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Objective: The purpose of this study was to determine if substitution of a heparin-coated oxygenator and salvaged autologous blood for cardiotomy suction would improve platelet function., Design: A prospective, randomized trial., Setting: A large academic medical center., Participants: Sixty adult patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass (CPB)., Interventions: Patients were randomized into 1 of 4 groups in a 2 x 2 factorial design by oxygenator (heparinized v nonheparinized) and blood salvage during CPB (cardiotomy suction v salvaged autologous blood)., Measurements and Main Results: Primary outcome measures were platelet function, glass-bead retention, platelet dense-body adenosine triphosphate secretion, platelet-rich plasma (PRP) aggregometry, Plateletworks platelet-function analyzer (Helena Laboratories Corp, Allen Park, MI), and platelet count. Secondary outcome measures were chest-tube drainage and allogeneic blood transfusion requirements. All platelet-function tests except thrombin-receptor activator peptide-induced PRP aggregometry showed a reduction in platelet function during and immediately after CPB (all p < 0.05). The only statistically significant difference in platelet-function tests between the groups was the glass-bead assay at 5 minutes before discontinuation of CPB (p < 0.05). This difference resolved 10 minutes after protamine administration. There were no differences between the groups in the amount of blood transfused, chest-tube drainage, and routine laboratory test results., Conclusions: The authors concluded that the effects of these changes to the CPB circuit were small and inconsequential in this cohort of patients.

Endogenously activated protein C is evaluated for antithrombotic activity in porcine carotid arteries subjected to mechanical trauma. Protein C is activated by intravenous administration of guanidinobenzoyl-thrombin, which binds to thrombomodulin and there deacylates to yield thrombin. The bound, transiently active thrombin yields a peak of anticoagulant activity between 5 and 10 min after infusion of the latent thrombin. Inhibition of thrombin binding in vivo by co-infusing and active-site-blocked thrombin preparation elicits acute and lethal systemic thrombosis. Nearly occlusive platelet thrombosis, which occurs within 30 min of crushing 1 cm segments of carotid arteries with a standard hemostat, is blocked by endogenous protein C activation initiated 2 min before the crush injury. It is concluded that activated protein C blocks thrombosis is deeply injured musculo-elastic arteries, and that activation of latent thrombin bound to thrombomodulin in vivo is a practical means for delivery of pharmacologically effective concentrations of activated protein C.

Factor Xa modified by reductive methylation (greater than 92%) loses the capacity to bind heparin as determined both by gel chromatography and by sedimentation equilibrium ultracentrifugation. The kinetic properties of methylated factor Xa differ, with respect to KM and Vmax for a synthetic tripeptide substrate and for antithrombin III inhibition rate constants, from those of the unmodified enzyme. The 10,000-fold rate enhancement elicited by the addition of heparin to the antithrombin III inhibition reaction, however, is the same. The observed second-order rate constants (k"obs) for antithrombin III inhibition of factor Xa and methylated factor Xa are 3000 and 340 M-1 s-1, respectively, whereas k"obs values for the inhibition of factor Xa or methylated factor Xa with antithrombin III-heparin are 4 X 10(7) and 3 X 10(6) M-1 s-1, respectively. These findings provide direct evidence that the interaction of factor Xa with heparin is not involved in the heparin-enhanced inhibition of this enzyme.

A heparin preparation obtained by gel chromatography is compared to unfractionated heparin with respect to the effects of heparin on the reaction between thrombin and heparin cofactor. Whereas both preparations enhance the rate of inhibition of thrombin by heparin cofactor, the extent of inhibition is decreased by the unfractionated, but not by the fractionated heparin. The decreased extent of inhibition is accounted for by residua of unreacted and undegraded heparin cofactor and thrombin, as demonstrated by gel electrophoresis in dodecyl sulfate. However both heparin preparations enhance the rate of degradation by thrombin of the thrombin-heparin cofactor complex.

The interaction of heparin with chemically modified thrombin and heparin cofactor is studied. Amidinated heparin cofactor does not bind to heparin-agarose and the reaction rate of the amidinated inhibitor with unmodified thrombin is not affected by heparin. Likewise, thrombin modified with 1,2--cyclohexanedione does not bind to heparin agarose and the reaction rate of the modified enzyme with unmodified inhibitor is not affected by heparin. In the absence of heparin, the modified and unmodified proteins react at the same rate in all possible combinations. Affinity chromatography of diisopropylphosphoryl thrombin on heparin cofactor coupled to Sephadex G--50 is used to study the binding of heparin cofactor and thrombin to heparin. The thrombin for all experiments is tritium-labeled and then inactivated with diispropylfluorophosphate. Thrombin is not bound to heparin cofactor-Sephadex columns. However, after treatment of the columns with a heparin solution, thrombin binds tightly, and is eluted at high ionic strength. Bound thrombin can also be eluted with either excess non-radioactive thrombin or excess free heparin. Heparin-dependent binding of thrombin does not occur if the heparin cofactor-Sephadex is heat-denatured. The ability of heparin to couple solution-phase thrombin to solid-phase heparin cofactor indicates that a ternary complex is formed. Analysis of the binding of the proteins to heparin by a dye displacement method suggests that at least one site on heparin binds to thrombin but not to heparin cofactor. Further support for a catalytic role for heparin derives from the ability of catalytic concentrations of heparin to enhance the rate of hydrolysis of prothrombin by thrombin, another protein pair which bind mutually to heparin.

A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.

This assay for heparin is based on the heparin-accelerated rate of alpha-thrombin III. The rate or product formation from the residual active thrombin is inversely proportional to plasma heparin content. The assay can be performed manually, but our results were obtained with a discrete analyzer, the ABA-100. The assay is insensitive to concentrations of antithrombin III in plasma. Precision studies gave CVs of less than 10%. This assay was compared to the Dade Protopath heparin assay and a correlation coefficient of 0.90 was obtained (n = 62). The correlation between activated partial thromboplastin times and heparin concentrations (r = 0.67) was calculated frm results on 78 plasma specimens from 10 patients.