Platelet Clearance Research Articles

Autoantibodies to platelets: Roles in thrombocytopenia

Circulating platelets are targeted by autoantibodies in various pathologic conditions, such as immune thrombocytopenic purpura (ITP). Anti-platelet antibodies cause thrombocytopenia through enhanced platelet clearance via Fcγ receptor-mediated platelet destruction by the reticuloendothelial system and impaired platelet production. Moreover, functional blockade of platelet surface receptors by autoantibodies may further promote bleeding tendency. The major targets of these autoantibodies are platelet membrane glycoproteins, including GPIIb/IIIa and GPIb/IX, receptors for fibrinogen and other platelet-activating ligands, but some patients with ITP have antibodies to a receptor for thrombopoietin, which is a growth factor required for megakaryocytogenesis and platelet production. Several antigen-specific assays have been developed to measure anti-glycoprotein antibodies, whereas we have recently established an enzyme-linked immunospot assay for the detection of circulating B cells secreting IgG anti-GPIIb/IIIa antibodies, which is a sensitive, specific, and convenient method for evaluating the presence or absence of ITP. Production of pathogenic anti-platelet antibodies is maintained by a continuous loop, in which B cells produce anti-platelet antibodies, antibody-coated platelets are phagocytosed and GPIIb/IIIa-derived cryptic peptides presented by splenic macrophages, and GPIIb/IIIa-reactive CD4+ T cells exert their helper activity. Important discoveries on cellular and molecular mechanisms for anti-platelet autoantibody production contribute to development of diagnostic assays and therapeutic strategies for ITP.

Fc-Independent Phagocytosis: Implications for Intravenous IgG Therapy in Immune Thrombocytopenia

Phagocytes were first described 120 years ago. Although the molecular mechanisms involved in initiating phagocytosis (through Fc or other receptors) are still not fully understood, the roles of phagocytes in innate and adaptive immunity have been well studied. Phagocytes in the reticuloendothelial system, particularly macrophages, have been implicated in the clearance of senescent blood cells. The destruction of these cells may be primarily mediated through an Fc-independent pathway. Fc-independent phagocytosis may also play an important role in platelet clearance, including immune thrombocytopenia (ITP). The two major platelet antigens targeted in ITP are GPIIbIIIa and the GPIb complex. It has been demonstrated that anti-GPIbalpha antibodies, in contrast to anti-GPIIbIIIa, can induce thrombocytopenia in an Fc-independent manner. We further demonstrated in an animal model that intravenous IgG is not able to ameliorate thrombocytopenia caused by most anti-GPIbalpha antibodies, though it is effective in anti-GPIIbIIIa mediated thrombocytopenia. Our data was supported by a recent retrospective study of ITP patients. Therefore, identification of antibody specificity (e.g. anti-GPIIbIIIa (Fc-dependent) versus anti-GPIbalpha (Fc-independent)) in patients may be important for ITP therapy.

Drug Dependent Immune Destruction of Human Platelets in the NOD/SCID Mouse.

Animal models have been of limited value for studying survival and immune destruction of human blood cells because of xenoantibodies that mediate rapid elimination of the transfused cells. The NOD/SCID mouse is a genetic variant that lacks immunoglobulins, including xenoantibodies, and may therefore be useful for studying survival and immune destruction of human blood cells (Newman, PJ et al. J Thromb Haemost. 2007; 5 Suppl 1:305–9). Human platelets, infused into the retroorbital plexus of NOD/SCID mice survive about three days, nearly as long as mouse platelets themselves. Injection of IgG from a patient with quinine-induced immune thrombocytopenia (TP) and a newly developed quinine-dependent murine monoclonal antibody (mAb) were without effect on platelet survival. However, both antibodies caused rapid platelet clearance after IP injection of quinine. 500 μg, but not 50 μg, of IgG from serum containing an anti-HPA-1a (PlA1) antibody also caused rapid clearance of HPA-1a positive platelets. These findings indicate that the NOD/SCID mouse can be valuable for the study of immune clearance of human platelets and perhaps other unresolved problems in transfusion medicine. However, it is not known whether mice recognize cells opsonized with human IgG or mouse IgG equally well. To examine this, we compared the survival of human platelets sensitized with 1) mAb 7E3 (IgG1) specific for an epitope in the beta A domain of GPIIIa or 2) c7E3 (from Centocor Inc), which recognizes the same epitope but possesses a human IgG1 Fc. In titration studies, we found that about four times as much c7E3 as 7E3 is needed to produce equivalent clearance of human platelets, indicating that mouse IgG is a more efficient opsonin than human IgG. However, this difference should not limit use of the model for studies of antibody-mediated blood cell destruction, since mg quantities of IgG can easily be isolated from small amounts (0.1– 0.2 ml) of human serum.

