On-pump Cardiac Surgery Enhances Platelet Renewal and Impairs Aspirin Pharmacodynamics: Effects of Improved Dosing Regimens.

On-pump cardiac surgery may trigger inflammation and accelerate platelet cyclooxygenase-1 renewal, thereby modifying low-dose aspirin pharmacodynamics. Thirty-seven patients on standard aspirin 100 mg once-daily were studied before surgery and randomized within 36 hours postsurgery to 100 mg once-daily, 100 mg twice-daily, or 200 mg once-daily for 90 days. On day 7 postsurgery, immature and mature platelets, platelet mass, thrombopoietin, glycocalicin, leukocytes, C-reactive protein, and interleukin-6 significantly increased. Interleukin-6 significantly correlated with immature platelets. At day 7, patients randomized to 100 mg once-daily showed a significant increase in serum thromboxane (TX)B2 within the 24-hour dosing interval and urinary TXA2 metabolite (TXM) excretion. Aspirin 100 mg twice-daily lowered serum TXB2 and prevented postsurgery TXM increase (P < 0.01), without affecting prostacyclin metabolite excretion. After cardiac surgery, shortening the dosing interval, but not doubling the once-daily dose, rescues the impaired antiplatelet effect of low-dose aspirin and prevents platelet activation associated with acute inflammation and enhanced platelet turnover.

Qualitative and quantitative modifications of von Willebrand factor in patients with essential thrombocythemia and controlled platelet count.

BACKGROUND: Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleeding in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. OBJECTIVES: We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. PATIENTS/METHODS: Sixty-nine ET patients (M = 29; median age, 62 [48-70] years; platelets, 432 [337-620] × 10(3)  µL(-1) ), 69 matched controls and 10 subjects with reactive thrombocytosis (RT) were studied. VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoproteinV (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. RESULTS: In ET patients, VWF:Ag was increased by 31 ± 13% vs. controls (P < 0.01), without dependence of blood groups, while VWF:act was reduced by 21 ± 12% vs. controls and by 50 ± 24% vs. RT (P < 0.01). The VWF:act/VWF:Ag ratios in ET were reduced by 35 ± 17% vs. controls and RT patients (P < 0.001) and significantly associated with: immature or total platelet counts, GC, GpV and TXB2 . In multivariable analysis, only GC inversely predicted ET patients' VWF:act/VWF:Ag ratios (ß = -0.42, P = 0.01). By electrophoresis analyses, high-molecular-weight VWF multimers were variably reduced with atypical cleavage bands in ET only. VWF:propeptide, ADAMTS-13 and elastase levels were normal in ET patients. Platelet-associated ADAM-10 and ADAM-17 hydrolyzed VWFm in vitro, showing patterns similar to those in ET samples. CONCLUSIONS: In ET patients with controlled platelet counts, the VWF:act/VWF:Ag ratio is decreased and predicted by GC, a product of platelet activation. ADAM-10 and/or ADAM-17 might be involved. In vivo platelet activation, which characterizes ET, might contribute to disease-specific VWF alterations.

Novel path to IL-6 trans-signaling through thrombin-induced soluble IL-6 receptor release by platelets.

Interleukin (IL)-6 is a multifunctional cytokine with a critical role in inflammatory, immunoregulatory and haemopoietic responses. Its receptor consists of an ubiquitously expressed membrane transducing element (gp130) and of the specific element IL-6R-alpha (gp80), present only on hepatocytes and some leukocyte subsets. IL-6R-alpha also exists as soluble protein (sIL-6R) that, in the presence of IL-6, forms a complex able to bind gp130 and, thanks to the mechanism called trans-signaling, transduces IL-6 effect through tyrosine phosphorylation and activation of the signal transducer and transcription activator (STAT)-3. The aim of this study was to analyze the bidirectional relationships between platelet aggregation and IL-6-dependent effects. While platelets do not produce IL-6, we found that resting platelets express gp130, but not gp80, on their membranes. Upon activation by thrombin or calcium ionophore A23187, but not by ADP, the IL-6R-alpha is released in soluble form, while cangrelor, the specific inhibitor of P2Y12 receptor, can partially inhibit sIL-6R release. This sIL-6R is biologically active and, in the presence of IL-6, can trigger IL-6 trans-signaling, inducing an autocrine activation loop (as measured by an increase in gp80 and gp130 content) and STAT3 phosphorylation. On the other hand, IL-6 trans-signaling has no effect on platelet degranulation or aggregation by itself, nor on thrombin-induced platelet aggregation. Our data add an important piece to the puzzle of thrombosis and inflammation: in the presence of IL-6, which can be produced by stressed endothelial cells, the platelet-derived IL-6 trans-signaling could be crucial for the evolution of inflammation within a damaged vessel.

