thromboplastin and Graft-Occlusion--Vascular

Because of its unique position at the convergence point of the intrinsic (contact) and extrinsic (tissue factor/factor VIIa) pathways in the coagulation system, coagulation factor Xa (FXa) has been a theoretically interesting therapeutic target for antithrombotic drugs for many years. More recently, the discovery of naturally occurring FXa inhibitors, such as tick anticoagulant peptide and antistasin, has helped substantiate FXa as a desirable target by demonstrating the efficacy and potential safety advantages of FXa inhibition over conventional antithrombotic therapy. These discoveries led to the design and development of many small-molecule inhibitors of FXa, which have provided potent and selective tools for evaluating the potential role of FXa in various diseases. In addition, these advances have been instrumental in defining the biology of FXa and have aided in the discovery of specific receptors and intracellular signaling pathways for FXa that may be important in the progression of, or the response to, various diseases.

Topics: Animals; Cell Adhesion; Clinical Trials as Topic; Coronary Restenosis; Cysteine Endopeptidases; Cytokines; Factor Xa; Factor Xa Inhibitors; Graft Occlusion, Vascular; Humans; Hyperplasia; Inflammation; MAP Kinase Signaling System; Neoplasm Proteins; Neoplasms; Platelet-Derived Growth Factor; Receptor, PAR-2; Receptors, Platelet-Derived Growth Factor; Receptors, Thrombin; Sepsis; Thromboplastin; Up-Regulation

Intimal hyperplasia at the venous anastomosis of dialysis grafts causes early failure. We developed a sheep model of arteriovenous prosthetic grafts that fail rapidly due to intimal hyperplasia with histologic features nearly identical to human access grafts. A prominent feature of lesion development in this model is formation of luminal thrombus that becomes organized into stenosing lesions by macrophage and myofibroblast infiltration. To better understand this process, we examined the presence and activity of tissue factor (TF) in this system. This protein is the physiological initiator of coagulation in vivo and is known to contribute to development of intimal hyperplasia after vascular injury.. Expanded polytetrafluorethylene (ePTFE) grafts were placed between the carotid artery and external jugular vein in sheep. Grafts were examined for luminal TF activity using a novel ex vivo assay. In a separate series of grafts, immunohistochemistry was used to localize smooth muscle cells, monocytes, and TF protein.. At 2 days, luminal TF activity already was higher in the venous and arterial end of the graft than in the adventitia. This high level of activity persisted at 8 weeks. TF activity was higher in the venous end of the grafts than in the arterial end at 2 and 8 weeks (40% and 47% increase, n = 5, n = 3, respectively, P < 0.05). Immunohistochemistry revealed TF protein localized in regions with or adjacent to fibrin accumulation and often in regions close to the lumen.. This study further examines the development of intimal hyperplasia in ePTFE dialysis access grafts. In this model, TF levels on the luminal surface were increased throughout the arteriovenous grafts and the adjacent vessels as early as 2 days after engraftment and for as long as 8 weeks thereafter. The highest levels of activity were found in the venous end of the graft, where hyperplasia is most robust. Increased activity of TF is associated with luminal thrombus, which provides a scaffolding for development of intimal hyperplasia. These findings present an opportunity to develop strategies to limit TF activity within the graft. Further studies using agents delivered locally or incorporated into the graft matrix to block the luminal activity of TF are warranted.

Topics: Animals; Arteriovenous Shunt, Surgical; Blood Vessel Prosthesis; Blood Vessel Prosthesis Implantation; Carotid Arteries; Female; Graft Occlusion, Vascular; Hyperplasia; Immunohistochemistry; Jugular Veins; Male; Models, Animal; Neointima; Prosthesis Design; Renal Dialysis; Sheep; Thromboplastin; Time Factors

