Interleukin-8 administration enhances venous thrombosis resolution in a rat model.

BACKGROUND: Therapy for deep vein thrombosis (DVT) resolution in those patients in whom a complication or contraindication to anticoagulation occurs is limited. As prior work suggests that thrombus maturation involves early influx of neutrophils (PMN) and neovascularization, we hypothesized that administering the proinflammatory/proangiogenic chemokine interleukin (IL)-8 might accelerate thrombus resolution. MATERIALS AND METHODS: An established rodent model of DVT (inferior vena cava [IVC] ligation) was used whereby daily intravenous recombinant human IL-8 (1 microg) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and at harvest, duplex ultrasound of the DVT and femoral venous pressure measurements were performed. Thrombi were analyzed by immunohistochemical techniques for PMN, monocytes, and neovascularization; for chemokines, by enzyme-linked immunoassay; and fibrosis, by hydroxyproline assay and trichrome staining. RESULTS: IL-8 accelerated thrombus dissolution 4 days after IVC ligation, with 6-fold increased thrombus blood flow by duplex ultrasound and a 23% increased absolute femoral venous pressure compared with controls (both P < 0.05). These findings may be partially explained by the fact that animals receiving IL-8, as compared with controls, had 2.5-fold greater thrombus neovascularization (with a trend continuing to 8 days) and increased PMN at 4 days. Thrombus vascular endothelial growth factor was significantly reduced at 8 days postligation, while monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha were not altered by IL-8 administration. At 8 days post-IVC-ligation, fibrosis was 12-fold greater with IL-8 treatment compared with controls. CONCLUSIONS: A proinflammatory/proangiogenic thrombus milieu, as conferred by IL-8, enhances thrombus resolution and underscores the important relationship between neovascularity and inflammation.

Viral IL-10 gene transfer decreases inflammation and cell adhesion molecule expression in a rat model of venous thrombosis.

Post-thrombotic inflammation probably contributes to chronic venous insufficiency, and little effective treatment exists. IL-10 is an anti-inflammatory cytokine that previously has been shown to decrease perithrombotic inflammation and thrombosis. We investigated in a rat model whether local expression of viral IL-10 (vIL-10) in a segment of vein that undergoes thrombosis would confer an anti-inflammatory effect and how this effect might be mediated. Rats underwent inferior vena cava isolation, cannulation, and instillation of saline or adenovirus encoding either beta-galactosidase or vIL-10. Two days after transfection, thrombosis was induced, 2 days after this the rats underwent gadolinium (Gd)-enhanced magnetic resonance venography exam, and the vein segments were harvested. Tissue transfection was confirmed by either RT-PCR of vIL-10 or positive 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-Gal) staining. vIL-10 significantly decreased both leukocyte vein wall extravasation and area of Gd enhancement compared with those in controls, suggesting decreased inflammation. Immunohistochemistry demonstrated decreased endothelial border staining of P- and E-selectin, while ELISA of vein tissue homogenates revealed significantly decreased P- and E-selectin and ICAM-1 levels in the vIL-10 group compared with those in controls. Importantly, native cellular IL-10 was not significantly different between the groups. However, neither clot weight nor coagulation indexes, including tissue factor activity, tissue factor Ag, or von Willebrand factor levels, were significantly affected by local vIL-10 expression. These data suggest that local transfection of vIL-10 decreases venous thrombosis-associated inflammation and cell adhesion molecule expression, but does not directly affect local procoagulant activity.

Venous thrombosis prophylaxis by inflammatory inhibition without anticoagulation therapy.

