Monocyte procoagulant activity and membrane-associated D dimer after knee replacement surgery

We have recently shown that monocyte membrane-associated cross-linked fibrin derivatives (D dimer) can be evidenced by immunogold staining. Using this method, the procoagulant activity (PCA) expressed in vitro by endotoxin-stimulated monocytes has been found to correlate significantly with the number of D dimer-positive monocytes. The incidence of postoperative thrombosis in patients undergoing total knee replacement has been reported by Stulberg et al to be 57%. Since monocytes can play a role, via increased PCA, in the activation of intravascular coagulation, we sought to determine the level of monocyte PCA ex vivo after knee replacement surgery and its possible correlation with the number of D dimer-positive monocytes. Finally, we examined the possible link between these modifications and the occurrence of postoperative deep vein thrombosis (DVT). The PCA expressed by monocytes with or without suboptimal stimulation, the number of D dimer-positive monocytes and the plasma level of D dimer were measured pre- and post-operatively in 11 patients undergoing total knee replacement. Phlebography was performed on day 10 after surgery. A significant increase in the PCA of stimulated monocytes was observed on day 10 after surgery. Moreover, both the number of D dimer-positive monocytes and the plasma level of D dimer increased significantly post-operatively. The number of D dimer-positive monocytes correlated with both monocyte PCA and the plasma D dimer level. The relation between these parameters is discussed. However, neither monocyte PCA nor the number of D dimer-positive monocytes was found to correlate with the occurrence of deep vein thrombosis.     

All trans-retinoic acid modulates the procoagulant activity of human breast cancer cells

All trans-retinoic acid (ATRA) induces apoptosis and/or differentiation in solid tumors, including breast cancer, and has become a therapeutic tool in this disease. In human promyelocytic leukemia ATRA reduces the expression of cellular procoagulant activities (PCA), i.e. Tissue Factor (TF) and Cancer Procoagulant (CP). There are no studies on the effects of ATRA on the PCA of solid tumors, i.e. breast cancer cells. We analyzed different human breast cancer cell lines in order to: 1. characterize the expression of TF and CP; 2. evaluate whether these activities are affected by ATRA; and 3. verify whether a reduction in tumor cell procoagulants may occur in association to apoptosis and growth inhibition induced by ATRA. Two estrogen receptor positive (ER-positive; i.e. MCF7 and ZR75.1) and one estrogen receptor negative (ER-negative; i.e. MDA.MB.231) cell lines were included into the study. The results show that ATRA affected TF in a dose-dependent fashion only in ER-positive cell lines. In particular, at 1uM ATRA, TF significantly (p < 0.05) decreased by 57%, 44% in MCF7, ZR75.1 cells, respectively. Differently the results show that ATRA dose-dependently affected CP expression in all three cell lines. Specifically, at 1uM ATRA, CP significantly decreased by 44%, 50% and 25% in MCF7, ZR75.1, and MDA.MB.231. Only in ER-positive cell lines, there was a dose-dependent inhibition of cell growth that became statistically significant at 1uM ATRA, which was associated to a slight but significant increase in the percentage of apoptotic cells. In conclusion, this study demonstrates for the first time that ATRA downregulates the expression of TF and CP in breast cancer cells. Due to the pivotal role of coagulation activation in tumor progression, the capacity of ATRA to affect also tumor procoagulants, in parallel to cell apoptosis, open new perspectives in tumor therapy.     

Heparanase procoagulant activity

Heparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis and angiogenesis. We have recently demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. Heparanase was shown to up-regulate tissue factor (TF) expression and interact with tissue factor pathway inhibitor (TFPI) on cell surface, leading to dissociation of TFPI from cell membrane of endothelial and tumor cells, resulting in increased cell surface coagulation activity. We have lately shown that heparanase directly enhances TF activity resulting in increased factor Xa production and activation of the coagulation system. Data indicate increased plasma levels of heparanase suggesting its possible involvement in pregnancy vascular complications. Elevation in heparanase levels and procoagulant activity was also documented in orthopedic surgery patients receiving prophylactic doses of enoxaparin. Taking into account the pro-metastatic and pro-angiogenic functions of heparanase, with over-expression in human malignancies and abundance in platelets and placenta, its involvement in the coagulation machinery is an intriguing novel platform for further research.     

