Fibrinolytic Factors Research Articles

Marked Impairment of Protease-Activated Receptor Type 1-Mediated Vasodilation and Fibrinolysis in Cigarette Smokers: Smoking, Thrombin, and Vascular Responses In Vivo

We sought to test the hypothesis that cigarette smoking adversely alters protease-activated receptor type 1 (PAR-1)-mediated vascular effects in vivo in humans. Distinct from its role in the coagulation cascade, thrombin exerts its major cellular and cardiovascular actions via PAR-1. The activation of PAR-1 causes endothelium-dependent arterial vasodilation and the release of endogenous fibrinolytic factors. Forearm blood flow was measured with venous occlusion plethysmography in 12 cigarette smokers and 12 age- and gender-matched nonsmokers during intrabrachial infusions of PAR-1-activating-peptide (SFLLRN; 5 to 50 nmol/min), bradykinin (100 to 1,000 pmol/min), and sodium nitroprusside (2 to 8 mug/min). Plasma tissue plasminogen activator (t-PA) and plasminogen-activator inhibitor 1 antigen and activity concentrations were measured throughout the experiment. All agonists caused dose-dependent increases in forearm blood flow (p < 0.0001 for all). Although bradykinin and sodium nitroprusside caused similar vasodilation, SFLLRN-induced vasodilation was attenuated in smokers (p = 0.04). Smokers had modest reductions in bradykinin-induced active t-PA release (reduced by 37%, p = 0.03) and had a marked impairment of SFLLRN-induced t-PA antigen (p = 0.02) and activity (p = 0.006) release, with a 96% reduction in overall net t-PA antigen release. The use of SFLLRN also caused similar (p = NS) increases in inactive plasminogen-activator inhibitor 1 in both smokers and nonsmokers (p <or= 0.002 for both). Cigarette smoking causes marked impairment of PAR-1-mediated endothelial vasomotor and fibrinolytic function. Relative arterial stasis and near abolition of t-PA release will strongly promote clot propagation and vessel occlusion. These findings suggest a major contribution of impaired endothelial PAR-1 action to the increased atherothrombotic risk of cigarette smokers.

Studies of TGF-β 1-3 in Serosal Fluid During Abdominal Surgery and Their Effect on In Vitro Human Mesothelial Cell Proliferation

Increased transforming growth factor-beta (TGF-beta) levels are associated with fibrosis, affected cell proliferation, and postsurgical adhesion development, but the knowledge regarding TGF-beta response to the surgical trauma is limited. This study investigated TGF-beta(1-3) isoforms and fibrinolytical factors in peritoneal serosal fluid during abdominal surgery, together with the in vitro effect of TGF-beta(1-3) on human mesothelial cell proliferation. Total as well as biologically active TGF-beta(1-3) and fibrinolytic factors: t-PA, uPA, and PAI-1 were measured in serosal fluid and plasma from 23 patients undergoing colorectal cancer surgery. In vitro proliferation of human primary mesothelial cell cultures upon TGF-beta(1-3) stimulation was also investigated. Total TGF-beta1 and TGF-beta2 levels were similar in serosal fluid and plasma while active fractions were increased in serosal fluid. In contrast, total fraction of TGF-beta3 was higher in serosal fluid compared with plasma, while levels of active fractions did not differ. Plasminogen activators (t-PA, uPA) were elevated while the inhibitor (PAI-1) was decreased in serosal fluid compared with plasma. The in vitro mesothelial cell proliferation studies revealed that high TGF-beta(1-3) concentrations decreased cell proliferation, while lower concentrations of TGF-beta1 increased mesothelial cell proliferation. This human study shows increased active TGF-beta levels in peritoneal serosal fluid, compared with plasma, during abdominal surgery and that TGF-beta1 at physiological concentrations increased human mesothelial cell proliferation in vitro. TGF-beta cytokines may be involved in postsurgical adhesion formation.

Response of fractional synthesis rate (FSR) of fibrinogen, concentration of D-dimer and fibrinolytic balance to physical activity-based intervention in obese children.

