Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline.

Light transmission aggregometry (LTA) is considered the gold standard method for evaluation of platelet function. However, there are a lot of variation in protocols (pre-analytical procedures and agonist concentrations) and results. The aim of our study was to establish a national LTA protocol, to investigate the effect of standardization and to define national reference values for LTA. The SSC guideline was used as base for a national procedure. Almost all recommendations of the SSC were followed e.g. no adjustment of PRP, citrate concentration of 109 mM, 21 needle gauge, fasting, resting time for whole blood and PRP, centrifugation time, speed and agonists concentrations. LTA of healthy volunteers was measured in a total of 16 hospitals with 5 hospitals before and after standardization. Results of more than 120 healthy volunteers (maximum aggregation %) were collected, with participating laboratories using 4 different analyzers with different reagents. Use of low agonist concentrations showed high variation before and after standardization, with the exception of collagen. For most high agonist concentrations (ADP, collagen, ristocetin, epinephrine and arachidonic acid) variability in healthy subjects decreased after standardization. We can conclude that a standardized Dutch protocol for LTA, based on the SSC guideline, does not result in smaller variability in healthy volunteers for all agonist concentrations.

Successful treatment of fulminant postoperative bleeding due to acquired haemophilia.

Acquired haemophilia is a rare but life-threatening phenomenon in patients who have undergone surgical treatment. We describe a patient with a history of pancreatic cancer and a conventional pancreaticoduodenectomy, who underwent elective resection of an enterocutaneous fistula, complicated by fulminant haemorrhagic shock, caused by acquired haemophilia A. Eventually, the bleeding was controlled by a combination of aggressive haemostatic and immunosuppressive therapy. Prompt diagnosis of acquired haemophilia is crucial to allow early and appropriate haemostatic treatment and reduce the period of increased bleeding risk by eradicating the inhibitor with immunosuppressive therapy.

Implementation of a new platelet pooling system for platelet concentrates led to a higher corrected count increment after transfusion: a comparative observational study of platelet concentrates before and after implementation.

OBJECTIVES: To study the effect of extended storage of platelet concentrates (PCs) and the implementation of a new platelet pooling system for PCs on corrected count increment (CCI) after transfusion. BACKGROUND: Due to new developments and changes in processes or procedures, one should remain alert for the effects of these changes. Besides in vitro studies and validation, in vivo studies are also important, as it has been shown that in vitro results do not always predict in vivo outcomes. METHODS/MATERIALS: After introduction of extended storage of PCs for 5-7 days prepared from five buffy coats and plasma, transfusion monitoring for transfusions of PCs in haemato-oncological patients was set up. After 9 months, a new pooling system for PCs was implemented, Composelect instead of Optipure PLT, and transfusion monitoring was continued for another 8 months. The CCI was used as primary outcome. RESULTS: In total, 93 patients were included and transfused with PCs prepared in the Optipure PLT system (262 transfusions) or in the Composelect system (127 transfusions). Extended storage of PCs for 7 days had no significant effect on CCI. Although the implementation of the Composelect system did not influence the CCI1 h (13.8 ± 6.0 vs. 13.0 ± 5.8; n.s.), it seemed to have a positive effect on CCI24 h (7.0 ± 4.9 vs. 4.7 ± 4.5; P < 0.05). CONCLUSION: Although the influence of confounders could not be excluded, it seemed that implementation of the Composelect system for PCs led to an improved CCI24 h and that extended storage of PCs did not influence the CCI.

A positive effect of immune suppression on corrected count increment after platelet transfusion at 1 but not at 24h.

The effects of patient characteristics on corrected count increment (CCI) in hemato-oncology patients were studied. CCI values were 13.6 ± 6.5 (1h, n=79) and 6.3 ± 5.3 (24h, n=69). With concomitant immune suppression CCI was higher at 1h (20.0 ± 7.9 versus 13.2 ± 6.3, p=0.023), but not at 24h (5.9 ± 6.7 versus 6.3 ± 5.2; p=0.88). The observed effect is short lived, potentially benefiting bleeding patients, but may not increase intervals between transfusions. Further, CCI1h was lower if fever was present (9.7 ± 3.6 versus 14.3 ± 6.7; p=0.002), and corresponding CCI24h values were 3.7 ± 6.3 versus 6.7 ± 5.0 (p=0.09). At 24h an effect for previous transfusions was observed, 6.7 ± 5.1 (with) versus 1.6 ± 5.4 (without p=0.02).

[Acute anaemia in a Vietnamese patient with alpha-thalassaemia and a parvovirus infection]. / Acute anemie bij een Vietnamese patiënt met alpha-thalassemie en een parvovirus-infectie.

