Application of thrombin based fibrin glue and non-thrombin based batroxobin glue on intact human blood vessels: evidence for transmural thrombin activity.

An alternative method of uniting small diameter vessels to obtain tissue union while limiting the thrombogenic effect of suture placement at a vessel anastomosis involves the use of a thrombin based fibrin glue as a surgical sealant. This investigation addresses whether the in vitro application of a thrombin based glue (TG), or batroxobin glue (BG), a non-thrombin based glue made with the snake venom enzyme batroxobin, alters intravascular platelet deposition (PD) or cleaves blood fibrinogen, as measured by fibrinopeptide A (FPA) production, when the respective glue is applied to the external surface of an intact human placental artery or an artery with an anastomosis. When TG was applied to the adventitial surface of an intact vessel or an anastomosis (n = 7) of control and experimental vessels, there was a significant increase in intraluminal platelet deposition, an effect not realized with BG (n = 12, intact vessel TG p = 0.01, BG p = 0.66, anastomosis TG p <0.01, BG p <0.01). Both TG and BG significantly increased FPA levels when human whole blood was perfused through both intact vessels or vessels containing an anastomosis when compared to control vessels (intact vessel TG and BG p <0.01, anastomosis TG and BG p <0.01). Labelled thrombin studies document the rapid passage of thrombin through an intact vessel wall or vessels with an anastomosis when TG was applied to the adventitial surface of the vessel. The data suggest that TG and BG are drug delivery systems for their respective enzymes that either pass through or transfer a message across not only a surgically created anastomosis, but also an intact vessel wall.

The effect of airway maneuvers on the unstable C1-C2 segment. A cadaver study.

STUDY DESIGN: This is a cadaver study in which video fluoroscopy is used to measure motion of the unstable spine at C1-C2 during intubation maneuvers. OBJECTIVES: To quantify the amount of motion that occurs at an unstable C1-C2 spinal segment during the use of various intubation techniques using a cadaver model. SUMMARY OF BACKGROUND DATA: In previous work by the authors, a methodology and measurements for the unstable C5-C6 segment in a cadaver model were developed. These studies showed that the most motion was created by a chin lift and jaw thrust and that oral techniques created more motion than nasal intubation. The potential motion that occurs during intubation with instability at C1-C2 is yet unstudied. Therefore, a study to determine the effects of intubation on the spine with an unstable C1-C2 segment was designed. METHODS: Six human cadavers were used for the study. Measurements before and after transoral osteotomy of the odontoid were performed using video fluoroscopy. Pre-intubation maneuvers and oral and nasal intubation were studied. RESULTS: Oral intubation and nasal intubation caused similar diminution of space available for the cord. Chin lift and jaw thrust caused a larger diminution of space available for the cord than either nasal or oral intubation techniques. CONCLUSIONS: Although nasal intubation is the accepted procedure for intubation of the unstable spine, nasal and oral intubation seemed to have the same ability to narrow the space available for the cord in the model in this study. Great care should be taken while performing the chin lift/jaw thrust maneuvers in preparation for intubation, because these pre-intubation techniques caused the most motion and hence narrowed the space available for the cord in the unstable cervical spine.

Thrombin activity on dissected and anastomosed human arteries.

The mechanism of anastomotic thrombosis in microvascular surgery remains poorly understood. We hypothesized that thrombin activity at anastomoses plays a major role in this process. To study this, a surgically relevant human artery anastomosis model was used to (i) measure surface thrombin activity on anastomoses and on intact vessel, (ii) determine the inhibitability of surface thrombin by heparin and recombinant hirudin (r-hirudin), and (iii) determine the anastomotic and intact vessel binding capacity for additional thrombin. Human placental artery segments were placed in chambers in which 0.2 cm2 of luminal surface was exposed to citrated platelet-poor plasma for 10 min at 37 degrees C. The fibrinopeptide A (FPA) concentration (indicating the action of thrombin on fibrinogen) in the supernatant was then measured using an ELISA assay. Intact vessels and anastomoses expressed equivalent thrombin activity that could not be inhibited by heparin at a concentration (0.3 U/ml) that is sufficient to prolong the activated partial thromboplastin time two-fold. Conversely, the concentration of heparin routinely used in intraoperative vessel irrigation solutions (50 U/ml) was able to completely block thrombin activity at both sites. r-Hirudin (0.3 heparin equivalent anti-IIa U/ml) was able to inhibit nearly all of the thrombin activity on each site. Each site was able to bind and express the activity of additional thrombin, indicating the potential for increased vessel thrombogenicity after local clot has formed and has been removed. These data indicate the presence of thrombin on dissected human vessels and its presence in equal amounts on intact and anastomosed vessels when measurement is made before blood flow resumes. Furthermore, vessel-associated thrombin is resistant to a standard systemic concentration of heparin but is susceptible to the much higher heparin concentration that can be delivered locally by the surgeon during vessel irrigation.

