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The thrombopoietin receptor agonist romiplostim is approved for second-line use in chronic immune thrombocytopenia (ITP), but its effects in patients with ITP for ≤1 year are not well characterized. This analysis of pooled data from 9 studies included patients with ITP for ≤1 year (n = 311) or >1 year (n = 726) who failed first-line treatments and received romiplostim, placebo or standard of care. In subgroup analysis by ITP duration, patient incidences for platelet response at ≥75% of measurements were higher for romiplostim [ITP ≤1 year: 74% (204/277); ITP >1 year: 71% (450/634)] than for placebo/standard of care [ITP ≤1 year: 18% (6/34); ITP >1 year: 9% (8/92)]. Of patients with ≥9 months on study, 16% with ITP ≤1 year and 6% with ITP >1 year discontinued romiplostim and maintained platelet counts ≥50 × 109 /l for ≥6 months without ITP treatment (treatment-free remission). Independent of ITP duration, rates of serious adverse events and bleeding were lower with romiplostim than placebo/standard of care and thrombotic events occurred at similar rates. In this analysis, romiplostim and placebo/standard of care had similar safety profiles and romiplostim increased platelet counts in patients with either ITP ≤1 year or ITP >1 year, with more treatment-free remission in those with ITP ≤1 year.

A working group of clinicians and scientists was formed to review the clinical considerations for use of low-molecular-weight heparin (LMWH) biosimilars. LMWH are biological molecules of significant complexity; the full complexity of chemical structure is still to be elucidated. LMWH biosimilars are products that are biologically similar to their reference product and rely on clinical data from a reference product to establish safety and efficacy. The complex nature of LMWH molecules means that it is uncertain whether a LMWH biosimilar is chemically identical to its reference product; this introduces the possibility of differences in activity and immunogenicity. The challenge for regulators and clinicians is to evaluate the level of evidence required to demonstrate that a LMWH is sufficiently similar to the reference product. The consensus opinion of the working group is that prior to clinical use a LMWH biosimilar should have proven efficacy and safety, similar to the reference product with prospective studies, which should be confirmed with a proactive post-marketing pharmacovigilance programme.

Poly(N-isopropylacrylamide)-block-poly(l-lactic acid)-block-poly(N-isopropylacrylamide) (PNIPAAM-b-PLLA-b-PNIPAAM) and PNIPAAM-b-PDLA-b-PNIPAAM triblock copolymers with varying polylactic acid (PLA) lengths are synthesized using a combination of ring-opening polymerization and atom-transfer radical polymerization. Results of (1) H NMR and gel permeation chromatography analyses show that the copolymers have a well-defined triblock structure and the PLA segment lengths can be readily controlled with monomer feed ratio. Stereocomplexation between the enantiomeric PLA segments is confirmed with differential scanning calorimetry and wide-angle X-ray scattering. Dynamic light scattering experiments show that (1) the LCST of PNIPAAM in water could be tailored from 32 °C up to 38.5 °C by increasing the length of PLA segments and mixing copolymers of similar molecular weight with enantiomeric PLA segments to induce stereocomplexation, and (2) the LCST of each mixed copolymer system could be tailored within a 2-3 °C range of body temperature by manipulating the ratio of the enantiomeric copolymers in solution.

Immune thrombocytopenia can be a therapeutic challenge with multiple first- and second-line treatment options. A change in idiopathic thrombocytopenic purpura (ITP) definition and classification in recent consensus guidelines suggests that past descriptions of ITP presentation and outcome may be outdated. In this single centre retrospective analysis of patients with thrombocytopenia from 1 January 2005 to 1 June 2010, 139 patients met current ITP diagnostic criteria. About 54/139 were new presentations of primary ITP. Six- and 24-month response rates were 39% and 30% respectively. About 26/54 patients did not require treatment at presentation: 15 were followed up for at least 6 months and none required treatment subsequently. These results suggest that almost half of all new primary ITP do not need treatment.

