dose, 100 mg) for at least 10 days before in vitro experiments. Luminol-enhanced luminescence of platelet-rich plasma samples mixed with a PAF solution was measured. After stimulation of platelets with PAF, we did not find a luminol-enhanced chemiluminescent response either in the non-coronary artery disease volunteers or in eight out of the 10 coronary artery disease patients examined. However, in samples from two patients where platelets were stimulated with PAF reactive oxygen species were formed. This ability was expressed as an intensive luminol-enhanced luminescence of activated platelets. Such a reaction was observed against the background of the administration of aspirin. The addition of aspirin to a test tube considerably enhanced the intensity of chemiluminescence. In one case, the cancellation of aspirin was accompanied by diminution of the intensity of luminol-enhanced chemiluminescence of platelets. The clinical significance of this phenomenon is unknown.     

Development of a computer program to analyze the parameters of platelet-vessel wall interaction

The use of the Baumgartner perfusion system allows the morphometric quantification of platelets interacting with vessel wall, however it presents the basic difficulties of morphometrical measurements. In order to facilitate the procedure of evaluation we developed a semiautomated method to avoid the complexity of the classical evaluation. Our system consists on an optical picture analysis system connected with a specially developed computer program which allows fast quantification. Simultaneously to the outlining of interacting platelets the computer program recognizes, corrects, selects and stores the information, in order to perform the final calculations as previously established. This system has been demonstrated to be as effective as the classical morphometric evaluation in the measure of platelets interacting with subendothelium. Potential sources of error such as subjectivity of the observers in selecting the class of interacting platelets are avoided. The use of this combined method opens the possibility to adapt the Baumgartner perfusion system to clinical routine and to the screening of drugs that modify platelet adherence.