Physical activity-induced reduction in obesity-related hyperfibrinogenemia in children has been reported. The underlying mechanisms remain elusive. Further, the effect of such interventions on fibrinolysis in children is scarce. To investigate in obese children, before and after a physical activity-based intervention: (i) the mechanistic role of fractional synthesis rate (FSR) of fibrinogen in the reduction of hyperfibrinogenemia; and (ii) the changes in fibrinolytic factors. Subjects included 21 (age > 14 < 18 years; Tanner stage, IV-V) children (15 obese, BMI >95%tile for age and sex and six lean, BMI <85%tile). After baseline measurements of FSR of fibrinogen, and concentrations of fibrinogen, D-dimer, PAI-1 and t-PA in all children, studies were repeated after a 3-month randomized controlled physical activity-based lifestyle intervention in obese children only. FSR of fibrinogen was higher (P = 0.002) in the obese (vs. lean) group, which was reduced (P = 0.001) after intervention. This almost completely accounted for the reduction in obesity-related hyperfibrinogenemia. High levels of D-dimer decreased (P = 0.001) after intervention, whereas fibrinolysis was not enhanced. The direct reduction in the FSR of fibrinogen and the remarkable correlation between the magnitudes of reduction in fibrinogen FSR and concentration signify a mechanistic role for FSR in the regulation of physical activity-induced reversal of hyperfibrinogenemia in obese children. The congruent reductions in the FSR of fibrinogen and the concentrations of fibrinogen and D-dimer in response to intervention despite depressed fibrinolysis suggest an overall improvement in the hypercoagulable state in obese children with physical activity-based lifestyle intervention.

Role of the Endothelium in the Vascular Effects of the Thrombin Receptor (Protease-Activated Receptor Type 1) in Humans

The purpose of this study was to determine the role of the endothelium in the vascular actions of protease-activated receptor type 1 (PAR-1) activation in vivo in man. Thrombin is central to the pathophysiology of atherothrombosis. Its cellular actions are mediated via PAR-1. Protease-activated receptor type 1 activation causes arterial vasodilation, venoconstriction, platelet activation, and tissue-type plasminogen activator release in man. Dorsal hand vein diameter was measured in 6 healthy volunteers before and after endothelial denudation. Forearm arterial blood flow, plasma fibrinolytic factors, and platelet activation were measured in 24 healthy volunteers during venous occlusion plethysmography. The effects of inhibition of prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor on PAR-1 responses were assessed during coadministration of aspirin, the "NO clamp" (L-N(G)-monomethyl arginine and sodium nitroprusside), and tetraethylammonium ion, respectively. Endothelial denudation did not affect PAR-1-evoked venoconstriction (SFLLRN; 0.05 to 15 nmol/min). Although aspirin had no effect, SFLLRN-induced vasodilation (5 to 50 nmol/min) was attenuated by the NO clamp (p < 0.0001) and tetraethylammonium ion (p < 0.05) and abolished by their combination (p < 0.01). The NO clamp augmented SFLLRN-induced tissue-type plasminogen activator and plasminogen activator inhibitor type 1 antigen (p < 0.0001) release, but tetraethylammonium ion and aspirin had no effect. SFLLRN-induced platelet activation was unaffected by NO or prostacyclin inhibition. Acting via PAR-1, thrombin causes contrasting effects in the human vasculature and has a major interaction with the endothelium. This highlights the critical importance of endothelial function during acute arterial injury and intravascular thrombosis, as occurs in cardiovascular events including myocardial infarction and stroke.

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 ( P < 0.006), plasminogen activator inhibitor-1 ( P < 0.04), and tissue plasminogen activator ( P < 0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.

Effect of obesity and physical activity on D‐dimer – A Randomized controlled study in Adolescents

We studied the effects of obesity and physical activity on D‐dimer (DD), a marker of increased turnover of intravascular cross‐linked fibrin, in children and its relationship with fibrinolytic factors, fibrinogen &amp; fibrinogen fractional synthetic rate (FSR) in 21 children (15 obese and 6 lean;Tanner stage, IV–V). Obese subjects underwent a 3‐month randomized controlled physical activity‐based intervention. New data show that the higher DD levels in obese vs. lean controls (112.3±11.8 vs. 63.7±12.9 ng/mL; p=0.02) were reduced after the intervention (p&lt;0.01). We previously reported that fibrinogen levels and FSR decreased (p&lt;0.01), while fibrinolytic factors were unaffected by the intervention. Parallel reductions in DD and fibrinogen levels and the FSR of fibrinogen after intervention indicated a putative decrease in the production and availability of fibrinogen for fibrin formation, thus ‘indirectly’ lowering DD, the primary degradation product of fibrin. The lack of change in fibrinolysis reflects a lack of degradation of preexistent fibrin, but may also argue against generation of new fibrin from fibrinogen representing a potentially beneficial change in coagulation function. Therefore, it appears that despite depressed fibrinolysis, the balance between coagulation and fibrinolysis was maintained after intervention suggesting a state of overall lower atherogenesity.