OBJECTIVE: This study was performed to determine the effectiveness of recombinant P-selectin glycoprotein ligand Ig (rPSGL-Ig) pretreatment to decrease thrombosis and inflammation in experimental venous thrombosis. rPSGL-Ig, a unique mucin-like glycoprotein, has a high affinity for P-selectin. METHODS: Twelve juvenile baboons underwent inferior vena cava (IVC) thrombosis with temporary 6-hour IVC balloon occlusion. Before balloon placement, the animals received rPSGL-Ig (4 mg/kg; n = 8) or saline solution for control (n = 4). The animals underwent evaluation with duplex ultrasound scan imaging, magnetic resonance venography (MRV), phlebography, coagulation profile, and tissue analysis at death for cytokines and vein wall leukocyte morphometrics. With the MRV results, thrombus development, thrombus resolution, and inflammation (gadolinium; square millimeters of enhancement) were assessed. RESULTS: Each animal provided two time points for evaluation (days 2 and 6 after balloon occlusion). A significant decrease in IVC thrombosis between balloons was found in the rPSGL-Ig animals (1 of 16) versus the control animals (5 of 8; P <.01). The MRV results showed significantly less enhancement in the rPSGL-Ig animals at days 2 and 6 (P <.05). Spontaneous thrombus resolution (including balloon sites) was significantly greater from day 2 to day 6 in the rPSGL-Ig animals versus the control animals (23% vs 2%; P <.001), without pulmonary embolism. Lower interleukin-8, platelet factor IV, and monocyte chemotactic protein-1 levels were found in rPSGL-Ig vein walls without significant differences in vein wall leukocyte morphometrics. There were significantly lower D-dimer levels in the rPSGL-Ig-treated animals (P <.05), but there were no differences in measurements of coagulation. Adequate circulating rPSGL-Ig levels were documented. CONCLUSION: Pretreatment with rPSGL-Ig results in: (1) a significant inhibition of thrombosis and vein wall inflammation; (2) a decrease in vein wall cytokine expression; and (3) a promotion of thrombus resolution. Inflammatory inhibition by rPSGL-Ig without anticoagulation therapy provides effective venous thrombosis prophylaxis in experimental venous thrombosis.

Neovascularization during venous thrombosis organization: a preliminary study.

PURPOSE: Thrombus organization after venous thromboembolism leading to recanalization occurs at a variable rate. The angiogenic chemokine interleukin-8 (IL-8) has been found in thrombus months after thrombus initiation. We hypothesize that thrombus organization involves neovascularization and leukocyte influx and that IL-8 administered at thrombus induction will promote thrombus organization. METHODS: A group of rats underwent inferior vena caval occlusive thrombosis. At thrombus induction and every 24 hours, the rats were administered IL-8 (1 microgram) or serum albumin. The rats were killed at either day 4, day 8, or day 12, and, at death, colloidal carbon was perfused via the heart. The inferior vena cava was isolated, measured, weighed, and formalin fixed. The sections were stained with anti-polymorphonuclear leukocyte antibody, the endothelial marker factor VIII-related antigen, and with hematoxylin and eosin. Thrombus neovascularization (colloidal carbon) with morphometric analysis was normalized to the total thrombus area. In addition, the rats underwent perfusion with fluorescein isothiocyanate dextran (molecular weight, 150,000) at death to correlate with colloidal carbon perfusion, and thrombus fluorescence was determined. RESULTS: Thrombus cellularity initially involved neutrophils, followed by monocytes. Significantly more neutrophils, monocytes, and cells that were defined as spindle shaped (fibroblasts and endothelial cells) were noted in the animals treated with IL-8. Neovascularization was significantly increased at day 4 in the animals treated with IL-8 versus the animals treated with serum albumin and was corroborated with a significant increase in thrombus fluorescein isothiocyanate dextran fluorescence at day 4 in the rats treated with IL-8. Colloidal carbon perfusion was noted within vascular channels without extravasation and colocalized with factor VIII-related antigen. CONCLUSION: This study shows that thrombus organization involves neovascularization and that IL-8 augments thrombus organization.

Detection of perivenous inflammation in a rat model of venous thrombosis using MRV.

Venous thrombosis is associated with a significant inflammatory response in the vein wall, which can be imaged noninvasively with gadolinium (Gd)-enhanced magnetic resonance venography (MRV). Interleukin-10 (IL-10), a naturally occurring anti-inflammatory cytokine, has been found to decrease the inflammatory response at the proper dosage and timing of administration. The present study determines if MRV with Gd is useful in a rat model of stasis-induced venous thrombosis to document the anti-inflammatory effects of rIL-10. Rats underwent laparotomy and ligation of the inferior vena cava (IVC). Animals were infused with rIL-10 at 2.5 microg (n = 6), rIL-10 at 10 microg (n = 6), or rIL-10 at 40 microg (n = 6). Six animals without IVC ligation or drug infusion served as controls. Two days after thrombosis induction, the rats underwent MRV with both time-of-flight imaging and pre/post-Gd T1-weighted imaging. Inflammation was analyzed by measuring the area of Gd enhancement at the point of IVC thrombosis. Enhancement area was also measured in the distal IVC where flow persisted. All animals with IVC ligation developed thrombosis, and all control rats were free of thrombus. In areas where flow remained, the area of enhancement was 1.8 +/- 0.4 mm2, while controls demonstrated 3.8 +/- 1.0 mm2 enhancement. Enhancement was significantly greater in all groups at the level of thrombus compared to the area of distal IVC flow and control IVCs (p < .001). Animals receiving rIL-10 at 40 microg revealed the most enhancement, 32.7 +/- 6.2 mm2, while the least enhancement was noted with 2.5 microg, 14.7 +/- 1.5 mm2 (p < .05). In conclusion, Gd-enhanced MRV was found useful in this rat model of stasis-induced venous thrombosis to document inflammation noninvasively and to evaluate the effects of anti-inflammatory interventions during stasis-induced IVC venous thrombosis.

