Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall.

BACKGROUND: Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). METHODS: Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. RESULTS: An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). CONCLUSION: SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

Effect of miR-223 on thrombophlebitis rats through regulating Toll-like receptor signaling pathway.

OBJECTIVE: To explore the effect of the micro ribonucleic acid (miR)-223 on the thrombophlebitis rats by regulating the Toll-like receptor (TLR) signaling pathway. MATERIALS AND METHODS: The rat model of thrombophlebitis was established, and miR-223 was silenced or overexpressed through lentiviral transfection. The rats were divided into miR-223 inhibitors group (Inhibitors group), miR-223 mimics group (Mimics group), and normal group (Control group). The transfection efficiency of miR-223 in venous tissues was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR), the hemorheological indexes plasma viscosity (PV) and hematocrit (HCT) were observed, and the content of the serum inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-ß (TNF-ß) were detected via enzyme-linked immunosorbent assay (ELISA). Moreover, the fibrinolytic indexes plasminogen activator inhibitor (PAI) and the tissue-type plasminogen activator (t-PA) were detected, the morphological changes in the venous tissues were observed via hematoxylin-eosin (HE) staining, and the gene and protein expressions of the TLR signaling pathway were detected via RT-PCR and Western blotting. RESULTS: The expression of miR-223 was significantly increased in the Mimics group (p<0.05) and significantly decreased in the Inhibitors group (p<0.05). The high-shear and low-shear whole blood viscosity and HCT in the Inhibitors group were significantly higher than those in the Mimics group (p<0.05). The levels of serum IL-6, IL-1ß, and TNF-ß in the Inhibitors group were remarkably higher than those in the Mimics group (p<0.05). The Inhibitors group had a remarkably lower level of t-PA (p<0.05) and a remarkably higher level of PAI than the Mimics group (p<0.05). Besides, the inferior vena cava wall shed and disappeared due to complete necrosis in the Inhibitors group. In the Mimics group, the vascular lumen was slightly expanded, and the vascular wall had intact contour. It was found in the gene detection that the mRNA levels of TLR2, myeloid differential protein-88 (MyD88) and c-Jun N-terminal kinase (JNK) were evidently increased in the Inhibitors group, and the significant increases in the protein levels of TLR2 and MyD88 were also observed in the protein detection. CONCLUSIONS: The overexpression of miR-223 can inhibit the TLR signaling pathway, thereby promoting the recovery of thrombophlebitis rats.

Specific uptake of radioiodinated fragment E1 by venous thrombi in pigs.

Fragment E1, a product of plasmic digestion of cross-linked fibrin, binds specifically in vitro to polymerized fibrin but not to fibrinogen. Purified human Fragment E1 was radiolabeled with 125I or 131I by the Iodogen technique. The uptake of radioiodinated Fragment E1 in vitro into forming or preformed clots was demonstrated. Animal biodistribution studies of radioiodinated Fragment E1 showed its rapid removal from the circulation; radioactive catabolites did not reside long in any organ and were excreted in the urine. The uptake in vivo was evaluated in pigs with preexisting venous thrombi of various ages from 1 h up to 5 d at the time of intravenous systemic injection of the tracer. Radioiodinated fibrinogen was also injected into the same animals to compare the uptake of the two tracers. Thrombus-to-blood ratios for Fragment E1 averaged 43:1 (range 10-108) and 29:1 (range 8-107) in thrombi 1-6 h and 1-5 d old, respectively. In contrast, mean thrombus-to-blood ratios for fibrinogen were, in the same time intervals, 26:1 (range 17-41) and 2:1 (range 0.5-3.9), respectively. It is concluded that radioiodinated Fragment E1 is a specific marker of thrombi in vivo: its uptake by fresh thrombi is better than that of labeled fibrinogen and, in contrast to radioiodinated fibrinogen, this fragment is incorporated into old thrombi as well.

Effects of heparin and epsilon-aminocaproic acid in dogs on plasmin-125-I generation in response to urokinase injections and venous injury.