Role of coagulation and fibrinolysis in lung and renal fibrosis

Elevated procoagulant and suppressed fibrinolytic activities are regularly encountered in different forms of clinical and experimental fibrosis of the lungs and the kidneys. Although primarily serving to provide a provisional matrix of repair largely consisting of fibrin and fibronectin, the involved procoagulant serine proteases and protease inhibitors may also exert distinct cellular downstream signaling events modifying the fibrotic response. In this review, evidence for an impaired regulation of coagulation and fibrinolysis factors in clinical and experimental lung and renal fibrosis is provided and the role of PAR (protease activated receptor) induced profibrotic and HGF (hepatocyte growth factor) elicited antifibrotic cellular events is worked out. In view of experiments obtained in animal models of lung and renal fibrosis, the potential therapeutic usefulness of anticoagulant or profibrinolytic strategies is discussed.

Effect of Shear Stress on the Expression of Coagulation and Fibrinolytic Factors in Both Smooth Muscle and Endothelial Cells in a Co-Culture Model

Blood vessels are subjected to forces due to the flow. Endothelial cells (EC) are recipients, cross-talk with smooth muscle cells (SMC), and regulate physiology. It was hypothesized that both EC and SMC respond to shear stress, which alters the expression of factors in coagulation and fibrinolysis. Methods: A co-culture of human saphenous vein EC (HSVEC) and human saphenous vein SMC (HSVSMC) was exposed to shear, following which the cells were separated. Gene expression of tissue factor, thrombomodulin (TM), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) were analyzed with real-time RT-PCR. Protein expression was studied with ELISA. In HSVEC, the expression of PAI-1 (×2.1), tPA (×1.8), uPA (×1.6), tissue factor (×2.5) and TM (×1.9) was upregulated after 4 h of shear compared to controls. After 24 h of shear, expression was still upregulated in tPA (×2.3) and TM (×1.6). In HSVSMC, change in expression of PAI-1 (×2.1) was present after 4 h and in uPA (×2.1), and TM (×0.4) after 24 h. Both HSVEC and HSVSMC responded to shear, which led to altered expression of coagulation and fibrinolytic factors. This indicates that SMC, and interactions between EC and SMC, are more important in the regulation of vascular wall hemostasis than earlier studies have reported.

Blood coagulation and fibrinolytic factors

Blood coagulation and fibrinolytic factors

A new possible strategy of prevention and preventive treatment of age-related macular degeneration

The beneficial effect achieved by the treatment of endothelial dysfunction in chronic cardiovascular diseases is already an evidence belonging to the basic treatment of the disease. Given the fact that the vascular system is uniform and consubstantial both physiologically, pathophysiologically and in terms of therapy, and that it plays a key role in age-related macular degeneration (AMD)--a disease leading to tragic loss of vision with its etiology and therapy being unknown--endothelial dysfunction should be treated. The pleiotropic effects of ACE-inhibitors, AR-blockers and statins help to restitute the balance between vasodilators and vasoconstrictors in endothelial dysfunction caused by oxidative stress, the balance of growth factors and their inhibitors, pro- and anti-inflammatory substances and prothrombotic and fibrinolytic factors, inhibit the formation of oxidative stress and its harmful effects; while aspirin with its pleiotropic effects acting as an antiaggregation substance on platelets helps to set the endothelial layer back to its normal balance regarding its vasodilating, antithrombotic, antiadhesive and anti-inflammatory functions; trimetazidine as an adjuvant agent helps to normalize, to restore the disturbed metabolism of the retinal tissue functioning insufficiently, in the end. For the above reasons it is suggested that, as a part of long term primary and/or secondary prevention, the following groups of patients with AMD receive--taking into consideration all possible side effects--ACE-inhibitor and/or AR blocker and statin and aspirin treatment, and trimetazidine as adjuvant medicine 1. those without maculopathy but being above the age of 50 and having risk factors inducing endothelial dysfunction; 2. those, who already developed AMD in one eye as a prevention in the second, unaffected eye; and 3. those patients who developed AMD in both eyes in order to ameliorate or merely slow the progression of the disease. Besides, it is advisory to eliminate AMD risk factors inducing oxidative stress with consecutive endothelial dysfunction.