Ultrasound echogenicity in experimental venous thrombosis.

This study characterizes the echogenicity of experimentally induced venous thrombosis. Venous duplex imaging (Diasonics Spectra) was performed of the rat (n 12) and primate (n 3) inferior vena cava (IVC). Thrombosis was induced by IVC ligation at the level of the renal veins (rat, baboon) or balloon occlusion (baboon) of the IVC at the renal vein and iliac vein bifurcation level. Sham-treated rats served as controls. B-mode images were stored for off-line computer analysis. Fixed depth gain control curves allowed for measuring gain-corrected echogenicity units over the IVC in both a longitudinal and transverse orientation. In rat studies, thrombus was removed at time of euthanasia and dissolved, allowing for fibrin monomer determination using a chromogenic assay. Echogenicity values generally increased over time in both rat and primate studies. Significant differences between ligated and sham-treated rats were noted at each time point measured (6 h, 2 days, and 6 days after IVC ligation) and fibrin monomer values correlated (p < 0.05) with echogenicity units. In primate studies, echogenicity values significantly were different from baseline values at all time points measured (6 h, 2 days, 6 days, and 13 days after thrombus induction). Duplex ultrasound can be used to quantitate thrombus echogenicity, which correlates to fibrin content. Such measurement may potentially allow for improved thrombus age determination and the noninvasive quantitation of thrombus progression/resolution.

Low-dose low-molecular-weight heparin is anti-inflammatory during venous thrombosis.

PURPOSE: Venous thrombosis results in a vein wall inflammatory response initiated by thrombus. Although anticoagulation with standard heparin (SH) and low-molecular-weight heparin (LMWH) is known to limit further thrombosis, their anti-inflammatory properties are poorly defined. The anti-inflammatory properties of these heparins were studied. METHODS: Sprague-Dawley rats were divided into groups and underwent inferior vena caval (IVC) ligation just below the renal level producing IVC thrombosis. One hour before ligation, animals received subcutaneous SH or LMWH at either high or low dose; normal saline (NS) was used as control. Six hours after ligation, animals were killed, and the IVCs were analyzed for clot presence, vein wall morphometrics, and vein wall permeability (VP) to define injury. RESULTS: Animals in both low-dose groups had no measurable anticoagulation, whereas those in both high-dose groups were adequately anticoagulated. There were statistically less IVC neutrophils for all groups compared with the control group, with low-dose LMWH showing the least cells (low-dose LMWH, 16 +/- 3; high-dose LMWH, 37 +/- 10; low-dose SH, 37 +/- 6; high-dose SH, 32 +/- 9; NS control, 63 +/- 2). Similar results were noted for total inflammatory cells. The lowest VP was noted for low-dose LMWH. CONCLUSION: Although both SH and LMWH inhibited vein wall neutrophils and total inflammatory cells, low-dose LMWH was most effective limiting neutrophil extravasation and was the only intervention to decrease VP below control levels. This occurred without preventing thrombus formation or causing a state of anticoagulation. Low-dose LMWH possesses anti-inflammatory properties distinct from its anticoagulant properties.

IL-10 regulates thrombus-induced vein wall inflammation and thrombosis.

Vein wall inflammation associated with venous thrombosis is mediated by an imbalance in proinflammatory as compared with antiinflammatory molecules. We hypothesize that IL-10 is an important antiinflammatory cytokine that influences vein wall inflammation and thrombus propagation during venous thrombosis. To test this hypothesis a model of inferior vena caval thrombosis was used. Studies were performed at sacrifice 2 days after thrombus induction and included leukocyte morphometrics, myeloperoxidase activity, vein wall permeability, thrombus weight, and IL-10 ELISA analysis from the vein wall. IL-10 was elevated in the vein wall during venous thrombosis. Neutralization of IL-10 increased inflammation, while supplementation with rIL-10 demonstrated a dose- and time-dependent decrease in inflammation. Interestingly, a low 2.5-microg rIL-10 dose given at time of initiation of thrombosis most significantly decreased inflammation. Thrombus weight was importantly diminished by reconstitution of IL-10. These studies support an important role for IL-10 in the regulation of thrombus-associated inflammation and thrombosis and suggest that IL-10 could be used as a therapeutic agent in the treatment of venous thrombosis.