High mobility group box 1 is a novel substrate of dipeptidyl peptidase-IV.

AIMS/HYPOTHESIS: High mobility group box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. Previous studies have shown that topical application of HMGB1 to skin wounds of mouse models of diabetes enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. Dipeptidyl peptidase IV (DPP-IV/CD26) is a protease whose activity is increased in diabetes and whose inhibition improves glucose tolerance. Since HMGB1 contains potential DPP-IV cleavage sites, we determined whether HMGB1 may be a substrate for DPP-IV and whether DPP-IV-mediated cleavage may alter the biological activity of HMGB1. METHODS: Reversed phase HPLC, mass spectrometry and western blot analyses were performed to analyse and identify HMGB1 peptides generated following DPP-IV digestion. HMGB1 angiogenic functions in the presence of DPP-IV were evaluated in vitro and in vivo. HMGB1 protein was detected in the serum of type 2 diabetic patients before and after treatment with DPP-IV inhibitors. RESULTS: DPP-IV cleaved HMGB1 at its N-terminal region and affected its angiogenic functions. Specifically, DPP-IV inhibited HMGB1-induced endothelial cell migration and capillary-like structure formation, as well as HMGB1-mediated vascular network formation in Matrigel implants in mice. We had previously found that HMGB1 promoted endothelial cell migration through activation of extracellular regulated kinase signalling pathway. Here we showed that such an effect was abolished in the presence of DPP-IV. Finally, the N-terminal truncated form of HMGB1 was detected in the serum of type 2 diabetic patients, in whom DPP-IV inhibitors enhanced the levels of full-length HMGB1. CONCLUSIONS/INTERPRETATION: DPP-IV cleaves HMGB1 and, via this mechanism, inhibits HMGB1 angiogenic activity. Treatment with DPP-IV inhibitors may enhance HMGB1 activity in diabetic patients, thereby improving angiogenesis in this condition.

Targeting indoleamine 2,3-dioxygenase (IDO) to counteract tumour-induced immune dysfunction: from biochemistry to clinical development.

The enzyme indoleamine 2,3-dioxygenase (IDO) regulates immune responses through the capacity to degrade the essential amino acid tryptophan into kynurenine and other downstream metabolites that suppress effector T-cell function and favour the differentiation of regulatory T cells. Considerable experimental evidence indicates that IDO can be expressed by dendritic cells, by tumour cells or by surrounding stromal cells, either within proximity of the tumour or at distal sites. Recent advances in the biochemistry of IDO and in our understanding of the biological relevance of IDO-mediated tryptophan consumption to the establishment of dominant immune tolerance to cancer will be summarised and discussed. Within the wider context of cancer immunotherapy, this Review also delineates how IDO could be exploited as a molecular target for therapeutic intervention in order to boost anti-cancer immunity.

Defective platelet responsiveness to thrombin and protease-activated receptors agonists in a novel case of gray platelet syndrome: correlation between the platelet defect and the alpha-granule content in the patient and four relatives.

BACKGROUND: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly. METHODS AND RESULTS: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. CONCLUSIONS: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.

Low-grade exercise enhances platelet aggregability in patients with obstructive coronary disease independently of myocardial ischemia.

