Initiator Of Blood Coagulation Research Articles

The use of thermal analysis in assessing the effect of bound water content and substrate rigidity on prevention of platelet adhesion

Implant surface-induced initiation of blood coagulation leading to thrombus formation is a major problem. It is an important challenge in biomedical research to enhance thromboresistance of polymer coatings of blood-contacting implants and devices by the reduction of platelet adhesion to their surfaces. Currently, the molecular mechanisms responsible for interfacial processes related to regulation of protein adsorption and platelet adhesion in antithrombotic coatings are not yet clearly understood. We studied the role of water binding energy in chitosan molecules using differential scanning calorimetry and thermogravimetric analysis and the role of chitosan coating rigidity using dynamic mechanical thermal analysis in the prevention of platelet adhesion. It was found that the presence of loosely bound water in chitosan coatings increases platelet adhesion. The high molecular weight (HMW) chitosan coating that contains only tightly bound water prevents platelet adhesion. The high molecular weight (HMW) chitosan coating is more rigid and has higher platelet adhesion compared with more soft LMW chitosan coatings. The degree of hydration of thromboresistant coatings is significant parameter that must be taken into account in the design of blood-contacting surfaces.

Heparanase and coagulation-new insights.

Heparanase, a β-D-endoglucuronidase abundant in platelets that was discovered 30 years ago, is an enzyme that cleaves heparan sulfate side chains on the cell surface and in the extracellular matrix. It was later recognized as being a pro-inflammatory and pro-metastatic protein. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. We had shown that heparanase up-regulated the expression of the blood coagulation initiator tissue factor (TF) and interacted with the tissue factor pathway inhibitor (TFPI) on the cell surface membrane of endothelial and tumor cells, leading to dissociation of TFPI and resulting in increased cell surface coagulation activity. Moreover, we have demonstrated that heparanase directly enhanced TF activity which led to increased factor Xa production and subsequent activation of the coagulation system. Recently, heparanase inhibitory peptides derived of TFPI-2 were demonstrated by us to inhibit heparanase procoagulant activity and attenuate sepsis in mouse models.

Ebola hemorrhagic fever: genetic biomarkers and vaccine development.

Ebola hemorrhagic fever: genetic biomarkers and vaccine development.

Abstract 502: Sp1 and HIFs mediate synergistic activation of ICAM1 gene under serum starved hypoxia and promotes growth of ovarian cancer

Abstract Cancer tissues are generally exposed to severe hypoxia conditions due to sparse and aberrant vasculature. Hypoxia-inducible factors HIF-1α and HIF-2α (HIF1 and HIF2, respectively) are major transcription factors required for adaptive responses to hypoxia. HIFs complexed with arylhydrocarbon receptor nuclear translocator bind target genes and act as enhancer of transcriptional regulation. This conventional mechanism regulates genes such as VEGF, GLUT1, and HO to improve mild to severe hypoxia conditions. It is likely that various oxygen concentrations and their duration exist within tumor tissue. It appears that adaptive response by HIFs is a major way to overcome stresses under milder hypoxia. However, majority of tumor tissues of many cancer types are known to undergo more severe hypoxic conditions. Thus, additional molecular mechanisms supporting HIFs signaling may be required to enhance survival activity of cancer cells. We have previously demonstrated that FVII gene, which is essential for initiation of extrinsic blood coagulation can be induced in ovarian clear cell carcinoma (CCC) cells in response to hypoxia. Activation of this gene potentially results in thrombotic events frequently observed in cancer patients. We have demonstrated that a physical interaction between Sp1 and HIF2 is a major cause of the activation of this gene under hypoxia. Furthermore, FVII activation is synergistically enhanced when cells are cultured under ischemic condition, in which hypoxia and serum-free conditions coexist. We envisioned that genes regulated by this mechanism are responsible for adaptive response to hypoxia with severity and/or duration characteristic to CCC tissues. Furthermore, ovarian cancer is a most malignant among gynecological diseases. CCC is highly tolerant to chemotherapy and the prognosis is poor. Thus, we first explored genes those are regulated by both Sp1 and HIFs in CCC cells by cDNA microarray analysis. We found that Sp1 and hypoxia inducible factors activate multiple genes under the ischemic condition in CCC cells. Among genes which meet this criteria, ICAM1 is one of a most dramatically induced gene. This transcriptional induction involves activation of NFκB associated with mTOR and autonomously produced TNFα. We further performed xenograft tumor experiments to test whether ICAM1 can be induced and contributes to CCC progression in vivo. It was found that ICAM1 is dramatically induced within xenograft tumor tissues. Tumor growth was evidently suppressed when ICAM1 inducton was abrogated by shRNA transfection. Thus, interplay among transcription factors associated with serum deficiency results in cancer progression. This study highlights importance of adaptive response to serum-starved condition as to how cancer cells overcome severe hypoxic stress. Citation Format: Shiro Koizume, Shin Ito, Yoshiyasu Nakamura, Mitsuyo Yoshihara, Yasuo Takano, Yohei Miyagi. Sp1 and HIFs mediate synergistic activation of ICAM1 gene under serum starved hypoxia and promotes growth of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2014-502