Tissue factor and its inhibition at the human microvascular anastomosis.

The surgically created vascular anastomosis is a thrombogenic zone of uncertain etiology. This study was designed to investigate the importance of tissue factor as a cause of human microvascular thrombogenicity. The ability of tissue factor pathway inhibitor (TFPI) to block the effect of tissue factor was also tested in this whole-vessel model system. Tissue factor activity in the presence of absence of TFPI was assayed on the luminal surface of dissected human placental arteries, on the advential surface, and also at the site of a microvascular anastomosis. Vessel wall thrombin activity was measured in the presence and absence of TFPI. Platelet deposition onto a vessel surface using a perfusion system was measured with and without TFPI. Tissue factor activity was greater on the adventitia (4.6 +/- 2.8 x 10(-4) units factor Xa generated/min) than on the endothelium (1.8 +/- 1.6 x 10(-4), P < 0.03) or at a surgically created anastomosis (2.1 +/- 1.2 x 10(-4), P < 0.04). TFPI reduced Xa generation to undetectable levels in 21 of 23 endothelial, adventitial, and anastomotic segments (P < 0.002). TFPI significantly reduced vessel wall thrombin activity in comparison to control anastomoses (control, 3.2 +/- 1.7 ng fibrinopeptide A (FPA)/(ml x min); TFPI, 1.4 +/- 1.2 ng FPA/(ml x min); P < 0.0001). TFPI reduced the platelet deposition on vessel segments with intact endothelium (no TFPI, 0.88 +/- 0.69 x 10(6) platelets/cm2; TFPI, 0.49 +/- 0.29 x 10(6) platelets/cm2; P < 0.06) and on vessel segments with anastomoses (no TFPI, 1.3 +/- 0.70 x 10(6) platelets/cm2; TFPI, 0.76 +/- 0.35 x 10(6) platelets/cm2; P < 0.02). This study demonstrates the importance of tissue factor as a thrombogenic element in a human whole-vessel model system. TFPI is effective in reducing this thrombogenicity.

Insulin therapy increases low plasma growth hormone binding protein in children with new-onset type 1 diabetes.

This study was undertaken (1) to evaluate growth hormone binding protein (GHBP) levels in newly diagnosed patients with Type 1 diabetes before and after insulin therapy and (2) to determine the relationship of GHBP to glycaemic control, C-peptide level and blood pH. GHBP, expressed as a percentage of (125I)GH bound, was determined in 33 patients with Type 1 diabetes (M/F = 19/14, 12.3 +/- 0.4 years) before (day 0), after 5 days (day 5) and after 3 months (month 3) of insulin therapy. At day 0, GHBP was lower in Type 1 diabetes compared with 38 matched healthy control subjects (3.9 +/- 0.4 vs 8.2 +/- 0.4%, p < 0.001). There was no significant improvement in GHBP at day 5 (4.4 +/- 0.3%). At month 3, GHBP increased to (6.0 +/- 0.4%, p < 0.001 vs day 0), but was still lower than controls, p < 0.001. At day 0 GHBP correlated with BMI (r = 0.50, p = 0.001), blood glucose (r = -0.43 p = 0.006) and pH (r = 0.48, p = 0.004), but not HbA1. GHBP at month 3 correlated with day 0 C-peptide (r = 0.41, p = 0.02). Thus, (1) circulating GHBP is low in newly diagnosed patients with Type 1 diabetes, and increases after 3 months of insulin therapy but does not normalize and (2) the severity of biochemical derangement and residual beta-cell function at diagnosis may determine GHBP status and its recovery. We conclude that insulin is an important modulator of GH binding protein in newly diagnosed children with Type 1 diabetes.

Hepatic insulin action in adolescents with insulin-dependent diabetes mellitus: relationship with long-term glycemic control.

The relationship between hepatic insulin action and long-term glycemic control was assessed in 20 adolescents with insulin-dependent diabetes mellitus (IDDM) and five healthy matched controls using a two-step (0.8 and 1.6 mU/kg/min) hyperinsulinemic-euglycemic clamp and [6,6-2H2]glucose. The night before the study, diabetic patients received variable-rate intravenous insulin in an attempt to normalize fasting plasma glucose concentrations. In the postabsorptive state, hepatic glucose production (HGP) was similar in IDDM patients and controls (593 +/- 40 v 518 +/- 27 mumol/m2/min); however, plasma glucose and free insulin concentrations were higher in IDDM patients than in controls (6.5 +/- 0.4 v 5.4 +/- 0.1 mmol/L [P = .01], and 207 +/- 21 v 104 +/- 10 pmol/L [P < .001], respectively). There was a positive correlation (r = .62 P = .002) between basal HGP and glycohemoglobin level (HbA1). Separation of the patients at the median HbA1 (group no. 1 < and group no. 2. > 11.4% HbA1) revealed two distinct patient populations with regard to hepatic and peripheral insulin action and plasma free fatty acid (FFA) suppression. During hyperinsulinemia, the percent suppression of HGP was lower in group no. 2 compared with group no. 1 (65.7% +/- 9.8% v 94.4% +/- 3.8%; P = .018). Rates of glucose disposal were lower in group no. 2 compared with controls and with group no. 1. Postabsorptive FFA levels were similar between group no. 2 and group no. 1 (0.45 +/- 0.03 and 0.43 +/- 0.04 mmol/L) despite higher free-insulin concentrations in group no. 2 (260 +/- 30 v 171 +/- 25 pmol/L; P = .04).(ABSTRACT TRUNCATED AT 250 WORDS)

Synergy between tetrandrine and FK506 in prevention of diabetes in BB rats.

