Morphologic and molecular study on the lens anterior capsule in systemic sclerosis / Relato sobre estudo morfológico e molecular da cápsula anterior do cristalino na esclerose sistêmica

ABSTRACT This study aimed to analyze the anterior lens capsule specimens from both eyes of a patient with systemic sclerosis and compare them to the eyes of a control patient. No significant differences between systemic sclerosis and control eyes were observed in the results from the hematoxylin-eosin and picrosirius staining. In the samples obtained from both systemic sclerosis and control eyes, there were expressions of caspase, a molecule expressed in cell death by apoptosis. Heparanase was overexpressed in the systemic sclerosis sample compared to the control sample. Therefore, the anterior lens capsule of the patient with systemic sclerosis is probably affected by the disease since it showed marked expression of heparanase 1.(AU)

RESUMO Analisamos as amostras das cápsulas anteriores do cristalino de uma paciente com esclerose sistêmica e comparamos com as de um paciente controle. Não foram observadas diferenças significativas entre esclerose sistêmica e controle nos resultados da coloração com hematoxilina-eosina e picrosirius. Nas amostras obtidas da esclerose sistêmica e do controle, obtivemos expressão de caspase, uma molécula expressa na morte celular por apoptose. A heparinase foi expressa de forma mais marcante na amostra de esclerose sistêmica quando comparada ao controle. Portanto, a cápsula anterior do cristalino da paciente com esclerose sistêmica provavelmente foi afetada pela doença, uma vez que mostrou expressão aumentada de heparinase 1.(AU)

Heparinase-modified thromboelastography in cats.

BACKGROUND: Evaluation of underlying hemostatic function is challenging when feline patients are receiving an anticoagulant medication. Discontinuing the anticoagulant to obtain accurate results for hemostatic testing may lead to thrombotic complications. The addition of heparinase to blood samples may mitigate the effects of exogenous heparin and allow hemostatic testing. METHODS: Tissue factor (TF)-activated thromboelastography (TEG) was performed using citrated whole blood from 19 cats. Assays were performed using citrated whole blood, with and without addition of unfractionated heparin to a concentration of 0.2 U/mL. For each blood sample, TEG assays were performed using a standard cup and a heparinase-coated cup. KEY FINDINGS: For TEG variables R, k, α-angle, and MA, mean values were not statistically different when citrated blood was used with standard or heparinase-coated cups. Heparinized blood assayed in standard cups displayed a significantly increased R and k, and significantly decreased α-angle and MA when compared to heparinized blood assayed in heparinase-coated cups. TEG variables for heparinized blood assayed in heparinase cups was not statistically different from those of the citrated whole blood without added heparin. SIGNIFICANCE: Heparinase modified, TF-activated, TEG reverses heparin effects in feline-citrated blood.

Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes.

The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10(-)(7)M fMLP. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 (D(Dx70)), and a smaller sublayer 173nm above the EC surface (D(173)), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D(Dx70) was twice that of D(173) suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D(Dx70) and D(173). However, heparinase decreased D(Dx70), and did not induce any significant change for the D(173). These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.

Systemic anticoagulant effect of low-dose subcutaneous unfractionated heparin as determined using thrombelastography.

In an observational study using heparinase-modified thrombelastography, we investigated the percentage of elective cardiothoracic surgical patients receiving low-dose unfractionated heparin (5000 IU 12 hourly subcutaneously) who had a demonstrable systemic heparin effect. Blood samples were obtained at induction from 40 adult elective cardiothoracic surgical patients who had received 5000 IU unfractionated heparin subcutaneously within six hours. Simultaneous kaolin and heparinase-modified thrombelastographies were run on all samples. Fourteen patients (35%; 95% CI: 20 to 50%) had a demonstrable heparin effect (defined as a kaolin thrombelastography R time >25% longer than the heparinase-modified control). Their mean +/- SD kaolin thrombelastography R time was 13.6 +/- 5.9 minutes (normal range 4 to 8 minutes) vs. 7.1 +/- 2.0 minutes for the heparinase-modified controls. In 10 patients the thrombelastography R times were >50% longer and in four patients >100% longer than their respective heparinase-modified controls. In a post hoc analysis, there was little correlation between the extent of the prolongation and patient age (r = 0.02), weight (r = -0.31), preoperative creatinine (r = -0.17), or time since administration of heparin (r = 0.14). These results indicate that about one third of patients who have received low-dose unfractionated heparin subcutaneously within six hours have a demonstrable heparin effect. The potential for this effect should be considered if central neural blockade is planned.

Antimetastatic activities of modified heparins: selectin inhibition by heparin attenuates metastasis.