Role of Sialic Acid for Platelet Lifespan and Function

Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.

Inhibition of Syk Activity Is Sufficient for Inhibitory Effect in Animal Models of Autoimmune Diseases

Spleen tyrosine kinase (Syk) serves as a key mediator of Fc receptor-mediated signaling in immunologic signaling cascades. Human Phase II trials have reported that dual inhibition of Syk and Janus kinases (JAK) by agents such as R788 (tamatinib fosdium) result in clinical benefit in rheumatoid arthritis (RA) and idiopathic thrombocytopenia purpura (ITP) patients. In order to delineate if inhibition of Syk alone was sufficient to produce a treatment benefit, we utilized compounds from two chemically diverse series of oral Syk inhibitors with differing kinase specificity in mouse models of RA and ITP. Compounds from the first series (P142–76, P459–72) were potent inhibitors of purified Syk (IC50 = 4 to 43nM) and when tested at a ten fold higher concentration (300–500nM), lacked JAK inhibitory activity. Even at a concentration of 5μM, P142–76 did not inhibit JAK activity. Compounds from the second series (P420–1, P420–89) were potent Syk inhibitors (IC50 = 15 to 31nM) and also exhibited comparable inhibitory potency against purified JAK (IC50 JAK1 = 6–7nM, JAK2 = 2–3nM, JAK3 = 0.6–0.8nM). The compounds inhibited B cell receptor-induced activation of primary mouse splenocytes; P420–89 (IC50 ~ 50nM) being somewhat more potent than P459–72 (IC50 = 125–250nM). Importantly, phorbol 12-myristate 13-acetate (PMA)-induced B cell activation was not inhibited by any of the compounds at the tested concentration (1μM). Further, oral dosing of these compounds inhibited B cell receptor-induced B cell activation in mice. The RA model (n=12/group) investigated collagen antibody induced arthritis over a seven to ten day time course. The ITP model (n=10/group) utilized rapid clearance and destruction of antibody-coated platelets by phagocytic cells such as spleen macrophages. In this model, mice injected with a rat anti-mouse CD41 antibody became thrombocytopenic (average of 49% reduction in number of circulating platelets) over the 8h time course of experimentation.ModelKinase specificityAgent, Dose per dayAverage peak plasma concentration (μM)Average inhibitory activity (%)RASykP459–72, 100mg/kg10.288*RASykP459–72, 30mg/kg4.427*RASyk/JAKP420–89, 30mg/kg6.957*RASyk/JAKP420–89, 10mg/kg0.80ITPSykP142–76, 30mg/kg2.182*ITPSyk/JAKP420–1, 10mg/kg2.850*ITPSyk/JAKP420–1, 3mg/kg0.417*ITPSyk/JAKP420–89, 30mg/kg5.444*ITPSyk/JAKP420–89, 10mg/kg0.64* denotes statistically significant reduction (p<0.05) in inflammation score (RA) or platelet clearance (ITP) compared to vehicle by two-tailed, unpaired Student's t test.In the RA studies, in addition to macroscopic signs of arthritis and assessment of clinical score, positive results were also verified histologically by reductions in peri-articular edema and neutrophil count and by scoring of intra-articular neutrophils and fibrin. In the ITP studies, mice pre-treated with P142–76, P420–1 or P420–89 were protected from loss of platelets, while mice pre-treated with vehicle alone displayed no protection. Our results highlight the role of Syk inhibition by small molecule oral inhibitors and suggest that inhibition of Syk activity, without dual inhibition of Syk/JAK, may be a promising strategy for therapeutic intervention in human autoimmune and inflammatory diseases.