A 14-year-old girl of Vietnamese descent with an unremarkable medical history presented with haemodynamic shock due to severe anaemia. This was caused by an aplastic crisis resulting from the combined effects of a Parvovirus infection and HbH disease. The HbH disease was a result of compound heterozygosity for the South East Asia (SEA) deletion and the Constant Spring mutation in the genes coding for alpha-globin chains (HbH/Hb Bart's). The girl had multiple blood transfusions and recovered. Family investigation revealed that, in addition to these 2 mutations in the alpha-globin gene, some family members also carried the 3.7-kb deletion of the alpha-globin gene, a mutation in the beta-globin gene resulting in HbE, and a novel mutation of unknown clinical significance in the beta-globin gene. This case demonstrates that essentially asymptomatic carriership of thalassaemia can have serious consequences when coupled with a concurrent infection.

Adenoviral expression of a urokinase receptor-targeted protease inhibitor inhibits neointima formation in murine and human blood vessels.

BACKGROUND: Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as alpha(2)-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. METHODS AND RESULTS: The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5x10(9) pfu/mL) resulted in 87.5+/-3.8% (mean+/-SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9+/-2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. CONCLUSIONS: These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.

Antibodies to PAI-1 alter the invasive and migratory properties of human tumour cells in vitro.

Recent reports suggest that elevated levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to tumour progression. The studies reported here were designed to help elucidate PAI-1's contribution to the invasive and migratory phenotype. Antibodies to PA-1 dose-dependently, and significantly, inhibited the invasive and migratory potential of human HT1080 fibrosarcoma cells, as did an antibody to uPA and the plasmin inhibitor aprotinin. Invasion of the human melanoma cell line, BLM, was also attenuated by the anti-PAI-1 monoclonal antibody MAI-12. The non-invasive human melanoma cell line, IF6, which does not express uPA, provided further confirmation of PAI-1 and uPA's role as, upon transfection with uPA, this cell line attained an invasive phenotype, which was again attenuated by MAI-12. Although antibodies to PAI-1 did not affect the adhesion of HT1080 cells to vitronectin, the antibody to uPA reduced their attachment. Addition of exogenous PAI-1, however, prevented HT1080 cell adhesion (IC50 180 nM) and promoted cell detachment from vitronectin. Furthermore melanoma cells transfected with a uPA variant, which had an impaired interaction with PAI-1, were not invasive and had impaired binding to vitronectin. These data highlight the importance of a balanced proteolysis and suggest an additional role for PAI-1 distinct from its role in proteolysis. These data also suggest that uPA and PAI-1 may co-operate in the migratory process by respectively facilitating the attachment to, and subsequent detachment from, vitronectin in the extracellular matrix. These results support the clinical findings and indicate that modulation of PAI-1 activity may be of therapeutic benefit for the treatment of cancer.

Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor.

The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.

Cytokeratin expression in human arteries pertinent to intimal thickening formation in the ductus arteriosus.

Expression of epithelial cytokeratins type 8, 18 and 19 can be used to study smooth muscle cell differentiation during development. We studied the differentiation of smooth muscle cells in the ductus arteriosus before and during intimal thickening and compared the changes occurring in this vessel with the adjoining elastic ascending and descending aorta and the pulmonary trunk. The ductus arteriosus, a vessel connecting the pulmonary trunk and the aorta during fetal life, constricts shorty after birth and eventually closes. Effective closure occurs only in the case of well developed intimal thickening. Cytokeratin expression during fetal development was greatest in the media of the ascending aorta and pulmonary artery, while in the ductus and descending aorta cytokeratin staining was slight. These results suggest that ductus smooth muscle cells and the smooth muscle cells of the descending aorta show a more advanced differentiation as compared to the ascending aorta and pulmonary artery. At neonatal stages cytokeratin expression in the descending aorta, pulmonary artery and the ascending aorta had disappeared as was expected with increased differentiation. In the neonatal ductus arteriosus reexpression of cytokeratins was found in cell clusters in the hyaluronic acid rich environment of the intimal thickening and in the inner media. Reexpression of cytokeratins, especially when organized in clusters, may reflect changes in gene regulation. Therefore the clusters of cytokeratin positive cells in the ductus may be indicative of extensive changes, occurring during closure of this vessel in the neonatal period, in which inner media and intima are especially involved.

Differentiation, dedifferentiation, and apoptosis of smooth muscle cells during the development of the human ductus arteriosus.

Differentiation of vascular smooth muscle cells (SMCs) is characterized by several molecular transitions. As differentiation proceeds, proteins of the cytoskeletal and contractile apparatus, such as alpha-smooth muscle actin, smooth muscle myosin, calponin, and heavy caldesmon, and the expression of the membrane-related protein smooth muscle phosphoglucomutase-related protein increase, whereas the expression of other proteins, such as fibronectin splice variants with extradomains A (EDA) and B (EDB), decreases. In this study, we investigated the differentiation of the SMCs of the ductus arteriosus during the development of intimal thickening. Ascending and descending aortas of the same age were used for comparison because these vessels lack intimal thickening. In the fetal ductus arteriosus, a relatively early differentiation of the contractile apparatus was observed compared with the ascending and descending aortas. EDA and EDB expression was already low, being similar in the ductus and descending aorta and even lower in the ascending aorta. In the neonatal ductus, SMCs of the media and outer intima were well differentiated and comparable with SMCs of the ascending aorta. Dedifferentiated SMCs, with a low expression of cytoskeletal and contractile proteins and a high expression of EDA and EDB, were found in regions in the inner intima that show features of progression of intimal thickening and in areas of cytolytic necrosis in the media. With a technique using in situ end labeling of DNA fragments, we found extensive apoptosis in the area of cytolytic necrosis and to a lesser extent in these areas of the inner intima. In conclusion, SMCs of the fetal ductus arteriosus have an advanced differentiation of the contractile apparatus compared with the adjacent aorta. Reexpression of fetal characteristics is seen in a number of cells in inner intima and media of the neonatal ductus arteriosus. The finding of apoptosis in these areas suggests that dedifferentiation and apoptosis are associated processes that may play a role in vascular remodeling.

