Tinzaparin for venous thromboembolism in patients with renal impairment: a single-centre, prospective pilot study.

BACKGROUND: Low molecular weight heparins (LMWH) are used extensively for prophylaxis and treatment of venous thromboembolism (VTE), bridging therapy for warfarin and standard of care in cancer-associated VTE (CA-VTE). Tinzaparin has the highest molecular weight of all LMWH and relies least on renal clearance to Cockcroft-Gault creatinine clearance (CrCl) of 20 mL/min. Previous pharmacological studies have demonstrated safety and effectiveness in elderly patients. Prospective clinical trials have confirmed these findings to CrCl 20 mL/min and in CA-VTE. We describe the pilot program developed at Concord Repatriation General Hospital for tinzaparin. AIMS: We aim to confirm the deliverability of tinzaparin in patients with renal insufficiency. METHODS: Twenty patients were established on tinzaparin as therapeutic anticoagulation with CrCl or CKD-EPI estimated glomerular filtration rate (eGFR) 20-50 mL/min with an indication for anticoagulation. Tinzaparin was given as a subcutaneous injection at 175 units/kg as a single daily dose, rounded to the nearest vial size. Tinzaparin anti-Xa levels were tested at Days 2, 7 and 14 (±1 day) and transition to oral anticoagulants were allowed at clinician discretion. RESULTS: No accumulation of tinzaparin was seen into Day 14. Two patients required dose-adjustment, five patients had bleeding complications (two major, three minor) and four patients died during follow-up, all attributable to patients' comorbidities. CrCl and body surface area-standardised CrCl were significantly correlated with tinzaparin anti-Xa level only on Day 2, and this effect was lost when patients with CrCl >50 mL/min were excluded. Data from our cohort confirm the deliverability of therapeutic tinzaparin in patients with CrCl or CKD-EPI eGFR 20-50 mL/min. Bleeding and death outcomes were also comparable to other trials using tinzaparin in CA-VTE. CONCLUSION: For patients with renal insufficiency, tinzaparin represents an attractive alternative anticoagulant with once-daily administration in a range of potential indications.

Necrotic platelets provide a procoagulant surface during thrombosis.

A subpopulation of platelets fulfills a procoagulant role in hemostasis and thrombosis by enabling the thrombin burst required for fibrin formation and clot stability at the site of vascular injury. Excess procoagulant activity is linked with pathological thrombosis. The identity of the procoagulant platelet has been elusive. The cell death marker 4-[N-(S-glutathionylacetyl)amino]phenylarsonous acid (GSAO) rapidly enters a subpopulation of agonist-stimulated platelets via an organic anion-transporting polypeptide and is retained in the cytosol through covalent reaction with protein dithiols. Labeling with GSAO, together with exposure of P-selectin, distinguishes necrotic from apoptotic platelets and correlates with procoagulant potential. GSAO(+) platelets form in occluding murine thrombi after ferric chloride injury and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. These platelets form a procoagulant surface, supporting fibrin formation, and reduction in GSAO(+) platelets is associated with reduction in platelet thrombus size and fibrin formation. Analysis of platelets from human subjects receiving aspirin therapy indicates that these procoagulant platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway. These findings indicate that the major subpopulation of platelets involved in fibrin formation are formed via regulated necrosis involving cyclophilin D, and that they may be targeted independent of platelet activation.

Platelets as Biomarkers of Coronary Artery Disease.

Primary and secondary prevention of cardiovascular disease remain a public health priority. Effective risk stratification of patients is a central requisite for effective preventative care and several scoring systems incorporating biomarkers have been used for prognostication in patients to guide intervention decisions. Thrombosis of atherosclerotic coronary arteries is the main mechanism behind the acute coronary syndromes and since platelets play a pivotal role in the pathogenesis of thrombosis, atherogenesis, and angiogenesis, platelet-derived biomarkers are an attractive concept. This review assesses the potential and the limitations of a range of platelet-based assays as biomarkers for coronary artery disease.

Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis.

Extracellular vesicles (EVs) play a role in a variety of physiological and pathological processes. However, use of EVs as biomarkers has been hampered by limitations of current detection and enumeration methods. We compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for enumeration of cell culture-derived EVs. FT-FC and NTA utilising fluorescence mode (F-NTA) enumerated similar numbers of EVs stained with a membrane dye PKH67. Both methods were sufficiently sensitive to detect cell-derived EVs above the background of culture medium. Light scatter NTA (LS-NTA) detected 10-100× more particles than either fluorescence-based method but demonstrated poor specificity. F-NTA appeared to have better sensitivity for <100nm vesicles, however, the FT-FC method combined direct enumeration of EVs with high sensitivity and specificity in the >100nm range. Due to wider availability and higher degree of automation and standardisation, FT-FC is a reasonable surrogate to F-NTA for quantification of EVs. FROM THE CLINICAL EDITOR: Extracellular vesicles are small particles, which can act as tools for intercellular communication. One recent area of interest in EVs is their potentials as biomarkers. In this article, the authors investigated and compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for the detection of EVs and showed that FT- FC method could be more advantageous. This technique should provide a new alternative for the future.

In vitro characterization of rare anti-αIIbß3 isoantibodies produced by patients with Glanzmann thrombasthenia that severely block fibrinogen binding and generate procoagulant platelets via complement activation.