Hyaluronan in wound healing: Rediscovering a major player

Wound healing involves a series of carefully modulated steps, from initial injury and blood clot to the final reconstituted tissue or scar. A dynamic reciprocity exists throughout between the wound, blood elements, extracellular matrix, and cells that participate in healing. Multiple cytokines and signal transduction pathways regulate these reactions. A major component throughout most of the process is hyaluronan, a straight-chain carbohydrate extracellular matrix polymer. Hyaluronan occurs in multiple forms, chain length being the only distinguishing characteristic between them. Levels of hyaluronan in its high-molecular-weight form are prominent in the earliest stages of wound repair. Progressively more fragmented forms occur in a manner not previously appreciated. We outline here steps in the wound healing cascade in which hyaluronan participates, as well as providing a review of its metabolism. Although described by necessity in a series of quantum steps, the healing process is constituted by a smooth continuum of overlapping reactions. The prevalence of hyaluronan in the wound (initially termed "hexosamine-containing mucopolysaccharide"), particularly in its early stages, was pointed out over half a century ago by the Harvard surgeon J. Engelbert Dunphy. It appears we are now returning to where we started.

Ca2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation.

Calcium (Ca2+) plays a pivotal role in cellular and organismal physiology. The Ca2+ ion has an intermediate protein-binding affinity and thus it can serve as an on/off switch in the regulation of different biochemical processes. The serum level of ionized Ca2+ is regulated with normal ionized Ca2+ being in the range 1.10-1.3mM. Hypocalcaemia (free Ca2+<1.1mM) in critically ill patients is commonly accompanied by haemostatic abnormalities, ranging from isolated thrombocytopenia to complex defects such as disseminated intravascular coagulation, commonly thought to be due to insufficient functioning of anticoagulation pathways. A small amount of fXa (Factor Xa) produced by Factor VIIa and exposed tissue factor is key to initiating blood coagulation by producing enough thrombin to induce the later stages of coagulation. fXa must bind to PS (phosphatidylserine)-containing membranes to produce thrombin at a physiologically significant rate. In the present study, we show that overall fXa activity on PS-containing membranes is sharply regulated by a 'Ca2+ switch' centred at 1.16mM, below which fXa is active and above which fXa forms inactive dimers on PS-exposing membranes. Our data lead to a mathematical model that predicts the variation of fXa activity as a function of both Ca2+ and membrane concentrations. Because the critical Ca2+ concentration is at the lower end of the normal plasma ionized Ca2+ concentration range, we propose a new regulatory mechanism by which local Ca2+ concentration switches fXa from an intrinsically active form to a form requiring its cofactor [fVa (Factor Va)] to achieve significant activity.