Anti-P-selectin antibody decreases inflammation and thrombus formation in venous thrombosis.

PURPOSE: Venous thrombosis and inflammation are interrelated. P-selectin contributes to activation of leukocyte-mediated inflammation. Therefore, we hypothesized that the neutralization of P-selectin would decrease vein wall inflammation and thrombosis. METHODS: Twelve baboons underwent infrarenal inferior vena caval balloon occlusion to induce thrombosis. Two groups of four baboons received neutralizing intravenous anti-P-selectin antibody (PSab) GA6 or CY1748 before occlusion and at days 2 and 4. Four baboons received saline control injections. One baboon per group was killed at days 2, 6, and 13, and at 2 months. Analysis included phlebography, ultrasound, gadolinium (Gd)-enhanced magnetic resonance venography (reflecting vein wall inflammation), and histologic, morphometric, and protein evaluation of the vein wall. Thrombus presence or absence was assessed. RESULTS: By day 2 in PSab baboons, vein wall Gd enhancement was decreased in the mid-inferior vena cava and the right iliac vein (p < 0.05; GA6 vs control baboons), normalizing by 2 months. The mid-inferior vena cava revealed fewer neutrophils and total leukocytes in PSab baboons; however, for GA6 in the right iliac vein these decreases were not present despite the absence of Gd enhancement; they were decreased with CY1748. PSab baboons demonstrated significantly less thrombus than control baboons (p < 0.01, GA6 and CY1748 vs control baboons). CONCLUSIONS: Anti-P-selectin antibody decreases vein wall inflammation and thrombus formation. Inhibition of P-selectin may be useful in venous thrombosis prophylaxis.

Comparison of the hemodynamic and hematologic toxicity of a protamine variant after reversal of low-molecular-weight heparin anticoagulation in a canine model.

Using the dog as an animal model, we developed an experimental preparation to compare hemodynamic and hematologic toxicity of anticoagulation reversal. Currently, protamine sulfate reversal of standard unfractionated heparin and low-molecular-weight heparin (LMWH) anticoagulation causes adverse side effects, including decreased systemic mean arterial pressure (MAP), decreased cardiac output (CO), decreased oxygen consumption (VO2), and thrombocytopenia. In addition, standard protamine is only marginally effective at reversing the factor Xa inhibition induced by LMWHs. We have produced protamine-like variant peptides to decrease the adverse responses attributed to standard protamine. The hemodynamic, hematologic, and coagulation effects of standard protamine and the protamine variant (+18RGD) were assessed after reversal of LMWH anticoagulation in anesthetized dogs. Flow probes and vascular catheters were surgically implanted for measurement of hemodynamic parameters including MAP, CO, VO2, and heart rate (HR). Hematologic studies (platelet and white blood cell counts) and coagulation studies (activated clotting time [ACT], activated partial thromboplastin time [aPTT], thrombin clotting time [TCT], antifactor Xa and antifactor IIa values) also were performed. The protamine variant +18RGD was less toxic, induced less thrombocytopenia, and was more effective in anticoagulation reversal than was standard protamine sulfate. Results of this study indicate that the dog may be a useful model for investigating important hemodynamic, hematologic, and coagulation parameters during reversal of LMWH anticoagulation by use of synthetic protamine variants.

Neutrophils are the initial cell type identified in deep venous thrombosis induced vein wall inflammation.

