Pre-treatment with compound Danshen dripping pills prevents lipid infusion-induced microvascular dysfunction in mice.

CONTEXT: Recent studies have shown compound Danshen dripping pills (CDDP) could improve microcirculation in ischemic/reperfusion injury and other microvascular disorders. The mechanism for CDDP's role in microcirculation is not clear. OBJECTIVE: To explore the protective effects of CDDP on microvascular dysfunction. MATERIALS AND METHODS: C57BL/6 male mice (6-8 weeks) were randomized into control, model and CDDP groups (n = 10), which were treated with normal saline or CDDP (105.30 mg/kg), respectively. Then, lipid emulsion and heparin were infused via mice jugular vein to establish systemic microvascular dysfunction model. Coronary flow reserve (CFR) and leukocytes adhesion on microvascular wall were measured. Relative CD11b and CD62L expression levels on neutrophils were measured by flow cytometric analysis. Expression level of forkhead box transcription factor O1 (FOXO1) mRNA was identified by real-time PCR. RESULTS: Lipid infusion significantly attenuated the CFR (1.84 ± 0.14 vs. 2.65 ± 0.02) and increased the number of leukocytes adherent to microvascular wall in cremaster (4067.00 ± 581.20 cells/mm2 vs. 10.67 ± 4.81 cells/mm2). The expression level of CD11b and FOXO1 in neutrophils was also up-regulated by lipid infusion. Pre-treatment with CDDP significantly improved CFR (2.57 ± 0.29 vs. 1.84 ± 0.14), decreased the number of leukocytes adherent to microvascular wall (2500.00 ± 288.70 cells/mm2 vs. 4067.00 ± 581.20 cells/mm2) and down-regulated CD11b and FOXO1 expression. Discussion and conclusions: Pre-treatment with CDDP could prevent lipid infusion-induced systemic microvascular disorder including coronary and peripheral microvascular dysfunction. Down-regulated FOXO1 and decreased leukocyte adhesion might play an important role in the mechanisms of CDDP's efficacy.

Comparison of the Effects of Contractubex Gel and Benzothiazole After Topical Application in an Experimental Model of Epidural Fibrosis in Rats.

BACKGROUND: Postoperative epidural adhesion is a frequent cause of failed back surgery syndrome, manifesting with back and leg pain or neurologic deficits. Development of preventive measures for epidural adhesion after laminectomy is critical to improve outcomes of lumbar surgery. We hypothesized that positive effects of topical application of Contractubex (CTX) gel and benzothiazole (BT) individually and in combination could aid in preventing epidural fibrosis in a rat laminectomy model. METHODS: Rats were randomly assigned to 2 control and 5 experimental groups (n = 8 for each group). The control(-) group received no surgery, whereas the control(+) group underwent laminectomy without any drug administration. In experimental groups, study agents applied to dura mater after laminectomy were 100mgCTX, 2.5%BT, 5%BT; 100mgCTXplus2.5%BT, and 100mgCTXplus5%BT. Laminectomy was performed at the L3 level for all rats. The extent of epidural fibrosis was assessed 4 weeks later macroscopically and histopathologically. Hepatic and renal toxicity of study drugs was assessed histopathologically. RESULTS: Topical CTX and BT individually and in combination reduced epidural fibrosis after laminectomy in rats. Although a meaningful decrease of epidural fibrosis with individual application of CTX and BT (2.5% or 5%) was obtained (P < 0.05), the effect of their combination was more pronounced without meaningful hepatic and renal toxicity (P < 0.05). CONCLUSIONS: Combined use of topical CTX and BT could be a potential therapy for epidural fibrosis. Further research with this agents for the prevention of epidural fibrosis is warranted.

In Vitro and In Vivo Biocompatibility Evaluation of Polyallylamine and Macromolecular Heparin Conjugates Modified Alginate Microbeads.

Host reactivity to biocompatible immunoisolation devices is a major challenge for cellular therapies, and a human screening model would be of great value. We designed new types of surface modified barium alginate microspheres, and evaluated their inflammatory properties using human whole blood, and the intraperitoneal response after three weeks in Wistar rats. Microspheres were modified using proprietary polyallylamine (PAV) and coupled with macromolecular heparin conjugates (Corline Heparin Conjugate, CHC). The PAV-CHC strategy resulted in uniform and stable coatings with increased anti-clot activity and low cytotoxicity. In human whole blood, PAV coating at high dose (100 µg/ml) induced elevated complement, leukocyte CD11b and inflammatory mediators, and in Wistar rats increased fibrotic overgrowth. Coating of high dose PAV with CHC significantly reduced these responses. Low dose PAV (10 µg/ml) ± CHC and unmodified alginate microbeads showed low responses. That the human whole blood inflammatory reactions paralleled the host response shows a link between inflammatory potential and initial fibrotic response. CHC possessed anti-inflammatory activity, but failed to improve overall biocompatibility. We conclude that the human whole blood assay is an efficient first-phase screening model for inflammation, and a guiding tool in development of new generation microspheres for cell encapsulation therapy.