Stent implantation into atherosclerotic coronary arteries releases particulate debris and soluble substances that contribute to impaired microvascular perfusion. Here we addressed the potential for microvascular obstruction in patients with stenotic native right coronary arteries (nRCA) compared with saphenous vein grafts on right coronary arteries (SVG-RCA). We enrolled symptomatic, male patients with stable angina pectoris and a flow-limiting stenosis in their nRCA or SVG-RCA (n = 18/18). Plaque volume and composition were analyzed using intravascular ultrasound before stent implantation. Coronary aspirate was retrieved during stent implantation under protection with a distal occlusion/aspiration device and divided into particulate debris and plasma. The release of catecholamines, endothelin, serotonin, thromboxane B2, and tumor necrosis factor-α was measured. The response of rat mesenteric arteries with intact (+E) and denuded (-E) endothelium to aspirate plasma (without and with selective endothelin receptor blockade) was normalized to that by potassium chloride (KClmax = 100%). Plaque volume and composition were not different between nRCA and SVG-RCA. There was less particulate debris (65 ± 8 vs. 146 ± 23 mg; P < 0.05) and more endothelin release (5.8 ± 0.8 vs. 1.3 ± 0.7 pg/ml; P < 0.05) in nRCA than in SVG-RCA, whereas the release of the other mediators was not different. Aspirate from nRCA induced stronger vasoconstriction than that from SVG-RCA [nRCA, 78 ± 6% (+E)/84 ± 5% (-E); SVG-RCA, 59 ± 6% (+E)/68 ± 3% (-E); P < 0.05 nRCA vs. SVG-RCA], which was attenuated by a nonspecific endothelin and a specific endothelin receptor A antagonist. Thus coronary aspirate from stented nRCA is characterized by less debris but more endothelin and stronger vasoconstrictor response than that from SVG-RCA.

Topics: Aged; Angina, Stable; Animals; Coronary Stenosis; Endothelins; Epinephrine; Graft Occlusion, Vascular; Humans; Male; Mesenteric Arteries; Middle Aged; Norepinephrine; Rats; Saphenous Vein; Serotonin; Stents; Thromboplastin; Thromboxane B2; Tumor Necrosis Factor-alpha; Vasoconstriction

Atherosclerotic coronary arteries are more calcified in patients with than without chronic kidney disease (CKD). We addressed the potential for coronary microvascular obstruction in patients with and without CKD during stenting for saphenous vein aorto-coronary graft (SVG) stenosis under protection with a distal occlusion/aspiration device. In patients with and without CKD (n = 20/20), SVG plaque composition was analyzed from virtual histology using intravascular ultrasound analysis before stent implantation. There was more dense calcium and more necrotic core in patients with than without CKD (14 ± 3 vs. 3 ± 1 % and 21 ± 3 vs. 12 ± 2 % of plaque volume, respectively). Coronary aspirate was retrieved during stent implantation and divided into particulate debris and plasma. Patients with CKD had more particulate debris and calcium release than patients without CKD. In contrast, the release of serotonin was less in patients with than without CKD (0.4 ± 0.1 vs. 1.2 ± 0.3 μmol/L), whereas that of catecholamines, endothelin, tissue factor, thromboxane, tumor necrosis factor α, and C reactive protein was not significantly different. Confirming the biochemical results, aspirate plasma from patients with CKD induced less vasoconstriction of rat mesenteric arteries than that from patients without CKD (with endothelium (+E), 26 ± 7 %; without endothelium (-E): 28 ± 7 % vs. +E, 68 ± 12 %; -E: 95 ± 16 % of maximum KCl-induced vasoconstriction). Graft atherosclerosis of patients with CKD is more degenerated and releases more particulate debris and calcium, but the aspirate has surprisingly less serotonin and vasoconstrictor potential.

Topics: Adult; Aged; Aged, 80 and over; alpha-2-HS-Glycoprotein; Animals; Atherosclerosis; Blood Vessel Prosthesis Implantation; C-Reactive Protein; Calcium; Coronary Artery Disease; Graft Occlusion, Vascular; Humans; Male; Middle Aged; Necrosis; Plaque, Atherosclerotic; Rats; Renal Insufficiency, Chronic; Saphenous Vein; Stents; Thromboplastin; Tumor Necrosis Factor-alpha; Ultrasonography, Interventional; Vascular Calcification; Vasoconstriction

The resistance of internal mammary artery (IMA) toward thrombotic occlusion and accelerated atherosclerosis is not well understood. This study analyzed gene expression profiles of vascular smooth muscle cells (VSMCs) from IMA versus saphenous vein (SV).. 54'675 probe sets were examined by Affymetrix microarrays. Thirty-one genes belonged to the coagulation system; 2 were differentially expressed, namely tissue factor (TF) and tissue-type plasminogen activator (tPA). TF was 3.1-fold lower in IMA than SV (P=0.006), whereas tPA was 9.0-fold higher (P<0.001). TF mRNA expression was lower in IMA than SV (P<0.05); tPA was higher (P<0.001). TF protein expression was 4.2+/-0.5-fold lower in IMA than SV (P<0.001); tPA was 2.6+/-0.4-fold higher (P<0.01). In IMA VSMC supernatant, TF protein and activity was lower (P<0.05), TFPI and tPA protein higher (P<0.05 and P<0.005), and clotting time of human plasma prolonged (P<0.05) as compared to SV. Migration to TF/FVIIa (10(-9) mol/L) was 3-fold lower in IMA than SV (P=0.01); PAR-2 protein expression was similar (P=NS), PAR-2 blockade without effect (P=NS).. Among the genes of the coagulation system, TF and tPA are differentially expressed in VSMCs from IMA versus SV. This is consistent with protection of IMA from thrombus formation and vascular remodeling.