Venous thrombosis and inflammation are interrelated. The authors hypothesized that inferior vena cava thrombosis results in a predictable vein wall inflammatory response, characterized by early neutrophil infiltration. Thrombosis was induced in rats by placement of an inferior vena cava ligature with branch ligation. Animals were killed at baseline, 6 hrs, day 2, and day 6. Analysis included vein wall morphometrics, myeloperoxidase activity, and fluorescence activated cell sorting. At 6 hrs, there was an increase in neutrophils and lymphocytes as compared to sham animals (p < 0.0001 for neutrophils, p < 0.05 for lymphocytes). By day 2, only neutrophils were elevated in the experimental groups (experimental = 75.5 cells/5 high power fields vs. 9.6 cells/ 5 high power fields in shams, p < 0.0001). Myeloperoxidase activity in the experimental group was greater than shams on day 2(34.7 delta optical density/min vs. 5.9 delta optical density/ min, p < 0.0001). Fluorescence activated cell sorting of the neutrophil marker at 6 hrs confirmed the increase in neutrophils (experimental = 63.1%, shams = 39.1%, p < 0.0001), and peaked on day 2 (71.9%). This study suggests that 1) neutrophils are elevated early during the inflammatory response due to thrombus initiation, and 2) neutrophils, because of their early predominance, likely contributed to vein wall injury during venous thrombosis.

P-selectin and TNF inhibition reduce venous thrombosis inflammation.

Venous thrombosis induces a detrimental inflammatory response in the vein wall. The cytokine tumor necrosis factor-alpha (TNF) and the adhesion molecules, selectins, have been found to be important in mediating inflammatory cell stimulation and leukocyte-endothelial cell adhesion, respectively. This study assesses the role of TNF and P-selectin in the inflammatory events associated with venous thrombosis. Rats were passively immunized with neutralizing anti-TNF serum alone, anti-TNF plus anti-P-selectin antibody, anti-P-selectin antibody alone, control serum, or control anti-P-selectin antibody. Antibodies or control sera were given prior to occlusion and at Days 2 and 4 postocclusion. Rats were sacrificed at Days 1-6 and Day 13 after occlusion for inferior vena caval (IVC) wall histopathology and TNF analysis. Differences in the extent of inflammatory cell infiltrate into the vein wall were found on Days 2, 6, and 13. TNF levels were elevated in the vein wall of the three groups not given anti-TNF antibody. The levels of TNF at Day 6 positively correlated with both total inflammatory cell (r = 0.53, P < 0.05) and neutrophil presence (r = 0.72, P < 0.01). The lowest IVC wall neutrophil and total inflammatory cell count at Days 2 and 6 and the lowest neutrophil count at Day 13 were found in the anti-TNF plus anti-P-selectin antibody group. Monocyte influx was also inhibited at Day 13 in this group. These results suggest a role for combined neutralization of TNF and P-selectin in the attenuation of inflammation induced by venous thrombosis.

A [+18RGD] protamine variant for nontoxic and effective reversal of conventional heparin and low-molecular-weight heparin anticoagulation.

Protamine sulfate reversal of heparin anticoagulation causes adverse side effects. Additionally, protamine sulfate is relatively ineffective at reversing factor Xa inhibition caused by low-molecular weight heparin (LMWH, Enoxaparin). Previously, a +18 compound partially reversed heparin and LMWH with minimal toxicity. In the present study, a new +18 protamine-like variant, [+18RGD], with an added RGD sequence [acetyl-EA(R2A2R2A)4R2GRGDSPA-amide], was compared to a previously developed compound, [+18BE,Acetyl-EAA-(K2A2K2A)4K2-Amide] and standard protamine [Prot +21] regarding the reversal of conventional unfractionated heparin (Hep) and LMWH. These three agents were given at 1 mg per 100 IU activity of Hep or LMWH rapidly over 10 sec. Hemodynamic toxicity was based on maximum declines in blood pressure, heart rate, cardiac output, and oxygen consumption over the first 5 min after reversal (calculated as a total toxicity score, TTS). The more negative the TTS, the more toxic the agent. Degrees of toxicity (TTS) of [+18RGD], [+18BE],and[Prot +21] for reversal of Hep were -1.19, -2.00, and -7.32, respectively; and for reversal of LMWH they were -2.85, -3.98,and -6.17, respectively. These differences were significant for Hep (P < 0.01) and approached significance for LMWH (P = 0.07). Maximum hemodynamic perturbations paralleled the TTS pattern. [+18RGD] provided equal reversal efficacy to [Prot +21] for Hep, with a statistically significant (P < 0.05) lessening of platelet count declines (Plt 27, -46, and -55%, respectively). [+18RGD] improved reversal efficacy for LMWH, at 3, 10, and 30 min following its administration. At 3 min, antifactor Xa reversal was 72, 40, and 30%, respectively, for [+18RGD], [+18BE], and [Prot +21]; [+18RGD] effects were significantly better (P < 0.01). [+18RGD] reversed both Hep and LMWH anticoagulation with minimal toxicity. Such a compound should decrease clinical complications attending the use of standard protamine for reversal of conventional heparin or LMWH anticoagulation.