Moderate and strenuous exercise is known to enhance platelet aggregability in patients with obstructive coronary artery disease (CAD), but the effect of low-grade exercise is not known. We assessed shear-induced platelet aggregability before and after mild exercise (less than or equal to stage III of the modified Bruce protocol) in 27 patients with documented CAD who were receiving aspirin and in 12 subjects without CAD (controls). Ex vivo platelet aggregability was assessed in flowing whole blood as the time to occlude a collagen and adenosine diphosphate-coated ring; shorter times indicated greater aggregability. Aggregability, plasma von Willebrand factor (vWF) antigen, platelet and white cell counts, and hematocrit were measured at baseline, immediately after exercise (peak), and at 30 and 180 minutes after exercise. Exercise of similar workloads induced myocardial ischemia in 14 patients (group 1), but not in the other 13 (group 2) nor in controls. Both patient groups showed a reduction in aggregation time at peak exercise compared with baseline (group 1: 84+/-17 seconds at peak vs 96+/-22 seconds at baseline; group 2: 84+/-20 seconds at peak vs 99+/-20 seconds at baseline; p <0.03 for both comparisons), with a return to baseline values within 180 minutes. No significant variation occurred in controls (89+/-18 seconds at peak vs 85+/-21 second at baseline). Changes in vWF antigen did not differ significantly among groups. Aggregation times did not correlate with hematocrit or platelet and white cell counts. Thus, even low-grade exercise transiently enhances whole blood platelet aggregability in patients with obstructive CAD, but not in controls. The effect is independent of myocardial ischemia, occurs despite aspirin, and is likely dependent on hemodynamic factors interacting with coronary obstructions or dysfunctional endothelium.

Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects.

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.

Thrombin interaction with platelet GpIB: role of the heparin binding domain.

The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Complications and surgical indications in 144 cases of nonmetastatic osteosarcoma of the extremities treated with neoadjuvant chemotherapy.

From September 1986 to December 1989, 144 patients with osteosarcoma of the extremities were treated with combined surgery and neoadjuvant chemotherapy. The disease-free survival was 79% for good responders (necrosis greater than 90%) and 72% for poor responders (necrosis less than 90%), and the local recurrence rate was low. Improvement in long-term prognosis and the increase of limb-sparing surgery determine a higher rate of immediate and late complications. Most of the complications were observed in limb-salvage procedures; 63% of these procedures presented one or more complications. In nine rotationsplasties, there were four complications, and in 13 amputations no complications were observed. Therefore, 55% of patients were affected by surgical complications. Twenty-eight complications were considered minor (not requiring surgery), whereas 77 complications were major. Functional results, evaluated according to Enneking's new system, were higher than 50% in two thirds of the limb-salvage procedures. Complications in limb-salvage procedures are more influenced by the type of reconstruction than by the surgical procedure used. Probably the most troublesome consequence of surgical complications in osteosarcoma is the deviation or delay in administering postoperative chemotherapy, which jeopardizes survival.

Selective arterial embolization in the treatment of aneurysmal bone cyst and angioma of bone.

Nineteen aneurysmal bone cysts and five angiomas of bone were treated by selective arterial embolization. The median follow-up was 22 months. In 17 patients healing occurred with complete relief of symptoms; in 11 of these almost complete ossification of the lesion resulted. In the remaining cases, little or no ossification was apparent but ossification may take 1 year or more to occur. No recurrence was observed in any of these cases. Recurrence occurred only in two cases. In one, growth of the recurrence stopped after a second embolization, and the X-rays showed no change. Selective arterial embolization represents a treatment of choice in aneurysmal bone cyst and angioma of bone especially of the spine, sacrum, or pelvis. In these sites embolization replaces surgery which might be hazardous due to intraoperative bleeding.

Complications in the surgical treatment of lumbar stenosis.

The authors analyze the complications which may occur in the surgical treatment of lumbar stenosis. They report 4 cases of cauda equina syndrome and 8 dural tears in 96 patients aged from 21 to 81 years submitted to multiple bilateral laminectomy. Based on a review of the patients some considerations on surgery for the treatment of lumbar stenosis are discussed. The advanced age of the patients, hypertension, diabetes, vasculopathies in general, severe neurological deficit dating back some time contraindicate surgery. When surgery is indicated a correct preoperative evaluation by MRI from T12 to the sacrum is required to determine the extent of the laminectomy and a safe and accurate intra- and postoperative bleeding control is mandatory. Dural laceration may be repaired by a thoracolumbar fascia patch.

Neoadjuvant chemotherapy for nonmetastatic osteosarcoma of the extremities.

