Platelet Clearance Research Articles

Regulation of Platelet Survival by Protein Phosphatase 1 Gamma

AbstractAbstract 2026Platelets are key players in hemostasis and their senescence is intrinsically associated with the activation of apoptotic pathways that shows similarities to the apoptosis of nucleated cells. Anti-apoptotic protein Bcl-xl restrains the pro-apoptotic Bak activity and maintains platelet survival in circulation. In nucleated cells, serine phosphorylation of a pro-apoptotic protein Bad can also promote cell survival. Serine phosphorylation of Bad is regulated by the action of serine/threonine (Ser/Thr) protein kinases and Ser/Thr protein phosphatases. Although alterations in the activities of either enzyme can change the rate of apoptosis, whether Ser/Thr phosphatases participate in regulating platelet apoptosis is unknown. In this study, we report that the mice lacking the catalytic subunit of protein phosphatase 1 gamma (PP1cγ) exhibit a moderate but significant increase in the number of circulating platelets [596 × 103/μL ± 17.9 in WT compared with 694 × 103/μL ± 19.8 in PP1cγ–/– mice; P = .0003]. Examination of the bone marrow from the PP1cγ–/– mice revealed a non-significant increase in the number of morphologically matured megakaryocytes. A trend towards decreased plasma thrombopoietin levels were also noticed in PP1cγ–/– mice. These observations suggest that an increased megakaryopoiesis/thrombopoiesis may not fully account for the increased platelet numbers in PP1cγ–/– mice. In vivo platelet survival studies revealed that the loss of PP1cγ modestly increased platelet half life in circulation (t1/2 ∼69 hours in WT compared to ∼78 hours in PP1cγ–/– mice). PP1cγ–/– platelets had decreased mean platelet volume, suggesting the PP1cγ–/– mice may harbor a greater proportion of older circulating platelets. These studies are consistent with the delayed clearance of platelets from PP1cγ–/– mice. Pro-apoptotic Bad possess PP1c binding motif and mechanistically, PP1cγ interacts with Bad protein in platelets. Phosphorylation of Bad Ser112, which promotes cell survival, was enhanced ∼50% in PP1cγ–/– platelets. Consistent with the increased BAD phosphorylation, co-immunoprecipitation studies revealed increased BAD-14-3-3 protein complexes from the PP1cγ–/– platelets. It is reported that in the phosphorylated state, BAD can interact with 14-3-3 and is sequestered in the cytoplasm, thereby preventing the binding of Bad with the mitochondrial anti-apoptotic Bcl-xl. Interaction of Bad with Bcl-xl has the potential to displace the pro-apoptotic Bak from the Bcl-xl-Bak protein complex to trigger apoptosis. Finally, immunoblotting with anti-caspase 9 antibody revealed decreased caspase 9 cleavage product (∼37 kDa) in the PP1cγ–/– platelets, suggesting decreased activation of caspase 9 dependent apoptotic pathway. These data indicate that the increased platelet counts in PP1cγ–/– mice could be in part due to delayed platelet apoptosis. Loss of PP1cγ leads to the hyperphosphorylation of BAD, which via an interaction with 14-3-3, delays caspase mediated apoptosis to prolong the life span of platelets. Disclosures:No relevant conflicts of interest to declare.

A Critical Role of CD4+CD25+ Regulatory T Cells In Prevention of Murine Autoantibody-Mediated Thrombocytopenia