Nutrition and hemostasis: A focus on urbanization in South Africa

South Africa is experiencing a rapid urbanization of its African population characterized by a demographic, nutrition, lifestyle, and health transition. The resultant high prevalence of high cardiovascular disease, in particular of stroke, is of concern. In this narrative review it is suggested that, together with hypertension, changes in the hemostatic system may be one of the major contributors to stroke in this population. It is further suggested that these changes are related to increased fat and animal protein intakes, decreased intakes of total carbohydrate and dietary fiber, as well as persistent suboptimal micronutrient intakes of Africans in transition. The effects of this nutrition transition on plasma fibrinogen, fibrin network structures, plasminogen activator inhibitor 1 activity levels and some other clotting and fibrinolytic factors are discussed. It is concluded that despite indications of present protective mechanisms against the development of coronary heart disease (CHD) in this population the observed changes in diet and hemostatic profiles may eventually lead to a high prevalence of both stroke and CHD in urban black South Africans. It is further suggested that timely nutritional interventions and research of effects thereof on the hemostatic system are urgently needed.

Relation of depression to various markers of coagulation and fibrinolysis in patients with and without coronary artery disease

It has been suggested that changes in blood coagulation and fibrinolysis might explain the observed association between depression and coronary artery disease (CAD). So far, only a few coagulation factors have been investigated in this regard, and the results were not consistent. The aim of our study was to analyse a broad range of coagulation and fibrinolytic factors, with emphasis on factors directly involved in clot formation and degradation or reflecting coagulation activation, in patients with CAD and controls without CAD, as assessed by coronary angiography, who also underwent a diagnostic procedure for depression. We screened 306 patients with CAD and controls without CAD for depression using the Hospital Anxiety and Depression Scale and Allgemeine Depressions Skala-L questionnaires. In participants with positive screening result, diagnosis of major depression was confirmed or excluded by a structured interview. We analysed the following coagulation and fibrinolytic factors: fibrinogen, prothrombin fragment F1+2, factor XIII A-subunit, factor XIII B-subunit, tissue plasminogen activator, plasminogen activator inhibitor-1, thrombin-activable fibrinolysis inhibitor, and D-dimer. We did not observe significant associations between depression and CAD, nor between depression and cardiovascular risk factors. Coagulation and fibrinolytic factors showed no differences between patients with CAD and controls, but they were associated with several cardiovascular risk factors. Depression was not associated with coagulation and fibrinolytic factors. No associations were found either when both CAD and depression were taken into account. Our study gives no evidence that there is a significant relation among depression, CAD, and blood coagulation and fibrinolysis.

The Plasminogen Fibrinolytic Pathway Is Required for Hematopoietic Regeneration

Hematopoietic stem cells within the bone marrow exist in a quiescent state. They can differentiate and proliferate in response to hematopoietic stress (e.g., myelosuppression), thereby ensuring a well-regulated supply of mature and immature hematopoietic cells within the circulation. However, little is known about how this stress response is coordinated. Here, we show that plasminogen (Plg), a classical fibrinolytic factor, is a key player in controlling this stress response. Deletion of Plg in mice prevented hematopoietic stem cells from entering the cell cycle and undergoing multilineage differentiation after myelosuppression, leading to the death of the mice. Activation of Plg by administration of tissue-type plasminogen activator promoted matrix metalloproteinase-mediated release of Kit ligand from stromal cells, thereby promoting hematopoietic progenitor cell proliferation and differentiation. Thus, activation of the fibrinolytic cascade is a critical step in regulating the hematopoietic stress response.

Changes in the haemostatic system after thermoneutral and hyperthermic water immersion

Warm water bathing is a popular recreational activity and is frequently used in rehabilitation medicine. Although well tolerated in most cases, there are reports indicating an increased risk of thrombotic events after hot tub bathing. The effects of a 45 min thermoneutral bath followed by a 50 min bath with increasing water temperature (maximum 41 degrees C) until reaching a body core temperature of 39 degrees C on factors of blood coagulation and fibrinolysis were studied in eight healthy male volunteers. Blood was obtained after a 45-min resting period as control and after the thermoneutral and hyperthermic bath as well as after another 45 min recovery period at the end of the study. Hyperthermic immersion (HI) lead to a shortening of activated partial thromboplastin time (aPTT) (P < 0.05). Fibrinogen concentration decreased immediately after HI (P < 0.05) but increased during recovery (P < 0.05). Plasminogen activator inhibitor (PAI) activity decreased during HI (P < 0.05), D-dimer concentration was not found to change. Thrombocyte count increased (P < 0.05) during HI. The increases in tissue-type plasminogen activator concentration as well as leucocyte count during HI were due to haemoconcentration. Prothrombin time, PAI-activity and granulocyte count decreased during thermoneutral immersion (P < 0.05). Warm water bathing leads to haemoconcentration and minimal activation of coagulation. The PAI-1 activity is decreased. A marked risk for thrombotic or bleeding complications during warm water bathing in healthy males could not be ascertained.