Effective and less toxic reversal of low-molecular weight heparin anticoagulation by a designer variant of protamine.

PURPOSE: This investigation assessed protamine reversal of heparin anticoagulation by formation of a protamine-heparin alpha-helix by use of a new designer-variant protamine [+18BE] that was made from an existing protamine variant [+18B] whose non-alpha-helix-forming amino acid proline (P) was replaced by an alpha-helix-forming glutamic acid (E). The rate of administration of the new [+18BE] variant protamine on efficacy and toxicity in comparison to that of [+21] standard protamine and [+18B] was also studied. METHODS: Acetyl-EAA(K2A2K2A)4K2-Amide [+18BE] was administered intravenously in a 1:1 dose to low-molecular-weight heparin (LMWH)-anticoagulated (intravenous 150 IU antifactor Xa/kg) dogs over 10 seconds or 3 minutes (n = 7, each group). Reversal efficacy was documented by measuring activated clotting time, thrombin clotting time, antifactor Xa, and antifactor IIa. Toxicity was defined by measuring systemic blood pressure, heart rate, cardiac output, pulmonary artery pressure, and oxygen consumption. Measurements were made at baseline, after administration of LMWH, before its reversal, and for 30 minutes thereafter. Results were compared with those after LMWH reversal with [+21] standard protamine and the [+18B] variant. A total toxicity score (TTS) was calculated for each compound from maximal declines in blood pressure, heart rate, cardiac output, and oxygen consumption. RESULTS: LMWH anticoagulation reversal was significantly (p < 0.01) less toxic over 10 seconds and 3 minutes with the [+18BE] designer variant (TTS -2.3, -2.2) compared with the [+21] standard protamine (TTS -6.4, -7.2). Percent LMWH reversal at 3 minutes revealed [+18BE] to have antifactor Xa activity as high as 91%, compared with 68% for protamine [+21], when given over 3 minutes (p < 0.05). CONCLUSIONS: This investigation documents that a new designer variant of protamine [+18BE] has superior efficacy compared with [+21] standard protamine for reversal of LMWH anticoagulation and that this occurs with a highly favorable toxicity profile.

Venous thrombosis-associated inflammation and attenuation with neutralizing antibodies to cytokines and adhesion molecules.

Thrombosis and inflammation are closely related. However, the response of the vein wall to venous thrombosis has been poorly documented. This study examines the hypothesis that venous thrombosis is associated with an inflammatory response in the vein wall. In a rat model of inferior vena caval thrombosis, vein wall was temporally examined for inflammation by assessment of histopathology, leukocyte morphometrics, and cytokine levels. Animals were killed 1 hour and 1, 3, and 6 days after thrombus induction. Our findings demonstrated an early (day 1) neutrophil infiltration into the vein wall followed by a later (days 3 and 6) monocyte/macrophage and lymphocyte response. Cytokines were elevated only under conditions of venous thrombosis. Levels of epithelial neutrophil activating protein-78 (ENA-78), tumor necrosis factor-alpha (TNF), interleukin-6, and JE/monocyte chemoattractant protein-1 (JE/MCP-1) increased over the 6-day period, while macrophage inflammatory protein-1 alpha (MIP-1 alpha) peaked at day 3 after thrombus induction. Additionally, rats were passively immunized with neutralizing antibodies to TNF, ENA-78, MIP-1 alpha, JE/MCP-1, intercellular adhesion molecule-1 (ICAM-1), and CD18 compared with control antibodies. The most effective antibody early after thrombus induction for attenuating vein wall neutrophil extravasation was anti-TNF (P < .01). The monocyte/macrophage extravasation was inhibited most by anti-ICAM-1 followed by anti-TNF (P < .01). These findings demonstrate that venous thrombosis is associated with significant vein wall inflammation that is partially inhibited by neutralizing antibodies to cytokines and adhesion molecules.

Reversal of low-molecular-weight heparin anticoagulation by synthetic protamine analogues.