Elevated numbers and altered subsets of procoagulant microparticles in breast cancer patients using endocrine therapy

Introduction

Microparticles (MP) can be elevated in cancer and thromboembolic disease. We hypothesized a role for MP in the hypercoagulable state in breast cancer patients using endocrine therapy, in whom both cancer and the use of endocrine therapy are independent risk factors for the development of thrombosis.

Design and methods

Plasma samples were collected from 40 breast cancer patients using endocrine therapy (20 patients without metastases receiving adjuvant therapy and 20 patients with metastatic disease treated in a palliative setting) and from 20 female healthy controls. The endocrine therapy used was either an anti-estrogen or an aromatase inhibitor. Numbers and cellular origin of MP subsets were analyzed by flowcytometry. MP-associated procoagulant activity was measured using a thrombin generation assay using conditions that allow analysis of MP induced thrombin generation.

Results

Breast cancer patients using endocrine therapy had higher levels of MP positive for Annexin V (median 10000 vs 6500 × 10E6/l), P-selectin (330 vs 200 × 10E6/l), tissue factor (33 vs 15 × 10E6/l), and of MP derived from platelets (CD41) and leukocytes (CD45). Thrombin generation in plasma was dependent on the presence of MP and thrombin generation performed after addition of isolated MP to normal plasma showed a higher endogenous thrombin potential (1105 vs 1029 nM.min) in breast cancer patients. No differences were observed in MP levels and thrombin generation parameters between the metastatic and adjuvant group.

Conclusion

Breast cancer patients using endocrine therapy have an increased MP number and a higher MP-dependent thrombin generation, irrespective of the presence of metastatic disease. Altered MP subset characteristics in these patients, especially the higher number of (activated) platelet derived MP and leukocyte derived MP, may in part explain a heightened procoagulant state in breast cancer patients using endocrine therapy.     

Heparanase procoagulant activity is elevated and predicts survival in non-small cell lung cancer patients

Background

Heparanase is implicated in angiogenesis and tumor progression. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. It forms a complex and enhances the activity of the blood coagulation initiator- tissue factor (TF). Although increased heparanase antigen level in the plasma and biopsies of cancer patients was previously demonstrated, in the present study we evaluated, for the first time, the heparanase procoagulant activity in the plasma of patients with lung cancer.

Materials and Methods

Sixty five patients with non-small cell lung cancer at presentation and twenty controls were recruited. Plasma was studied for TF / heparanase procoagulant activity, TF activity and heparanase procoagulant activity using chromogenic assay and heparanase antigen levels by ELISA.

Results

Heparanase antigen levels were higher in the study group compared to control (P = 0.05). TF / heparanase activity, and even more apparent, heparanase procoagulant activity were significantly higher in the study group compared to controls (P = 0.008, P < 0.0001, respectively). No significant difference was observed in the TF activity between the groups. Survival of patients with heparanase procoagulant activity higher than 31 ng/ml predicted a mean survival of 9 ± 1.3 months while heparanase procoagulant activity of 31 ng/ml or lower predicted a mean survival of 24 ± 4 months (P = 0.001). Heparanase procoagulant activity was higher than 31 ng/ml in the four cases of thrombosis detected during the follow-up period.

Conclusions

Elevated heparanase procoagulant activity in patients with lung cancer reveals a new mechanism of coagulation system activation in malignancy. Heparanase procoagulant activity can potentially be used as a predictor for survival.     

An assay to evaluate heparanase procoagulant activity

Background

Heparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is directly involved in the regulation of the hemostatic system. Heparanase was shown to form a complex and enhance tissue factor (TF) activity, resulting in increased factor Xa production (Nadir et al. Haematologica, 2010). The present work suggests a novel assay to evaluate heparanase procoagulant activity.

Methods

Heparanase procoagulant activity was studied using purified proteins of heparanase, TF, factor VIIa and factor X. The assay was verified in 55 plasma samples and compared to heparanase and tissue factor pathway inhibitor (TFPI) levels by ELISA and factor Xa, thrombin levels and antithrombin activity by chromogenic substrates. Thirty five samples were of third-trimester pregnant women (weeks 36–41) who were in labor or came for appointed elective cesarean section and 20 control samples were of non-pregnant healthy women.