Omni-Stat (Chitosan) arrests bleeding in heparinised subjects in vivo: an experimental study in a model of major peripheral vascular injury.

OBJECTIVE: To explore the effectiveness of Omni-Stat (Chitosan) in a model of major haemorrhage in the presence of clotting dysfunction. METHOD: A total of 12 major femoral artery bleeds in moderately heparinised swine treated with Omni-Stat (Chitosan) were compared with five control bleeds. RESULTS: Haemostasis was successfully achieved at first treatment in 10 of 12 bleeds and at second treatment in the remaining two bleeds. CONCLUSION: The study supports the evidence that Omni-Stat (Chitosan) acts independently of classical clotting pathways and should be effective in patients with clotting dysfunctions, who suffer major haemorrhage. It also suggests the potential for a role in cardiac surgery.

The effect of transcatheter injections on cell viability and cytokine release of mononuclear cells.

BACKGROUND AND PURPOSE: Several studies suggest that various types of cellular therapies enhance recovery after stroke in animal models. IA-based delivery of cells to the brain is under investigation for stroke, but it is unknown whether cells are injured as a result of being injected through a catheter or exposed to iodinated contrast medium or solutions containing heparin. MATERIALS AND METHODS: We assessed the effect of catheterization with the Excelsior SL-10 catheter or exposure to heparin or iodine contrast on human bone marrow MNCs. Viability and cell injury were assessed by trypan blue exclusion, caspase-3 activity, and lipid peroxidation. Cellular function of MNCs was assessed by their production and release of VEGF, IL-10, and IGF-1. RESULTS: Flow rates of 10 million cells from 0.5 to 2 mL/min did not alter MNC viability; however, 5 mL/min of MNCs did reduce viability by 19%. Iodine and low-dose heparin exposure did not affect cell viability; however, high-dose heparin was cytotoxic. Catheter delivery at 2 mL/min did not affect levels of VEGF, IL-10, or IGF-1. CONCLUSIONS: MNCs do not appear to be damaged by heparin, iodine contrast, and the Excelsior SL-10 catheter at flow rates up to 2 mL/min. However, higher flow rates did reduce viability, and high-dose heparin did cause cell death.

Development of a non-human primate sub-clinical model of heparin-induced thrombocytopenia: platelet responses to human anti-heparin-platelet factor 4 antibodies.

The purpose of this study was to characterize the responses of human and non-human primate (Macaca mulatta) platelets to anti-heparin-platelet factor 4 (AHPF4) antibodies. Due to the variations observed in the functionality and immunoglobulin isotypes in patients with heparin-induced thrombocytopenia (HIT), we used highly characterized human AHPF4 antibodies to study platelet activation responses. Using ELISA and 14C-serotonin release assay (SRA) systems, three patients' plasmapheresis fluid with similar responses to these assays were pooled. This pool was then used to study the platelet activation responses of human and primate platelets in the HIT platelet aggregation assay, a flow cytometry assay, and a variation of the aggregation assay in which glycoprotein IIb/IIIa inhibitors were supplemented. In the plasmapheresis fluid from three patients, the most significant AHPF4 immunoglobulin isotype present (based on optical density readings) was IgG, with less IgM (p < 0.001) and IgA (p < 0.001). The SRA yielded equivalent platelet activation results in all three patients. Using this pool in the platelet aggregation assay, without any heparin present, there was less percent aggregation (p < 0.001) with human platelets (11.8 +/- 2.35, n = 5) compared to the primate platelets (54.3 +/- 10.2, n = 9). In presence of 0.4 U/ml heparin, both platelet types had similar percent aggregations (p > 0.05). Three glycoprotein IIb/IIIa receptor inhibitors were used to evaluate the similarities in platelet activation. Eptifibatide was found to be a strong inhibitor of both species' platelet types at concentrations greater than 0.01 microg/ml. This was not the case with tirofiban which inhibited both human and monkey platelets at concentrations greater than 0.025 microg/ml. Abciximab inhibited aggregation at concentrations greater than 6.25 microg/ml. These data indicate that phylogenetic similarities in platelets of humans and primates may be used to further characterize the pathophysiology of HIT syndrome.