Psychosocial factors and venous thromboembolism: a long-term follow-up study of Swedish men.

The link between psychosocial factors and coronary heart disease is well established, but although effects on coagulation and fibrinolysis variables may be implicated, no population-based study has sought to determine whether venous thromboembolism is similarly related to psychosocial factors. To determine whether venous thromboembolism (deep vein thrombosis or pulmonary embolism) is related to psychosocial factors. A stress questionnaire was filled in by 6958 men at baseline from 1970 to 1973, participants in a cardiovascular intervention trial. Their occupation was used to determine socio-economic status. After a maximum follow-up of 28.8 years, 358 cases of deep vein thrombosis and/or pulmonary embolism were identified through the Swedish hospital discharge and cause-specific death registries. In comparison with men who, at baseline, had no or moderate stress, men with persistent stress had increased risk of pulmonary embolism [hazard ratio (HR)=1.80, 95% CI: 1.21-2.67]. After multivariable adjustment, the HR decreased slightly to 1.66 (95% CI: 1.12-2.48). When compared with manual workers, men with white-collar jobs at intermediate or high level and professionals showed an inverse relationship between occupational class and pulmonary embolism (multiple-adjusted HR=0.57, 95% CI: 0.39-0.83). Deep vein thrombosis was not significantly related to either stress or occupational class. Both persistent stress and low occupational class were independently related to future pulmonary embolism. The mechanisms are unknown, but effects on coagulation and fibrinolytic factors are likely.

IL-8 induces imbalances between nitric oxide and endothelin-1, and also between plasminogen activator inhibitor-1 and tissue-type plasminogen activator in cultured endothelial cells

Interleukin-8 (IL-8), a member of the CXC chemokine family, plays an important role in the modulation of multiple biological functions in endothelial cells containing the receptors CXCR1 and CXCR2. It has previously been shown that IL-8 directly enhances endothelial cell survival, and stimulates the production of matrix metalloproteinases, which in turn regulates angiogenesis. However, its role in the regulation of the production of vasoactive substances in endothelial cells is less well defined. In this study, we investigate the effects of IL-8 on the proliferation of human umbilical vein endothelial cells (HUVECs). In addition, we also study the effects of IL-8 on the production of vasodilator, vasoconstrictor and fibrinolytic factors in these cells. The results show that recombinant IL-8 (50–200 ng/ml) induces neither HUVEC proliferation nor nitric oxide (NO) release. However, it significantly increases the production of endothelin-1 (ET-1) in a concentration-dependent manner. Furthermore, incubation of endothelial cells with IL-8 (200 ng/ml) up-regulates the plasminogen activator inhibitor-1 (PAI-1) in HUVECs, while it down-regulates the tissue plasminogen activator (t-PA). These findings suggest that IL-8 offsets the balance between endothelial vasoconstrictors and vasodilators. Furthermore, IL-8 also leads to an imbalance between PAI-1 and t-PA, which causes the ECs to become procoagulative and hypofibrinolytic.

A redox-silent analogue of tocotrienol inhibits hypoxic adaptation of lung cancer cells

We have previously reported that a redox-silent analogue of α-tocotrienol (T3), 6- O-carboxypropyl-α-tocotrienol (T3E) shows more potential anti-carcinogenic property than T3 in a lung cancer cell (A549 cell). However, the mechanisms by which T3E exerts its potential anti-carcinogenic effect is still unclear. As tumor malignancy is associated with hypoxia adaptation, in this study, we examined whether T3E could suppress survival and invasion in A549 cells under hypoxia. Hypoxia treatment drastically-induced activation of the protein tyrosine kinase, Src, and its regulated signaling required for hypoxia adaptation of A549 tumor cells. The survival and invasion capacity of the tumor cells under hypoxia was suppressed by T3E via the inactivation of Src. More specifically, T3E-dependent inhibition of Src-induced Akt activation contributed to suppression of cell survival under hypoxia, and the reduction of fibrinolytic factors such as plasminogen activator-1(PAI-1) via the decrease of hypoxia-inducible factor-2α by T3E led to inhibition of hypoxic invasion. Overall these results suggest that T3E suppresses hypoxia adaptation of A549 cells by the inhibition in hypoxia-induced activation of Src signaling.