AbstractAbstract 381Immune thrombocytopenic purpura (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. Since CD4+CD25+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens, it has been believed that Treg dysfunction contributes to the development of a various forms of human autoimmune disorders. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. However, it remains unclear how Treg alteration is involved in the pathophysiology of ITP.Recently, we have found that a group of Treg-deficient mice develop autoantibody-mediated thrombocytopenia. For preparation of Treg-deficient mice, Treg-depleted T cells were prepared from BALB/c splenocytes by serial purification steps consisting of a positive selection of CD4+ T cells and a negative selection of CD25+ cells using magnetic bead-based cell sorting, and were transferred into syngeneic T cell-deficient nude mice via tail vein. Treg-depleted T cell fraction transferred contained >99% CD4+CD25− cells, and was confirmed to lack expression of Foxp3, a typical Treg marker. Three weeks after transfer, approximately one third of the recipient mice spontaneously developed thrombocytopenia, which sustained for > 20 weeks. Thrombocytopenic mice represented elevated platelet-associated IgG and increased proportion of reticulated platelets, but non-thrombocytopenic mice did not. In addition, platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contained IgG antibodies capable of binding to intact platelets, which were not detected in non-thrombocytopenic mice. The presence of anti-platelet antibodies and increased platelet turnover observed in thrombocytopenic Treg-deficient mice are analogous to ITP patients. Treg-deficient mice prepared by transfer of a less number of Treg-depleted T cells resulted in reduced prevalence of thrombocytopenia, suggesting that onset of thrombocytopenia depends on the number of conventional T cells transferred. Treg-deficient mice are known to frequently develop autoimmune gastritis, another autoimmune disease mediated by IgG anti-parietal cell antibodies, but anti-parietal cell antibodies were almost equally detected in plasma from thrombocytopenic and non-thrombocytopenic mice (70% versus 60%). Transplantation of Tregs together with Treg-depleted T cells completely prevented the onset of thrombocytopenia, but Treg transplantation was not effective as a treatment once thrombocytopenia occurred. To further investigate how Tregs exert the regulatory function, Treg-depleted T cells and Tregs were simultaneously transferred in the presence of antibodies that blocked engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). This treatment cancelled Treg function and resulted in development of thrombocytopenia in recipient nude mice, while mock treatment with control antibodies had no effect.In summary, these results together indicate that CD4+CD25+Foxp3+ Tregs play a critical role in preventing the development of murine autoantibody-mediated thrombocytopenia, in part, through CTLA4 engagement. Disclosures:No relevant conflicts of interest to declare.

Expression of the Neonatal Fc Receptor FcRn Is Not Required for the Amelioration of Murine ITP by IVIg or a Monoclonal Antibody

AbstractAbstract 2529There are several theories as to the mechanism of intravenous immunoglobulin (IVIg) in the treatment of autoimmune diseases such as immune thrombocytopenia (ITP). One prominent theory involves accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc Receptor (FcRn). FcRn is an IgG receptor, and FcRn within endosomes binds endocytosed IgG and diverts IgG from degradation. In the treatment of ITP, it has been theorized that high concentrations of IVIg saturate FcRn, reducing the ability of the pathogenic anti-platelet antibodies to bind FcRn, increasing their catabolism, and thus rapidly decreasing their serum concentration which results in a decrease in their ability to induce platelet clearance. Mice lacking FcRn have only 20–30% of the level of endogenous IgG as compared with wild-type mice and have an accelerated clearance rate of both endogenous and injected IgG. We have utilized these mice in a murine model of ITP to help understand the role, if any, that FcRn plays in IVIg's therapeutic activity.Both IVIg (2 g/kg body weight) and monoclonal antibodies to CD44 (2 mg/kg body weight; please see submitted abstract ID# 29504, “Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP?”) successfully ameliorate murine ITP. To definitively determine if FcRn is required for the acute amelioration of ITP by these two therapeutics, we employed FcRn deficient mice in the murine ITP model. Here, we demonstrate that FcRn deficient mice (B6.129X1-Fcgrttm1Dcr/DcrJ) injected with an anti-platelet antibody exhibit a slightly more profound degree of thrombocytopenia than wild-type mice. FcRn deficient mice treated with IVIg or the anti-CD44 antibody KM114 (at a 3 log fold lower dosage than IVIg) were protected from ITP to the same extent as wild-type mice.FcRn has an absolute requirement for the protein β2 microglobulin (β2M) to be functionally expressed. Specifically, β2M deficient mice do not possess functional FcRn and also show low endogenous IgG levels and increased clearance of IgG. To verify and substantiate the results found with FcRn deficient mice, we next employed β2M deficient mice in the murine ITP model and found that β2M deficient mice (B6.129P2-B2mtm1Unc/J) treated with IVIg or KM114 were also protected from ITP to the same extent as wild-type mice.These data suggest that for both high dose IVIg as well as low dose monoclonal CD44 antibody treatment in an acute ITP model, FcRn expression is dispensable. Disclosures:No relevant conflicts of interest to declare.