Microparticle-associated tissue factor activity in patients with acute unprovoked deep vein thrombosis and during the course of one year

BackgroundTissue factor (TF) is the main in-vivo initiator of blood coagulation. Microparticles (MPs) are small procoagulant membrane vesicles. Elevated TF-bearing MPs have been found in different prothrombotic conditions and MP-associated TF activity may contribute to the pathogenesis of unprovoked deep vein thrombosis (DVT). ObjectiveTo determine MP-TF activity levels at diagnosis of DVT and at four additional time points during the course of one year in a well-defined group of patients with unprovoked DVT of the lower limb. Patients/MethodsIn this study, 41 patients with acute unilateral symptomatic and unprovoked DVT of the lower limb were included and followed for 1year. Venous blood samples for determination of MP-TF activity were drawn at diagnosis of acute DVT, and 1-, 3-, 6-, and 12months later. In addition, 10 young and healthy control subjects were included. ResultsThe median MP-TF activity was 0.06pg/mL (25th-75th percentile: 0.0-0.53) in patients with acute DVT and 0.18pg/mL (0.07-0.33) in healthy controls, and did not differ significantly (p=0.35). No significant changes in MP-TF activity were found in the follow-up measurements. MP-TF activity did also not differ significantly between patients with proximal- or distal DVT and between those with- or without residual DVT after 6months. ConclusionsMP-TF activity is low at the acute event in patients with unprovoked DVT of the lower limb and remains unchanged during the course of the disease. Our data do not support the hypothesis that TF-bearing MPs play a determining role in the pathogenesis of unprovoked DVT.

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

BackgroundHeparanase 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 MethodsSixty 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. ResultsHeparanase 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 31ng/ml predicted a mean survival of 9±1.3months while heparanase procoagulant activity of 31ng/ml or lower predicted a mean survival of 24±4months (P=0.001). Heparanase procoagulant activity was higher than 31ng/ml in the four cases of thrombosis detected during the follow-up period. ConclusionsElevated 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.

Soluble phosphatidylserine binds to two sites on human factor IXa in a Ca2+ dependent fashion to specifically regulate structure and activity.

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

Heparanase multiple effects in cancer

Heparanase is an endo-β-D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Among these are the up-regulation of vascular endothelial growth factor (VEGF)-A, VEGF-C and activation of intra-cellular signaling involved in cell survival and proliferation. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. We had shown that heparanase up-regulated the expression of the blood coagulation initiator- tissue factor (TF) and interacted with the tissue factor pathway inhibitor (TFPI) on the cell surface membrane of endothelial and tumor cells, leading to dissociation of TFPI and resulting in increased cell surface coagulation activity. Moreover, we have demonstrated that heparanase directly enhanced TF activity which led to increased factor Xa production and subsequent activation of the coagulation system. Taking into account the prometastatic, pro-angiogenic and pro-coagulant functions of heparanase, over-expression in human malignancies and abundance in platelets, implies that heparanase is potentially a good target for cancer therapy.

Ca 2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation

Ca 2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation

Novel peptides that inhibit heparanase activation of the coagulation system.

Heparanase is implicated in cell invasion, tumour metastasis and angiogenesis. It forms a complex and enhances the activity of the blood coagulation initiator - tissue factor (TF). We describe new peptides derived from the solvent accessible surface of TF pathway inhibitor 2 (TFPI-2) that inhibit the heparanase procoagulant activity. Peptides were evaluated in vitro by measuring activated coagulation factor X levels and co-immunoprecipitation. Heparanase protein and/or lipopolysaccharide (LPS) were injected intra-peritoneally and inhibitory peptides were injected subcutaneously in mouse models. Plasma was analysed by ELISA for thrombin-antithrombin complex (TAT), D-dimer as markers of coagulation activation, and interleukin 6 as marker of sepsis severity. Peptides 5, 6, 7, 21 and 22, at the length of 11-14 amino acids, inhibited heparanase procoagulant activity but did not affect TF activity. Injection of newly identified peptides 5, 6 and 7 significantly decreased or abolished TAT plasma levels when heparanase or LPS were pre-injected, and inhibited clot formation in an inferior vena cava thrombosis model. To conclude, the solvent accessible surface of TFPI-2 first Kunitz domain is involved in TF/heparanase complex inhibition. The newly identified peptides potentially attenuate activation of the coagulation system induced by heparanase or LPS without predisposing to significant bleeding tendency.