Protamine reversal of unfractionated and low-molecular-weight heparin (LMWH) causes hypotension, bradycardia, pulmonary artery hypertension, and declines in oxygen consumption. Furthermore, protamine incompletely reverses the anti-Xa activity of LMWH. The present study assesses the efficacy and toxicity of three protamine variants having +16 and +18 charges in reversal of LMWH (Logiparin, LHN-1): [+16] P(AK2A2K2)4, [+18] PK(K2A2K2A)3K2AK3, and [+18B] acetyl-PA(K2A2K2A)4K2-amide. The [+18B] compound was made by acetylating and amidating the [+18] to decrease in vivo degradation and to increase the alpha-helix forming propensity. Variants were examined in a canine model (n = 7, each variant) and compared to controls (n = 7, each variant) and compared to controls (n = 7) reversed with standard protamine with a +21 charge. Animals were anesthetized, anticoagulated with LMWH (150 IU factor Xa activity/kg), and reversed with protamine variants (1.5 mg/kg with 100 IU/mg). Blood pressure (BP), heart rate (HR), cardiac output (CO), pulmonary artery pressures, oxygen saturations, and oxygen consumption (VO2) were continuously monitored. Comparisons were undertaken at baseline, after heparin, before variant administration, and for 30 min thereafter. A total toxicity score (TTS) was calculated for each variant, accounting for maximal declines in BP, HR, CO, and VO2 during the first 5 min after reversal. Protamine [+21] was most toxic, TTS -7.6, with the variants being less toxic (P < 0.01, ANOVA): TTS = [+16] -2.8, [+18] -1.3, and [+18B] -4.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue distribution, circulating half-life, and excretion of intravenously administered protamine sulfate.

Intravenous protamine reversal of heparin anticoagulation may cause adverse hemodynamic side effects, but little is known about protamine's tissue distribution, circulating half-life (t/2), and excretion. The latter were assessed by examining 125I Bolton-Hunter (125I BH) radiolabeled protamine kinetics in a rat model. Three groups were studied: Group I controls (n = 5) received intravenous 125I BH label alone; Group II (n = 10) received intravenous 125I BH radiolabeled protamine (0.15 mg/100 g); and Group III (n = 10) received intravenous heparin (15 IU/100 g) followed by intravenous 125I BH radiolabeled protamine (0.15 mg/100 g). Five animals in each group were killed at 3 min, and tissue radioactivity was quantitated. An additional five animals each in Groups II and III were followed up for 60 min to determine protamine's circulating t/2 and its renal excretion. The lungs, heart, and kidneys, compared with other organs, retained the most 125I BH radiolabeled protamine per gram tissue at 3 min. Retention of 125I BH radiolabeled protamine (Groups II & III) was greater (p < 0.05, Kruskal-Wallis) than control 125I BH label alone (Group I). Higher tissue 125I activity was observed in Group II than in Group III rats, suggesting that tissue retention of protamine was greater in the absence of prior heparin administration. Circulating t/2 was shorter (18 vs. 24 min) and urinary protamine 125I excretion was higher (34 vs. 24%) in Group III than in Group II, respectively, suggesting more rapid renal clearance of protamine in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy and toxicity of differently charged polycationic protamine-like peptides for heparin anticoagulation reversal.

PURPOSE: The role of total cationic charge of synthetic protamine-like peptides in heparin anticoagulation reversal and accompanying adverse hemodynamic effects was studied. METHODS: Five protamine variants having specific total charges of [+8], [+16], [+18], [+20], and [+21] were synthesized by fluorenylmethoxycarbonyl procedures. Each of these lysine-containing peptides plus arginine-containing control salmine native protamine (n-protamine, [+21] charge) was studied in five dogs who received heparin 150 IU/kg intravenously followed by 1.5 mg/kg (intravenously during a 10-second period) of the synthesized peptide or control n-protamine. RESULTS: Anticoagulation reversal as assessed by a number of coagulation tests was more effective with peptides of greater cationic charge. In this regard, activated clotting time reversal 3 minutes after peptide administration was 7%, [+8]; 54%, [+16]; 81%, [+18]; 92%, [+20]; 81%, [+21]; and greater than 100% [n-protamine]. Reversal of heparin anticoagulation at 3 and 30 minutes, respectively, correlated significantly (*p < or = 0.05, p < or = 0.01 [see corresponding symbols within abstract]) with total cationic charge as assessed by activated clotting time (r = 0.97, 0.99 ), prothrombin time (r = 0.98, 0.87*), activated partial thromboplastin time (r = 0.99, 0.78), thrombin clotting time (r = 0.84,* 0.85*), heparin anti-Xa activity (r = 0.87,* 0.85*), and heparin anti-IIa activity (r = 0.79 at 3 minutes, p = 0.06). Maximum declines in systemic mean arterial pressure (MAP) were greater with more positively charged peptides: -1 mm Hg, [+8]; -3 mm Hg, [+16]; -31 mm Hg; [+18]; -31 mm Hg, [+20]; -35 mm Hg, [+21]; and -34 mm Hg [n-protamine]. Maximum decreases in MAP, cardiac output, and systemic oxygen consumption were highly correlated (p < or = 0.05) with total cationic charge: MAP, r = 0.87; cardiac output, r = 0.87; and systemic oxygen consumption, r = 0.86. A total toxicity score, reflecting adverse hemodynamic effects, was greater with increasing charge: -1.9 +/- 1.1, [+8]; -2.7 +/- 0.8, [+16]; -6.6 +/- 3.3, [+18]; -6.1 +/- 3.5, [+20]; -6.9 +/- 3.8, [+21]; and -7.0 +/- 5.2 [n-protamine]. The correlation of mean peptide total toxicity score to total cationic charge was significant (r = 0.89, p < 0.05). CONCLUSIONS: These data suggest for the first time that effective alternatives to salmine protamine for reversal of heparin anticoagulation can be synthesized. Furthermore, total cationic charge appears to be an important determinant for both anticoagulation reversal and toxicity of protamine-like peptides.

