L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Transfusion efficacy of ABO major-mismatched platelets (PLTs) in children is inferior to that of ABO-identical PLTs

BACKGROUND: ABO major compatibility is essential in transfusions of red blood cells but is not requisite in PLT transfusions. In adults there is some evidence that transfusion efficacy of ABO blood group-identical platelets (PLTs) is superior to major-mismatched PLTs. However, in children this question has not been investigated for more than 30 years. STUDY DESIGN AND METHODS: In a prospective study, the efficacy (based on the 1-hour percentage of PLT recovery [PPR(1hr)]) of 400 eligible ABO blood group-identical or out-of-group apheresis PLT concentrates (APCs), transfused mainly prophylactically to 50 children with hematologic malignancies, solid tumors, or aplastic anemia was investigated. The primary objective was to compare PPR(1hr) between ABO-identical and major-mismatched transfusions. RESULTS: After ABO major-mismatched transfusions, PPR(1hr) was significantly lower than after ABO blood group-identical transfusions (median 21% vs. 32%; p = 0.034). Multivariate analysis showed major-mismatched transfusions to be significantly more often unsuccessful than identical transfusions (odds ratio [OR], 3.97; 95% confidence interval [CI], 1.52-10.39; p = 0.005). Using flow cytometry and fluorescent microscopy, it could be demonstrated that PLTs of subgroup A(1), significantly expressing A antigen on their surface, were rapidly cleared from the circulation of group O or B recipients. In contrast, major-mismatched transfusions of A(2) PLTs, expressing no detectable A antigen, were as successful as identical transfusions (OR, 1.13; 95% CI, 0.16-7.88; p = 0.90). CONCLUSION: These data clearly indicate that in children ABO major-mismatched PLT transfusions result in inferior transfusion efficacy, with the only exception of group A(2) PLTs. ABO minor-mismatched PLTs showed comparable efficacy to identical transfusions.

Fecal calprotectin more accurately reflects endoscopic activity of ulcerative colitis than the Lichtiger Index, C-reactive protein, platelets, hemoglobin, and blood leukocytes

BACKGROUND The correlation between noninvasive markers with endoscopic activity according to the modified Baron Index in patients with ulcerative colitis (UC) is unknown. We aimed to evaluate the correlation between endoscopic activity and fecal calprotectin (FC), C-reactive protein (CRP), hemoglobin, platelets, blood leukocytes, and the Lichtiger Index (clinical score). METHODS UC patients undergoing complete colonoscopy were prospectively enrolled and scored clinically and endoscopically. Samples from feces and blood were analyzed in UC patients and controls. RESULTS We enrolled 228 UC patients and 52 healthy controls. Endoscopic disease activity correlated best with FC (Spearman's rank correlation coefficient r = 0.821), followed by the Lichtiger Index (r = 0.682), CRP (r = 0.556), platelets (r = 0.488), blood leukocytes (r = 0.401), and hemoglobin (r = -0.388). FC was the only marker that could discriminate between different grades of endoscopic activity (grade 0, 16 [10-30] μg/g; grade 1, 35 [25-48] μg/g; grade 2, 102 [44-159] μg/g; grade 3, 235 [176-319] μg/g; grade 4, 611 [406-868] μg/g; P < 0.001 for discriminating the different grades). FC with a cutoff of 57 μg/g had a sensitivity of 91% and a specificity of 90% to detect endoscopically active disease (modified Baron Index ≥ 2). CONCLUSIONS FC correlated better with endoscopic disease activity than clinical activity, CRP, platelets, hemoglobin, and blood leukocytes. The strong correlation with endoscopic disease activity suggests that FC represents a useful biomarker for noninvasive monitoring of disease activity in UC patients.

Platelets increase while serum reduces the differentiation and activity of osteoclasts in vitro

Platelets modulate formation of osteoclasts and osteoblasts, but research with different preparations of platelets remains inconclusive. Here, we assessed whether serum components modulate the effect of platelet preparations. In murine bone marrow cultures, osteoclastogenesis was investigated in the presence of platelet-released supernatant (PRS), serum containing PRS (SC-PRS), and serum. Osteoclastogenesis was quantified by the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, TRAP activity and resorption assays. Also human osteoclastogenesis assays were performed. Viability and proliferation were tested by MTT and (3) [H]thymidine incorporation assays, respectively. Osteoblastogenesis was assessed by histochemical staining for alkaline phosphatase-of murine bone marrow cultures and human MG63 cells. We found PRS to increase the number of TRAP(+) multinucleated cells in the early phase and TRAP activity in the later phase of osteoclastogenesis. SC-PRS and serum decreased the number and activity of TRAP(+) multinucleated cells. Both serum containing preparations reduced viability and proliferation of hematopoietic progenitors. PRS decreased the numbers of alkaline phosphatase-positive colonies while SC-PRS and serum increased osteoblastmarkers in MG63. Proliferation of MG63 was stimulated by all preparations. These results show that activated platelets support osteoclastogenesis, while platelet preparations that contain serum components decrease osteoclastogenesis and increase osteoblastogenesis in vitro, suggesting that serum components modulate the effects of platelets on osteoclastogenesis and osteoblastogenesis.

Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis

Background: A clinically relevant bleeding diathesis is a frequent diagnostic challenge, which sometimes remains unexplained despite extensive investigations. The aim of our work was to evaluate the diagnostic utility of functional platelet testing by flow cytometry in this context. Methods: In case of negative results after standard laboratory work-up, flow cytometric analysis (FCA) of platelet function was done. We performed analysis of surface glycoproteins (GP) Ibα, IIb, IIIa; P-selectin expression and PAC-1 binding after graded doses of ADP, collagen and thrombin; content/secretion of dense granules; ability to generate procoagulant platelets. Results: Out of 437 patients investigated with standard tests between January 2007 and December 2011, we identified 67 (15.3%) with high bleeding scores and non-diagnostic standard laboratory work-up including platelet aggregation studies. Among these patients FCA revealed some potentially causative platelet defects: decreased dense-granule content/secretion (n=13); decreased alpha-granule secretion induced by ADP (n=10), convulxin (n=4) or thrombin (n=3); decreased fibrinogen-receptor activation induced by ADP (n=11), convulxin (n=11) or thrombin (n=8); decreased generation of COAT-platelets, i.e. highly procoagulant platelets induced by simultaneous activation with collagen and thrombin (n=16). Conclusion: Our work confirms that storage pool defects are frequent in patients with a bleeding diathesis and normal coagulation and platelet aggregations studies. Additionally, flow cytometric analysis is able to identify discrete platelet activation defects. In particular, we show for the first time that a relevant proportion of these patients has an isolated impaired ability to generate COAT-platelets - a conceptually new defect in platelet procoagulant activity, that is missed by conventional laboratory work-up. © 2014 Clinical Cytometry Society.

The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects.

1-deamino-8-d-arginine vasopressin (desmopressin [DDAVP]) is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na(+)/Ca(2+) fluxes, before and 2 and 4 hours after DDAVP (0.3 µg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, δ-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of α-granules and GPIIb-IIIa activation induced by adenosine 5'-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na(+)/Ca(2+) concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na(+)/Ca(2+) mobilization. This report indicates that the beneficial hemostatic effect of DDAVP is not limited to an increase in large VWF multimers. An enhancement of platelet procoagulant activity appears to be an additional and (at least in platelet disorders) -possibly clinically relevant mechanism of DDAVP's action.

Transfusion efficacy of apheresis platelet concentrates irradiated at the day of transfusion is significantly superior compared to platelets irradiated in advance.

BACKGROUND Gamma irradiation is currently the standard care to avoid transfusion-associated graft-versus-host disease. Guidelines on gamma irradiation of blood components state that platelets (PLTs) can be irradiated at any stage in their 5-day storage and can thereafter be stored up to their normal shelf life of 5 days after collection. In this study, we explored whether the timing of irradiation has an effect on transfusion efficacy of apheresis PLT concentrates (APCs). METHODS Based on the 1-hour percent PLT recovery (PPR1h), transfusion efficacy of 1,000 eligible APCs transfused to 144 children were evaluated retrospectively. PPR1h was compared in transfused APCs irradiated at the day of transfusion and APCs irradiated in advance. RESULTS In univariate analysis, transfusion efficacy of APCs irradiated in advance was significantly lower than that of APCs irradiated at the day of transfusion (mean PPR1h 27.7 vs. 35.0%; p = 0.007). This was confirmed in multivariate analysis (p = 0.030). Compared to non-irradiated APCs, transfusion efficacy of APCs irradiated at the day of transfusion was not significantly inferior (mean difference -2.8%; 95% CI -6.1 to 0.5%; p = 0.092), but APCs irradiated in advance were clearly less efficient (mean difference -8.1%; 95% CI -12.2 to -4.0%; p < 0.001). CONCLUSION Our data strongly support that APCs should not be irradiated in advance, 1.e., ≥24 h before transfusion.

Improvement of platelets after SVR among patients with chronic HCV infection and advanced hepatic fibrosis.