Between September 1986 and December 1988, 125 patients with osteosarcoma of the extremities entered the second neoadjuvant study at the authors' institution. Patients received preoperatively two cycles of methotrexate (MTX) intravenously, followed by cisplatinum (CDP) intraarterially, plus adriamycin (ADM) intravenously. After surgery, the patients classified as "good responders" (more than 90% tumor necrosis) received ADM, MTX, and CDP, while the "poor responders" (less than 90% tumor necrosis) had a longer chemotherapy that included ifosfamide and etoposide (VP-16) in addition to MTX, ADM, and CDP. Limb salvage was possible in 85% of patients, 8% had an amputation, and 7% had a rotationplasty. The surgical margins were adequate (radical or wide) in 88% of cases and inadequate (marginal or intralesional) in 12%. At an average follow-up period of 28 months (range, 13 to 41), 109 patients (87%) remained continuously disease free, 15 (12%) relapsed with pulmonary metastases, and one patient (0.8%) had a local recurrence. Compared with the first neoadjuvant study at the authors' institution that used only MTX and CDP preoperatively, the percentage of limb salvages, "good responders," and continuously disease-free survival at two years was significantly higher in the second Rizzoli neoadjuvant study (85%, 74%, and 87% versus 77%, 52%, and 59%). Systemic toxicity because of chemotherapy was superimposable. A retrospective analysis of the real dose intensity for each patient demonstrated a correlation between the intensity of chemotherapy and prognosis.

Systemic mastocytosis with skeletal involvement.

Systemic mastocytosis is a rare disease. The observation of one case characterized by clinical and radiographic bone involvement encouraged the authors to review the literature on the subject. The classification and most recent pathogenetic hypotheses correlated with various radiographic pictures, the different histological aspects, and most frequent differential diagnoses are reported.

Effect of fibrinogen concentration on the velocity of platelet aggregation.

The relationship between fibrinogen binding to its receptor and platelet aggregation has been investigated by comparing 125I-fibrinogen binding and aggregation velocities of gel-filtered platelets in the presence of adenosine diphosphate (ADP). Aggregometric responses at various fibrinogen concentrations are found to be bell-shaped and show a maximum at fibrinogen concentrations (Fmax) similar to the 125I-fibrinogen hemisaturating doses. At higher and lower fibrinogen concentrations, platelet aggregation velocities decrease in a parallel manner. Lowering ADP concentration increases Fmax, in agreement with the modulatory effect of ADP on fibrinogen binding to platelets. Variations of fibrinogen in the range of physiopathologic plasma concentrations affect platelet aggregation induced by any ADP dose. These results clarify the relationship between the fibrinogen binding process and aggregation and demonstrate that plasma fibrinogen concentration has a major influence on the velocity of platelet aggregation.

Selective arterial embolization in the treatment of lesions of the musculoskeletal apparatus.

The authors describe 42 cases of lesions of the musculoskeletal apparatus (traumatic, pseudoneoplastic or tumorous) in which selective arterial transcatheter percutaneous embolization (SAE) was indicated. In 3 patients SAE was not performed because the angiography had shown it to be too dangerous for the spinal cord. Out of 39 patients 2 were embolized in order to stop unrestrainable hemorrhaging (1 post-traumatic and 1 post-bioptic), 7 in order to reduce intraoperative bleeding, while in 7 cases (aneurysmal bone cyst, angioma of bone) the aim was curative. In the remaining 23 patients SAE was performed for adjuvant (8) or palliative (15) purposes in association with radio-and/or chemotherapy (11). In these last 15 cases the clinical results obtained were good in 67% of the cases, with partial or total regression of pain. Healing was obtained in 100% of the patients treated for curative purposes.

Multicentric osteosarcoma. Report of 5 cases.

Multiple foci of osteosarcoma are found in several pathological conditions: skip metastases, late bone metastases from osteosarcoma, so called metachronous osteosarcoma and multicentric osteosarcoma. The authors describe five cases with multicentric osteosarcoma of the skeleton. These lesions differ from classic osteosarcoma for their clinical and radiographical features. They generally arise in younger patients and are always sclerotic on X-rays and histological evaluation. Our data, as reported in literature, underline the poor prognosis of this disease.

Tumours of the patella.

We report 15 cases of primary tumours and pseudotumours of the patella. The radiographic features and the surgical treatment of the different histological types are described in relation to Enneking's surgical staging and data derived from the literature.