The isotopic method described previously for quantification of plasmin- (125)I by disc gel electrophoresis was modified by inclusion of euglobulin precipitation to expand its applicability to plasmas containing low radioactivity of plasmin- (125)I and plasminogen- (125)I. It was found that the euglobulin precipitation method precipitates 72.4+/-2.1 (sd)% of both plasmin- (125)I and plasminogen- (125)I. Using this method and plasminogen- (125)I as a tracer, studies were first made of the effects of heparin and epsilon-aminocaproic acid in dogs on plasmin- (125)I generation in responese to a single injection of urokinase and to venous injury; second, of the effects of venous occlusion and thrombosis on plasmin- (125)I generation; and third, in vitro studies of plasminogen- (125)I affinity to fibrin and its activation in blood clots. The venous injury was produced by the damage of venous endothelium by an injection of 90% phenol and the thrombosis by a thrombin injection into an occluded vein. Heparin and epsilon-aminocaproic acid under the present experimental conditions inhibited about 78 and 100%, respectively of plasmin- (125)I generation by the urokinase injection. Similar inhibitory effects of heparin and epsilon-aminocaproic acid were observed on plasmin- (125)I generation in response to venous injury. The venous occlusion caused a small degree of plasmin- (125)I generation, but thrombin thrombosis did not seem to stimulate the generation of plasmin- (125)I. The in vitro studies showed that plasminogen- (125)I does not have a specific affinity to fibrin and is incorporated into blood clots in approximately equal concentrations as those in serum during clotting processes, and that blood clots per se do not stimulate plasmin- (125)I generation. These results suggest that injured veins release considerable amounts of vascular plasminogen activators into circulation and that these play an important role in thrombus dissolution in vivo.

Plasminogen- 125 I responses in dogs to a single injection of urokinase and typhoid vaccine and to vascular injury.

In vivo plasminogen responses to various stimuli were studied. Plasminogen-(125)I was prepared and used first for metabolic studies of plasminogen in control dogs. The average results were: the plasma plasminogen, 29.3+/-4.1 (SD) mg/kg; the interstitial plasminogen, 8.79+/-4.47 (SD) mg/kg; the half-life of plasma plasminogen-(125)I, 2.81+/-0.24 (SD) days; the fractional direct catabolic rate of plasminogen (j(3)), 0.295 day(-1); and the catabolic (synthetic) rate of plasminogen, 8.61+/-1.35 (SD) mg/kg per day. Studies were then made of the plasminogen-(125)I responses in dogs to a single injection of urokinase (A) and typhoid vaccine (B), and to vascular injury (C), which was produced by the damage of venous endothelium by a phenol injection. Effects of heparin were also studied in dogs given the phenol injection (D). Disc electrophoretic analysis of plasma showed generation of plasmin-(125)I in all except the control experiments. The duration of plasmin-(125)I generation was about 6 hr in A, 6 hr in B, and at least 5 days in C. Heparinization (D) shortened the duration of generation to about 6 hr. For further quantitative analysis of the tracer data, a model for coexistent plasminogen-(125)I and plasmin-(125)I was proposed and validated, from which some new analytical methods were derived. Using these methods, the average fractional rate of plasmin-(125)I generation from plasminogen-(125)I (j(4)) was 0.41 day(-1) in A, 0.30 day(-1) in B, 0.324 day(-1) in C, and 0.382 day(-1) in D. Further mathematical consideration showed that j(3) was zero at least in C during plasmin generation. Plasminogen synthesis was unchanged in all experiments. The average fractional breakdown rate of plasmin-(125)I (j(5)) in A, B, C, and D was 1.19, 1.13, 1.35, and 1.11 day(-1), respectively, and were closely similar. These results indicate that under normal conditions a major portion of plasminogen is directly catabolized without the formation of plasmin, but that significant amounts of plasmin were generated under the conditions described, that the normal process of direct breakdown of plasminogen is abolished during plasmin generation at least in C, and that the potential value of j(5) determination should be further explored.

Selectin-mucin interactions as a probable molecular explanation for the association of Trousseau syndrome with mucinous adenocarcinomas.