Apoptosis in Platelets from Pediatric Patients with Acute Immune Thrombocytopenic Purpura (ITP) Is Ameliorated by IV Ig.

Interaction of auto-antibodies with platelet surface glycoproteins (GP) has been implicated in the accelerated clearance of platelets in ITP. It was recently shown, using a murine model, that the ITP induced by injection of anti-GPIIb is associated with apoptotic-like processes in platelets. These apoptotic-like processes in platelets were similar to those observed during apoptosis in nucleated cells: activation of caspase-3 (aCASP3), loss of mitochondrial inner transmembrane potential and externalisation of phosphatidylserine (PS). Administration of IVIg, that ameliorates the ITP in this model, also inhibited aCASP3 and PS externalization (Leytin et al., 2006, Br J Haematol ; 133:78; Song et al., 2003, Blood ; 101:3708). Whether these observations in mice are applicable to patients with ITP had not been investigated. Thus, the aim of our study was to investigate whether platelets from paediatric patients with acute ITP show an enhancement of apoptotic-like processes. Further, we examined whether the increase in platelet count in response to IVIg in these patients is accompanied by an inhibition of apoptotic-like processes in platelets. Children with clinical and laboratory diagnosis of acute ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a paediatric bleeding score for ITP at the time of diagnosis and after IVIg therapy. Blood samples were obtained at these time points for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. Specifically, platelet aCASP3 and PS externalisation were measured in citrated platelet-rich plasma. Platelets were identified as CD42 positive events; aCASP3 was measured as % platelets with bound FLICA-FITC fluorescence; and PS exposure as % platelets with bound annexin A5-FITC fluorescence. The fraction of young, reticulated platelets (RP) was analysed using thiazole orange. All patients (median age 7.5, n = 12) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3. 10 patients had platelet counts below 10'000/micro l, and 2 had platelets counts over 10'000/micro l. ITP patients had increased levels of platelets with aCASP3 (n = 11) and PS exposure (n = 9) compared with healthy control children (see Figure) and increased reticulated platelets (15.6 ± 2.7 %, n = 10; controls: 1.2 ± 0.8, n = 11). The 10 patients with platelet counts below 10,000/micro l were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose). All patients showed a rise in platelet counts to above 20'000/micro l and amelioration of bleeding symptoms 24 – 72 hours after IVIg administration. Concomitantly, platelet aCASP3 and PS exposure decreased (see Figure), as did RP, to 3.8 ± 1.5 % (n = 9). One patient with an initial platelet count of 18,000/micro l was not treated. This patient showed 5% positive aCASP3 platelets, 3.5 % platelets with PS exposure and 3.5% RP, similar to controls; one month later, the platelet count increased spontaneously to 60'000/micro l with 10% aCASP positive platelets, 4% platelets with PS exposure and 3 % RP. One patient was lost to follow-up. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP3 and PS externalisation in paediatric acute ITP, similar to the reported murine model. Consistent with acute ITP, platelet counts were low and levels of RP were high; after administration of IVIg, platelet counts increased whereas levels of RP decreased. IVIg administration also led to a decrease of aCASP3 and PS exposure in the patients' platelets. Thus, amelioration of acute ITP in children by IVIg is accompanied by inhibition of enhanced apoptosis-like events in platelets. [Display omitted]

Platelet Death.