Novel Functions for a Fibrinolytic Pathway in Controlling Hematopoiesis.

The adult mammalian hematopoietic system maintains a well-regulated supply of mature and immature hematopoietic cells within the circulation throughout life. The system is capable of rapid recovery and compensation following injury, or physiological stress. There is still an incomplete understanding of which factors regulate such a stress response. We show that plasminogen (Plg), a classical fibrinolytic factor, is a key regulator controlling the hematopoietic stress response.The fibrinolytic factors Plg and tPA are found within the BM after myelosuppression, where they could activate another protease cascade, namely matrix metalloproteinases (MMPs). Activation of MMP-9 releases e.g. Kit ligand (KitL), which in turn control hematopoietic cell proliferation and differentiation. This process helps to replenish the hematopoietic cells within the BM by promoting hematopoietic stem and progenitor cell proliferation and differentiation. We were able to demonstrate that deletion of Plg in mice prevented hematopoietic stem cells from entering the cell cycle and undergoing multi-lineage differentiation after myelosuppression, leading to the death of the mice. Activation of Plg by tissue type plasminogen activator (tPA) activated MMPs and stimulated the release of KitL from stromal cells.The tPA-mediated release of KitL from stromal cells took place via MMP activation as addition of a MMP inhibitor inhibited KitL release from stromal cells.tPA administration in vivo augmented the number of BM cells in wild-type controls, but this was attenuated in both Plg deficient and MMP-9 deficient mice. tPA increased the number of immature spleen colony forming units (colony forming units-spleen), and long term culture-initiating cells and the c-Kit+/ Sca-1+/lin− (KSL) cell fraction in BM cells of treated animals. To test if tPA-mediated hematopoietic cell proliferation is dependent on KitL in vivo, we treated KitL deficient Sl/Sld mice and WBB6F1+/+ control mice with tPA. BM cellularity and the number of immature CFU-S progenitor cells increased in WBB6F1+/+ animals by day 2 after tPA injections, but not in Sl/Sld (KitL deficient) mice. To elucidate whether the observed tPA effects on hematopoietic cells were mediated directly by tPA or indirectly, e.g., via BM stromal cells, we examined the effect of tPA on hematopoietic cell expansion in vitro. tPA improved proliferation of lin− cells only in stromal-cell based cultures (MS-5 feeder layer), but not in the absence of a feeder layer. In synergy, tPA and KitL expanded the number of progenitors in stromal-cell based cultures. If c-Kit/KitL signaling following tPA treatment promotes hematopoietic cell proliferation, blocking the signaling should prevent tPA-induced progenitor proliferation. Indeed, addition of neutralizing antibodies against c-Kit and/or KitL prevented the tPA-mediated generation of CFU-Cs from lin− cells in stromal cell-based cultures. Our data indicate that fibrinolytic factors can be found in the regenerative phase after myelosuppression in the BM. Here, fibrinolytic co-assemble and are activated result in an orderly, protease cascade, like MMPs. This proteolytic growth factors bioavailable, which in a fine-tuned hematopoietic cell proliferation and differentiation. The involvement of the fibrinolytic system in the regulation of adult stress hematopoiesis represents a new paradigm with important implications for cancer therapy and regenerative medicine.

Defibrotide Counteracts the Modifications of Anti-Thrombotic Phenotype of Endothelial Cells Induced by Thalidomide.