Heparin, which is traditionally used as an anticoagulant but has a variety of additional biological activities, was shown in several retrospective and prospective clinical trials to have an effect on cancer survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. To clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. Cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. Heparin is also a potent inhibitor of selectin-mediated interactions. P- and L-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet-tumor cell thrombi formation. To delineate the biological activities of heparin contributing to metastasis inhibition, modified heparins with specific activities were evaluated. Low anticoagulant heparin preparations still inhibited metastasis efficiently, indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. Modified heparins characterized for heparanase inhibitory activity are also potential inhibitors of selectins. Selectin inhibition is a clear component of heparin inhibition of metastasis. The contribution of selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity.

Heparinase enhances collateral vessel development in the ischemic limb.

Several angiogenic factors are bound to heparin and stored in the extracellular matrix of diverse tissues; thus, the controlled release of these factors can provide a novel approach of angiogenic stimulation to ischemic tissues. The purpose of this study was to test the hypothesis that heparinase, when administered in vivo, might enhance collateral vessel development in the ischemic limb by the controlled release of angiogenic factors such as basic fibroblast growth factor (bFGF). Eleven male New Zealand White rabbits underwent ligation and excision of the common and superficial femoral arteries in the left hindlimb. In the heparinase group (n = 6), 9.7 IU of heparinase (in 3 ml saline) was injected intramuscularly to the left thigh daily for 10 days, beginning 11 days after surgery. In the control group (n = 5), inactivated heparinase (also in 3 ml saline) was administered following the same experimental protocol. Calf systolic pressure was measured in both hindlimbs and was expressed as a ratio of left to right (L/R ratio) before injections (on day 10) and after injections (on days 20, 30, and 40). Vascularization was quantified by comparing the number of vessels along a line drawn across the mid-thigh on angiograms taken 4 seconds after contrast injection at day 40 when the study was terminated. The intramuscular injection of heparinase improves perfusion to the ischemic limb through the process of enhanced collateral vessel development. The angiogenic effect of heparinase on the ischemic tissue may result from release of endogenous angiogenic factors, such as bFGF.

A dose-determining trial of heparinase-I (Neutralase) for heparin neutralization in coronary artery surgery.

UNLABELLED: Heparinase-I, a specific heparin-degrading enzyme, may represent an alternative to protamine. We explored the dose of heparinase-I for efficacy and safety in patients undergoing coronary artery surgery. At the conclusion of cardiopulmonary bypass, subjects received 5, 7, or 10 microg/kg of open-label heparinase-I instead of protamine. Activated clotting time (ACT) and its difference from a contemporaneous heparin-free sample (DeltaACT) at 3 min before and 3, 6, and 9 min after heparinase-I determined reversal efficacy. After surgery, we recorded hourly chest tube drainage. Systemic and pulmonary arterial blood pressure and cardiac output measurements before and immediately after heparinase-I were used to evaluate hemodynamic safety. Coagulation measurements included anti-factor Xa and anti-factor IIa activities. Forty-nine patients from seven institutions participated: 12 received 5 microg/kg, 21 received 7 microg/kg, 4 received two doses of 7 microg/kg, 8 received 10 microg/kg, and 4 received two doses of 10 microg/kg. Treatment groups did not differ demographically. Median DeltaACT 9 min later was 11, 7, and 4 s for the 5, 7, and 10 microg/kg groups, respectively. No adverse hemodynamic changes occurred with heparinase-I administration. The authors conclude that heparinase-I effectively restored the ACT after cardiopulmonary bypass. This effect appeared to be dose dependent. IMPLICATIONS: Heparinase-I (Neutralase(TM)) successfully restored activated coagulation time with no adverse hemodynamic events in patients undergoing coronary artery surgery with cardiopulmonary bypass in an open-label dose-determining trial.

Thromboelastography with heparinase in orthotopic liver transplantation.

OBJECTIVE: To investigate the role of heparin in the postreperfusion coagulopathy during liver transplantation with heparinase-guided thromboelastography. DESIGN: A prospective, interventional study. SETTING: A university-affiliated hospital. PARTICIPANTS: Twenty-six patients undergoing orthotopic liver transplantation (OLT). INTERVENTIONS: Blood drawn at five intervals for thromboelastography assessment with native (12 patients) or celite blood (14 patients) compared with simultaneous thromboelastography traces with added heparinase. MAIN RESULTS: In the native samples, the prolonged R (reaction) and K (coagulation) time and decreased alpha angle were corrected in heparinase thromboelastograph traces immediately before reperfusion and 10 minutes postreperfusion. In the celite-accelerated samples, the heparinase traces showed correction of the R and K times and alpha angle only at the 10-minute postreperfusion stage. In seven patients who had thromboelastography performed after protamine administration, there were no differences between celite and heparinase-celite traces. CONCLUSIONS: Heparinase-treated thromboelastography offered compelling evidence for the presence of heparin-like activity after liver graft reperfusion. The objective evidence provided by this modification of thromboelastography-guided protamine administration and was useful in identifying one of the many potential causes of postreperfusion bleeding in patients undergoing OLT.