Background: Glanzmann thrombasthenia (GT) is a rare bleeding disorder caused by inherited defects of the platelet αIIbß3 integrin. Platelet transfusions can be followed by an immune response that can block integrin function by interfering with fibrinogen binding. Objectives: In this study, we aimed to determine the prevalence of such isoantibodies and better characterize their pathogenic properties. Methods: Twelve patients with GT were evaluated for anti-αIIbß3 isoantibodies. Sera from patients with GT with or without anti-αIIbß3 isoantibodies were then used to study their in vitro effect on platelets from healthy donors. We used several approaches (IgG purification, immunofluorescence staining, and inhibition of signaling pathways) to characterize the pathogenic properties of the anti-αIIbß3 isoantibodies. Results: Only 2 samples were able to severely block integrin function. We observed that these 2 sera caused a reduction in platelet size similar to that observed when platelets become procoagulant. Mixing healthy donor platelets with patients' sera or purified IgGs led to microvesiculation, phosphatidylserine exposure, and induction of calcium influx. This was associated with an increase in procoagulant platelets. Pore formation and calcium entry were associated with complement activation, leading to the constitution of a membrane attack complex (MAC) with enhanced complement protein C5b-9 formation. This process was inhibited by the complement 5 inhibitor eculizumab and reduced by polyvalent human immunoglobulins. Conclusion: Our data suggest that complement activation induced by rare blocking anti-αIIbß3 isoantibodies may lead to the formation of a MAC with subsequent pore formation, resulting in calcium influx and procoagulant platelet phenotype.

Tissue factor de-encryption, thrombus formation, and thiol-disulfide exchange.

Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

Procoagulant platelets: Laboratory detection and clinical significance.

Platelets play a critical role in both haemostasis and thrombosis, and it is now evident that not all platelets behave the same when they are called to action. A functionally distinct subpopulation of platelets forms in response to maximal agonist stimulation: the procoagulant platelet. This platelet subpopulation is defined by its ability to expose phosphatidylserine on its surface, allowing for coagulation factor complexes to form and generate bursts of thrombin and fibrin to stabilize platelet clots. Reduced levels of procoagulant platelets have been linked to bleeding in Scott's syndrome and haemophilia A patients, and elevated levels have been demonstrated in many thrombotic disorders, including identifying patients at higher risk for stroke recurrence. One obstacle for incorporating an assay for measuring procoagulant platelets into clinical management algorithms is the lack of consensus on the exact definition and markers for this subpopulation. This review will outline the biological characteristics of procoagulant platelets and the laboratory assays currently used to identify them in research settings. It will summarize the findings of clinical research demonstrating the relevance of measuring the procoagulant platelet levels in patients and will discuss how an appropriate assay can be used to elucidate the mechanism behind the formation of this subpopulation, facilitating novel drug discovery to improve upon current outcomes in cardiovascular and other thrombotic disorders.

Extramedullary presentation of acute leukaemia: a case of myeloid/natural killer cell precursor leukaemia.

AIM: Myeloid/natural killer (NK) cell leukaemia is characterised by coexpression of myeloid with natural killer cell antigens, a high incidence of extramedullary disease and an aggressive clinical course. METHODS: We report a case of a 28-year-old woman with myeloid/NK cell precursor acute leukaemia. Clinical presentation was correlated with leukaemic blast morphology, immunophenotype, cytogenetic analysis, molecular studies for clonal rearrangements and histological review. RESULTS: The patient had noted skin lesions and a breast infiltrate 4 months prior to the diagnosis. Bone marrow biopsy at the time of presentation revealed characteristic morphological features with a dense infiltrate of bizarre, pleomorphic blast cells with marked nuclear invagination and reniform shapes. Immunophenotypic analysis of the blasts displayed coexpression of myeloid and natural killer cell antigens with a relatively immature phenotype: CD34-,HLADR+, CD33+, CD56+, CD16-, CD57-, MPO-. Cyto- genetic analysis revealed a complex karyotype: del(6)(q21);-12 and add(19)(p13). Histological review of the previous breast biopsy was consistent with granulocytic sarcoma of the breast with a phenotype corresponding to the circulating blasts (positive cytoplasmic staining for myeloid markers, CD68 and CD31, and the NK cell marker CD56, with negative staining for MPO). Skin biopsy confirmed leukaemia cutis. CONCLUSION: Although nodal extramedullary disease is common in the myeloid/NK cell leukaemias, this is the first description of myeloid/NK cell leukemia primarily involving breast and skin. We speculate that CD56 may predispose to extramedullary localisation of tumour.

An immediate hemolytic reaction induced by repeated administration of oxaliplatin.

BACKGROUND: Platinum-based chemotherapy agents have been associated with potentially fatal acute immune-mediated hemolytic anemia. The target antigen, cause of the positive direct antiglobulin test (DAT) and mechanism of hemolysis have been the subject of controversy. CASE REPORT: We report a patient who developed a DAT-positive hemolytic episode after a red cell (RBC) transfusion was delivered during the infusion of her 17th cycle of oxaliplatin. Standard pretransfusion testing was uncomplicated; however, after infusion, the serum was no longer compatible with the transfused units and a strong (4+) panreactive IgG antibody was detected. RESULTS: The patient's serum from 10 days after the episode, only when therapeutic concentrations of oxaliplatin were added, reacted with all RBCs tested using the indirect antiglobulin test (IAT) (3+). The effect was retained with a purified IgG fraction and almost eliminated with IgG-depleted serum. Immunoprecipitation analysis revealed a band with the molecular weight of the Band 3 anion channel only in the presence of the patient's serum and oxaliplatin. CONCLUSION: Our investigations indicated that oxaliplatin interacted with both an IgG antibody and a RBC membrane epitope probably located on the Band 3 anion channel.