Delayed administration of tetrandrine, a novel broad-spectrum anti-inflammatory agent, to BB rats at a dosage schedule of 20 mg kg-1 day-1 from 79 days of age reduced the cumulative incidence of diabetes from 73.1 to 41.7% (p < 0.01). Brief treatment with the potent immunosuppressive agent FK506 at a dosage schedule of 0.5 mg kg-1 day-1 from 79 days of age for 5 days had no significant effect on the cumulative incidence of diabetes (66.7%, p > 0.1). However, the combination of tetrandrine and FK506 in the afore-mentioned dosage schedules reduced the incidence of diabetes to only 3.6% (p < 0.001). These results suggest that the strong synergy between tetrandrine and FK506 may offer a safe and effective therapeutic strategy for the treatment of patients with recent onset or imminent IDDM.

Thrombin activity resides on LVAD Dacron inflow and outflow grafts.

Patients on left ventricular assist devices (LVADs) are at increased risk for thromboembolism, and they experience elevations in platelet release and thrombin activity indices during device implantation. The Dacron grafts that lead from the ventricle to the LVAD and back to the aorta may harbor thrombin activity and protect this thrombin from anticoagulant action. To investigate this possibility, specimens isolated from LVAD grafts at the time of device explantation or implantation were incubated with citrated platelet poor plasma (cPPP), cPPP and 2 U/ml of heparin, cPPP and 2 U/ml of recombinant hirudin (r-hirudin), or cPPP and 50 U/ml of heparin. Thrombin activity was measured by the increase in incubated cPPP fibrinopeptide A (FPA) concentrations over cPPP FPA levels, without material contact. A chromogenic substrate for thrombin verified the effects seen. Thrombin activity was found at comparable levels on both the inflow and outflow LVAD grafts at explantation. This activity was not inhibited by low concentrations of heparin, but 50 U/ml of heparin and 2 U/ml of r-hirudin reduced the activity. At graft implantation (after preclotting), thrombin activity was higher than at explantation, but susceptibility to inhibition by heparin was also significantly greater. These results confirm that the LVAD Dacron grafts harbor surface thrombin activity resistant to anticoagulation that may be a primary source for LVAD thrombogenicity.

Sexual dimorphism in insulin sensitivity in adolescents with insulin-dependent diabetes mellitus.

Sex-related differences in insulin sensitivity were evaluated in male and female adolescents with insulin-dependent diabetes mellitus (IDDM). They were matched for age, pubertal staging, body mass index, and glycohemoglobin levels. During a 1.7 mU/kg.min hyperinsulinemic-euglycemic clamp, the insulin-mediated glucose disposal rate was lower (26.9 +/- 2.1 vs. 47.1 +/- 3.7 mumol/kg.min; P less than 0.001), and GH levels were higher (6.5 +/- 1.2 vs. 2.9 +/- 0.8 micrograms/L; P = 0.03) in females than in males. Plasma glucagon, cortisol, epinephrine, and norepinephrine levels during the clamp were similar in the two sexes, except for pancreatic polypeptide, which showed a tendency to be higher in females (19 +/- 4 vs. 11 +/- 1 pmol/L; P = 0.07). During insulin-induced hypoglycemia, the rate of drop in plasma glucose was faster (0.16 +/- 0.01 vs. 0.11 +/- 0.01 mmol/L.min; P = 0.001), and the rate of glucose recovery was slower in males than in females (0.04 +/- 0.01 vs. 0.06 +/- 0.01 mmol/L.min; P = 0.05). Plasma glucose concentrations were lower in males than in females (glucose nadir, 2.3 +/- 0.2 vs. 3.3 +/- 0.2 mmol/L; P = 0.002; glucose peak, 3.7 +/- 0.2 vs. 5.3 +/- 0.4 mmol/L; P = 0.002), with similar plasma free insulin concentrations. Despite a greater degree of hypoglycemia, GH responses were lower in males than in females. The remaining counterregulatory hormone responses were similar in both sexes. We conclude that there is a distinct sexual dimorphism in insulin sensitivity in adolescents with IDDM. This is likely to be due to sex-related differences in GH levels. Furthermore, male patients with IDDM are apt to develop greater degrees of hypoglycemia accompanied by lower GH responses.