Results

heparanase procoagulant activity assay was shown to differentiate heparanase procoagulant effect from TF activity, in purified proteins. Heparanase procoagulant activity was significantly higher in the plasma of pregnant women compared to non-pregnant (p < 0.005). Heparanase relative contribution to the TF / heparanase complex activity was significantly higher in the plasma of pregnant women compared to non-pregnant (29% increase, p < 0.0001). Differences in heparanase procoagulant activity were more prominent than changes in heparanase levels by ELISA, TF activity, factor Xa, thrombin and free TFPI levels.

Conclusions

Heparanase procoagulant activity can be determined by the suggested assay. The assay revealed a significant contribution of heparanase to the procoagulant state in late third-trimester pregnancy and at delivery.     

Analysis of serum cancer procoagulant activity and its possible use as a tumor marker

The two objectives of the study were to determine whether a procedure could be developed for measuring cancer procoagulant (CP) activity in human serum and if this procedure provided a method for distinguishing people with cancer from those without cancer. A procedure was developed for processing human serum such that the activity of other coagulation enzymes would be minimized and the activity of cancer procoagulant could be measured. In a blinded study, we collected serum from 61 individuals in serum separator tubes, removed the clot by centrifugation, extracted the serum with a simple, single step procedure and analyzed the extract for CP activity. The results indicate that this test could correctly identify about 92% of the cancer patient serum samples and about 75% of the non-cancer patients serum samples, for an overall accuracy of about 85%.     

Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay

Introduction

Circulating microparticles are reported to play a role in cancer hypercoagulability. The procoagulant properties of microparticles derive from the amount of tissue factor and/or phosphatidylserine that they can expose. The aim of our study is to assess the procoagulant activity, including microparticles’ activity, in the plasma of newly diagnosed cancer patients with a simple assay, easy to implement in the laboratory.

Material and methods

Newly diagnosed cancer patients (n = 31) before the start of anticoagulant or chemotherapy were compared to matched controls. We used a thrombin generation assay in four conditions: 1: addition of 1pM tissue factor and 4 μM procoagulant phospholipids, 2: without any trigger, 3 and 4: addition of tissue factor or procoagulant phospholipids alone respectively.

Results

When we added only phospholipids, so that thrombin generation is dependent upon endogenous tissue factor, the lag time was significantly shorter in cancer patients. When we added only tissue factor, i.e. made the results dependent upon phospholipids, the endogenous thrombin potential, the peak, and the velocity index were significantly higher and the time-to-peak was significantly shorter. This suggests that the plasma of cancer patients contained a higher activity of endogenous phospholipids and/or tissue factor which may be borne by microparticles.

Conclusion

This new simple methodology can demonstrate a procoagulant activity in the plasma of newly diagnosed cancer patients which can be explained by higher procoagulant phospholipids and tissue factor activity and thus, brings potentially useful information that current coagulation tests cannot provide.     

Molecular weight of human factor VIII procoagulant activity

Low molecular weight factor VIII procoagulant activity has been prepared by agarose gel chromatography of highly purified human factor VIII using 0.24 M CaCl₂ buffer. The sedimentation properties of this procoagulant activity in sucrose density gradient centrifugation studies carried out at physiologic ionic strength were consistent with a 6.7S protein. These experiments demonstrate that the elution pattern of factor VIII activity in agarose gel chromatography with high ionic strength buffers is due to separation of low molecular weight material — rather than artifactual retention of large molecules within the gel bed — and that the low molecular weight portion of the large factor VIII complex is sufficient for procoagulant function.     

Induction of endothelial cell procoagulant activity by cytomegalovirus infection

Endothelial cells isolated from rat aorta were infected in vitro with rat cytomegalovirus. Viral antigens appeared in nucleus and cytoplasma and newly made extracellular virus was detected in the supernatant. Furthermore, the viral infection caused the appearance of procoagulant activity on the endothelial cells.     