Trousseau described spontaneous, recurrent superficial migratory thrombophlebitis associated with occult cancers, and this was later correlated with disseminated microangiopathy (platelet-rich clots in small blood vessels). Trousseau syndrome often occurs with mucinous adenocarcinomas, which secrete abnormally glycosylated mucins and mucin fragments into the bloodstream. Since carcinoma mucins can have binding sites for selectins, we hypothesized that selectin-mucin interactions might trigger this syndrome. When highly purified, tissue-factor free carcinoma mucin preparations were intravenously injected into mice, platelet-rich microthrombi were rapidly generated. This pathology was markedly diminished in P- or L-selectin-deficient mice. Heparin (an antithrombin-potentiating agent that can also block P- and L-selectin recognition of ligands) ameliorated this platelet aggregation, but had no additional effect in P- or L-selectin-deficient mice. Inhibition of endogenous thrombin by recombinant hirudin also did not block platelet aggregation. Mucins generated platelet aggregation in vitro in hirudinized whole blood, but not in platelet-rich leukocyte-free plasma nor in whole blood from L-selectin-deficient mice. Thus, Trousseau syndrome is likely triggered by interactions of circulating carcinoma mucins with leukocyte L-selectin and platelet P-selectin without requiring accompanying thrombin generation. These data may also explain why heparin ameliorates Trousseau syndrome, while vitamin K antagonists that merely depress thrombin production do not.

Hyperglycemia accelerates arterial thrombus formation and attenuates the antithrombotic response to endotoxin in mice.

Recent human studies reveal that hyperglycemia induces procoagulant and antifibrinolytic effects in blood that may contribute to a greater risk of arterial thrombosis, but the direct relationship between high blood glucose levels and thrombosis has not yet been investigated. We performed a number of experiments to clarify whether hyperglycemia was causally related to arterial thrombosis and whether the combined stimulus of hyperglycemia and inflammation would enhance the thrombotic effect. In a model of ferric-chloride-induced carotid artery thrombosis, hyperglycemia did not influence the time to occlusion in mice pretreated with streptozotocin, but the rate of thrombus formation was accelerated. This effect was associated with increased thrombin generation and could not be explained by changes in vessel-wall tissue factor activity. The prothrombotic effect of hyperglycemia was assessed in a separate experiment, showing that collagen/thrombin-induced platelet procoagulant activity was increased in hyperglycemic mice. The effect of inflammation was studied by injecting a low dose of endotoxin that caused a systemic inflammatory state after 24 h (increased plasma levels of tumor necrosis factor alpha, interleukin-6 and monocyte chemotactic protein 1 in diabetic and nondiabetic mice) associated with a mild delay in thrombus formation. This reduced rate of thrombus formation was attenuated by hyperglycemia. Together, these data establish a discrete but clear contribution of hyperglycemia in experimental arterial thrombosis.

Assessment of apoptotic cells in the wall of thrombophlebitic saphenous vein.

INTRODUCTION: Programmed cell death plays a critical role in various physiological processes. In the present study, we investigated its possible pathogenic role in primary varicose veins. We studied histological changes in surgical specimens from thrombophlebitic saphenous veins. In thrombophlebitic saphenous, varicose, and healthy veins, we also determined the number of apoptotic cells, and investigated apoptosis in the role of the pathogenesis of varicose veins. METHODS: Forty-four specimens of thrombophlebitic saphenous veins and simple varicose veins were collected. Thirteen samples of normal great saphenous veins were also collected (control group). Apoptosis of venous walls was determined by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and immunofluorescence methods. The corpuscular number per high-power field was counted under light microscopy. RESULTS: A significantly higher apoptotic ratio of the intima and media were observed in control veins as compared with thrombophlebitic saphenous veins and simple varicose veins (p < 0.01). A significant difference was not observed between thrombophlebitic saphenous veins and simple varicose veins (p > 0.05). A significant difference was not seen between the intima and media of the three groups (p > 0.05). CONCLUSION: In the walls of thrombophlebitic saphenous veins and varicose veins, the apoptotic indices were clearly decreased. The results suggest that the process of programmed cell death was inhibited in walls of thrombophlebitic saphenous veins and varicose veins.

Fibrinogen uptake by thrombi: effect of thrombus age.

