RECOMBINANT HIRUDIN INHIBITS EXPERIMENTAL VENOUS THROMBOSIS INDUCED BY INJECTION OF TISSUE FACTOR AND STASIS

1987 ◽  
Author(s):  
M Freund ◽  
J-P Cazenave ◽  
M-L Wiesel ◽  
C Roitsch ◽  
N Riehl-Bellon ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Tissue Factor ◽  
Large Scale ◽  
Recombinant Dna ◽  
Specific Activity ◽  
Thrombus Formation ◽  
Linear Regression Analysis ◽  
Vena Cava ◽  
Cloning And Expression ◽  
Clotting Factors

Hirudin (HIR), a polypeptide of 65 aminoacids, is the most potent natural inhibitor of coagulation by forming rapidly a very stable and specific non covalent 1:1 complex with α-thrombin, independent of antithrombin III. Although natural HIR has in vivo anticoagulant and antithrombotic properties, its limited availability for large scale purification has prevented further clinical testing and potential use; this can now be solved by recombinant DNA technology. We have previously reported the cloning and expression of a cDNA encoding one variant (called HV-2) of Hirudo medicinalis HIR (Proc. Natl. Acad. Sci. USA. 1986, 83, 1084-1088). The main factors responsible for venous thrombosis are stasis and thrombin generation secondary to tissue factor liberation from vascular cells and monocytes by injury, endotoxin, interleukin-1 or cachectin and the subsequent activation and circulation of activated clotting factors. We have studied the antithrombotic properties of recombinant HIR, HV-2, in a rat experiemental model of venous thrombosis. HV-2 was expressed in yeast, extracted from culture supernatant and purified by HPLC. Pure HV-2 had an isoleucine NH2-terminus and a specific activity of 13000 ATU/mg.30 male Wistar rats (225-300g) were anesthetized with pentobarbital. At time t (0 min) an i.v. (penis) injection of 0.4 ml of saline or HV-2 (2000 to 8000 ATU/kg) was given, followed at t (5min) by 25 mg/kg tissue factor (Thromboplastin C, Dade) i.v. ; 10 s later stasis of the exposed vena cava between 2 sutures 0.7 cm apart and at t (15 min) removal, blotting, fixation and weighing of the thrombus. Linear regression analysis showed a correlation (r=0.99) between the dose of HV-2 and thrombus weight and a calculated IC50 = 3000 ATU/kg. Total inhibition of thrombus formation was seen after injection of 6000 ATU/kg HV-2 and lasted up to 15 min of circulation, HV-2 being completely eliminated from blood in 60 min and accumulated in the kidneys as shown by gamma imaging with 131I-HV-2. In conclusion, the recombinant HIR HV-2 is a potent immediate antithrombin which inhibits venous thrombosis induced by tissue factor and stasis.

scholarly journals Importance of platelets in experimental venous thrombosis in the rat

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2281-2286 ◽  
Author(s):  
JM Herbert ◽  
A Bernat ◽  
JP Maffrand
Keyword(s):  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Progressive Increase ◽  
High Dose ◽  
Experimental Conditions ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Tissue Thromboplastin ◽  
Dose Dependent

Abstract Venous thrombosis was induced by ligature of the inferior vena cava in rats whose blood was made hypercoagulable by intravenous (IV) administration of tissue thromboplastin. From a dose-response showing that the administration of increasing doses of tissue thromboplastin resulted in a subsequent progressive increase of thrombus weight, two concentrations of tissue thromboplastin were chosen: a high dose (550 microL/kg, IV) where thrombus formation was optimal and a concentration (7 microL/kg, IV) where tissue thromboplastin-hypercoagulability was intermediate. In both experimental conditions, leukopenia provoked by a myelotoxic drug did not influence the development of venous thrombosis. However, after thrombocytopenia induced by an antiplatelet antiserum, a dramatic decrease in thrombus formation was observed in animals that had been pre-challenged with the lower dose of tissue thromboplastin, whereas decrease in platelet count did not affect venous thrombosis under high thrombogenic challenge. When administered orally 2 hours before thrombosis induction, the ticlopidine analogue clopidogrel showed dose-dependent inhibition of thrombus formation in animals that were pre-challenged with a low dose of tissue thromboplastin (ED50 = 7.9 +/- 1.5 mg/kg, orally) but remained ineffective against high tissue thromboplastin-induced venous thrombosis. We further determined the effect of heparin and hirudin, and showed that both of these drugs exhibited a more potent antithrombotic activity after injection of the lower dose of tissue thromboplastin than after injection of a high dose of tissue thromboplastin. Therefore, using our model of stasis and hypercoagulability, platelet activation played a major role in the development of venous thrombosis when the thrombogenitic stimulus was mild.

Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 192-198 ◽  
Author(s):  
Sharlene M. Day ◽  
Jennifer L. Reeve ◽  
Brian Pedersen ◽  
Diana M Farris ◽  
Daniel D. Myers ◽  
...  
Keyword(s):  
Blood Vessel ◽  
Tissue Factor ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Vascular Wall ◽  
Blood Vessel Wall ◽  
Wild Type

Abstract Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

The Role of gas6 in Venous Thrombosis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3057-3057
Author(s):  
Richard Robins ◽  
Peter Carmeliet ◽  
Mark Blostein
Keyword(s):  
Bone Marrow ◽  
Venous Thrombosis ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Recovery Period ◽  
Vena Cava ◽  
Specific Gene ◽  
Combined Genotypes

Abstract Abstract 3057 Poster Board II-1033 Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

Blood ◽  
2012 ◽  
Vol 119 (23) ◽  
pp. 5543-5552 ◽  
Author(s):  
Jian-Guo Wang ◽  
Julia E. Geddings ◽  
Maria M. Aleman ◽  
Jessica C. Cardenas ◽  
Pichika Chantrathammachart ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Tissue Factor ◽  
Cell Lines ◽  
Nude Mice ◽  
Culture Medium ◽  
Vena Cava ◽  
Human Pancreatic Cancer ◽  
Tumor Bearing ◽  
Mouse Xenograft Model ◽  
Increased Risk

Abstract Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

scholarly journals Protective Effects of Kininogen-1 Gene Deficiency in Mouse Models of Venous Thrombosis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 289-289
Author(s):  
Aatira Vijay ◽  
Mohamad B Kassab ◽  
Young Jun Shim ◽  
Shadi Swaidani ◽  
Adam Mauskapf ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Continuous Light ◽  
Light Irradiation ◽  
Fluorescence Angiography ◽  
Contact Activation ◽  
Tail Vein ◽  
Platelet Accumulation

Abstract Background- High molecular weight Kininogen (HK) is a nonenzymatic co-factor of the contact activation system. HK binds prekallikrein (PK) and FXI to surfaces in proximity to FXII, amplifying PK activation by FXIIa and the reciprocal activation of FXII by activated PK (PKa), as well as FXI activation of FXIIa. PKa cleavage of HK also liberates bradykinin-a proinflammatory and vasoactive nanopeptide. The aim of this study was to define the pro-thrombotic role of kininogen in venous thrombosis (VT) and to use in vivo serial analysis of thrombus development to understand the recruitment and retention of platelets in the growing thrombus in the absence and presence of kininogen. Methods- The development of VT in mice deficient in kininogen (mKng1-/-) was compared to that in their wild-type littermates. A femoral-saphenous stasis VT model was prepared by ligating both saphenous and femoral veins. Next VT formation, growth, and dissolution (n=3 for each group) was monitored using intravital microscopy (IVM) via a multichannel epifluorescence microscope (Nikon Eclipse 90i). To induce stasis VT, FITC-dextran (10 mg/kg, ex/em 488/520 nm) was injected retro-orbitally, and then continuous light irradiation (20x objective, 475nm/35nm) of the saphenous vein was applied for 5 minutes. FITC-dextran fluorescence angiography monitored thrombus formation and dissolution. Immediately after VT formation, platelet accumulation at the thrombus site was monitored in the Cy5 channel (630/38 nm) via injection of a GPIbβ antibody conjugated with Dylight-649 (150nmol/kg), over time. All images were identically windowed in each channel, and thrombus area was measured using NIH ImageJ software. To corroborate IVM studies, we also evaluated a complete stasis model of inferior vena cava (IVC) ligation (n=7-8 per group). Thrombi were harvested after 48 hours and thrombus weight and length were measured to estimate thrombus mass. FXI circulates in blood as a homodimer along with HK. We determined the effect of kininogen deficiency on FXI activity. FXI activity assay used a combination of inhibitors, serially, to monitor the cleavage of substrate specific to activated FXI and release of chromogen, as a function of FXI activity. Finally, to determine the effects of Kng1 deficiency on bleeding, tail vein bleeding times were also determined (n=8 per group). Results- In femoral-saphenous stasis VT, thrombus developed in both groups immediately following FITC-channel light irradiation. However, thrombus size was smaller in Kng1-/- as compared to WT (Figure 1). Results from serial IVM of VT indicated faster thrombus dissolution in the Kng1-/- group. Lower platelet signals, as shown at 2 and 6 hours in the Kng1-/- mice may be consistent with this hypothesis. Thrombus area analysis suggested decreased thrombus formation in the Kng1-/- animals, and temporal analysis indicated faster dissolution by 6 hours (Figure 2). IVC ligation results corroborated the findings of femoral-saphenous DVT model, demonstrating that thrombus weight was significantly lower in Kng1-/- mice as compared to WT (p&lt;0.001, Figure 3). FXI activity was also decreased in the Kng1-/- group (p&lt;0.10). Tail vein bleeding times, however, showed no increased bleeding in Kng1-/- mice. Conclusion- These initial results suggest a pro-thrombotic role of kininogen and a protective role of kininogen deficiency in two murine venous thrombosis models, without incurring a bleeding penalty. Thrombus dissolution was faster and platelet accumulation was inhibited in Kng1-/- mice. These findings suggest that targeting kininogen may provide a new approach to prevent and treat venous thrombosis. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria. Jaffer: Mercator, Inc.: Other: Sponsred research.