Topics: Atherosclerosis; Blood Coagulation; Coronary Artery Bypass; Gene Expression Profiling; Graft Occlusion, Vascular; Humans; Lipoproteins; Mammary Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oligonucleotide Array Sequence Analysis; Plasminogen Activator Inhibitor 1; RNA, Messenger; Saphenous Vein; Thromboplastin; Thrombosis; Tissue Distribution; Tissue Plasminogen Activator; Transplantation, Autologous

Activation of inflammatory and procoagulant mechanisms is thought to contribute significantly to the initiation of restenosis, a common complication after balloon angioplasty of obstructed arteries. During this process, expression of tissue factor (TF) represents one of the major physiologic triggers of coagulation that results in thrombus formation and the generation of additional signals leading to vascular smooth muscle cell (VSMC) proliferation and migration. In this study, we have investigated the mechanisms by which inhibition of coagulation at an early stage through overexpression of tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF, might reduce restenosis. In a rabbit femoral artery model, percutaneous delivery of TFPI using a recombinant adenoviral vector resulted in a significant reduction of the intimamedia ratio 21 days after injury. Investigating several markers of inflammation and coagulation, we found reduced neointimal expression of monocyte chemoattractant protein-1 (MCP-1), lesional monocyte infiltration, and expression of vascular TF, matrix metalloproteinase-2 (MMP-2), and MMP-9. Moreover, overexpression of TFPI suppressed the autocrine release of platelet-derived growth factor BB (PDGF-BB), MCP-1, and MMP-2 in response to factors VIIa and Xa from VSMCs in vitro and inhibited monocyte TF activity. These results suggest that TFPI exerts its action in vivo through not only thrombotic, but also nonthrombotic mechanisms.

Topics: Adenoviridae; Angioplasty; Animals; Becaplermin; Cell Division; Cell Movement; Chemokine CCL2; Cloning, Molecular; Constriction, Pathologic; DNA, Complementary; Factor VIIa; Factor Xa; Graft Occlusion, Vascular; Humans; Immunohistochemistry; In Vitro Techniques; Inflammation; Lipoproteins; Matrix Metalloproteinase 2; Microscopy, Confocal; Monocytes; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Platelet-Derived Growth Factor; Precipitin Tests; Proto-Oncogene Proteins c-sis; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Time Factors; Transfection; Transgenes; U937 Cells

Tissue factor (TF) is a membrane-bound glycoprotein that initiates the clotting cascade. Inhibition of the TF pathway has been shown to prevent thrombosis and restenosis after arterial injury in a variety of animal models.. We describe a novel approach to inhibiting the expression of the TF protein that involves the targeted destruction of cellular TF mRNA with the use of a tetraloop hairpin ribozyme. After construction of the ribozyme and determination of its optimal length and kinetic parameters, a ribozyme expression vector that used the retroviral vector pMV12 was constructed. The ability of this expression vector to generate anti-TF ribozyme was further augmented by positioning of the anti-TF ribozyme downstream of a rat tRNA val (RNA polymerase II) promoter. The resultant construct containing the anti-TF ribozyme was then used to transfect vascular smooth muscle cells and generate a variety of clonal cell lines. Northern blot analyses performed on 3 transfected and 3 untransfected clones demonstrated markedly reduced TF mRNA levels in the transfected clones both during quiescence and after serum stimulation. Cell lysates analyzed for total TF activity by monitoring factor Xa generation similarly demonstrated a statistically significant and concordant reduction in TF activity in smooth muscle cells transfected with the ribozyme expression vector compared with both untransfected clones and clones transfected with the empty vector.. These results demonstrate the feasibility of an antithrombotic strategy based on ribozyme technology.