Abstract Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite existence is circumscribed by members of the Bcl-2 family of proteins, which control the intrinsic apoptosis pathway. Pro-survival Bcl-xL is the critical regulator of platelet lifespan, functioning to keep pro-death Bak and Bax in check, thereby maintaining platelet viability. After 5–10 days in the circulation, platelets not consumed in hemostatic processes initiate a Bak and Bax-dependent cell death program and clearance from the bloodstream. Mutations in Bcl-xL reduce platelet lifespan in a dose-dependent fashion, while deletion of Bak and Bax extend it. Studies with the BH3 mimetic compound ABT-737, which inhibits pro-survival Bcl-xL, have shown that platelets induced to undergo cell death in vitro exhibit many of the hallmarks of apoptosis in nucleated cells, including mitochondrial damage, caspase activation and externalization of membrane phosphatidylserine (PS). Whether any of these features occur during physiological platelet clearance remains unclear. Certainly, mitochondrial damage can reduce the recovery of transfused platelets, but whether PS – which is known to promote the pro-coagulant activity of agonist-activated platelets – also acts as a clearance signal for dying platelets in vivo is yet to be established. Conversely, Bak and Bax may play a role in mediating PS exposure triggered by activation. Supporting the idea that there may be crosstalk between classical platelet signaling pathways and the intrinsic apoptosis pathway is recent evidence that platelet agonists can also activate caspases. Intriguingly, elements of the intrinsic pathway may also contribute to the generation of platelets by megakaryocytes. Several groups have demonstrated that megakaryocytes contain activated caspases and that their inhibition can block platelet shedding by cultured cells. Preliminary evidence we have generated suggests that Bcl-2 family proteins may be required for platelet production in vivo. Thus, it appears that there is much to be understood about the role of the intrinsic apoptosis pathway in the regulation of platelet biogenesis, function, and death.

Glycans and glycosylation of platelets: current concepts and implications for transfusion

Platelet products are currently stored at room temperature, because refrigeration causes their rapid clearance from the circulation upon transfusion. Glycans have recently been emphasized as important determinants for the clearance of refrigerated platelets. The present review addresses the current knowledge of platelet glycans and the potential of glycosylation for improving platelet storage. Removal of refrigerated platelets from the circulation is partly mediated by recognition of clustered beta-N-acetylglucosamine on platelet surface glycoproteins by the alphaMbeta2 hepatic lectin receptor. Capping the exposed beta-N-acetylglucosamine residues by enzymatic galactosylation restored the circulation of short-term chilled murine platelets, introducing a novel method that allows for cold storage of platelet. Recent studies have, however, shown that galactosylation is not sufficient to restore circulation of long-term refrigerated platelets. Additional data indicate that differential carbohydrate-mediated mechanisms may exist for clearance of short-term and long-term cold-stored platelets. Room temperature storage of platelet products increases the risk of transfusion-mediated sepsis and accelerates platelet deterioration, limiting platelet shelf life. Recent evidence suggests that glycoengineering of platelets might allow for their cold storage, significantly improving the quality of platelet products.

Genetics of type 2B von Willebrand Disease: “True 2B,” “tricky 2B,” or “Not 2B.” What Are the Modifiers of the Phenotype?

Type 2B von Willebrand disease (VWD) is a qualitative type of VWD with a unique feature among VWD types, resulting from an increased binding of von Willebrand factor (VWF) to its platelet receptor glycoprotein 1b-alpha (GP1BA). This heightened responsiveness takes place in vivo without endothelial injury or shear stress induction, typically resulting in loss of the hemostatically most active high-molecular-weight VWF multimers and leading to a bleeding diathesis. This process also typically leads to clearance of platelets and thus usually mild thrombocytopenia. At least this describes the classic representation of type 2B VWD (i.e., the typical picture we have come to know since the description/classification of this disorder in the early 1990s). Over more recent years, several reports have described individual cases, groups of patients, and families diagnosed with type 2B VWD where this picture was not typical in one or more aspects. This review discusses type 2B-like disorders and sheds light on potential phenotypic modifiers that might be responsible for the variation encountered with the classic picture of type 2B VWD as well as the impact on the diagnostic certainty of type 2B VWD.