scholarly journals Pathogenetic approach to venous thrombosis markers examination

2013 ◽  
Vol 94 (5) ◽  
pp. 685-691 ◽  
Author(s):  
L D Zubairova ◽  
I G Mustafin ◽  
R M Nabiullina
Keyword(s):  
Blood Flow ◽  
Venous Thrombosis ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Blood Platelets ◽  
Anticoagulant Activity ◽  
Venous Blood ◽  
Local Concentration ◽  
Clotting Factors ◽  
Blood Constituents

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

scholarly journals MicroRNA-145 Impedes Thrombus Formation via Targeting Tissue Factor in Venous Thrombosis

EBioMedicine ◽  
2017 ◽  
Vol 26 ◽  
pp. 175-186 ◽  
Author(s):  
Anita Sahu ◽  
Prabhash Kumar Jha ◽  
Amit Prabhakar ◽  
Heisnam Dinesh Singh ◽  
Neha Gupta ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Tissue Factor ◽  
Thrombus Formation

scholarly journals Diagnosis of plant viruses by nucleic acid hybridization

1987 ◽  
Vol 59 (3) ◽  
pp. 179-191
Author(s):  
Reijo Karjalainen ◽  
Leo Rouhiainen ◽  
Hans Söderlund
Keyword(s):  
Nucleic Acid ◽  
Large Scale ◽  
Recombinant Dna ◽  
Specific Activity ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Molecular Probes ◽  
Nucleic Acid Hybridization ◽  
Leaf Extracts ◽  
Sandwich Hybridization

Nucleic acid hybridization is a powerful technique for the diagnosis of many plant viruses not easily detected by serological techniques. It is particularly effective in the detection of viruses occurring in low amount in plant tissue, viruses that are poor immunogens or contain satellites. Molecular probes with desired specificities can be prepared by recombinant DNA techniques for large scale use. cDNA probes of potato virus X(PVX) RNA were made by molecular cloning, and the clones were 32P labelled by nick translation. Hybridization of cDNA to PVX RNA revealed 1 ng of purified virus in 2 µl spots dried onto nitrocellulose filter. Infected samples of crude leaf extracts were easily detected by hybridization, while probes did not react with healthy leaf samples. Nucleic acid hybridization research aims at replacing radiometric probes with nonradioactive methods involving enzymes which are directly or indirectly coupled to the probe and whose presence is observed with the aid of a colour changing substrate. Hybridization assay formats that can easily be automatized are under development. Sandwich hybridization is a simple test format developed for analyzing unpurified biological material, and it appears to be a powerful tool for microbial diagnostics. Sensitivity can be improved by using detection systems in which the specific activity of the probe is increased. Procedures such as ’polymerase chain reaction’, in which the amount of detectable nucleic acid sequences can be increased, are promising alternatives for increasing sensitivity. It is concluded that even if probe-based assays are in their infancy, they will no doubt develop towards such easy use as have immunological test kits.