Bortezomib for ITP: First Report of Successful Treatment of Refractory ITP In a Patient with Otherwise Asymptomatic Monoclonal Gammopathy

AbstractAbstract 4667Current concepts of the pathophysiology of primary immune thrombocytopenia (ITP) reflect a complex interplay between antiplatelet antibodies, cytotoxic T-lymphocytes (CTL), T regulatory cells, and impaired bone marrow megakaryopoiesis. The mechanisms of ITP in the presence of monoclonal gammopathy of unknown significance (MGUS), a form of secondary ITP, are less well understood, yet treatment has been essentially the same as for primary ITP. Inhibition of antiplatelet CTL responses through apoptosis induction in clonal antiplatelet-antibody producing plasma cells and proteasome inhibition in CTLs may be achieved with bortezomib. However, bortezomib may cause transient isolated thrombocytopenia via presumed inhibition of marrow thrombopoiesis or enhanced platelet clearance peripherally.We used bortezomib in a 55 year-old female with IgG lambda monoclonal gammopathy and ITP refractory to previous treatment with corticosteroids, intravenous immunoglobulin, splenectomy, danazole, and rituximab, none of which induced a durable increase in her platelet counts to hemostatic concentrations. She suffered from repeated episodes of prolonged epistaxis, and when the platelet count fell below 10K/mm3, she experienced a life-threatening gastrointestinal hemorrhage. Otherwise, she had no anemia, her creatinine and calcium remained normal throughout, and skeletal surveys showed no evidence for myeloma. Repeated bone marrow examinations, reviewed at our institution, demonstrated lambda monotypic plasma cells always below 10%, and a normal female karyotype on metaphase cytogenetics. Her thrombocytopenia and bleeding episodes improved transiently on dexamethasone/IMiD combinations (thalidomide followed by lenalidomide), with concurrent reduction in total IgG by almost 50% (M-spike was not measured prior to IMiDs), however she became intolerant to thalidomide due to peripheral neuropathy and experienced diarrhea on lenalidomide. Her platelets decreased and her epistaxis worsened as her m-spike increased on dexamethasone single agent. We thus focused our treatment approach on more robust suppression of the monoclonal protein and initiated a course of weekly bortezomib (1.3 mg/m2), with dexamethasone (20 mg) on the day of and the day after bortezomib. After one month of therapy, the platelet count normalized, and she had no further bleeding episodes. The bortezomib dose was reduced to 1 mg/m2 and dexamethasone reduced to 20 mg weekly after 2 months, with persistently normal platelet counts thereafter. Her ITP resolution on bortezomib was accompanied by a decrease of her serum M-protein from 1.04 to 0.39 g/dL.This case illustrates that bortezomib can be remarkably effective in refractory ITP with concurrent monoclonal gammopathy. Assuming similar pathophysiology, it might also have efficacy in primary refractory ITP. This case also raises the question of whether refractory ITP in the presence of a clonal plasma cell disorder should be considered as a myeloma defining symptom. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Hepatic Asialoglycoprotein Receptor Regulates Platelet Homeostasis

AbstractAbstract 2025The human body produces and removes more than a 100 billion of platelets every day. The mechanisms responsible for platelet homeostasis are subject to speculation since the 1950's. The most popular hypothesis to date has been antibody-mediated clearance, platelet consumption due to massive blood loss and an internal “senescence timer”. We and others have recently demonstrated that sialic acid deficient platelets due to external triggers such as sepsis or chilling are cleared by hepatic asialoglycoprotein receptors (ASGPR) independently of macrophages. Here, we investigated whether loss of sialic acid mediates platelet clearance in vivo. We show that 1) Injection of the specific sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lengthened the survival of biotinylated platelets by ∼50% (T1/2 of 72h), compared to mock treated (PBS injected) control mice (T1/2 of 49h); 2) Similarly, biotinylated platelet survival in ASGPR-null mice was prolonged by ∼ 50% (T1/2 of 74h) compared to platelet survival in wild type (WT) mice (T1/2 of 48h); 3) ASGPR-null mice have significantly increased platelet counts, compared to WT (p=0.0004) and platelets isolated from ASGPR-null mice are ∼15% smaller than WT (p=0.03); 4) Platelets isolated from ASGPR-null mice showed significant increased in b-galactose exposure (∼50% increase, i.e. decrease of sialic acid), compared to WT, as evidenced by binding of the b-galactose specific lectin (RCA-I). These data show that the ASGPR not only removes desialylated platelets due to sepsis or chilling, but also regulates platelet homeostasis. Disclosures:No relevant conflicts of interest to declare.

GP IIb/Iiia-Dependent Complement Activation Is Common In Patients with Immune Thrombocytopenic Purpura.