Introduction: Patients with Multiple myeloma are at relatively high risk of developing thromboembolic events, usually deep vein thromboses (DVT). There are numerous contributing factors, including therapy, such as thalidomide, where DVT has been identified as a major toxicity, especially when thalidomide is used in combination with other treatments such as dexamethasone. The mechanisms by which thalidomide predisposes to thrombosis are not well understood. Defibrotide (DF) is an orally biovailable polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic and anti-adhesive properties. Previously, DF has been shown to dose-dependently counteracted the increase in Plasminogen Activator Inhibitor-1 (PAI-1) expression and decrease on tissue plasminogen activator (t-PA) activity after lipopolysaccharide (LPS) stimulation of endothelial cells in vitro.Methods and Results: We have conducted in vitro studies using human microvascular endothelial cells (HMEC) in order to investigate the effect of different doses of thalidomide on various fibrinolytic factors. In addition, we evaluated whether DF modulates changes of fibrinolysis induced by thalidomide. HMEC were treated with 50 and 100μg/ml of thalidomide for 24 hours in presence and absence of DF (at a dose of 150μg/ml). t-PA and PAI-1 gene expression were evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Release of t-PA and PAI-1 were evaluated by imunoenzymatic assay (ELISA). Furthermore, we evaluated the fibrinolytic activity of cell surpernatant using a fibrin clot plate assay. In this method the fibrin clot was formed by mixing fibrinogen, plasminogen and thrombin. The plasmin generated by the cell surpernatant was able to digest fibrin and also hydrolyzed the chromogenic substrate S-2251. The RT-PCR results showed that thalidomide reduces t-PA (2.2 fold) and increases PAI-1 gene expression (4.0 fold) in HMEC cells, whereas DF was able to counteract this effect by up-regulating the t-PA and down-regulating PAI-1 gene expression induced by thalidomide (8.8 and 2.0 fold, respectivielly). Similar results was observed analyzing t-PA release by HMEC cells treated with different concentrations of thalidomide with and without DF. Thalidomide significantly reduces the t-PA released in both concentrations (p<0.001) and DF significantly increase the release of t-PA reduced by thalidomide (p<0.01). The changes of fibrinolytic activity in HMEC by thalidomide and the capacity of DF to restore the fibrinolysis was confirmed by analyzing the lyses of fibrin clots with endothelial cell surpernatant (p<0.01).Conclusions: These results show that DF is able to counteract the alterations of fibrinolytic factors in HMEC treated with thalidomide. Whilst further studies in preclinical MM models are underway, these data suggest a potential role for DF in the prevention of DVT induced by thalidomide and support ongoing clinical trials of DF in combination with thalidomide-based treatment.

Genomic, Proteomic and Functional Screens Identify PAI-1 as an Important Factor Influencing Mesenchymal Stromal Cell Survival Post-Transplantation In Vivo.

MSCs have robust reparative properties through their ability to limit apoptosis, enhance angiogenesis and direct positive tissue remodelling. However, a major limitation on the widespread application of MSC transplantation is that transplanted cells have low survival rates in vivo. Thus, enhancing MSCs survival post-transplantation is critical for the successful implementation of cellular therapy. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue - such as infarcted myocardium - where there is poor blood supply and low oxygen tension. How these factors influence MSC survival is still unclear, therefore the objective of this project was to better understand how hypoxia under low nutrient conditions affects the behaviour and survival of MSCs. To answer this question, we performed the following series of experiments:1.Isolate and immunophenotype murine MSCs.2.Assess impact of in vitro ischemic conditions (hypoxia and serum deprivation) on MSCs by microarray analysis and ITRAQ proteomics for secreted proteins.3.Confirm changes of specific factors in vitro.4.Isolate and immunophenotype MSCs from knock-out mice for specific factors.5.Determine whether knock-out MSCs have altered survival in vitro and in vivo.As confirmed by PCR and Western blotting, microarray and proteomic screens, identified plasminogen activator inhibitor-1 (PAI-1) as one factor consistently upregulated in hypoxia (3% oxygen) treated MSCs. In vitro migration studies demonstrated that PAI-1 blocks migration of MSCs towards a chemotactic gradient, while isolation of PAI-1 knock-out MSCs, revealed an enhanced survival of PAI-1null MSCs over wild-type MSCs. In vivo subcutaneous transplantation of beta-galactosidase engineered wild-type and PAI-1null MSCs, recapitulated our in vitro results, such that 14 days post-implantation PAI-1null MSCs had a five fold increase in survival compared to wild-type MSCs. In conclusion, PAI-1 is a multifunctional protein. As an inhibitor, PAI-1 acts as ‘bait' regulating the conversion of plasminogen to plasmin thus functioning as a major control point in the regulation of systemic fibrinolysis and local growth factor availability. In addition, PAI-1 can also act as a ligand for vitronectin to influence cellular migration/adhesion. Up-regulation of PAI-1 by MSCs after transplantation impacts MSC survival. Determining how to block PAI-1 following transplantation of MSCs is currently under investigation.