A smart and recyclable oil absorber based on pH-responsive block copolymer modified magnetic nanoparticles is constructed for effective separation of oil-in-water emulsions. Superparamagnetic iron(III) oxide (Fe3 O4 ) nanoparticles are designed as the core materials for providing the separation force, and surface modification with poly(4-vinylpyridine-b-dimethyl siloxane-b-4-vinylpyridine) (P4VP-PDMS-P4VP) block copolymer is performed to supply switchable oil wettability. The mean hydrodynamic radius of the nanoparticles increases from 43.8 ± 3.6 to 75.2 ± 5.3 nm when the neutral pH is reduced to 3, due to the protonation and subsequent swelling of P4VP segments at lower pH. The nanoparticles show excellent separation performance and can absorb octadecene up to 78.2 times of their own weight, simply by switching the pH of the oil-in-water emulsion treated with the nanoparticles and using a magnetic field. The different dipole of P4VP-PDMS-P4VP and protonated P4VP-PDMS-P4VP, as confirmed by density functional theory calculations, is responsible for the different oil miscibility and wettability of the nanoparticles.

A qualitative platelet defect was demonstrated in a patient with heparin-induced thrombocytopenia. His platelets showed impaired aggregation to collagen and arachidonate and absent second wave aggregation to adenosine diphosphate and epinephrine but normal response to ristocetin. These aggregation abnormalities were similar to those reported in idiopathic thrombocytopenia and systemic lupus erythematosus. This platelet function defect may contribute to haemorrhagic complications which appear to be common in heparin-induced thrombocytopenia.

Patients with immune heparin-induced thrombocytopenia (HIT) possess antibodies that bind to a complex of platelet factor 4 (PF4) and heparin. We observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, we are able to provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd = 7-30 nM) and polystyrene adsorbed PF4 alone (Kd = 20-70 nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. We conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and therefore most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (suboptimal) promotes recognition of this epitope.

Heparin binds to platelets and can cause platelet proaggregating and potentiating effects, possibly causing thrombocytopenia, particularly in patients in intensive care with hyperaggregable platelets. In this study we compared the platelet proaggregating and potentiating effects of unfractionated heparin (UH), 2 low molecular weight (LMW) heparins, enoxaparin and dalteparin, and a heparinoid, danaparoid sodium (orgaran), to platelets of an ICU patient population and a normal control group. In both populations UH caused platelet aggregation in a dose-dependent manner. This occurred in the therapeutic range of the drug, with as little as 0.5 U/ml UH. The LMW heparins caused less and the heparinoid least platelet aggregation. Generally, the aggregation observed in ICU patients was greater than in the normal population. The potentiating effects of the 4 drugs in association with physiological agonists was examined. Similar patterns of potentiation were observed in both populations, with UH causing significant enhancement of platelet aggregation, the LMW heparins intermediate and heparinoid least enhancement. There was substantial variability in the individuals' platelets' reactions to the drugs, in particular to UH. Our findings suggest that UH has the greatest effect, the low molecular weight heparins an intermediate effect and the heparinoid the least propensity to cause platelet activation.

Although the physiological role of beta2-glycoprotein (B2GPI) is unknown, in vitro evidence indicates that B2GPI may be a natural anticoagulant. In this study we have examined whether fluctuations of plasma B2GPI occur in in vivo coagulation. Serial measurements of B2GPI and other anticoagulant proteins were performed in 51 patients with thrombotic (group 1: six patients with disseminated intravascular coagulation (DIC), group 2: venous (n = 4) or arterial (n = 170 thrombosis) and non-thrombotic disease (group 3: 24 patients undergoing elective surgery). Reductions in plasma B2GPI levels were seen in most patients which were roughly proportional to the severity of their illness. Particularly striking reductions of B2GPI, protein C (PC) and antithrombin III (AT-III) (mean +/- 95% CI: 42.7 +/- 8.6%, 42.1 +/- 14.8%, 39.1 +/- 28.4% respectively) were seen in group 1. The reductions in plasma B2GPI were significantly greater in group 1 than in the other groups. Dilutional factors explain most of the reductions in B2GPI, PC and AT-III in groups 2 and 3, but contribute little to group 1. In conclusion, although B2GPI behaves as a 'negative acute phase reactant', the magnitude of reduction of plasma B2GPI levels, accompanied by reductions in other anticoagulant proteins in patients with DIC, suggests specific consumption of B2GPI in in vivo coagulation. This study provides further evidence that B2GPI is an anticoagulant of physiological importance.