The uptake of radiolabeled fibrinogen in canine thrombi was determined at varying times after thrombus induction by electric current. The greatest thrombus/blood ratio was achieved when fibrinogen was administered 4 hr after thrombus induction but definite thrombus fibrinogen uptake was still observed when the tracer was administered up to 72 hr after thrombus induction. There was continued fibrinogen accumulation despite a decrease in weight of older thrombi suggesting that net thrombus propagation is not necessary for labeled fibrinogen uptake. Our results suggest that the fibrinogen uptake test may be useful for the diagnosis of deep vein thrombosis for several days after the onset of thrombosis.

Organ distribution of canine leukocytes labeled with 99mTc-sulfur colloid.

Previous reports have shown that scintigraphic localization of acute inflammation can be achieved using autologous leukocytes labeled in vitro with 99mTc-sulfur colloid (TcSC). The technique is limited, however, by a marked accumulation of radioactivity in the lungs and liver of normal animals. A modified procedure was developed using preparations of TcSC of small particle size to label blood leukocytes in vitro. Markedly decreased levels of lung and liver radioactivity and elevated levels of blood radioactivity were found after intravenous infusion of autologous canine leukocytes labeled by this method. These leukocytes could be used to image areas of acute inflammation resulting from induction of septic or sterile venous thrombi.

A randomised, double-blind, placebo-controlled trial of dermatan sulphate for prevention of deep vein thrombosis in hip fracture.

Dermatan sulphate (MF 701) is a natural glycosaminoglycan that catalyses thrombin inhibition by heparin cofactor II. The aim of the study was to evaluate the efficacy and safety of MF 701 for prevention of deep vein thrombosis (DVT) in patients with hip fracture. A randomised, double-blind, placebo-controlled design was used to assess two dose regimens of MF 701 in two consecutive study phases. Treatment was started within 48 h from the trauma and continued for 14 days for non-operated patients or until the 10th postoperative day. Bilateral mandatory venography was used to assess the end-point. Eighty patients were included in the first phase (40 MF 701, 40 placebo). MF 701, 100 mg IM b.i.d., did not reduce incidence of DVT from that on placebo and did not induce any bleeding. In the second phase 126 patients were included, with a randomisation ratio of 2:1 (84 MF 701, 300 mg IM b.i.d., 42 placebo). Bilateral venography was obtained for 110 patients. The incidence of DVT was 64% (23/36) in the placebo group and 38% (28/74) in the MF 701 group (p = 0.01; odds ratio [OR] = 0.34, 95% confidence limits [CL] = 0.15-0.80p; proximal DVTs were 42% (15/36) and 20% (15/74), respectively (p = 0.02; OR = 0.36, CL = 0.15-0.89). No significant differences were found in haemorrhagic complications (2.4% in each group), blood loss from drains, blood transfusions, haemoglobin and haematocrit values. This study is the first demonstration that dermatan sulphate is a clinically effective antithrombotic agent without bleeding effects. It also provides evidence of the biological role of heparin cofactor II.

Superficial vein thrombosis of lower limbs: influence of factor V Leiden, factor II G20210A and overweight.

Superficial vein thrombosis (SVT) has been reported in patients with thrombophilia. In the present unmatched case-control study, the two most common thrombophilic abnormalities (factor V Leiden and factor II G20210A) were searched for in 112 consecutive patients with SVT of lower limbs and in 180 healthy donors. FV Leiden was present in 16/112 (14.3%) SVT patients and 11/180 (6.1%) controls (odds ratio 2.51, 95% CI 1.04-6.24) and FII G20210A in 4/112 (3.6%) patients and 2/180 (1.1%) controls (OR 3.28, 95% CI 0.46-36.84). In addition, body mass index (BMI) > or =28 kg/m2 was also associated with SVT (OR 2.81, 95% CI 1.60-5.00). After adjustment for BMI > or =28 kg/m2, the association between FV Leiden and SVT remained strong though no longer statistically significant. Among patients with SVT, the presence of FV Leiden was independently associated with the absence of varicose veins (OR 4.62, 95% CI 1.25-18.0) and with a BMI > or =28 kg/m2 (OR 3.74, 95% CI 1.05-15.1). In conclusion, both FV Leiden and overweight seem to predispose to SVT, a finding that should be confirmed in larger studies.