Dual effects of sulfated D-galactans from the red algae Botryocladia occidentalis preventing thrombosis and inducing platelet aggregation.

Sulfated D-galactans occur on the red algae Botryocladia occidentalis as three fractions that differ in their sulfate content. Fractions F2 and F3 are potent anticoagulants. Like heparin, they enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. The inhibition potency increases simultaneously with the sulfate content of the fractions. The antithrombotic activity of these sulfated D-galactans was investigated on an experimental thrombosis model in which thrombus formation was induced by a combination of stasis and hypercoagulability. In contrast with heparin. the sulfated D-galactans showed a dual dose-response curve preventing thrombosis at doses up to approximately 0.5 mg/ kg body weight but losing the effect at higher doses. This unexpected behavior is probably due to a combined action of the sulfated D-galactan as anticoagulant and also as a strong inducer of platelet aggregation. In platelet-depleted animals the antithrombotic activity at higher dose of sulfated D-galactan is restored and almost total inhibition of thrombus formation is achieved. The sulfated D-galactan has no hemorrhagic effect even at high doses, possibly as a consequence of its effect on platelet aggregation. At comparable dose heparin has an intense bleeding effect. These results indicate that new polysaccharides, with well-defined structures, can help to distinguish events, such as antithrombotic and anticoagulant activities, bleeding and platelet-aggregating effects, which are obscure when induced simultaneously by a single compound.

Pharmacological effects of a novel recombinant hirudin, CX-397, in vivo and in vitro: comparison with recombinant hirudin variant-1, heparin, and argatroban.

The novel recombinant hirudin analog CX-397 was investigated with respect to its pharmacological activity and antithrombin profiles in vivo and in vitro. In three different types of thrombosis models in rats, including stasis and thrombin-induced venous, glass surface-activated arterio-venous shunt, and ferric chloride-induced arterial thrombosis models, CX-397 and rHV-1 elicited potent antithrombotic effects, where the minimum effective doses of rHV-1 tended to be higher than those of CX-397 in the arterio-venous shunt and arterial thrombosis models. The hemorrhagic risk of CX-397 in template bleeding in rats was not higher than that of rHV-1, indicating that CX-397 is superior to rHV-1 for treating the platelet-dominant type of thrombosis. However, no differences were detected between CX-397 and rHV-1 in their effects on in vitro coagulation times and thrombin-induced platelet aggregation, suggesting the possibility that some unknown mechanisms other than simple thrombin inhibition are also involved in their antithrombotic actions.

Effect of a synthetic factor Xa inhibitor, YM-60828, on blood vessel patency in combination with a thrombolytic agent and on blood loss from the operation site in a rat model of arterial thrombosis.

We examined the adjunctive effect of a novel factor Xa inhibitor, YM-60828, on vessel patency and blood loss from the operation site after successful thrombolysis with a modified tissue-type plasminogen activator (moPA) in an electrically-induced carotid artery thrombosis model in rats. Five minutes after the induction of occlusive thrombus, a test drug (YM-60828, argatroban, heparin or saline) was administered by i.v. bolus injection followed by continuous infusion. Thrombolysis was induced with moPA by i.v. bolus injection at a dose of 650,000 IU/ kg. YM-60828 at 1 mg/kg i.v. followed by 3 mg/kg/h significantly prevented reocclusion, increased the duration of patency, and improved vessel patency after successful thrombolysis without any significant increase in blood loss from the operation site. Argatroban at 1 mg/kg i.v. followed by 3 mg/kg/h and heparin at 300 U/kg i.v. followed by 150 U/kg/h also significantly improved these parameters, but were accompanied by a significant increase in blood loss. These results suggest that the factor Xa inhibitor YM-60828 may be a potent and useful adjunctive agent with a lower risk of bleeding complications than argatroban and heparin in thrombolytic therapy.

Comparison of enoxaparin, hirulog, and heparin as adjunctive antithrombotic therapy during thrombolysis with rtPA in the stenosed canine coronary artery.