Deficiency of decay-accelerating factor and complement receptor 1–related gene/protein y on murine platelets leads to complement-dependent clearance by the macrophage phagocytic receptor CRIg

AbstractComplement activation on human platelets is known to cause platelet degranulation and activation. To evaluate how normal platelets escape complement attack in vivo, we studied the fate of murine platelets deficient in 2 membrane complement regulatory proteins using an adoptive transfer model. We show here that deficiency of either decay-accelerating factor (DAF) or complement receptor 1–related gene/protein y (Crry) on murine platelets was inconsequential, whereas DAF and Crry double deficiency led to rapid clearance of platelets from circu-lation in a complement- and macrophage-dependent manner. This finding contrasted with the observation on erythrocytes, where Crry deficiency alone resulted in complement susceptibility. Quantitative flow cytometry revealed that DAF and Crry were expressed at similar levels on platelets, whereas Crry expression was 3 times higher than DAF on erythrocytes. Antibody blocking or gene ablation of the newly identified complement receptor CRIg, but not complement receptor 3 (CR3), rescued DAF/Crry-deficient platelets from complement-dependent elimination. Surprisingly, deficiency of CRIg, CR3, and other known complement receptors failed to prevent Crry-deficient erythrocytes from complement-mediated clearance. These results show a critical but redundant role of DAF and Crry in platelet survival and suggest that complement-opsonized platelets and erythrocytes engage different complement receptors on tissue macrophages in vivo.

Keeping blood clots at bay in sepsis

Clearance of platelets by the liver can help counteract the dangerous blood coagulation that can occur during sepsis. The mechanism involves clearance of platelets through the liver's Ashwell receptor, which binds to platelet glycoproteins altered by sepsis-causing bacteria (pages 648–655).

Thrombocytopenia and Platelet Abnormalities in High-Density Lipoprotein Receptor–Deficient Mice

High-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediated cellular uptake of lipoprotein cholesterol controls HDL structure and plasma HDL and biliary cholesterol levels. In SR-BI knockout (KO) mice, an unusually high plasma unesterified-to-total cholesterol ratio (UC:TC) and abnormally large HDL particles apparently contribute to pathology, including female infertility, susceptibility to atherosclerosis and coronary heart disease, and anemia. Here we examined the influence of SR-BI deficiency on platelets. The high plasma UC:TC ratio in SR-BI KO mice was correlated with platelet abnormalities, including high cholesterol content, abnormal morphologies, high clearance rates, and thrombocytopenia. One day after platelets from wild-type mice were infused into SR-BI KO mice, they exhibited abnormally high cholesterol content and clearance rates similar to those of endogenous platelets. Platelets from SR-BI KO mice exhibited in vitro a blunted aggregation response to the agonist ADP but a normal response to PAR4. In SR-BI KO mice abnormal circulating lipoproteins, particularly their high UC:TC ratio-rather than the absence of SR-BI in platelets themselves-induce defects in platelet structure and clearance, together with a mild defect in function.

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

AbstractCold 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 β-N-acetylglucosamine (βGlcNAc) on platelet surfaces. Glycosylation of βGlcNAc residues with uridine 5′-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0°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°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°C storage. We next developed a murine model of platelet storage for 48 hours at 4°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.