Topics: Animals; Base Sequence; Blotting, Northern; Cells, Cultured; Factor Xa; Feasibility Studies; Gene Expression Regulation; Genetic Vectors; Graft Occlusion, Vascular; Male; Molecular Sequence Data; Muscle, Smooth, Vascular; Nucleic Acid Conformation; Rats; Rats, Sprague-Dawley; RNA; RNA, Catalytic; RNA, Messenger; RNA, Transfer, Val; Structure-Activity Relationship; Thromboplastin; Thrombosis; Transcriptional Activation; Transfection

We investigated changes in blood coagulation in the coronary circulation after percutaneous transluminal coronary angioplasty (PTCA) and its clinical significance. We examined 43 patients with ischemic heart disease who underwent elective PTCA of isolated stenotic lesions in the left coronary artery. Ten patients underwent PTCA alone, 15 received percutaneous transluminal rotational atherectomy (PTRA) and 18 stent implantation. Blood samples were drawn from the coronary sinus before and immediately after PTCA, as well as 4 and 24 h later. Plasma levels of tissue factor (TF), thrombin-antithrombin III complex (TAT) and prothrombin fragment 1+2 (F 1+2) were measured by enzyme-linked immunosorbent assay. Follow-up coronary angiography was performed 6 months after PTCA. Minimal luminal diameter was assessed by quantitative coronary angiography to evaluate late loss index. TF, TAT and F 1+2 levels in the coronary sinus blood showed significant increases 24 h after PTCA. A significant positive correlation was found between changes in TF levels 24 h after PTCA and late loss index 6 months after the procedure. TF levels in the coronary sinus blood were significantly higher in patients with late restenosis than in those without restenosis. These results suggest that TF expression in the coronary circulation after PTCA is a prognostic factor for late restenosis.

Topics: Adult; Aged; Aged, 80 and over; Angioplasty, Balloon, Coronary; Biomarkers; Blood Coagulation; Coronary Circulation; Coronary Disease; Female; Graft Occlusion, Vascular; Humans; Male; Middle Aged; Prognosis; Recurrence; Thromboplastin

Healthy endothelium provides a nonthrombogenic surface. In this study the authors investigated the effect of arterial flow on the saphenous vein endothelial expression of proteins controlling thrombosis. Human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit with flow conditions shown to simulate arterial or venous circulations. In separate experiments, placement of an external polytetrafluoroethylene (PTFE) stent was used to differentiate the effects of pulsatile wall deformation and shear stress, while addition of drugs to the vein perfusate allowed study of the role of ion channels in transducing the response of the vein to arterial flow. Endothelial concentrations of thrombomodulin, nitric oxide synthase, tissue factor, and tissue plasminogen activator were assessed by quantitative immunohistochemistry and Western blotting of endothelial cell lysates, in paired vein samples, in comparison to control proteins. Arterial flow conditions caused a rapid and significant reduction in the endothelial concentration of thrombomodulin: The immunostaining area decreased from 80.1 +/- 7.0 to 48.3 +/- 5.0 and 32.9 +/- 3.0% at 45 and 90 minutes respectively, p = 0.01. These findings were confirmed by Western blotting. The reduction in thrombomodulin concentration was unaffected by eliminating vein wall deformation by placement of an external PTFE stent or by including the K+ channel blocker tetraethylammonium (TEA) in the vein perfusate. In contrast, thrombomodulin concentrations remained high when blockers of stretch-activated cation and calcium channels were included in the vein perfusate. The endothelial concentration of nitric oxide synthase increased after 90 minutes of arterial flow and this change was abolished when TEA was included in the vein perfusate. Arterial flow induced rapid changes in saphenous vein antithrombotic proteins. Different cation channels mediated the flow-induced changes in thrombomodulin and nitric oxide synthase.

Topics: Blood Flow Velocity; Blotting, Western; Endothelium, Vascular; Graft Occlusion, Vascular; Hemodynamics; Humans; Immunoenzyme Techniques; Ion Channels; Models, Cardiovascular; Nitric Oxide Synthase; Polytetrafluoroethylene; Pulsatile Flow; Saphenous Vein; Thrombomodulin; Thromboplastin; Tissue Plasminogen Activator; Veins