Nature and origin of the neointima in whole vessel wall organ culture of the human saphenous vein.

Intimal proliferation is a characteristic feature of arteriosclerosis. Whole vessel wall organ culture systems have been developed to study the early stages of neointima formation. We have cultured a large number of explants of human saphenous vein specimens for several weeks, and have identified the nature of the cells in the newly formed intima by a panel of monoclonal antibodies recognizing endothelial cells (von Willebrand factor, platelet endothelial cell adhesion molecule-1 and EN-4 antigen), smooth muscle cells (monoclonal antibodies HHF35 and CGA-7) and fibroblasts (5B5 antibody). In addition we determined the uptake of fluorescently labelled acetylated low density lipoprotein by the surface cells of the explants. We found that an apparent neointima was formed in the vein organ system, the cells of which were predominantly smooth muscle cells and originated from the cut edges and from the adventitia of the vein segment. The endothelial cells originally lining the luminal surface of the vessel segments became overgrown by these cells. They remained at the base of the newly formed neointima and a number of them reorganized into capillary-like structures. Our data suggest that explant culture of saphenous vein does not reflect the classical concept of neointima formation, in which intimal smooth muscle cells migrate through the internal elastic lamina and accumulate in the intima. Although it has this limitation, the model may serve well to study specific aspects of cell migration, smooth muscle cell differentiation and angiogenesis, and may reflect aspects of intimal thickening at surgical suture sites.

Anatomical and sonographic correlation of the fetal ductus arteriosus in first and second trimester pregnancy.

Ultrasonic visualization of the ductus arteriosus in first and second trimester pregnancies was compared with postmortem preparations. Twenty human fetal postmortem specimens from 8 to 19 weeks menstrual age were examined, 11 with microscopic reconstruction, nine with macroscopic dissection. The angle between ductus arteriosus and aortic isthmus (upstream) and ductus arteriosus and descending aorta (downstream) was determined. In 52 normally developing fetuses between 14 and 27 weeks, the angle between the ductus arteriosus and the thoracic spine as visualized in real-time ultrasound was determined. In a further 19 normally developing fetuses between 14 and 25 weeks, ductal blood flow was visualized by colour velocity imaging (CVI). In anatomical preparations, the upstream angle was always less than 90 degrees and the downstream angle was always 80 degrees or more. These angles were unrelated to menstrual age. In both real-time and CVI ultrasound, the angle between ductus arteriosus and thoracic spine remained at approximately 90 degrees. CVI showed highest blood flow velocities at the point of ductal insertion into the aorta. When performing Doppler ultrasound examinations in the fetal ductus arteriosus, no menstrual age dependent angle adjustment appears to be necessary.

Formation of intimal cushions in the ductus arteriosus as a model for vascular intimal thickening. An immunohistochemical study of changes in extracellular matrix components.

There is a great resemblance in the sequence of events that take place in the pathological development of intimal thickening, so called arteriosclerosis and the formation of intimal cushions in both the normal ductus arteriosus (DA) and the persistent ductus arteriosus (PDA). The human DA was used as a model to study the changes in the extracellular matrix during this process with immunohistochemistry. The formation of intimal cushions was studied in 4 normal fetal DA, 4 normal mature DA and 3 persistent DA. The process of intimal thickening in the fetus starts in the second trimester of pregnancy with an accumulation of glycosaminoglycans in the subendothelial region (SER), accompanied by separation of endothelial cells from the internal elastic lamina and followed by migration of smooth muscle cells into the subendothelial region. This phenomenon was also observed in the mature DA in the neonate, indicating that cushion formation is a continuous process. Intimal cushions had also developed in the persistent DA, although they were morphologically different from the cushions found in the normal mature DA. It was remarkable that two elastic lamellae could be distinguished: one at the original site on the borderline of intimal cushion and media and the other in a subendothelial position. The endothelial cells were firmly attached to this subendothelial lamina, which was wrapped in the basal lamina components laminin and type IV collagen. The main morphological difference between the normal mature DA and the persistent DA is the close relation between endothelial cells and the subendothelial elastic lamina, suggesting an altered elastin metabolism in the PDA. PGI2 synthase was increased in the wall of both the normal and persistent DA as compared with the aorta. It may be related to a role of PGI2 in the formation of intimal cushions.