Inferior vena cava branch variations in C57BL/6 mice have an impact on thrombus size in an IVC ligation (stasis) model.

BACKGROUND: Animal models of venous thrombosis (VT) are critical tools for those investigating the VT mechanism. Recently, inferior vena cava (IVC) branches have been subject to debate, causing controversy in the field. OBJECTIVES: To understand how the variability of IVC branches, in commonly used C57BL/6 mice, have an impact on thrombus formation in the IVC ligation model. METHODS: C57BL/6 male mice (n = 46), 20-25 g, were subjected to the IVC ligation model with various interruptions of the IVC branches. Control animals (n = 50) had all branches interrupted. Two days after IVC ligation, thrombus weight (TW), as a parameter of thrombus size, was assessed. RESULTS: We found four different anatomical patterns. Side branches were more prevalent on the mouse's right side (34%) compared with the left (20%). In mice where side branches were absent (21%), back branches appeared larger. Also, 25% of mice had both side branches. Controls that had all IVC branches interrupted had the most consistent and largest TW (32.6 mg to 34.7 mg) while groups that had no back branches interrupted had the smallest TW (3.6-9.7 mg), a 4 to 9-fold decrease. All groups with open back branches had significantly smaller TW (P < 0.05) than controls. CONCLUSIONS: Variations in TW were observed based on different branch interruption patterns, compared with the fully ligated controls. Having two back branches was the most consistent anatomy and open back branches had the largest negative impact on thrombus size. This work confirms that the IVC branches significantly affect thrombus burden in C57BL/6 mice and further studies should be conducted in order to standardize this and other animal models of VT.

Rosuvastatin reduced deep vein thrombosis in ApoE gene deleted mice with hyperlipidemia through non-lipid lowering effects.

INTRODUCTION: Statins, particularly rosuvastatin, have recently become relevant in the setting of venous thrombosis. The objective of this study was to study the non-lipid lowering effects of rosuvastatin in venous thrombosis in mice with hyperlipidemia. MATERIALS AND METHODS: An inferior vena cava ligation model of venous thrombosis in mice was utilized. Saline or 5mg/kg of rosuvastatin was administered by gavage 48hs previous to thrombosis. Blood, the inferior vena cava, thrombus, and liver were harvested 3, 6hours, and 2days post-thrombosis. Thrombus weight, inflammatory markers, and plasminogen activator inhibitor-1 expression and plasma levels were measured. Also, neutrophil migration to the IVC was assessed. RESULTS: Rosuvastatin significantly decreased thrombus weight, plasminogen activator inhibitor-1 expression and plasma levels, expression of molecules related to the interleukin-6 pathway, and neutrophil migration into the vein wall. CONCLUSIONS: This work supports the beneficial effects of rosuvastatin on venous thrombosis in mice with hyperlipidemia, due to its non-lipid lowering effects.

Decrease in fibrin content of venous thrombi in selectin-deficient mice.

The purpose of this study was to quantify the fibrin content of thrombi produced in a mouse model of venous thrombosis and correlate this to thrombus mass. The role of P-selectin, E-selectin, and IL-10 on thrombus fibrin content was analyzed using knockout (KO) mice. Five groups of mice were evaluated: control (N = 10), P-selectin KO (N = 7), E-selectin KO (N = 5), combined E-/P-selectin KO (N = 12), and IL-10 KO (N = 10). Venous thrombosis was induced by ligation of the infrarenal IVC. Mice were sacrificed on postoperative days (POD) 2 and 6. Thrombus mass was calculated. Sections of IVC were stained with an antibody that cross reacts with mouse fibrin. The distribution of RGB color pixels was generated from digitized micrographs of the thrombus of each animal. The mean pixel value for each group was compiled and analyzed using 2-way ANOVA. Mean pixel value per group was correlated with the mean thrombus mass per group. Color analysis demonstrated significant decreases in the analyzed fibrin content on POD-2 between the control vs E-/P-selectin KO (P < 0.05) and control vs IL-10 KO (P < 0.05) groups. In addition, significantly less fibrin staining was noted on POD-6 between the control vs E-selectin KO (P = 0.03), control vs P-selectin KO (P = 0.01), and control vs E-/P-selectin KO (P < 0.01). There was a strong overall correlation between the mean pixel value for each group and the thrombus mass (R = 0.964; P < 0.01). This study demonstrates a difference in fibrin content of thrombi produced in animals deficient in E-selectin, P-selectin, and IL-10, supporting their importance in thrombus amplification, fibrin formation, and the mass of thrombus formed.