AbstractAbstract 1430 Introduction:It is commonly accepted that antibody-mediated removal of platelets represents the major mechanism of platelet destruction in immune thrombocytopenic purpura (ITP). However, both the clinical course and the response to various treatments vary largely between ITP patients, suggesting a multi-factorial pathogenesis of thrombocytopenia in ITP. Ineffective thrombopoiesis and direct T-cell cytotoxicity have been described as additional immune-mediated mechanisms. In contrast, although some promising early studies were performed in the 1980s, the role of the complement system in ITP is still not well defined. The present pilot study investigated the possible contribution of the complement system in ITP. Material and Methods:We examined blood samples from 240 patients with ITP. First, all samples were assessed for the presence of free and bound platelet autoantibodies by a standard glycoprotein-specific assay (MAIPA). Second, the ability of all sera to fix complement to a panel of human platelets was investigated in a complement fixation assay. Third, fixation of C1q to immunobeads coated with platelet-derived GP IIb/IIIa as well as the generation of microparticles from test platelets were assessed by flow cytometry. The study was approved by the local research ethics committee. Results:Glycoprotein-specific autoantibodies were detected as platelet-bound antibodies in 129 (54%); as additional free antibodies in 26 (11%); and were undetectable in 111 (46%) of all patients. When sera from these patients where then assessed for their ability to fix complement to a panel of 5 test platelets, 103 (65%), 21 (81%), and 33 (30%) sera gave positive results. When studied in the presence of test platelets lacking either GP IIb/IIIa or GPIb/IX, 72% and 25% of all sera lost their ability to fix complement, respectively. Fixation of C1q to immunobeads covered with GP IIb/IIIa was observed in 70% of sera that had fixed complement to platelets. Half of these sera induced the production of platelet microparticles. In a group of 50 controls, platelet-reactive antibodies were undetectable, fixation of complement to platelets and fixation of C1q to immunobeads was observed in 2/50 (4%), and there were no sera inducing the production of micoparticles. Discussion:In a significant number of patients with chronic ITP, platelet autoantibodies are capable of activating the classical complement pathway. Complement fixation is even present in ITP sera without detectable autoantibodies, indicating that current techniques for autoantibody detection may be insufficient. The major target for complement-fixing autoantibodies in ITP is GP IIb/IIIa. In vitro, however, only 50% of sera that can fix complement in a GP IIb/IIIa-dependent manner are also capable of inducing platelet lysis. We conclude that complement fixation may contribute to thrombocytopenia by directly damaging platelets and/or by enhancing platelet clearance via complement-receptor mediated phagocytosis. It will thus be relevant to study the influence of complement-mediated platelet destruction on treatment responses, particularly in the light of newly developed approved and non-approved therapies, such as, thrombopoietin receptor agonists and inhibitors of complement activation. Disclosures:No relevant conflicts of interest to declare.

Novel clearance mechanisms of platelets

Blood platelets are involved in primary and secondary hemostasis and thus maintain the integrity of the vasculature. They circulate with an average lifespan of 5-9 days in humans. Thus, the body must generate and clear platelets daily to maintain normal physiological blood platelet counts. Known platelet clearance mechanisms include antibody-mediated clearance by spleen macrophages, as in immune thrombocytopenia, and platelet consumption due to massive blood loss. New concepts in the clearance mechanisms of platelets have recently emerged. New evidence shows that platelets desialyted due to chilling or sepsis are cleared in the liver by macrophages, that is Kupffer cells, as well as hepatocytes, through lectin-mediated recognition of platelet glycans. On the contrary, platelet-associated antibodies normalize the clearance of platelets in a mouse model for Wiskott-Aldrich syndrome. The goal of this review is to summarize the latest findings in platelet clearance mechanisms with a focus on lectin-mediated recognition of platelet glycans. Transfusion medicine and treatments of hematopoietic disorders associated with severe thrombocytopenia may benefit from a better understanding of these mechanisms.

Are there more tricks in the bag for treating thrombocytopenia?

Thrombocytopenia, an abnormally low number of circulating platelets, results from inadequate platelet production, splenic platelet sequestration, or accelerated platelet clearance. Platelet transfusions are now the cornerstone for treating thrombocytopenia. With an ever-expanding demand for platelets, and with many patients having an inadequate response to platelet transfusions, new strategies are needed to treat thrombocytopenia. In this issue of the JCI, Fuentes et al. present provocative data regarding the use of direct megakaryocyte infusions as a novel approach to manage this vexing clinical problem.