Studies of the effects of estradiol, progesterone, cortisol, thrombophlebitis, and typhoid vaccine on synthesis and catabolism of antithrombin III in the dog.

Effects of estradiol, progesterone, cortisol, thrombophlebitis and typhoid vaccine on the synthesis and catabolism of antithrombin III (AT) in dogs were studied, using I-125-labeled AT (I-125-AT) as a tracer. Five dogs were used for each study. A single intramuscular injection of 20 mg estradiol caused a 20% decrease of plasma AT concentration in 6 days without appreciable changes in the plasma half-lives of I-125-AT but with a significant decrease in the fractional catabolic rate of I-125-AT(j3u). A single intramuscular injection of 250 mg progesterone did not produce any appreciable changes of plasma AT concentration, the plasma half-lives of I-125-AT or j3u. On the other hand, intravenous and intramuscular injections of a total of 750 mg cortisol caused a 17% increase of plasma AT concentration in a day after the injections without alterations of the plasma half-lives of I-125-AT or j3u. Next, thrombophlebitis was produced in dogs by a single intravenous injection of 1 ml 90% phenol into a leg vein occluded for 1 min by a gauze tourniquet and the effects of thrombophlebitis were studied. The results indicated that it did not cause appreciable changes of plasma AT concentration, the plasma half-lives of I-125-AT or j3u. However, studies of the effects of a single intravenous injection of 3 ml typhoid vaccine showed a 25% decrease of plasma AT concentration in a day after the injection with a moderate acceleration of the decline rate of plasma I-125-AT and a 14% increase in j3u values. Further studies in heparinized dogs showed similar effects with typhoid vaccine. These results indicate that estradiol causes a decreased rate of AT synthesis, that progesterone has no appreciable effects on AT metabolism, that cortisol increases the rate of AT synthesis, that localized thrombophlebitis has no appreciable effects on AT metabolism and that typhoid vaccine causes an increased j3u by unknown mechanisms which is not an accelerated coagulation process.

Fibrinopeptide A in urine from patients with venous thromboembolism, disseminated intravascular coagulation and rheumatoid arthritis--evidence for dephosphorylation and carboxyterminal degradation of the peptide by the kidney.

Urinary fibrinopeptide A immunoreactivity was determined by radioimmunoassay using two anti-fibrinopeptide A sera with a different specificity in patients with venous thromboembolism, disseminated intravascular coagulation and rheumatoid arthritis. Elevated levels were frequently observed with both sera, and intravenous administration of heparin in patients with a thromboembolic disorder resulted in a decline of urinary fibrinopeptide A (FPA) concentrations to normal or nearly normal values. For both sera significant correlations with plasma levels were found although one of the sera reacted significantly better with the material in urine samples from these patients than the other (p less than 0.0001, n = 73). Analysis of urinary fibrinopeptide A immunoreactivity by high performance liquid chromatography (HPLC) provided evidence that A peptide material present in this body fluid was heterogeneous. In view of the characteristics of the antisera used in this study, data suggest that urinary FPA immunoreactivity consists to a large extent of carboxyterminally degraded FPA. Excretion of circulating FPA immunoreactive material through the kidneys apparently involves dephosphorylation and carboxyterminal breakdown of the A peptide. Since both synthetic and native phosphorylated or unphosphorylated fibrinopeptide A appeared to be stable in urine in vitro, an active role of the kidney in degrading the A peptide is likely.

Intramuscular dermatan sulfate MF701 in patients with hip fracture: relationship between pharmacokinetics and antithrombotic efficacy.