Atherosclerosis and venous thromboembolism — similarities

Copyright © Polskie Towarzystwo Kardiologiczne INTRODUCTION Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), and atherothrombosis have long been considered to be separate entities with distinct pathogenic mechanisms. The traditional rationale for this concept has been based on: (1) pathologic data showing platelet-rich thrombi in the arteries in contrast to ‘red clots’ observed in veins; (2) clinical data on a substantial efficacy of antiplatelet agents in the prevention of arterial thromboembolic events in contrast to negligible benefits from these drugs in patients with VTE; (3) experimental data demonstrating diffuse inflammatory infiltrates, extracellular cholesterol deposits, neovessel formation and calcification within advanced atherosclerotic lesions within the arterial wall in contrast to minor lesions largely within the endothelial layer of the thrombosed veins. A key component of this concept is the assumption that deranged local processes occurring inside the vessel wall account for arterial thrombosis with a minor contribution by blood-borne abnormalities, whereas a combination of extrinsic factors ranging from venous blood stasis to hypercoagulable blood that act on a normal wall of a vein determine the occurrence of venous thrombosis. Experimental data derived from biochemical laboratories, cell cultures and animal models has shown however many similarities in the pathophysiology of arterial and venous thrombosis [1]. Coagulation pathways and their regulation are in fact identical in arteries and veins. Blood circulating in the two types of vessels is the same in terms of coagulation proteins. All kinds of thrombosis are triggered with the same major physiological initiator of blood coagulation, i.e. tissue factor (TF) [2]. Finally, thrombus composition assessed following thrombectomy in patients with ST-segment elevation acute myocardial infarction (MI) is frequently hard to differentiate from that of thrombi obtained from thrombosed veins, as evidenced by the fact that fibrin represents the largest portion of the thrombotic mass in both types of vessels [3, 4]. Despite the key role of activated platelets as a driving factor of thrombus formation under high shear stress conditions, there is convincing evidence for a significant contribution of thrombin and blood coagulation proteins in arterial thrombosis, as well as for their expression in macrophages and other cells within atherosclerotic lesions [5]. Until the beginning of the current century, there was no clinical data to support basic science findings indicating that thrombus formation in most vessels follows the same pattern and once deranged haemostasis tilted toward thrombosis results in increased risk of thromboembolic events putting in danger both the arterial and venous beds. Within the last decade, accumulating evidence has indicated that patients following VTE are at risk of MI or ischaemic stroke and vice versa. However a lot of experimental and clinical questions are to be answered including those as to whether all affected patients are indeed prone to develop the other type of thrombosis and which diagnostic and therapeutic strategy should be adopted in such a clinical setting. This review summarises the most important clinical and experimental data on the association between arterial and venous thrombosis.

Identification of Novel Downstream Molecules of Tissue Factor Activation by Comparative Proteomic Analysis

Tissue factor (TF) is both an initiator of blood coagulation and a signaling receptor. Using a proteomic approach, we investigated the role of TF in cell signaling when stimulated by its ligand, activated factor VII (FVIIa). From a 2-D difference gel electrophoresis (DIGE) study we found forty one spots that were differentially regulated over time in FVIIa stimulated cells or in comparison to nonstimulated cells. Mass spectrometry identifies 23 out of these as 13 different proteins. One of them, elongation factor 2 (EF-2), was investigated in greater detail by Western blot, a protein synthesis assay and cell cycle analysis. When tissue factor was stimulated by FVIIa, the phosphorylation of EF-2 increased which inactivates this protein. Analyzing the effect using site inactivated FVIIa (FVIIai), as well as the protease activated receptor 2 (PAR-2) agonist SLIGKV, indicated that the inactivation was not PAR-2 dependent. A panel of tissue factor mutants was analyzed further to try to pinpoint what part of the cytoplasmic domain that is needed for this effect. Performing a protein synthesis assay in two different cell lines we could confirm that protein synthesis decreased upon stimulation by FVIIa. Cell cycle analysis showed that FVIIa also promotes a higher degree of cell proliferation.