BACKGROUND & AIMS Patients with chronic hepatitis C virus (HCV) infection may develop cirrhosis with portal hypertension, reflected by decreased platelet count and splenomegaly. This retrospective cohort study aimed to assess changes in platelet counts after antiviral therapy among chronic HCV-infected patients with advanced fibrosis. METHODS Platelet counts and spleen sizes were recorded in an international cohort of patients with Ishak 4-6 fibrosis who started antiviral therapy between 1990 and 2003. Last measured platelet counts and spleen sizes were compared to their pre-treatment values (within 6 six months prior to the start of therapy). All registered platelet count measurements from 24 week following cessation of antiviral therapy were included in repeated measurement analyses. RESULTS This study included 464 patients; 353 (76%) had cirrhosis and 187 (40%) attained sustained virological response (SVR). Among patients with SVR, median platelet count, increased by 35 x10(9) /L (IQR 7-62, p<0.001). In comparison, patients without SVR showed a median decline of 17 x10(9) /L (IQR -5-47, p<0.001). In a subgroup of 209 patients, median decrease in spleen size was 1.0 cm (IQR 0.3-2.0) for patients with SVR, while median spleen size increased with 0.6 cm (IQR -0.1-2.0, p<0.001) among those without SVR. The changes in spleen size and platelet count were significantly correlated (R=-0.41, p<0.001). CONCLUSIONS Among chronic HCV-infected patients with advanced hepatic fibrosis the platelet counts improved following SVR and the change in platelets correlated with the change in spleen size following antiviral therapy. These results suggest that HCV eradication leads to reduced portal pressure. This article is protected by copyright. All rights reserved.

The Role of Platelets in Venous Thromboembolism

Multiple factors contribute to the risk of venous thromboembolism (VTE). Platelets have attracted much interest in arterial cardiovascular disease, whereas their role in VTE has received much less attention. Recent evidence suggests that platelets may play a more important role in VTE than previously anticipated. This review discusses the mechanisms that link platelets with venous thrombotic disease and their potential applications as novel risk factors for VTE. In addition, animal studies and randomized clinical trials that highlight the potential effect of antiplatelet therapy in venous thrombosis are evaluated to assess the role of platelets in VTE. The clinical significance of platelets for VTE risk assessment in specific patient cohorts and their role as a suitable therapeutic target for VTE prevention is acknowledged. The role of platelets in VTE is a promising field for future research.

WASP plays a novel role in regulating platelet responses dependent on alphaIIbbeta3 integrin outside-in signalling

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent -yet- actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by alphaIIbbeta3 integrin outside-in signalling, we questioned whether its function might be linked to integrin. Agonist-induced alphaIIbbeta3 activation (PAC-1 binding) was normal for patient platelets, indicating normal integrin inside-out signalling. Inside-out signalling (fibrinogen, JON/A binding) was also normal for wasp-deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp-deficient murine platelets, indicating decreased integrin outside-in responses. Another integrin outside-in dependent response, fibrin clot retraction, involving contraction of the post-aggregation actin cytoskeleton, was also decreased for patient platelets and wasp-deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp-deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro-coagulant response, was enhanced for WASP-deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro-aggregatory and pro-coagulant responses downstream of integrin outside-in signalling.

von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models

Deep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here we show that 48-hour flow restriction in the inferior vena cava (IVC) results in the development of thrombi structurally similar to human deep vein thrombi. von Willebrand factor (VWF)-deficient mice were protected from thrombosis induced by complete (stasis) or partial (stenosis) flow restriction in the IVC. Mice with half normal VWF levels were also protected in the stenosis model. Besides promoting platelet adhesion, VWF carries Factor VIII. Repeated infusions of recombinant Factor VIII did not rescue thrombosis in VWF(-/-) mice, indicating that impaired coagulation was not the primary reason for the absence of DVT in VWF(-/-) mice. Infusion of GPG-290, a mutant glycoprotein Ib?-immunoglobulin chimera that specifically inhibits interaction of the VWF A1 domain with platelets, prevented thrombosis in wild-type mice. Intravital microscopy showed that platelet and leukocyte recruitment in the early stages of DVT was dramatically higher in wild-type than in VWF(-/-) IVC. Our results demonstrate a pathogenetic role for VWF-platelet interaction in flow disturbance-induced venous thrombosis.

CD36 and macrophage scavenger receptor a modulate foam cell formation via inhibition of lipid-laden platelet phagocytosis

CD34 (+) progenitor cells are a promising source of regeneration in atherosclerosis or ischemic heart disease. However, as recently published, CD34(+) progenitor cells have the potential to differentiate not only into endothelial cells but also into foam cells upon interaction with platelets. The mechanism of platelet-induced differentiation of progenitor cells into foam cells is as yet unclear. In the present study we investigated the role of scavenger receptor (SR)-A and CD36 in platelet-induced foam cell formation. Human CD34(+) progenitor cells were freshly derived from human umbilical veins and were co-incubated with platelets (2 x 10(8)/mL) up to 14 days resulting in large lipid-laden foam cells. Developing macrophages expressed SR-A, CD36, and Lox-1 as measured by fluorescent-activated cell sorting analysis. The presence of a blocking anti-CD36 or anti-SR-A antibody nearly abrogated foam cell formation, whereas anti-Lox-1 did not affect foam cell formation. Consistently blocking either anti-CD36 or anti-SR-A antibody significantly reduced the phagocytosis of lipid-laden platelets by macrophages. We conclude that CD36 and SR-A play an important role in platelet-induced foam cell formation from CD34(+) progenitor cells and thus represent a promising target to inhibit platelet-induced foam cell formation.