Regulation of tissue factor procoagulant activity by post-translational modifications

Post-translational modification of amino acid residues is a common way to regulate localization, stability and ultimately the function of the protein. Tissue factor (TF), the major initiator of blood coagulation cascade, receives several post-translational modifications, like glycosylation, phosphorylation, palmitoylation and nitrosylation. Recent studies have demonstrated that these processes play important roles in modulating biological functions of TF. The present review highlights the mechanisms of several common protein post-translational modifications of TF with the special reference on the recent knowledge about their roles in regulation of trafficking, stability as well as procoagulant and signaling functions of TF.     

Relationship of cardiac troponin T and procoagulant activity in unstable angina

OBJECTIVE: The present study sought to determine the incidence of increased procoagulant activity in patients with unstable angina (UAP), and to evaluate the relationship between cardiac troponin T (cTnT) and molecular markers of hemostatic activation. Method. We studied 44 patients with UAP further classified by plasma cTnT levels. All patients received an antithrombotic therapy consisting of therapeutic doses of unfractionated heparin and acetylsalicylic acid. Quantitative levels of cTnT and plasma concentrations of fibrin monomers (FM), prothrombin fragments F1+2, thrombin antithrombin III complexes (TAT), plasminogen and alpha2-antiplasmin were sampled serially within the first 48 h. RESULTS: Increased plasma concentrations of FM were detected in 45.5% of patients and were more frequently present among those with cTnT concentrations > or =0.1 ng/ml (13 of 18 vs 7 of 26 patients, p = 0.003). In these patients, mean plasma concentrations of FM were significantly higher than in patients with cTnT <0.1 ng/ml (7.93 +/- 2.3 vs 3.12 +/- 0.6 microg/ml, p = 0.02). There was a close relationship between plasma levels of cTnT and FM (r = 0.74, p <0.004), prothrombin fragments F1+2 (r = 0.71, p = 0.046) and a trend to significance was noted for TAT (r = 0.42, p = 0.055). No significant correlation was observed with markers of the fibrinolytic system (plasminogen and alpha2-anti-plasmin). Plasma levels of cTnT > or =0.1 ng/ml identified a concomitant increase of hemostatic markers with a sensitivity, specificity and positive predictive value of 65, 79, and 72% for FM, 63, 76, and 67% for prothrombin fragments F1+2, and 58, 66, and 39% for TAT, respectively. CONCLUSIONS: In patients with UAP, cTnT identifies patients with increased procoagulant activity and is closely related to plasma levels of molecular markers of hemostatic activation. Therefore, cTnT alone or in combination with one of these markers may be helpful to identify patients requiring more potent antithrombin or antiplatelet therapy.     

Vitamin E inhibition on platelet procoagulant activity: involvement of aminophospholipid translocase activity

Background

Activated platelets provide an important procoagulant surface, because exposed negatively charged phosphatidylserine (PS) is an important cofactor of the coagulation cascade. Aminophospholipid translocase (APLT) can transport PS from the outer to the inner membrane leaflet. Although vitamin E has been investigated for its anti-aggregating effect on platelets, its effect on platelet procoagulant activity has not been reported.

Methods

Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, and thrombin were used to induce PS exposure on platelet surface. The expression of PS was measured by annexin A5 binding with flow cytometry. Platelet procoagulant activity was measured by a prothrombinase assay. APLT activity was measured by flow cytometry by determining the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. Inhibition effects of vitamin E on platelet aggregation were simultaneously measured by a Multiplate aggregometer, a Chrono-log aggregometer, and a PFA-100 system.

Results

Vitamin E significantly attenuated PMA-induced conformational change of glycoprotein IIb/IIIa and P-selectin expression. Vitamin E significantly inhibited PMA and thrombin-induced PS externalization and reduced prothrombinase activity on platelet surfaces both in vitro and ex vivo. APLT activity was increased by vitamin E in a dose-dependent manner, indicating that reduced procoagulant activity may be attributed, at least in part, to this increased APLT activity. Vitamin E inhibited platelet aggregation induced by combined chemokine SDF-1 and low-dose ADP as well as by usual doses of ADP or collagen when measured by the Multiplate and Chrono-log aggregometers but not when measured by PFA-100.

Conclusions

These in vitro and ex vivo results showed that vitamin E inhibited platelet PS exposure and procoagulant activity partly by increasing APLT activity. These actions of vitamin E on platelet function provide new insights into the anticoagulation properties of vitamin E.