Background —Cardiopulmonary bypass (CPB) induces platelet activation with release of platelet factor 4 (PF4), and patients are exposed to high doses of heparin (H). We investigated whether this contributes to the development of antibodies to H-PF4 and heparin-induced thrombocytopenia (HIT). Methods and Results —CPB was performed with unfractionated heparin (UFH) in 328 patients. After surgery, patients received UFH (calcium heparin, 200 IU · kg −1 · d −1 ) (group 1, n=157) or low-molecular-weight heparin (LMWH, Dalteparin, 5000 IU once daily) (group 2, n=171). Eight days after surgery, antibodies to H-PF4 were present in 83 patients (25.3%), 46 in group 1 and 37 in group 2 ( P =0.12). Most patients (61%) had IgG1 to H-PF4, but only 8 samples with antibodies induced platelet activation with positive results on serotonin release assay. HIT occurred in 6 patients in group 1, but no thrombocytopenia was observed in subjects receiving LMWH, although 2 had high levels of antibodies with positive serotonin release assay results. When antibodies to H-PF4 were present, mean platelet counts were lower only in patients with FcγRIIA R/R 131 platelets. Conclusions —These results provide evidence that the development of antibodies to H-PF4 after CPB performed with UFH is not influenced by the postoperative heparin treatment. The antibodies associated with high risk of HIT are mainly IgG1, which is present at high titers in the plasma of patients continuously treated with UFH.

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The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xabased chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients. IntroductionHeparin-induced thrombocytopenia (HIT) is a common complication of unfractionated heparin (UFH) therapy, often associated with arterial or venous thrombosis. 1 Such thrombocytopenia results from platelet activation caused by antibodies specific for complexes composed of heparin and platelet factor 4 (FP4). [2][3][4][5] The platelet activation involves the binding of HIT IgG to specific Fc receptors (Fc␥RIIA), 6 partly explaining why HIT differs from most other types of drug-induced immune thrombocytopenia, which are mainly complicated by hemorrhage.The most frequent manifestation in HIT is thrombosis, sometimes associated with disseminated intravascular coagulation, and one of the mechanisms proposed to explain the hypercoagulability in HIT is the production of phospholipid platelet microparticles after platelet activation. 7 However, HIT antibodies might also bind to endothelial cells and trigger the synthesis of tissue factor (TF), 4,8,9 a transmembrane glycoprotein that initiates the coagulation pathway. 10,11 TF can be produced by monocytes when activated by lipopolysaccharides, cytokines, or immune complexes, 12 and it can be triggered in the presence of activated platelets. 13 We therefore investigated whether antibodies to PF4 developed in patients with HIT to induce the synthesis of TF by human monocytes. Study design Plasma and purification of immunoglobulin GPlasma from 5 patients with definite HIT complicated by venous (n ϭ 5) and arterial (n ϭ 1) thromboses was studied. Every sample contained significant levels of antibodies to H-PF4 complexes (mean A 492 , 1.7; range, 1.0-2.1) (Asserachrom HPIA, Stago; Asnières, France) 14 able to induce heparin-dependent platelet activation in serotonin release assay (SRA). 15 Normal human plasma without antibodies to H-PF4 complexes (mean A 492 Ͻ 0.2) was collected from 5 healthy volunteers not receiving heparin. All samples were heated at 56°C for 1 hour, centrifuged ...

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Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with nonsmall-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4 -120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P ¼ 0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P ¼ 0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P ¼ 0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC.

These results suggest that high TF expression in lung tumors may result from K-ras mutation and contribute to NSCLC progression, probably via mechanisms other than angiogenesis.

The proinflammatory chemokine CC chemokine ligand 5 (CCL5) is a potent chemoattractant of immature dendritic cells (iDCs). It remains to be elucidated whether CCL5 may also enhance iDC migration through the basement membrane by affecting matrix metalloproteinase (MMP)-9 secretion. In this study, iDCs were differentiated in vitro from human monocytes of healthy donors. Zymographic analysis of cellular membranes of nontreated iDCs revealed a basal secretion of the pro- and active MMP-9, whereas only pro-MMP-9 was detected in conditioned media. Increasing concentrations of CCL5 significantly enhanced MMP-9 secretion by iDCs, peaking at 100 ng/ml, which optimally increased iDC migration through a reconstituted basement membrane (Matrigel) in vitro. The CCL5-enhanced secretion of MMP-9 occurred early (2 h) and was maintained at least for 10 h. A significant increase in MMP-9 mRNA synthesis was detected by reverse transcriptase-polymerase chain reaction, only at 6 h of CCL5 treatment, which suggests that the early effect of CCL5 (0-4 h) on MMP-9 secretion was independent of mRNA synthesis, whereas the more delayed effect (6-10 h) could be mediated through an increase in MMP-9 gene expression. In a Matrigel migration assay, the CCL5-enhanced iDC migration was reduced significantly by specific inhibitors of MMP-9, such as tissue inhibitor of metalloproteinase-1 or an anti-MMP-9 antibody, which indicates that iDC migration through the basement membrane depends on MMP-9. These results suggest that under inflammatory conditions, the chemokine CCL5 may enhance iDC migration through the basement membrane by rapidly increasing their MMP-9 secretion.

Most housekeeping genes, tumor-suppressor genes, and approx 40% of tissue-specific genes contain G+C sequences in their promoter region that were very difficult to amplify. In this report, we propose an improved polymerase chain reaction (PCR) method to be used for successful amplification of the tissue factor pathway inhibitor (TFPI)-2 gene promoter region that exhibit >70% G+C content in a sequence of approx 300 bp and a complete CpG island region spanning exon 1, the three transcription initiation sites, and the translation start site. Therefore, this method can be recommended to amplify other GC-rich genomic templates.