PAR-4 Deficiency Rescues The Embryonic Lethal Phenotype Of TFPI Null Mice

BackgroundTissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor that dampens tissue factor (TF) initiated blood coagulation. It is produced by endothelial cells and megakaryocytes. Mice lacking TFPI activity die in utero from yolk sac hemorrhage or a presumptive consumptive coagulopathy. The embryonic lethal phenotype of TFPI null mice can be rescued by TF or factor VII deficiency. Our laboratory has shown that platelet TFPI limits platelet accumulation, but not fibrin formation, in an in vivo vascular injury model. Interestingly, mice lacking PAR4, the major thrombin receptor in mouse platelets, have decreased platelet accumulation but not fibrin formation following vascular injury. Therefore, we hypothesized that PAR-4 deficiency would counterbalance the absence of TFPI in platelets and rescue the embryonic lethal phenotype of TFPI null mice. Methods/ResultsTFPI heterozygous mice (TFPI+/-; C57/Bl6) were bred with PAR4 null mice (PAR4-/-; C57/Bl6) to generate TFPI+/-/PAR4-/- mice. These mice were bred to assess rescue of TFPI lethality. Of 76 pups closely monitored following birth, 12 (15.8%) were TFPI-/-/PAR4-/-, with 8 (10.5%) surviving to wean. TFPI-/-/PAR4-/- male mice were bred with TFPI+/-/PAR4-/- females for subsequent studies. Of 447 pups generated, 101 TFPI-/-/PAR4-/- mice (22.6%) were produced. Thus, TFPI-/-/PAR4-/- pups survive to wean at approximately one-half the expected frequency. TFPI-/-/PAR4-/- mice surviving to wean appeared healthy with no overt signs of thrombosis, and have apparently normal survival to over 12 months of age. Compared to TFPI+/-/PAR4-/- littermates, TFPI-/-/PAR4-/- have reduced body weight at 8 weeks. Female TFPI-/-/PAR4-/- mice can reproduce successfully. Approximately one-third of the TFPI-/-/PAR4-/- mice have short or totally absent tails. The short tail phenotype develops in utero and changes in tail:body proportion do not occur following birth. There was no difference in blood loss following tail clip between TFPI+/+/PAR4-/-, TFPI+/-/PAR4-/- or TFPI-/-/PAR4-/- mice. There also was no difference in fibrin or platelet accumulation between TFPI+/+/PAR4-/- or TFPI-/-/PAR4-/-mice following venous electrolytic injury. ConclusionsThis is the first reported rescue of the TFPI null embryonic lethal phenotype by a platelet activation defect. Almost one-half of the TFPI-/-/PAR4-/- mice survive through adulthood despite having normal tissue factor function not regulated by TFPI. This, and our inability, thus far, to identify a procoagulant state in the adult mice, suggests that an essential TFPI anticoagulant function is to modulate PAR-4 mediated platelet function; likely by limiting thrombin generation. TFPI may do this through inhibition of TF-fVIIa, or via inhibition of early forms of prothrombinase on the platelet surface, a very recently recognized inhibitory function of TFPI. The long term survival of these mice also demonstrates that unfettered TF-fVlla-fXa mediated signaling events through PAR-2 are not solely responsible for the embryonic lethality of TFPI null mice. However, the short or absent tails that arise during development of TFPI deficient mice suggests that TF and/or TFPI may have roles in notochord development that is separate from the coagulation system. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Downregulation Of Tissue Factor Expression In Human Pericytes In An Endothelial Cell Co-Culture Model