Characterization of IgG Fc receptors on CD34 antigen-expressing cell lines (KG-1 and KG-la)

Fc gamma receptors (Fc gamma Rs) are glycoproteins on platelet surface that bind IgG-containing immune complexes. However, excessive binding of immune complexes leads to platelet activation and thrombosis or increased platelet clearance and thrombocytopenia. In this study, Fc gamma R transcripts in platelets and megakaryoblastic cell line (Meg-01) were investigated using specifically designed oligonucleotides and a new quantitative in situ hybridization assay. Platelets and Meg-01 cells were found to express only Fc gamma RII transcripts. Of Fc gamma RIIA mRNA isoforms (Fc gamma RIIA, B and C), Fc gamma RIIA mRNA predominates in these cells. Platelets and Meg-01 cells contain both alternative spliced forms of Fc gamma RIIA mRNA, those with and without the transmembrane (TM) exon and both forms were present in near equal amounts. In contrast, Fc gamma RIIA transcript with the TM exon predominates in neutrophils and monocytes, suggesting that the splicing of the TM exon is under lineage-specific control.

Heparin-induced thrombocytopenia (HIT) is not only a common but also a potentially serious drug adverse effect. Unlike other drug-induced thrombocytopenias, HIT does not usually cause bleeding, but instead causes thrombosis. Thrombosis in HIT can lead to limb gangrene (requiring leg amputation) or even death. HIT is mediated by an antibody that recognizes an epitope on the platelet factor 4 (PF4)-heparin complex. The antibody-PF4-heparin complex binds to FcgammaRII receptors on the platelet surface and cross-links the receptors. This induces intense platelet activation and platelet aggregation, and simultaneously activates blood-coagulation pathways. These changes are probably the basis of the thrombotic events in HIT. Diagnosis of HIT should be made mainly on clinical criteria but should be confirmed whenever possible by laboratory tests, particularly in patients with comorbid conditions, in whom the diagnosis of HIT cannot be made with certainty without testing. The tests for HIT antibodies are either immunoassays (e.g. ELISA), or functional tests, (e.g. 14C-serotonin release assay). Once a clinical diagnosis of HIT is made, heparin should be ceased immediately and treatment with an alternative anticoagulant (such as danaparoid, r-hirudin or argatroban) commenced. This should continue for at least 5 days unless the diagnosis of HIT is subsequently proven to be incorrect. Warfarin should also be commenced when the patient is clinically stable and thrombosis is under control. There should be an overlap of a few days between warfarin and the alternative anticoagulant therapy.

Heparin-induced thrombocytopenia and thrombosis syndrome (HITTS) is an immune-mediated drug reaction that occurs 5-14 d after initiation of heparin therapy and is a potentially life-threatening thrombotic complication. The antibody-heparin-PF4 complexes cause platelet activation and generation of platelet microparticles. The need for anticoagulant treatment in asymptomatic thrombocytopenia is uncertain. However, treatment is warranted in HITTS, as illustrated in the case reported here. Danaparoid, r-Hirudin and argatroban are effective drugs. Danaparoid has a 10-50% in vitro cross-reactivity rate with the HIT antibodies, but has been proven to be clinically efficacious even in these cases. Here, we report a case of in vivo cross-reactivity with danaparoid, the patient showed an excellent recovery with r-Hirudin.