Low molecular weight protamine as nontoxic heparin/low molecular weight heparin antidote (III): preliminary in vivo evaluation of efficacy and toxicity using a canine model.

Heparin employed in cardiovascular surgeries often leads to a high incidence of bleeding complications. Protamine employed in heparin reversal, however, can cause severe adverse reactions. In an attempt to address this clinical problem, we developed low molecular weight protamine (LMWP) as a potentially effective and less toxic heparin antagonist. A homogeneous 1880-d peptide fragment, termed LMWP-TDSP5 and containing the amino acid sequence of VSRRRRRRGGRRRR, was derived directly from protamine by enzymatic digestion of protamine with thermolysin. In vitro studies demonstrated that TDSP5 was capable of neutralizing various anticoagulant functions of both heparin and commercial low molecular weight heparin preparations. In addition, TDSP5 exhibited significantly reduced crossreactivity toward mouse sera containing antiprotamine antibodies. TDSP5 showed a decrease in its potential in activating the complement system. All of these findings suggested the possibility of markedly reduced protamine toxicity for TDSP5. In this article, we conducted preliminary in vivo studies to further demonstrate the feasibility and utility of using LMWP as a nontoxic clinical protamine substitute. Dogs were chosen as test animals because they were known to magnify the typical human response to protamine. By using a full spectra of biological and clinical assays for heparin, including the anti-IIa and anti-Xa chromogenic assays and the activated partial, thromboplastin time and TCT clotting assays, TDSP5 showed that it could completely neutralize all these different anticoagulant functions of heparin in dogs. Although administration of protamine in dogs produced a significant reduction in mean arterial blood pressure (-14.9 mm Hg) and elevation in pulmonary artery systolic pressure (+5.0 mm Hg), the use of TDSP5 in dogs did not elicit any statistically significant change in any of the variables measured. Furthermore, the use of LMWP also significantly reduced the protamine-induced transient thrombocytopenic and granulocytopenic responses. The white blood cell counts and platelet counts decreased to 82.1% and 60.0% of baseline, respectively, in dogs given intravenous protamine compared to 97.8% and 88.6% of baseline in dogs receiving TDSP5. These preliminary findings indicated that LMWP could potentially provide an effective and safe means to control both heparin- and protamine-induced complications.

P-selectin antagonism causes dose-dependent venous thrombosis inhibition.

Inhibition of P-selectin by antibody or selectin antagonist decreases inflammation and thrombosis. This study evaluates the dose-response relationship using a selectin receptor antagonist. Eight male baboons (Papio anubis) underwent inferior vena caval thrombosis using a 6 h balloon occlusion model. Three animals received 500 microg/kg P-selectin antagonist (rPSGL-Ig) and five 1 mg/kg rPSGL-Ig with or without a non-anticoagulant dose of Dalteparin. These animals were compared to our published results in this model with 4 saline controls and 8 animals that received 4 mg/kg rPSGL-Ig. A statistically significant dose-response relationship existed between rPSGL-Ig dose and thrombosis (p < 0.01), and between rPSGL-Ig dose and spontaneous recanalization (p<0.05). Inflammatory assessment revealed decreased gadolinium enhancement in all rPSGL-Ig groups compared to previously reported control, despite no significant differences in inflammatory cell extravasation. No dose of rPSGL-Ig caused anticoagulation. Selectin antagonism results in a dose-dependent decrease in thrombosis and increase in spontaneous recanalization.