ROLE OF PLATELETS IN EXPERIMENTAL THROMBOSIS INDUCED BY VENOUS STASIS

1987 ◽  
Author(s):  
A Bernat ◽  
E Vallée ◽  
J P Maffrand ◽  
J Gordon
Keyword(s):  
Platelet Count ◽  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Venous Stasis ◽  
Severe Thrombocytopenia ◽  
Antiplatelet Effect ◽  
Experimental Thrombosis ◽  
Dense Granules

Venous stasis in the rat, induced by ligature of the vena cava, provokes thrombosis. This venous thrombosis was initially believed to be platelet-independent because severe thrombocytopenia (95 % reduction in platelet count), aspirin and dipyridamole had little effect. However, the model responded to other platelet anti-aggregators, such as Ticlopidine and its analogue PCR 4099, although these compounds had no effect on coagulation, fibrinolysis or leucocyte functions (Thromb. Res. 37, 279-285, 1985). Both these drugs are known to exert their main antiplatelet effect against aggregation induced by ADP.The aim of the present study was to re-evaluate the role of platelets in this model of venous thrombosis. We have been able to show that :1) complete thrombocytopenia (99 %), achieved with an antiplatelet anti-serum, dramatically inhibited thrombus formation (by 84 % ; p < 0.01).2) partial transfusion of platelets (23 %) from control animals to these thrombocytopenic rats re-established the thrombosis.3) transfusion (under identical conditions) of platelets from rats treated with PCR 4099 had no effect.4) vena cava ligature in Fawn Hooded rats (deficient in platelet dense granules) induced less thrombosis (64 % of control ; p < 0.05).We conclude that this venous stasis model is platelet-dependent. Furthermore, because thrombus formation was reduced in normal rats treated with anti-aggregants acting selectively against ADP, and in rats lacking ADP in their platelet dense granules, it appears that ADP plays a major role in this model of thrombosis.

scholarly journals Leukocyte- and platelet-derived microparticles correlate with thrombus weight and tissue factor activity in an experimental mouse model of venous thrombosis

2009 ◽  
Vol 101 (04) ◽  
pp. 748-754 ◽  
Author(s):  
Angela Hawley ◽  
Diana Farris ◽  
Nicole Ballard ◽  
Shirley Wrobleski ◽  
Daniel Myers Jr ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Tissue Factor ◽  
Time Course ◽  
Vena Cava ◽  
Lipid Vesicles ◽  
Wild Type ◽  
Tissue Factor Activity ◽  
Chromogenic Assay ◽  
Experimental Mouse Model ◽  
Experimental Mouse

SummaryMicroparticles (MP) are lipid vesicles from platelets, leukocytes and endothelial cells that are involved in early thrombogenesis. We evaluated a detailed time-course analysis of MPs on thrombogenesis and the associated tissue factor (TF) activity in wild-type, in gene-deleted for E- and P-selectins and with high levels of P-selectin expression after the initiation of venous thrombosis in mice.Inferior vena cava (IVC) ligation was performed on C57BL/6 mice (n =191, 59 = wild-type [WT], 55 = gene-deleted for E- and P – selectins [knock-outs, EPKO] and 77 = elevated levels of soluble P-selectin, named Delta Cytoplasmic Tail (ΔCT). Animals were euthanised at various time points to assess MP production, origin and thrombus weight. MPs were re-injected into separate mice at concentrations of 80,000 and 160,000 units, as well as from different ages. In addition, MPs from thrombosed animals were pooled and TF activity quantitated using a chromogenic assay. Thrombus weight correlated negatively with MPs derived from leukocytes, and positively with MPs derived from platelets for WT animals (p<0.05), while MPs from platelets presented a positive correlation to thrombus weight in the WT and EPKO groups (p<0.01). Total MPs correlated negatively with thrombus weight in the ΔCT group (p<0.05). MP re-injections led to greater thrombus weight, while older MP reinjections tended to form larger thrombus than younger. Finally, TF bearing MPs showed a significant correlation to MP concentrations (R=0.99). In conclusion, MPs appear to be an important element in venous thrombogenesis.

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