Implementing a Statistical Model for Protamine Titration: Effects on Coagulation in Cardiac Surgical Patients.

OBJECTIVES: To implement a statistical model for protamine titration. DESIGN: Prospective randomized trial. SETTING: University hospital. PARTICIPANTS: Sixty (n = 30+30) patients scheduled for elective coronary artery bypass surgery were randomly assigned to 2 groups. INTERVENTIONS: Protamine dose calculated according to an algorithm established from a statistical model or to a fixed protamine-heparin dose ratio (1:1). MEASUREMENTS AND MAIN RESULTS: Both groups demonstrated comparable patient demographics and intraoperative data. Coagulation effects were evaluated using rotational thromboelastometry. Using the statistical model reduced (p<0.01) the protamine dose from 426±43 mg to 251±66 mg, followed by significantly (p<0.01) shorter intrinsic clotting time (208±29 seconds versus 244±52 seconds) and stronger clot firmness (p = 0.01), and effects on indices of extrinsic or fibrinogen coagulation pathways were insignificant. Test of residual heparin was negative in all patients after protamine administration, aligned with insignificant (p = 0.27) intergroup heparinase-verified clotting time differences. CONCLUSIONS: The statistical model for protamine titration is clinically feasible and protects the patient from exposure to excessive doses of protamine, with advantageous effects on coagulation as measured using rotational thromboelastometry. Significance regarding clinical outcome is yet to be defined.

Reversal of heparin after cardiac surgery: protamine titration using a statistical model.

OBJECTIVE: To establish a statistical model for determination of protamine dose in conjunction with cardiopulmonary bypass. DESIGN: Prospective. SETTING: University hospital. PARTICIPANTS: Ninety consecutive cardiac surgical patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A series of clinically oriented variables were introduced into a statistical model for projection of the protamine dose after cardiopulmonary bypass. The following significant predictors were identified using multivariable regression analysis: The patient's body surface area, the administered dose of heparin, heparin clearance, and the preoperative platelet count. The statistical model projected the protamine dose within 3±23 mg of the point-of-care test used as reference. CONCLUSION: Protamine dosing based on statistical modeling represents an alternative to point-of-care tests.

Heparin-protamine balance after neonatal cardiopulmonary bypass surgery.

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

Heparanase modulates heparinoids anticoagulant activities via non-enzymatic mechanisms.

A key element for the physiological restriction of blood coagulation at the endothelial cell surface is its non-thrombogenic property, mainly attributed to cell surface heparan sulfate proteoglycans. Heparanase is an endo-beta-D-glucuronidase with specific heparan sulfate degrading activity, which is produced and stored in platelets, and is released upon their activation. We examined the effects of heparanase pro-enzyme on coagulation functions, predominantly under physiological conditions. While heparanase pro-enzyme does not directly affect coagulation protein activities, it has profound effects on heparinoid-mediated regulation of coagulation responses, apparently via mechanisms that do not involve its enzymatic activity. Heparanase pro-enzyme reverses the anti-coagulant activity of unfractionated heparin on the coagulation pathway as well as on thrombin activity. In addition, heparanase pro-enzyme abrogated the factor X inhibitory activity of low-molecular-weight heparin (LMWH). The pro-coagulant effects of the non-active heparanase were also exerted by its major functional heparin-binding peptide. Finally, the effects of heparanase on the activity of factor VII activating protease that is auto-activated by heparinoids indicated a complete antagonistic action of heparanase in this system. Altogether, heparanase pro-coagulant activities that were also demonstrated in plasma samples from patients under LMWH treatment, point to a possible use of this molecule as antagonist for heparinoid treatment.

A simple and rapid label-free fluorimetric biosensor for protamine detection based on glutathione-capped CdTe quantum dots aggregation.

A novel fluorescent biosensor is developed, based on glutathione-capped CdTe quantum dots aggregation, for the determination of trace amount of an important drug, protamine. In this method with increasing the protamine concentration, the fluorescence of the quantum dots was quenched due to their aggregation. Different parameters affect the sensitivity, such as pH and the amount of the quantum dots, were optimized. Using the new optical biosensor, under the optimized conditions, protamine could be measured in the range of 2.0-200 ng mL(-1) with a detection limit of 1.0 ng mL(-)(1). The relative standard deviation for five replicates determination of 30.0 ng mL(-)(1) protamine was 1.26%. The influence of common interfering species on the protamine detection was studied. The results showed that the biosensor is highly selective and sensitive for the detection of protamine. The optical biosensor was successfully used for the determination of protamine in real samples.

Biochemical and cellular effects of heparin-protamine injection in rabbits are partially inhibited by a PAF-acether receptor antagonist.

The origin of the thrombocytopenia and leucopenia induced by protamine-heparin complexes is unknown. We studied the biochemical and cellular effects of protamine (6 mg x kg-1, i.v.) injected after heparin (5 mg x kg-1, i.v.) in New Zealand rabbits. After protamine injection (0.5 min) increases in blood platelet-activating factor (PAF-acether, PAF) (27.6 +/- 27.6 to 148.2 +/- 48.9 pg x ml-1, P < 0.05), thrombocytopenia (403 +/- 64 to 166 +/- 13 cells x 10(-3) x mm-3, P < 0.05) and leucopenia (7650 +/- 930 to 4300 +/- 668 cells x mm-3, P < 0.05) were noted. Plasma thromboxane B2 increased at 1 min (125.6 +/- 24.4 to 879.7 +/- 141.0 pg x ml-1, P < 0.01). Protamine alone induced no change. Indomethacin (3 mg x kg-1, i.v.) did not counteract the effects of heparin-protamine. Pretreatment with the PAF receptor antagonist BN 52021 [9H1, 7a-(epoxymethano)-1 H,6aH-cyclopenta[c]furo[2,3-b]furo-[3',2',3,4]cyclopenta[1,2-d]fur an-5,9, 12(4H)trione,3-tert-butylhexahydro-4,7b,11 hydroxy-8 methyl] alone (3 mg x kg-1, i.v.) delayed thrombocytopenia and reduced plasma thromboxane B2 concentration but did not modify leucopenia. Thus thrombocytopenia and thromboxane B2 release triggered by heparin-protamine may be potentiated by the release of PAF.