Immune Thrombocytopenia (ITP): An Historical Perspective

In 1735, two young girls presented to the German physician Paul Gottlieb Werlhof with symptoms of epistaxis and purpura. One young patient had a spontaneous remission, while the second suffered from repeated relapses. Describing these cases in his “Disquisitio medica et philologica de variolis et anthracibus,” Werlhof named the disorder “Morbus haemorrhagicus maculosus.” Mueller-Eckhart in his historical review of immune thrombocytopenic purpura (ITP) found that Brohm and Kraus (1883) and Denys (1883) independently were the first to report the relationship between thrombocytopenia and the clinical syndrome described by Werlhof. A controversy soon emerged regarding the pathologic mechanisms responsible for the thrombocytopenia. Frank (1915) proposed that “Die Essentielle Thrombopenia” resulted from toxic suppression of the megakaryocyte by a substance produced in the spleen. A year later, Kaznelson, a Viennese medical student, reported on the beneficial effect of splenectomy in this disorder. However, in contrast to Frank, he proposed that thrombocytopenia resulted from increased platelet destruction in the spleen. Over the next 30 years, little evidence emerged to support either theory. In 1946, Dameshek and Miller studied bone marrow from patients with ITP and found an increase in the total number of megakaryocytes in the bone specimens, but the majority of cells appeared not to be producing platelets. Effective platelet production appeared to increase after splenectomy. A series of reports published in 1951 firmly documented an immunologic role for accelerated platelet destruction. Evans and co-workers reported an association between Coombs test–positive hemolytic anemia and ITP, suggesting an autoimmune mechanism for thrombocytopenia. In the same year, Harrington and Moore clearly demonstrated that a humoral factor was responsible for the rapid clearance of platelets in this disorder. With these publications, “idiopathic” thrombocytopenic purpura became “immune” thrombocytopenic purpura. Subsequent studies by a number of investigators confirmed that the “humoral anti-platelet factor” was an antibody directed against platelet glycoproteins. The dogma of ITP as a disorder exclusively due to accelerated platelet destruction was challenged again in the 1980s by investigators at the Puget Sound Blood Center. By using platelet kinetic studies with indium111–labeled autologous platelets, these investigators found not only evidence of increased platelet clearance, but also impaired platelet production. These studies were initially viewed with a good deal of skepticism; however, additional studies performed in the last decade have demonstrated inhibition of in vitro megakaryocyte growth and maturation by antibodies from ITP patients. In addition, more recent studies have strongly supported an important role for the thymic lymphocyte in the initiation, progression, and response to treatment in ITP. The recognition that a number of other medical conditions can be associated with the development of thrombocytopenia that is clinically indistinguishable from classic ITP has lead to a separation of the disease into primary (idiopathic) and secondary forms. There is increasing recognition that the secondary forms of ITP, such as observed with the recent reports of Helicobacter pylori–associated disease, may be more common than primary disease. Our greater understanding of the complexity of the syndrome of ITP has lead to new and promising therapies such as the thrombopoietin receptor agonists and improved outcomes for the affected patients.

Lack of Efficacy of High-Dose Intravenous Immunoglobulin Treatment of Severe Thrombocytopenia in Patients with Secondary Dengue Virus Infection

Because most cases of secondary dengue virus infection are associated with an increased level of platelet-associated IgG, a high dose of intravenous immunoglobulin (IVIG) may have an effect on the development of severe thrombocytopenia in this disease. A randomized, controlled study was conducted with two treatment groups consisting of a treatment (IVIG) group (n = 15) and a non-treatment (non-IVIG) group (n = 16) to determine whether a high dose of IVIG is effective in hastening the recovery from thrombocytopenia in patients with secondary dengue virus infection. No significant difference was found in the baseline demographic data between the two groups. No adverse effect of IVIG was observed, but no effect in hastening the recovery of platelet counts was found in patients with secondary dengue infections. The lack of efficacy of IVIG suggests that platelet clearance by macrophages through Fc gamma receptors is not a primary mechanism in this disease.

FcγRIIa and the Integrin β3 Cytoplasmic Domain: Two Prerequisites for Thrombocytopenia and Thrombosis Following Administration of the Fibrinogen Receptor Antagonist, Eptifibatide.