IntroductionTissue factor (TF) is a protein expressed by perivascular cells including pericytes (PCs) where it is responsible for initiation of blood coagulation. Many conditions have been associated with increased cellular TF expression, including inflammation, malignant transformation and tumor angiogenesis. However, our previous studies have shown that TF expression in PCs paradoxically decreases during wound healing.{McDonald:2008fy} To date, no studies have demonstrated the mechanism responsible for the downregulation of TF expression in any cell type. Because angiogenesis is a critical occurrence during wound healing, pro-angiogenic cytokines are likely candidates responsible for the downregulation of TF expression in PCs. Therefore, several well-characterized pro-angiogenic cytokines were studied for their effects on TF expression and activity in PCs. Materials and MethodsPCs (PromoCell, Heidielberg, Germany) were treated directly with each of five individual angiogenic factors: angiopoietin 2 (ANG2, 1-1000ng/mL), basic fibroblast growth factor (bFGF, 0.1-10 ng/mL), platelet derived growth factor (PDGF, 1-1000ng/mL), transforming growth factor beta (TGFB 0.2-200 ng/mL), and vascular endothelial growth factor (VEGF, 1-1000ng/mL). Serum rich media was used for all experiments and all treatments lasted 24 hours. PCs were assessed for TF activity using a coagulation factor X (FX) activation assay. In addition to direct treatment, PCs were assayed for TF protein expression following treatment in a co-culture model. Briefly, PCs were plated in a standard tissue culture treated plate. Human Dermal Microvessel Endothelial Cells (ECs) were placed inside a 0.4um polyester membrane transwell insert, which was placed into the well containing the PCs. This allowed for communication via soluble factors through the membrane, but not direct contact between the two cell types. Co-cultured ECs were treated with angiogenic factors as described above. Additionally, ECs cells were cultured for 24 hours with bFGF and the conditioned media was collected, cleared of cells, and applied to cultured pericytes. Following treatment PCs were lysed and whole cell lysate was separated by polyacrylamide gel electrophoresis and Western blot (WB). WB membranes were probed with anti-TF antibody for total protein determination. Actin was used as a loading control for all blots. ResultsFollowing direct stimulation for 24 hours, none of the angiogenic growth factors assayed produced a reduction in TF activity or antigen, even at supraphysiological concentrations. Following treatment of ECs in a co-culture model neither ANG2, PDGF, TGFB, nor VEGF produced a change in TF expression in pericytes. However, treatment of ECs with bFGF for 24 hours produced a marked decrease in total TF protein expression in co-cultured PCs. Four independent experiments confirmed the decreased expression of TF under these conditions. In contrast, bFGF conditioned EC media applied to PCs for 24 hours resulted in no change in TF protein expression. ConclusionsThe observation that bFGF stimulation of co-cultured ECs, but not direct stimulation, causes a decrease in TF protein expression in PCs suggests that a soluble mediator is responsible for the effect. The finding that a decrease in TF expression was not observed in PCs treated with bFGF-conditioned ECs media suggests that a labile mediator may be responsible. Disclosures:Hoffman:Novo Nordisk: Consultancy, Research Funding; CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding.