A canine model of electrolytic injury-induced coronary artery thrombosis and rtPA-induced thrombolysis was used to evaluate the relative antithrombotic efficacy of enoxaparin (a low molecular weight heparin), conventional therapy (heparin or heparin plus aspirin), and hirulog (a direct thrombin inhibitor), when used as adjunctive therapy during thrombolysis. After 60 min of clot aging, adjunctive therapy was begun at doses which elevated APTT approximately 2-fold over baseline. Fifteen minutes after the start of adjunctive therapy, recombinant tissue plasminogen activator (rtPA) was administered (100 microg/kg i.v. bolus + 20 microg/kg/min for 60 min). Adjunctive therapy continued for 1 h after termination of rtPA and blood flow was monitored for two additional hours. Enoxaparin (1 mg/kg i.v. bolus + 30 microg/kg/min, n = 10 for each treatment group) was the only adjunctive treatment that significantly increased the total minutes of flow (143 +/- 25 min out of a possible 240 min, vs 54 +/- 25 min for vehicle, p <0.05) and decreased thrombus mass (6.0 +/- 1.3 mg vs 11.8 +/- 3.2 mg for vehicle). Although hirulog (2 mg/kg i.v. bolus + 40 microg/kg/min) did not significantly increase the minutes of flow (120 +/- 27 min, p <0.06) or decrease thrombus mass (8.7 +/- 1.7 mg) compared to vehicle, these values were not significantly different than those measured in the enoxaparin group. However, the results with hirulog were achieved at the expense of a significantly greater increase in template bleeding time than that measured during enoxaparin treatment. Minutes of flow for heparin (50 U/kg i.v. bolus + 0.6 U/kg/min) and heparin plus aspirin (5 mg/kg i.v. bolus) were 69 +/- 20 and 60 +/- 23 min, respectively; thrombus masses were 8.2 +/- 1.3 and 7.3 +/- 1.0 mg, respectively. In summary, enoxaparin was more effective than conventional therapy in this model in terms of vessel patency and thrombus mass, and was as effective as hirulog, at least at a dose of hirulog that only modestly impaired hemostasis. Therefore, enoxaparin may prove to be a safe and effective alternative agent for adjunctive therapy during thrombolysis with rtPA.

Protamine sulphate inhibits pentasaccharide (SR80027)-induced bleeding without affecting its antithrombotic and anti-factor Xa activity in the rat.

The neutralization of a potent anti-factor Xa pentasaccharide (SR 80027) and heparin-induced bleeding enhancement by protamine sulphate was studied in vivo. Bleeding time, as measured by transection of the tail of anaesthetized rats, increased after the administration of standard heparin and SR 80027. Doses of 0.6 and 2.5 mg/kg of heparin and SR 80027, respectively, were required to enhance blood loss to the same extent (6-fold increase). Protamine sulphate (10 mg/kg i.v.) reduced blood loss induced by both compounds but also neutralized the anti-factor Xa activity as well as the antithrombotic activity of standard heparin measured in a venous thrombosis model. However, protamine sulphate did not affect the anti-factor Xa activity or the antithrombotic activity of SR 80027. These data suggest that protamine sulphate may be an effective antidote for the bleeding side-effects of SR 80027 but they also indicate that the bleeding tendency associated with this type of compounds cannot be attributed to their anti-factor Xa activity.

Comparison of hemorrhagic effect of heparin and human activated protein C with use of Thrombostat 4000.

The importance of bleeding as a complication of anticoagulant therapy is clearly recognized. We previously reported that amelioration of hemorrhage associated with disseminated intravascular coagulation by the human activated protein C (APC) was greater than that by heparin. In this study, we compared the bleeding complication of intravenously administered APC and heparin in rabbits, and also estimated primary hemostasis. When both anticoagulants were intravenously infused, the bleeding time from a punctured ear vein was prolonged dose-dependently. However, at doses which prolonged the activated partial thromboplastin time nearly equally, the prolongation of bleeding was greater in heparin-administered rabbits. Blood withdrawn from heparin-administered animals showed increases in in vitro bleeding parameters which correlated with the in vivo bleeding time. However, only small changes were observed in the blood withdrawn from APC-administered animals. Both drugs induced either no change or only a slight decrease in the platelet count, hematocrit and fibrinogen content. These observations suggest that APC may be a more useful anticoagulant than heparin since it causes less bleeding tendency.

Release of interleukin-1 beta by dermatan sulfate suppresses hepatocyte growth.

Among glycosaminoglycans, dermatan sulfate (DS) is the strongest inhibitor of DNA synthesis in adult rat hepatocytes in primary culture stimulated with insulin and epidermal growth factor. Hyaluronate also inhibited DNA synthesis, whereas chondroitin-6 sulfate, 4-sulfate or heparin had no effect on DNA synthesis in hepatocytes. Analysis of growth regulatory factors in hepatocyte culture medium treated with DS revealed that interleukin-1 (IL-1) was released into the medium. IL-1 beta mRNA was detected in DS-treated hepatocytes by reverse transcriptase-polymerase chain reaction, but not in untreated hepatocytes. For a marked inhibition of DNA synthesis, more than 10 hr of exposure to DS before cultured hepatocytes started DNA synthesis, was required. Similarly, more than 10 hr was required after the addition of DS before IL-1 beta mRNA was detected. These findings suggest that DS in the medium induced the production of IL-1 beta which, in turn, reduced DNA synthesis in hepatocytes.