Dextran sulfate included in factor Xa assay reagent overestimates heparin activity in patients after heparin reversal by protamine.

A lack of correlation between activated partial thromboplastin time (aPTT), thrombin time (TT) and anti-factor Xa (AXa) activity was observed in patients after cardiac surgery with cardiopulmonary bypass (CBP). Indeed, AXa activity measured by the chromogenic assay, Coamatic Heparin, was higher than expected with regard to results obtained in coagulation assays. To account for this discrepancy, another AXa chromogenic assay was tested. First, AXa activity was measured with two chromogenic assays (Coamatic Heparin and Rotachrom Heparin) in plasma samples of 25 patients undergoing cardiac surgery at two time points after heparin reversal by protamine. AXa activity was significantly higher when measured with Coamatic Heparin than with Rotachrom Heparin in samples collected just after protamine infusion (p<0.01). Next, since Coamatic( Heparin contains dextran sulfate (DXS) to reduce the influence of heparin antagonists such as platelet factor 4 (PF4), whereas Rotachrom Heparin does not, we hypothesized that the dextran sulfate contained in the reagent might explain this discrepancy. We therefore performed in vitro studies consisting in neutralizing unfractionated heparin (UFH) with protamine and measuring AXa activity with the two chromogenic assays. An AXa activity was still measurable with Coamatic Heparin after neutralization, thus strongly suggesting that dextran sulfate dissociates protamine/heparin complexes. We conclude that Coamatic Heparin assays should be avoided when measuring AXa activity in plasma samples immediately after protamine infusion, as inaccurate results may lead to inadequate management of heparin reversal.

Correlation of ACT as measured with three commercially available devices with circulating heparin level during cardiac surgery.

Automated activated clotting time (ACT) is utilized as the primary means of assessing anticoagulation status for cardiopulmonary bypass (CPB) procedures. Influences on the clotting cascade during CPB such as hypothermia, hemodilution, and platelet dysfunction are known to affect ACT. The recently introduced Thrombolytic Assessment System (TAS) has been reported to be less sensitive to changes in hemodilution and hypothermia during CPB than more conventional ACT devices. This study evaluated the ability of TAS, and two other commercially available automated ACT systems, the HemoTec and Hemochron, to correlate with circulating heparin levels. Reference standards for circulating heparin were determined by inactivation of factor Xa assay. Nineteen patients undergoing moderate hypothermic CPB served as subjects for this investigation. Blood samples were obtained for study at four time periods: 1) baseline (control), 2) post heparin administration (300-400 U/kg) prior to CPB, 3) during CPB, and 4) post protamine. Study results demonstrated a high correlation between the HemoTec and Hemochron (r = 0.99), increased heparin dose response on CPB compared to pre-CPB activity (p < 0.05), and a significant (p < 0.05) negative correlation between devices and patient hematocrit during CPB. Additionally, device correlation with anti-Xa assay during collection periods 2 and 3 showed negative correlations in each of the three devices evaluated. We conclude that all automated devices tested demonstrated an inability to predict circulating heparin at levels necessary for CPB, and that these discrepancies become magnified during CPB procedures.

[Mechanism of heparin inactivation by thromboplastin (factor III)]. / K mekhanizmu inaktivatsii geparina tromboplastinom (faktorom III).

Thromboplastin (a commercial one and that obtained from different tissues) is shown to inactivate heparin in proportion to the quantity of thromboplastin or to the heparin:thromboplastin ratio. A degree of inactivation of heparin changes after the modification of protein component of thromboplastin by proteases, however there is no dependence between the protein amount in the preparation and its antiheparin activity. Inactivation of heparin by thromboplastin is stipulated by the formation of associations due to electrostatic interactions between the clusters of amino acid protein residues (which dissociate under physiological conditions as bases) and heparin sulphogroups. It is suggested that factor III circulating in blood flow participates in the creation of hemostatic potential not only as a result of its ability to catalyze thrombinogenesis, but also due to the decrease of the anticoagulant blood activity.

[Complex compounds of heparin with blood proteins and their natural inhibitors in splenectomy in animals]. / Kompleksnye soedineniia geparina s belkami krovi i ikh estestvennye ingibitory pri splenéktomii u zhivotnykh.

In depression of the function of the anticoagulating system after splenectomy in rats the blood serum heparin level diminishes, the fibrinogen concentration increases, the total and nonenzymatic fibrinolytic activity decreases, and the activity of some heparin complexes (adrenaline-heparin and serotonin-heparin) increases. A low-molecular protein inhibiting nonenzymatic fibrinolytic activity was isolated from blood plasma of splenectomized animals, some of its properties were studied. No such protein inhibitor was found in blood plasma of false-operated and intact rats.