Inflammatory and procoagulant mediator interactions in an experimental baboon model of venous thrombosis.

Theoretic and in vitro evidence suggests that thrombosis and inflammation are interrelated. The purpose of the present study was to define the relationship between inflammation and deep venous thrombosis (DVT) in an in vivo model. Initiation of DVT was accomplished by administration of antibody to protein C (HPC4, 2 mg/kg) and tumor necrosis factor (TNF, 150 micrograms/kg); stasis; and subtle venous catheter injury. Thrombosis was assessed by thrombin-antithrombin assay (TAT), 125I-fibrinogen scanning (scan) over both the proximal and distal iliac veins, and ascending venography. Cytokines TNF, interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8) were measured along with differential white blood cell counts, platelet counts, fibrinogen (FIB), and erythrocyte sedimentation rates (ESR). Baboon pairs were sacrificed on day 3 (T + 3d), T + 6d, and T + 9d and veins removed. All animals developed inferior vena cava and left iliofemoral DVT by venography; no right DVT was found. TAT was elevated by T + 1hr and peaked at T + 3hrs. Left iliofemoral DVT was found at T + 1hr by scan and reached a 20% uptake difference between the affected left and nonaffected right side at T + 3hrs. TNF peaked at T + 1hr; MCP-1 peaked at T + 6hrs; IL-8 and IL-6 peaked on T + 2d; all cytokines declined to baseline. TNF and TAT elevations were found to correlate with all cytokines; elevations in IL-8 were correlated with elevations in MCP-1 and IL-6 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin-mediated reductions of the toxic effects of protamine sulfate on rabbit myocardium.

Protamine sulfate causes direct myocardial suppression when used to reverse heparin anticoagulation. Protamine's excessive positive charge accompanying its surface arginine groups appears to be responsible for this toxicity. This study was designed to assess the hypothesis that negatively charged heparin given after protamine exposure may enhance the recovery of protamine-induced myocardial dysfunction. Isolated rabbit hearts (n = 20) were perfused with physiologic saline solution at 80 to 90 mm Hg containing high dose protamine, 250 micrograms/ml, until heart contraction essentially ceased (baseline). Hearts were then randomly reperfused for 15 minutes with either physiologic saline solution (group I, n = 10) or heparin plus physiologic saline solution (group II, n = 10) at a dose of 0.1 IU/1.0 microgram of previously administered protamine. Developed left ventricular blood pressure, heart rate, pulmonary artery PaO2, contractility (+dp/dt), oxygen extraction (AvO2), oxygen consumption (VO2), and rate x pressure product were assessed. A protective, beneficial response accompanied heparin administration (group II) in all functions assessed except blood pressure. Maximum changes, comparing group I with II, were heart rate (beats/min) -72 versus -1, p less than 0.001; +dp/dt -64% versus -51%, p less than 0.01; PaO2 +86% versus +9%, p less than 0.001; AvO2 -37% versus -4%, p less than 0.001; VO2 -50% versus -28%, p less than 0.008; and rate x pressure product -73% versus -51%, p less than 0.001. These data suggest a separation of protamine's hemodynamic effects (blood pressure) and metabolic effects (VO2). Furthermore, these data support the tenet that heparin is able to markedly lessen the toxic myocardial effects of protamine.