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.

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.

A novel protamine variant reversal of heparin anticoagulation in human blood in vitro.

PURPOSE: Protamine reversal of heparin anticoagulation during cardiovascular surgery may cause severe hypotension and pulmonary hypertension. A novel protamine variant, [+18RGD], has been developed that effectively reverses heparin anticoagulation without toxicity in canine experiments. Heretofore, human studies have not been undertaken. This investigation hypothesized that [+18RGD] would effectively reverse heparin anticoagulation of human blood in vitro. METHODS: Fifty patients who underwent anticoagulation therapy during vascular surgery had blood sampled at baseline and 30 minutes after receiving heparin (150 IU/kg). Activated clotting times were used to define specific quantities of [+18RGD] or protamine necessary to completely reverse heparin anticoagulation in the blood sample of each patient. These defined amounts of [+18RGD] or protamine were then administered to the heparinized blood samples, and percent reversals of activated partial thromboplastin time, thrombin clotting time, and antifactor Xa/IIa levels were determined. In addition, platelet aggregation assays, as well as platelet and white blood cell counts were performed. RESULTS: [+18RGD] and protamine were equivalent in reversing heparin as assessed by thrombin clotting time, antifactor Xa, antifactor IIa levels, and white blood cell changes. [+18RGD], when compared with protamine, was superior in this regard, as assessed by activated partial thromboplastin time (94.5 +/- 1.0 vs 86.5 +/- 1.3% delta, respectively; p < 0.001) and platelet declines (-3.9 +/- 2.9 vs -12.8 +/- 3.4 per mm3, respectively; p = 0.048). Platelet aggregation was also decreased for [+18RGD] compared with protamine (23.6 +/- 1.5 vs 28.5 +/- 1.9%, respectively; p = 0.048). CONCLUSIONS: [+18RGD] was as effective as protamine for in vitro reversal of heparin anticoagulation by most coagulation assays, was statistically more effective at reversal than protamine by aPTT assay, and was associated with lesser platelet reductions than protamine. [+18RGD], if less toxic than protamine in human beings, would allow for effective clinical reversal of heparin anticoagulation.

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.

Effects of differing rates of protamine reversal of heparin anticoagulation.

BACKGROUND: Protamine sulfate reversal of heparin anticoagulation may be associated with adverse cardiovascular side effects. The purpose of this study was to determine whether diminished systemic oxygen consumption and hemodynamic changes were more likely to accompany rapid versus slow protamine administration. METHODS: Fifteen patients undergoing abdominal aortic aneurysm resection in a prospective randomized double-blinded study received intravenous protamine (1.5 mg/kg) rapidly during a 3-minute period (group I, n = 7) or slowly during a 15-minute period (group II, n = 8). Systemic oxygen consumption (VO2) and hemodynamic parameters were assessed for up to 20 minutes after protamine administration began. RESULTS: Blood pressure declines (millimeters of mercury) were greatest in group I with rapid protamine administration (-19 systolic and -9 diastolic) compared with group II with slow protamine administration (-12 systolic and -1 diastolic). Heart rate fell markedly in both groups I and II. Cardiac output (CO) declined in group I at virtually all time periods. Similar CO declines in group II occurred 10 minutes after protamine infusion had begun and persisted for 3 minutes after protamine administration was complete. Maximum VO2 decreases were -16% (60 seconds into protamine infusion) and -13% (1.5 minutes after protamine infusion) in groups I and II, respectively, with statistically significant declines (p < 0.05) occurring only in group I compared with baseline values. Statistically significant differences (p < 0.01), however, were found when mean declines during and after protamine infusion were compared with controls for both CO and VO2 in both groups. CONCLUSIONS: Significant declines in systemic VO2 and hemodynamic perturbations accompany protamine reversal of heparin anticoagulation during aortic surgery. Rapid protamine administration increases the magnitude of these adverse responses.

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.