Platelet production and destruction in liver cirrhosis

Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Drug-dependent clearance of human platelets in the NOD/scid mouse by antibodies from patients with drug-induced immune thrombocytopenia

AbstractDrug-induced immune thrombocytopenia (DITP) is a relatively common and sometimes life-threatening condition caused by antibodies that bind avidly to platelets only when drug is present. How drug-dependent antibodies (DDAbs) are induced and how drugs promote their interaction with platelets are poorly understood, and methods for detecting DDAbs are suboptimal. A small animal model of DITP could provide a new tool for addressing these and other questions concerning pathogenesis and diagnosis. We examined whether the nonobese diabetic/severe combined immunodeficient (NOD/scid) mouse, which lacks xenoantibodies and therefore allows infused human platelets to circulate, can be used to study drug-dependent clearance of platelets by DDAbs in vivo. In this report, we show that the NOD/scid model is suitable for this purpose and describe studies to optimize its sensitivity for drug-dependent human antibody detection. We further show that the mouse can produce metabolites of acetaminophen and naproxen for which certain drug-dependent antibodies are specific in quantities sufficient to enable these antibodies to cause platelet destruction. The findings indicate that the NOD/scid mouse can provide a unique tool for studying DITP pathogenesis and may be particularly valuable for identifying metabolite-specific antibodies capable of causing immune thrombocytopenia or hemolytic anemia.

Differenzialdiagnose und Differenzialtherapie der Thrombozytopenie

Thrombocytopenia is usually acquired. The laboratory artefact of pseudothrombocytopenia should always be excluded. Bone marrow insufficiency with impaired platelet production results from infiltrating tumor cells or from a myelodsplastic syndrome. In patients with splenomegaly, platelets are trapped by the spleen. An increased platelet turnover is caused by activation of the clotting cascade, e.g. due to sepsis or malignancy. Platelet binding antibodies cause thrombocytopenia by increased platelet clearance. Important differential diagnoses in patients with severe thrombocytopenia are: acute leukemia, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia and drug-dependent thrombocytopenia. Multifactorial causes are thrombocytopenia associated with pregnancy, chronic alcohol abuse, and liver cirrhosis. Treatment should focus on the underlying disease. In regard to low platelet counts only clinical bleeding and not platelet count numbers should be treated.

Drug-induced thrombocytopenia: development of a novel NOD/SCID mouse model to evaluate clearance of circulating platelets by drug-dependent antibodies and the efficacy of IVIG

AbstractDrug-induced immune thrombocytopenia (DITP) is an adverse drug effect mediated by drug-dependent antibodies. Intravenous immunoglobulin (IVIG) is frequently used to treat DITP and primary immune thrombocytopenia (ITP). Despite IVIG's proven beneficial effects in ITP, its efficacy in DITP is unclear. We have established a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of DITP in which human platelets survive for more than 24 hours, allowing platelet clearance by DITP/ITP antibodies to be studied. Rapid human platelet clearance was uniformly observed with all quinine-induced thrombocytopenia (QITP) patient sera studied (mean platelet lifespans: QITP 1.5 ± 0.3 hours vs controls 16.5 ± 4.3 hours), consistent with the clinical presentation of DITP. In contrast, clearance rates with ITP antibodies were more variable. IVIG treatment partially prevented platelet clearance by DITP and ITP antibodies. Our results suggest that the NOD/SCID mouse model is useful for investigating the efficacy of current and future DITP therapies, an area in which there is little experimental evidence to guide treatment.

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

AbstractType 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbα binding. These “gain of function” mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF−/− mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride–induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride–induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

Proteolysis of platelet receptors in humans and other species

In the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbalpha (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbalpha function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbalpha are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbalpha/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbalpha/GPVI shedding, evidence for the functional consequences of shedding in vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbalpha shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic shedding in vivo.

Management of chronic immune thrombocytopenic purpura: targeting insufficient megakaryopoiesis as a novel therapeutic principle

Traditionally, anti-platelet autoantibodies accelerating platelet clearance from the peripheral circulation have been recognized as the primary pathopysiological mechanism in chronic immune thrombocytopenia (ITP). Recently, increasing evidence supports the co-existence of insufficient megakaryopoiesis. Inadequate low thrombopoietin (TPO) levels are associated with insufficient proliferation and differentiation of megakaryocytes, decreased proplatelet formation, and subsequent platelet release. Recently two novel activators of TPO receptors have been made available: romiplostim and eltrombopag. In several phase III studies, both agents demonstrated increase of platelet counts in about 80% of chronic ITP patients within 2 to 3 weeks. These agents substantially broaden the therapeutic options for patients with chronic ITP although long-term results are still pending. This review will provide an update on the current conception of underlying mechanisms in ITP and novel, pathophysiologically based treatment options.