Two groups of 23 and 84 patients with hip fracture received intramuscularly 100 and 300 mg dermatan sulfate (MF701) b.i.d., respectively, for the prophylaxis of deep vein thrombosis. Median duration of treatment was 17 and 16 days, respectively. Four blood samples were collected from each patient while under treatment. Plasma levels of dermatan sulfate were determined by a chromogenic substrate assay. A one-compartment model for multiple doses was employed to estimate the pharmacokinetic parameters. Fitting was applied to mean plasma concentrations calculated for each sampling time and weighted according to the number of samples available at each time. Thrombin clotting time was measured on the same plasma samples. Antithrombotic efficacy was assessed by bilateral venography. Plasma levels of dermatan sulfate increased gradually throughout the treatment, indicating a marked accumulation process. Time to reach steady-state was 14 or 9 days with 100 or 300 mg b.i.d., respectively. This was due to an apparent prolonged terminal half-life (68 or 43 h), which actually reflected slow absorption from the injection sites. The clinical efficacy of MF701 in preventing DVT was found to be dependent on the plasma concentration of the drug and also, but less significantly, on the prolongation of thrombin clotting time. Dermatan sulfate plasma levels greater than 9 micrograms/ml are advisable to optimize efficacy in hip fracture patients.

A pilot study of subcutaneous recombinant hirudin (HBW 023) in the treatment of deep vein thrombosis.

BACKGROUND: Recombinant hirudin, a pure, specific antithrombin could be more effective than heparin in the treatment of deep vein thrombosis, but its short half-life requires constant intravenous infusion, whereas subcutaneous administration of recombinant hirudin can ensure stable and prolonged plasma levels. The aim of our study was to assess the pharmacokinetics, the results on the coagulation variables, and the safety of a recombinant hirudin (HBW 023) administered subcutaneously in patients suffering from deep vein thrombosis. METHODS: Recombinant hirudin (HBW 023) was administered subcutaneously to 10 patients with recent deep vein thrombosis, at a dose of 0.75 mg/kg of body weight twice daily for 5 days, after which standard heparin and acenocoumarol were introduced. Bilateral lower limb venography, and pulmonary angiography, and/or ventilation-perfusion lung scan were carried out on day 1 prior to recombinant hirudin injection and repeated on day 5. aPTT and recombinant hirudin plasma levels were serially assessed after the 1st and the 10th injections. Prothrombin fragments 1 + 2, thrombin-antithrombin III complexes, fibrin degradation products were collected on days 1 and 5. RESULTS: Clinical evolution was uneventful in all but one patient who had a probable recurrence of pulmonary embolism on day 4. No hemorrhagic complication, no untoward biological event was observed. On days 5, Marder score was unchanged or had decreased. Plasma levels of recombinant hirudin peaked in between 3 and 4 h following the injection. aPTT values paralleled, and were significantly correlated with plasma levels of recombinant hirudin on day 1 as well on day 5 (r = 0.903, r = 0.948 respectively). Fragment 1 + 2, and thrombin antithrombin complexes non-significantly decreased from day 1 to day 5. CONCLUSIONS: Subcutaneous administration of recombinant hirudin ensures prolonged stable plasma levels of recombinant hirudin which results in efficient anticoagulation. A dose-ranging study conducted with subcutaneous recombinant hirudin in comparison to conventional heparin therapy may answer the question as to efficacy.

The amount of plasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in human thrombi and the relation to ex-vivo lysibility.

Thrombolytic therapy successfully reopens obstructed blood vessels in the majority of cases. However, it is not known why a substantial amount of thrombi are resistant to lysis by a fibrinolytic agent. In vitro studies have demonstrated that tissue-type plasminogen activator (t-PA) and plasminogen incorporated in the clot (during formation) increase lysibility. To test whether lysibility of in vivo formed human thrombi is related to their composition, we studied 25 venous thrombi obtained at autopsy and 21 arterial thrombi obtained during embolectomy. Plasminogen activator inhibitor-1 (PAI-1) antigen was measured in a phosphate-buffered saline (PBS) extract of each thrombus; t-PA antigen and plasminogen antigen were determined in a 6 M urea extract of the thrombus, representing bound proteins. Lysibility was measured as weight reduction during 8 h of incubation in PBS containing streptokinase (SK) 100 U/ml, corrected for spontaneous lysis, reflected by weight loss in PBS without SK. In addition, lysibility in SK was compared with lysibility in urokinase (UK) 100 U/ml and in t-PA 200 U/ml. Spontaneous lysis amounted to 29 +/- 5% (mean +/- SEM) and 33 +/- 5% in venous and arterial thrombi, respectively, and inversely correlated with the PAI-1 content of thrombi (r = -0.43, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor in health and disease.