Pathogenetic approach to venous thrombosis markers examination

The review summarizes experimental and clinical findings decrypting the mechanisms that initiate venous thrombosis. It is still relevant to consider the pathogenesis of venous thrombosis within the frames of the classic Virchow’s triad, and the mechanisms of interrelation of its separate mechanisms - changes in blood composition, blood flow, or alterations of the blood vessel wall - becomes more clear. Changes in the blood constituents include the amount and functional state of proteins and hemostasis system cells. Among the important changes in blood flow are blood flow rate, affecting the cells and coagulation proteins transport to the site and from the site of thrombosis, and the local shear stress, modulating adhesion and procoagulant activity of endothelium and platelets. Vascular wall provides tissue factor, which is the initiator of blood coagulation; phospholipid surface of cell membranes and microvesicles for assembling coagulation enzyme complexes, as well as adhesion proteins for the blood platelets and leukocytes «capturing». Decreased venous blood outflow and stasis, causing the local hypoxia, are associated with procoagulant changes in blood cells: the expression of P-selectin on endothelium increases, leading to the accumulation of leukocytes and cell microvesicles containing the initiator of blood coagulation - tissue factor. The local concentration of activated clotting factors increases, which along with anticoagulant activity alterations initiates progressing fibrin formation and thrombogenesis. Marking out the key mechanisms allows using them as the potential markers for diagnosing venous thrombosis risk. Among them are cell derived microparticles, cytokines, P-selectin that are investigated as possible indicators of deep vein, pulmonary, cancer associated thrombosis.

The development of the blood clotting initiation conceptions: from A.A. Schmidt to D.M Zubairov

The objective of the review is to cover the formation of modern understanding of molecular mechanisms of blood coagulation initiation. It was provided mainly by the research of Professor D.M. Zubairov and his colleagues. Since 1963, he has established that blood coagulation initiation is not connected to the phenomenon of vascular wall moistening and contact complex factors activation. Research of the thromboplastic activity distribution in tissue cells, blood and in the serum phospholipid microparticles allowed to conclude that blood coagulation is initiated by long-term expression of tissue factor and rapid massive alterations in cellular membranes. This was confirmed by the detection of the turned phospholipids mesophases in tissue thromboplastin preparations and heterogeneity of vitamin К-depending factors binding. Based on the results of the research, a functional conception of blood coagulation initiation by phase alteration of bilayer structure of cellular membranes to a mesomorphic structure was developed. It is caused by different agonists through receptor dependant Са 2+-mobilizing cell signal systems or by massive migration of calcium ions into the cell at its damage. An initial bioimitating non-enzymatic proteolysis vitamin of К-dependant factors and their massive enzymatic activating in the ensembles of enzymatic complexes takes place on heterophase phospholipids surface. Clotting is limited by blood and tissue macrophages, removing cells and phospholipids particles with heterophase surface from cell circulation, and also by anticoagulant factors action. Based on this conception, the researches revealed the pathogenetic of role thrombogenic micro vesicles originating form the cellular membranes transformation in the development of disseminated intravascular coagulation syndrome, myocardial infarction, leucosis, autoimmune and infectious diseases. Finding out the basic concepts of blood coagulation initiation mechanism puts D.M. Zubairov in one row with scientists, pawning the bases of modern biology and medicine.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation

Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIβ and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIβ in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIβ acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

Intercellular transfer of tissue factor via the uptake of tumor-derived microvesicles

Coagulation proteins play a critical role in numerous aspects of tumor biology. Cancer cells express tissue factor (TF), the protein that initiates blood clotting, which frequently correlates with processes related to cell aggressiveness, including primary tumor growth, invasion, and metastasis. It has been demonstrated that TF gets incorporated into tumor-derived microvesicles (MVs), a process that has been correlated with cancer-associated thrombosis. Here, we describe the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. The highly invasive and metastatic MDA-MB-231 cells displayed higher surface levels of functional TF compared with the less aggressive MCF-7 cells. MVs derived from MDA-MB-231 cells were enriched in TF and accelerated plasma coagulation, but MCF-7 cell-derived MVs expressed very low levels of TF. Incubating MCF-7 cells with MDA-MB-231 MVs significantly increased the TF activity. This phenomenon was not observed upon pretreatment of MVs with anti-TF or annexin-V, which blocks phosphatidylserine sites on the surface of MVs. Our data indicated that TF-bearing MVs can be transferred between different populations of cancer cells and may therefore contribute to the propagation of a TF-related aggressive phenotype among heterogeneous subsets of cells in a tumor.