Thrombocytopenia and thrombosis following treatment with the glycoprotein αIibβ3 antagonist, eptifibatide, are rare complications that are thought to be caused by patient antibodies specific for ligand-occupied αIibβ3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both, is not well understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies cause platelet clearance, we incubated normal platelets with serum derived from a 73 year old patient who developed profound thrombocytopenia following eptifibatide administration. This patient's serum was found to contain an αIibβ3-specific eptifibatide-dependent antibody that induced significant platelet secretion and aggregation in the presence, but not in the absence, of eptifibatide. Interestingly, a number of signaling molecules recently shown to function downstream of integrin engagement, including the integrin β3 cytoplasmic domain, the protein-tyrosine kinase Syk, and phospholipase Cγ2 (PLCγ2) each became tyrosine phosphorylated in platelets subjected to patient serum plus eptifibatide. Because the Immunoreceptor Tyrosine-based Activation Motif (ITAM)-bearing platelet Fc receptor, FcγRIIa, plays a prominent role in antibody-induced platelet activation, we examined its role in eptifibatide-induced platelet activation and clearance. We found that (1) FcγRIIa became rapidly and strongly tyrosine phosphorylated following incubation of normal platelets with patient serum+eptifibatide and (2) pre-incubation of platelets with Fab fragments of the FcγRIIa-specific monoclonal antibody, IV.3, completely blocked both phosphorylation of FcγRIIa, Syk, and PLCγ2, as well as platelet secretion and aggregation induced by patient serum+eptifibatide. Because amounts of patient sera are often limiting, a model system comprised of the αIibβ3 complex-specific mAb, AP2, and the ligand mimetic peptide, RGDW was employed, and found to faithfully mimic these reactions. Thus, intact, but not Fab fragments of, AP2 caused tyrosine phosphorylation of FcγRIIa, Syk, PLCγ2, as well as platelet secretion and aggregation, and these reactions could be completely blocked with Fab fragments of mAb IV.3, further implicating FcγRIIa in eptifibatide-induced thrombocytopenia/thrombosis. Finally, to gain further insight into how integrins functionally link to FcγRIIa to activate platelets, we subjected platelets from a patient with a variant form of Glanzmann thrombasthenia whose platelets express normal levels of αIibβ3 and FcγRIIa, but lack most of the β3 cytoplasmic domain, to similar treatment. Neither patient serum+eptifibatide nor AP2+RGDW were able to activate this patient's platelets, implicating the β3 cytoplasmic domain and its associated tyrosine kinases in eptifibatide-induced platelet activation. Taken together, these data suggest a novel mechanism involving both FcγRIIa and the cytoplasmic domain of integrin β3 in thrombocytopenia and thrombosis following administration of fibrinogen receptor antagonists.

Metalloproteinase Inhibitors Increase the Survival of Long-Term Refrigerated Platelets in Mice.

The αMβ2 receptor on macrophages, which recognizes clustered β-N-acetylglucosamine (βGlcNAc) residues on glycoprotein (GP) Ib-V-IX, is one phagocytic receptor involved in the removal of transfused refrigerated platelets. Although coverage of the exposed βGlcNAc residues by galactosylation prevents the clearance of murine platelets refrigerated for less than 4h, it does not impact the clearance of platelets refrigerated for 48h, suggesting that further changes occur in platelets during prolonged refrigeration. We have now found that murine GPIbα and GPV are partially shed from the platelet surface after refrigeration for 48h and re-warming. Other members of the complex, GPIX and GPIbβ, were not affected. Platelets isolated from mice with inactive ADAM17 enzyme, a GPIbα and GPV sheddase, had preserved surface GPIbα and decreased GPV shedding compared to WT mice after long-term refrigeration. 48h refrigerated platelets with inactive ADAM17 or platelets treated with the metalloproteinase inhibitor GM 6001, had increased survival when transfused into WT mice. Metalloproteinase inhibitor-treated and galactosylated, 48h refrigerated and re-warmed mouse platelets exhibited survivals equivalent to fresh platelets. We conclude that 48h long-term refrigeration and re-warming of platelets induces shedding of GPIbα and GPV in murine platelets, and that metalloproteinase inhibitors increase the survival of long-term refrigerated platelets in mice. Further changes in GPIb-V-IX in long-term refrigerated platelets may recruit additional phagocytic receptors to promote clearance, in addition to the αMβ2 receptor.

Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.

Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.

Incidence of HPA-9b in Testing for Neonatal Alloimmune Thrombocytopenia.