Over the last years numerous studies have focussed on the in vivo expression of tissue factor (TF) in health and disease. The selective perivascular distribution of TF and the lethal effects of TF knockouts have added strong support to the widely accepted view that TF plays a pivotal role in the initiation of blood coagulation during physiological hemostasis. Inappropriate in vivo expression of TF, particularly by cells that do not express this protein under normal conditions (mainly monocyte-macrophages and endothelial cells), has been documented and is likely responsible for fibrin deposition in a variety of pathological conditions, among which sepsis-associated disseminated intravascular coagulation (DIC) and thromboembolic disease. In malignancy, in vivo expression of TF by tumor cells and/or by host cells has been implicated not only in intratumoral and systemic activation of blood coagulation but also in tumor growth and dissemination.

Low molecular weight heparin versus warfarin in the prevention of recurrences after deep vein thrombosis.

To evaluate the role of low-molecular weight heparin (LMWH) as an alternative to oral anticoagulants in the prevention of recurrent venous thromboembolism, we compared in a randomized trial conventional warfarin treatment with a three-month course of enoxaparin 4000 anti-Xa units once a day subcutaneously. 187 patients with symptomatic deep-vein thrombosis (DVT), diagnosed by strain-gauge plethysmography plus D-dimer latex assay and confirmed by venography in most cases, were treated with full-dose subcutaneous heparin for ten days and then randomized to secondary prophylaxis. During the 3-month treatment period, 6 of the 93 patients who received LMWH (6%) and 4 of the 94 patients on warfarin (4%) had symptomatic recurrence of venous thromboembolism confirmed by objective testing (p = 0.5; 95% confidence interval [CI] for the difference, -3% to 7%). Four patients in the LMWH group had bleeding complications as compared with 12 in the warfarin group (p = 0.04; 95% CI for the difference, 4% to 14%). In the 9-month follow-up period, during which 34 patients on warfarin prolonged treatment for other 3 months and 14 up to one year, 10 patients in the enoxaparin group and 4 patients in the warfarin group suffered a documented recurrence of venous thromboembolism. Of these 14 late recurrences, just one occurred in patients with postoperative DVT. After one year there were 16 recurrences (17%) in the LMWH group and 8 (9%) in the warfarin group (p = 0.07; 95% CI for the difference, 1% to 16%).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding properties of monoclonal antibodies against human fragment D-dimer of cross-linked fibrin to human plasma clots in an in vivo model in rabbits.

Two (MA-15C5 and MA-8D3) out of approximately 500 monoclonal antibodies, obtained by fusion of P3X63-Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified fragment D-dimer from human fibrin, demonstrated a more than 1,000-fold higher affinity for fragment D-dimer than for native fibrinogen. MA-15C5 was directed against a neoantigenic determinant only expressed in fragment D-dimer. MA-8D3 reacted equally well with fragment D-dimer of crosslinked fibrin and with fragment D of non-crosslinked fibrin but not with fragment D of fibrinogen. Both monoclonal antibodies did not crossreact with rabbit fibrin and its degradation products. The binding of 125I-labeled Fab fragments to human plasma clots, introduced and aged for 1 hr in the jugular vein of heparinized rabbits was studied. Following injection of an equimolar mixture of Fab fragments derived from MA-15C5 and MA-8D3, the clot to blood ratios of radioactivity increased from 3.2 +/- 1.2 (mean +/- SD) at 4 hr to 7.2 +/- 1.4 at 17 hr. The binding of Fab fragments of MA-15C5 and MA-8D3 was independent of the age (1 to 72 hrs) of the clot and of heparin anticoagulation and was only slightly decreased (by 20%) in the presence of circulating human fibrinogen (90 mg/kg body weight) and of human cross-linked fibrin degradation products at a plasma concentration of 10 micrograms/ml. The binding of Fab fragments of MA-15C5 and MA-8D3 to occlusive human plasma clots in the femoral artery of rabbits was comparable to that of the non-occlusive human plasma clots in the jugular vein.(ABSTRACT TRUNCATED AT 250 WORDS)