Is heparin the ideal anticoagulant for cardiopulmonary bypass? Dermatan sulphate may be an alternate choice.

Performance of cardiopulmonary bypass (CPB) during cardiac surgery requires the administration of high dose heparin to prevent CPB pump occlusion. However, this heparin use is associated with bleeding side-effects. Moreover, at the end of CPB, the heparin must be neutralized with protamine sulphate, which is also associated with adverse side-effects. A number of recent studies suggest that dermatan sulphate may be useful as an alternate anticoagulant to heparin. We determined whether CPB could be performed using dermatan sulphate instead of heparin, in an adult pig CPB model. When heparin was used, a high dose (> 200 U/kg, which generated > 3 anti-thrombin U/ml of plasma), was required to perform successful CPB and to maintain CPB pump patency. This dose was associated with a post CPB bleeding of approximately 600 ml/2 h. In contrast, successful CPB could be achieved when the pigs were given lower doses of dermatan sulphate than heparin, which in turn, were associated with less bleeding. We conclude that dermatan sulphate may be an alternate anticoagulant for cardiac surgery.

Hematological toxicity of adjuvant portal liver infusion with fluorouracil, mitomycin and heparin.

Adjuvant perioperative liver infusion chemotherapy with fluorouracil, mitomycin and heparin caused mild but significant myelosuppression in the first two postoperative weeks. This was associated with a slightly increased bleeding tendency. Mortality was not increased. Future trials might combine chemotherapy with hematopoietic growth factors in order to minimize myelosuppression.

A randomized blind study comparing standard heparin and a new low molecular weight heparinoid in cardiopulmonary bypass surgery in dogs.

Postoperative hemorrhage remains a serious complication in cardiopulmonary bypass (CPB) surgery. In our study, alternative anticoagulation with a new low molecular weight (LMW) heparinoid (Org 10172) was compared with a standardized heparin regimen. A preliminary dose-finding study indicated the minimal effective heparinoid dose to be 260 anti-Xa U/kg body weight, which was comparable to the standardized heparin regime, as revealed by similar plasma anti-Xa values. The following randomized open pilot study in 12 mongrel dogs undergoing CPB showed the heparinoid to be as effective as heparin, with an additional advantageous decrease in postoperative blood loss in the Org 10172 group. Our randomized blind study in 16 mongrel dogs undergoing CPB was performed to confirm previous results. Both antithrombotic agents were effective in the prevention of clot formation within the extracorporeal circuit. Hematocrit values and erythrocyte and platelet counts showed no significant intergroup differences. Post-CPB leukocyte counts revealed a significantly more rapid increase in the group given heparinoid (P less than 0.05). In the group given heparin, the expected prolongations of both the thrombin time (TT) and activated partial thromboplastin time (APTT) were noted, whereas in the group given heparinoid, only a transient peak prolongation of the TT after dose administration was revealed, and no significant prolongation of the APTT. Mean anti-Xa plasma levels were similar during CPB, showing a rapid decrease in the group given heparin on protamine administration, as did the APTT. Assessment of the operating field indicated an elevated intraoperative blood loss in the group given heparin. Postoperative blood loss measured over a period of 2.5 hours after closure of the thorax was significantly lower in the group given heparinoid than in the heparinized animals (625 +/- 100.0 ml, mean +/- SD, and 806 +/- 178.2 ml, respectively; P less than 0.05). Our observations suggest that the LMW heparinoid Org 10172 has an increased benefit/risk ratio over standard heparin and is effective in CPB in dogs. Additional investigations in humans should verify the possibility of use of this substance as an alternative means of anticoagulation during CPB in patients in whom heparin is relatively contraindicated.

Experimental colon neoplasia enhanced by extract of sperm (protamine).

An experimental study of colorectal tumorigenesis in inbred rats given the potent carcinogen dimethylhydrazine has shown that protamine, given before any tumours had developed, significantly enhanced the development of large bowel tumours (100% incidence). Those animals having no adjuvant therapy or who received either heparin or 5-fluorouracil (alone or in combination) had an approximate 50% incidence of such tumours.