Gemtuzumab ozogamicin as postremission treatment in AML at 60 years of age or more: results of a multicenter phase 3 study

In older patients with acute myeloid leukemia (AML), the prevention of relapse has remained one of the major therapeutic challenges, with more than 75% relapses after complete remission. The anti-CD33 immunotoxin conjugate gemtuzumab ozogamicin (GO) has shown antileukemic remission induction activity in patients with relapsed AML. Patients with AML or refractory anemia with excess blasts in first complete remission attained after intensive induction chemotherapy were randomized between 3 cycles of GO (6 mg/m(2) every 4 weeks) or no postremission therapy (control) to assess whether GO would improve outcome. The 2 treatment groups (113 patients receiving GO vs 119 control patients) were comparable with regard to age (60-78 years, median 67 years), performance status, and cytogenetics. A total of 110 of 113 received at least 1 cycle of GO, and 65 of 113 patients completed the 3 cycles. Premature discontinuation was mainly attributable to incomplete hematologic recovery or intercurrent relapse. Median time to recovery of platelets 50 x 10(9)/L and neutrophils 0.5 x 10(9)/L after GO was 14 days and 20 days. Nonhematologic toxicities were mild overall, but there was 1 toxic death caused by liver failure. There were no significant differences between both treatment groups with regard to relapse probabilities, nonrelapse mortality, overall survival, or disease-free survival (17% vs 16% at 5 years). Postremission treatment with GO in older AML patients does not provide benefits regarding any clinical end points. The HOVON-43 study is registered at The Netherlands Trial Registry (number NTR212) and at http://www.controlled-trials.com as ISRCTN77039377.

Role of complement and perspectives for intervention in ischemia-reperfusion damage

Reperfusion of an organ following prolonged ischemia instigates the pro-inflammatory and pro-coagulant response of ischemia / reperfusion (IR) injury. IR injury is a wide-spread pathology, observed in many clinically relevant situations, including myocardial infarction, stroke, organ transplantation, sepsis and shock, and cardiovascular surgery on cardiopulmonary bypass. Activation of the classical, alternative, and lectin complement pathways and the generation of the anaphylatoxins C3a and C5a lead to recruitment of polymorphonuclear leukocytes, generation of radical oxygen species, up-regulation of adhesion molecules on the endothelium and platelets, and induction of cytokine release. Generalized or pathway-specific complement inhibition using protein-based drugs or low-molecular-weight inhibitors has been shown to significantly reduce tissue injury and improve outcome in numerous in-vitro, ex-vivo, and in-vivo models. Despite the obvious benefits in experimental research, only few complement inhibitors, including C1-esterase inhibitor, anti-C5 antibody, and soluble complement receptor 1, have made it into clinical trials of IR injury. The results are mixed, and the next objectives should be to combine knowledge and experience obtained in the past from animal models and channel future work to translate this into clinical trials in surgical and interventional reperfusion therapy as well as organ transplantation.

Biocompatibility assessment of the first generation PediaFlow pediatric ventricular assist device

The PediaFlow pediatric ventricular assist device is a miniature magnetically levitated mixed flow pump under development for circulatory support of newborns and infants (3-15 kg) with a targeted flow range of 0.3-1.5 L/min. The first generation design of the PediaFlow (PF1) was manufactured with a weight of approximately 100 g, priming volume less than 2 mL, length of 51 mm, outer diameter of 28 mm, and with 5-mm blood ports. PF1 was evaluated in an in vitro flow loop for 6 h and implanted in ovines for three chronic experiments of 6, 17, and 10 days. In the in vitro test, normalized index of hemolysis was 0.0087 ± 0.0024 g/100L. Hemodynamic performance and blood biocompatibility of PF1 were characterized in vivo by measurements of plasma free hemoglobin, plasma fibrinogen, total plasma protein, and with novel flow cytometric assays to quantify circulating activated ovine platelets. The mean plasma free hemoglobin values for the three chronic studies were 4.6 ± 2.7, 13.3 ± 7.9, and 8.8 ± 3.3 mg/dL, respectively. Platelet activation was low for portions of several studies but consistently rose along with observed animal and pump complications. The PF1 prototype generated promising results in terms of low hemolysis and platelet activation in the absence of complications. Hemodynamic results validated the magnetic bearing design and provided the platform for design iterations to meet the objective of providing circulatory support for young children with exceptional biocompatibility.