Background: Neonatal alloimmune thrombocytopenia (NAT) is caused by maternal alloantibodies that cross the placenta and cause increased clearance of fetal platelets. The alloantibodies are directed to incompatible human platelet antigens (HPA) inherited from the father and expressed on fetal platelet glycoproteins. Most cases of NAT are caused by HPA-1a, -5a, -5b, -15a, or -15b. Some are associated with immunization to a low frequency antigen found only in the paternal lineage. We investigated the incidence of the low frequency antigen HPA-9b in a population with suspected NAT, and compared the frequency to that found in a normal control population. HPA-9b is caused by a Val837Met polymorphism on alpha-IIb and is expressed only in a rare subset of individuals with a corresponding serine at position 843 that defines the HPA-3b antigen.Methods: We used PCR-SSP for HPA-9b genotyping. Antibody studies were performed by incubating maternal serum with intact platelets using flow cytometry, monoclonal antigen capture enzyme immunoassay, and radioimmunoprecipitation.Results: Of 294 normal control blood donors, 4 typed positive for HPA-9b, yielding a gene frequency of 0.0068. However, if only HPA-3b positive donor controls are considered (n=177) the gene frequency for HPA-9b is 0.011. Archived DNA from HPA-3b positive (HPA-3a3b or -3b3b) fathers of children with NAT were tested for HPA-9b. Of 94 fathers, 3 (3.2%) typed HPA-9a9b, yielding a gene frequency for HPA-9b of 0.016, not significantly different from the frequency for HPA-9b found in the HPA-3b-positive healthy controls (p>0.1). Similarily, when DNA from HPA-3b positive mothers was tested in tandem, 3 of 60 were positive for HPA-9b, yielding a gene frequency of 0.026. Of the 3 cases of suspected NAT (platelet count range 15,000 to 63,000) in which the mother and father were incompatible for HPA-9b, 2 had no other platelet antigen incompatibility, and one was incompatible for HPA-15b. None of the 3 maternal sera was positive for anti-platelet reactivity using radioimmunoprecipitation and paternal platelets as a target, and no anti-platelet antibody was detected either by flow cytometry or by GPIIb/IIIa antigen capture immunoassay with platelets from a HPA-9a9b donor.Interpretation: These results support the findings that HPA-9b is distributed in the general population at a frequency higher than initially suspected. However, we also found that the frequency of HPA-9b in fathers of children with suspected NAT was similar to that in the normal control population when only HPA-3b-positive (HPA-3a3b or -3b3b) targets were analyzed. Therefore, in the absence of a positive anti-platelet antibody, the significance of a maternal/paternal incompatibility for HPA-9b alone is not predictive of an increased risk of NAT.

IVIg-Primed Splenocytes Ameliorate Murine ITP in an MHC Class II-Unrestricted Manner.

We have previously demonstrated that IVIg-primed leukocytes can, upon transfer to naïve mice, completley recapitulate the therapeutic effects of IVIg in treating immune thrombocytopenia in a murine model. Dendritic cells (DC) appear to be the primary cellular target of IVIg in this model. The exact pathway by which IVIg-primed DC inhibit platelet clearance remains, however, unknown. DC are professional antigen presenting cells of haematopoietic origin that are specialized for the capture, processing and presentation of antigens to T cells via their MHC Class II. This primary function of DC is absolutely dependent on MHC Class II expression. Herein, we have evaluated the efficacy of action of IVIg-primed splenocytes from MHC mismatched mice in the amelioration of murine ITP. Mice expressing the Class II haplotype I-Ab (C57BL/6) were injected intravenously with IVIg-primed splenocytes from syngeneic mice, or Balb/C mice (Class II haplotype I-Ad /I-Ed) or CD1 mice (outbred, mixed Class II haplotype I-A/E) prior to the induction of thrombocytopenia by an anti-platelet antibody. We found that IVIg-primed splenocytes from all strains of mice tested were able to successfully ameliorate thrombocytopenia in C57BL/6 mice, regardless of Class II haplotype. None of the splenocytes primed with a control protein, BSA, ameliorated thrombocytopenia. To confirm our findings that IVIg-primed splenocytes function independent of MHC haplotype, we next employed IVIg-primed splenocytes from MHC Class II deficient mice and found these splenocytes were also able to successfully ameliorate murine thrombocytopenia. These data demonstrate a non-MHC-restricted function for DC in IVIg function, and suggest that IVIg-primed DC function occurs independent of MHC Class II expression in the amelioration of murine ITP. In addition, since this potential new cell-based therapy for ITP is not MHC-restricted, we speculate that IVIg-primed DC from any donor (including autologous) could theoretically be used to treat individuals with ITP or other autoimmune diseases without the requirement for donor matching.