Summary. The 20210G!A mutation in the 3 0 -untranslated (UT) region of the prothrombin gene is extremely rare or absent in the Chinese population (0 in 449 subjects, 140 with a history of thromboembolism). This is in contrast to the results from 302 Caucasians from Australia in our study (4·6% in 153 patients with a thromboembolic history and 1·3% in 149 patients with no history). This rarity implies that the variant of the prothrombin gene is probably not the main cause of venous thromboembolism in the Chinese population. Even among Caucasians this mutation accounts for only a minor percentage of all patients with thromboembolism. The relatively low incidence of venous thromboembolism in the Chinese population compared with Caucasians is probably as a result of the low prevalence of factor V Leiden or other environmental or genetic factors.

The interaction of the heparin-dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin-induced thrombocytopenia. Both normal platelets and Bernard-Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib. GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV.3) strongly inhibited the heparin-dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti-GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti-GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C-serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin-dependent antibody with platelets and heparin is mediated by a Fc-dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti-GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.

The plasma of two patients with heparin-induced thrombocytopenia has been shown to cause platelet aggregation in the presence of heparin. The platelet aggregating factor was isolated in the IgG reaction of the patients' sera suggesting that it was an antibody. This heparin anti-platelet antibody (HAP-Ab) induced platelet aggregation and release but did not cause platelet lysis, although it fixed complement. Platelet aggregation was inhibited by EDTA and by inactivation of complement. There was a significant production of malondialdehyde (MDA) and thromboxane B2 (TXB2) implying a role of the prostaglandin synthesis pathway in HAP-Ab induced aggregation. ADP-release also appeared to be involved as apyrase blocked aggregation while hirudin, a thrombin inhibitor, had no effect. The thrombotic complications that have recently been reported in patients with heparin-induced thrombocytopenia may be explained by some effects of HAP-Ab on platelets, namely: the antibody mediated platelet factor 3 release, prostaglandin endoperoxides and thromboxane A2 (TXA2) production and platelet aggregation in vivo. These HAP-Ab mediated effects could be inhibited by anti-platelet drugs such as aspirin, indomethacin and dipyridamole and thus may have therapeutic implications.

A 60-yr-old female presented with typical thrombotic thrombocytopenic purpura (TTP). She remained in coma with frequent seizures for 1 wk, with persisting severe thrombocytopenia and microangiopathic haemolytic anaemia, despite treatment with prednisolone, plasma exchange, fresh frozen plasma, sulphinpyrazone and dipyridamole. Splenectomy induced haematological improvement within 1 d, there was cessation of fitting after 2 d, and full neurological recovery ensued over 3 wk. Laboratory studies did not reveal the presence of a platelet-aggregating factor (PAF), stated to be present in some two-thirds of cases. While plasma exchange and plasma infusion are beneficial in many cases, splenectomy appears still to be of value in unresponsive disease.

A patient with severe cardiac failure due to idiopathic congestive cardiomyopathy resistant to conventional therapy, responded to amrinone, a new non-glycosidic non-catecholamine positive inotropic agent. Amrinone produced significant haemodynamic improvement during both intravenous and oral administration. Thrombocytopaenia, however, was induced, necessitating cessation of the drug. Amrinone is a promising, orally effective inotropic agent for patients with refractory congestive cardiac failure, but is limited by its tendency to cause thrombocytopaenia.

A 62-year-old woman with an unusually protracted post-transfusion purpura failed to respond to three 3-litre plasma exchanges and prednisolone (50 mg/day) for 10 days but then had a good though transient response to two courses of high-dose immunoglobulin infusions with cessation of bleeding. The subsequent recurrent thrombocytopenia was mild and asymptomatic and only partially controlled by corticosteroids but finally resolved completely after 5 months.