Restored platelet function after romiplostim treatment in a patient with immune thrombocytopenic purpura

Whilst the precipitating aetiology of immune thrombocytopenic purpura (ITP) remains unclear, the predominant cause of the thrombocytopenia is the presence of circulating antiplatelet autoantibodies that coat platelets, leading to platelet destruction and clearance, primarily in the spleen. Traditional clinical management of patients with ITP, including treatment with corticosteroids, intravenous immunoglobulins, splenectomy, rituximab, and cyclophosphamide, aims to curb platelet destruction (Nurden et al, 2009a). Newer reagents approved for trial to treat ITP patients, such as thrombopoietin mimetics, romiplostim (Kuter et al, 2008) and eltrombopag, act primarily in the bone marrow to stimulate thrombopoiesis. Whilst neither plasma thrombopoietin levels nor platelet production kinetics are markedly altered in ITP patients, ITP anti-platelet autoantibodies interfere with megakaryocyte proliferation and platelet production in vitro (Chang et al, 2003). Here we describe a significant improvement to the nature and function of platelet immunoreceptor tyrosinebased activation motif (ITAM) receptors in an ITP patient with an autoantibody to platelet glycoprotein (GP)VI receiving romiplostim.

Cold treatment does not increase the rate of platelet clearance in 13‐lined ground squirrels.

Thirteen‐lined ground squirrels (Spermophilus tridecemlineatus)experience a dramatic decrease in body temperature and heart rate during hibernation. These extreme conditions would likely cause human platelets to clot due to low blood flow rate and pressure. We have found that ground squirrels drastically decrease their circulating platelet and leukocyte counts during hibernation, likely to reduce the risk of clotting. Previous research has shown that chilled human and mouse platelets are rapidly cleared from circulation. However, upon arousal from torpor, ground squirrel platelets are rapidly released into circulation from their storage site in the spleen and persist for several days until new platelet synthesis begins. Flourescently labeled platelets were incubated at 4°C or 37°C and reinjected into autologous ground squirrels. Clearance rates of both warm and chilled platelets were the same. Identification of protective factors that prevent rapid clearance of chilled platelets may ultimately lead to the ability to refrigerate human platelet concentrates. This work was supported by an NIH R15 grant.

P38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

AbstractPlatelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Platelets and viruses: an ambivalent relationship.

Thrombocytopenia is a frequent complication of viral infections providing evidence that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms are involved depending on the nature of the viruses involved. These include immunological platelet destruction, inappropriate platelet activation and consumption, and impaired megakaryopoiesis. Viruses bind platelets through specific receptors and identified ligands, which lead to mutual alterations of both the platelet host and the viral aggressor. We have shown that HIV-1 viruses are internalized specifically in platelets and megakaryocytes, where they can be either sheltered, unaltered (with potential transfer of the viruses into target organs), or come in contact with platelet secretory products leading to virus destruction and facilitated platelet clearance. In this issue, we have reviewed the various pathways that platelets use in order to interact with viruses, HIV and others. This review also shows that more work is still needed to precisely identify platelet roles in viral infections, and to answer the challenge of viral safety in platelet transfusion.

Impact of xenon anaesthesia in isolated cardiopulmonary bypass on very early leucocyte and platelet activation and clearance: a randomized, controlled study

Cardiopulmonary bypass (CPB) is associated with leucocyte and platelet activation and also organ dysfunction. Xenon has been found to have organ-protective effects. We therefore investigated the effect of isolated CPB on leucocyte and platelet activation and the efficacy of xenon in inhibiting these changes. Isolated CPB was conducted according to strict standardized clinical criteria using blood from healthy volunteers. They were randomized to an air-oxygen mixture (control group) vs xenon-oxygen mixture (xenon group). Blood samples were drawn at 5, 15, 30, 60, and 90 min from commencement of circuits and analysed for haemoglobin concentrations, white cell, neutrophil, monocyte, lymphocyte, and platelet counts. Leucocyte and platelet activation and also complex formation were determined by measuring levels of CD14++ monocytes, CD16+ monocytes, platelet-monocyte complexes, and platelet-neutrophil complexes (PNC). Differences between and within the groups were analysed with Student's t-test. Biomarker levels were not different between the groups. The data were pooled to identify the effects of isolated bypass. The neutrophils, monocytes, platelets, CD14++ monocytes, and CD16+ monocytes decreased within 5 min of the bypass experiments, whereas the percentage of platelet-CD++ monocyte complexes and PNC increased. Isolated CPB elicited rapid, substantial leucocyte and platelet activation, and xenon had no impact on inhibiting these changes.