Although saphenous vein is the most reliable conduit for arterial interposition procedures in the coronary circulation, graft thrombosis remains a clinical problem. We hypothesized that an important factor in early graft thrombosis is sudden change in the hemodynamic environment of the vein as it is placed in the coronary circulation.. We used an ex vivo perfusion system to study freshly excised segments of human saphenous vein (HSV) and pig internal jugular vein. For coronary graft (CAVG) simulation, sections of HSV were subjected to arterial pulsatile pressure and flow and twisting and stretching to mimic deformations caused by the beating heart. Using functional and immunohistochemical assays, we investigated the effect of these conditions on expression of tissue factor (TF), an important prothrombotic surface molecule.. In each of 11 experiments (6 human, 5 porcine), vein segments from a single donor were subjected to venous conditions (VEN), CAVG perfusion, or no perfusion. Expression of TF was measured as the amount of factor Xa generated per unit area of luminal vein surface. VEN perfusion did not cause a significant change in mean TF expression over nonperfused control values (human: 14.3 +/- 1.5 versus 11.4 +/- 2.3 U/cm2, p = 0.31; pig: 11.6 +/- 1.5 versus 12.5 +/- 1.4 U/cm2, p = 0.70). CAVG perfusion led to significant enhancement of TF expression over VEN perfusion (human: 36.8 +/- 6.2 versus 14.3 +/- 1.5 U/cm2, p < 0.05; pig: 40.0 +/- 9.9 versus 11.6 +/- 1.5 U/cm2, p < 0.05). Immunohistochemical analysis showed positive TF staining on the luminal side of a CAVG-stimulated HSV segment, but not on a VEN-stimulated segment. In four additional studies, HSV segments were subjected to arterial perfusion without twist and stretch to mimic lower extremity arterial interposition grafts. TF expression for lower extremity venous graft perfusion was significantly higher than for VEN perfusion (25.3 +/- 2.5 versus 14.3 +/- 1.5, p < 0.01) but not significantly different from CAVG perfusion.. Our studies in a unique perfusion system suggest that exposure of vein to coronary arterial hemodynamic conditions results in elevated expression of the important prothrombotic molecule TF. This phenomenon may contribute to early graft thrombosis.

Topics: Animals; Coronary Artery Bypass; Coronary Circulation; Factor Xa; Graft Occlusion, Vascular; Hemorheology; Humans; Immunohistochemistry; Jugular Veins; Models, Cardiovascular; Pulsatile Flow; Saphenous Vein; Swine; Thromboplastin; Thrombosis

Thrombolytic therapy is frequently used to manage vascular graft thrombosis. However, long-term patency after thrombolysis remains poor. The purpose of this study was to characterise the morphological and functional response of endothelial cells (EC) exposed to a thrombus and subsequently lytic therapy.. Human EC were exposed to human whole blood thrombus for 2, 6, 12, and 24 h. The thrombus was lysed with urokinase. Cell morphology was studied with electron microscopy. Northern blot analyses were performed with human c-DNA probes for endothelin-1, thrombomodulin, tissue factor, tissue plasminogen activator, plasminogen activator inhibitor, and triose phosphate isomerase.. EC retraction occurred for each period of incubation. Thrombomodulin expression was increased 2.2-fold at 6 h and 2.4-fold at 24 h. t-PA expression was depressed proportionally to the duration of thrombus exposure. PAI and TF expression transiently increased 1.5-fold at 2 h of exposure and returned to baseline at 6 h. Endothelin expression remained unchanged.. Except for a transient increase in TF expression and reversal of the tPA/PAI ratio, EC exposed to thrombus do not appear to become actively procoagulant. The increase in TM expression may reflect enhanced thromboresistance. However, EC retraction may be responsible for an increase thrombogenicity of saphenous vein graft after thrombosis and Urokinase therapy.

Topics: Cells, Cultured; Endothelin-1; Endothelium, Vascular; Graft Occlusion, Vascular; Humans; Microscopy, Electron, Scanning; Plasminogen Activator Inhibitor 1; Saphenous Vein; Thrombolytic Therapy; Thrombomodulin; Thromboplastin; Thrombosis; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

Primary antiphospholipid syndrome (PAPS) is an autoimmune disorder manifested by recurrent thrombosis in the venous and arterial system. We report a group of seven patients with lower limb ischaemia associated with PAPS. Four were male patients and three were females, with a mean age of 37 years. All had a previous deep vein thrombosis and the majority, five out of seven, had a prior cerebrovascular accident (CVA). Prolonged activated thromboplastin time was demonstrated in all our patients and PAPS was established by positive thromboplastin titration index, circulating anticoagulant index and increased anticardiolipin levels. Symptoms included claudication in three, rest pain in four and gangrene in five patients. Angiography demonstrated thrombosis of various segments of the arterial tree including: aorta, iliac, femoral and popliteal arteries. Two patients were treated conservatively and one by percutaneous transluminal angioplasty (PTA) of the distal aorta. A total of eleven vascular surgical procedures were performed in four patients resulting in early postoperative thrombosis (2h-30 days) in 10 cases. Only one graft remained patent, when full heparinisation (1000 units/h) was used perioperatively. We conclude that PAPS patients are at high risk for graft thrombosis and should only be operated upon on full anticoagulation, starting at operation and proceeding indefinitely.

Topics: Adolescent; Adult; Aged; Antiphospholipid Syndrome; Female; Graft Occlusion, Vascular; Humans; Ischemia; Leg; Male; Middle Aged; Partial Thromboplastin Time; Postoperative Complications; Thrombectomy; Thromboembolism; Thromboplastin; Ultrasonography; Whole Blood Coagulation Time