The pharmacological effects of amphetamine, procaine, procainamide, DOPA, isoproterenol, and atenolol upon activated partial thromboplastin time in the absence and presence of acetaldehyde have been investigated. In the absence of acetaldehyde, amphetamine and isoproterenol exhibit a procoagulant effect upon activated partial thromboplastin time, whereas atenolol and procaine display anticoagulant effects upon activated partial thromboplastin time. DOPA and procainamide do not alter activated partial thromboplastin time. Premixtures of procaine with acetaldehyde produce an additive anticoagulant effect on activated partial thromboplastin time, suggesting independent action of these compounds upon clotting factors. Premixtures of amphetamine with acetaldehyde, as well as atenolol with acetaldehyde, generate a detoxication of the anticoagulant effect of acetaldehyde upon activated partial thromboplastin time. A similar statistically significant decrease in activated partial thromboplastin time is seen when procainamide is premixed with acetaldehyde for 20 minutes at room temperature. Premixtures of DOPA and isoproterenol with acetaldehyde do not affect an alteration in activated partial thromboplastin time relative to acetaldehyde alone. Hence, a selective interaction of atenolol, procaine, and amphetamine with acetaldehyde to produce detoxication of the acetaldehyde is suggested, undoubtedly due to the presence of amino, hydroxyl, or amide groups in these drugs.

Polyamines such as protamine sulfate have been widely used clinically to neutralize the anticoagulant effect of excessive heparin. Protamine itself exhibits concentration-dependent anticoagulant and procoagulant effects. This laboratory has earlier reported that acetaldehyde exerts synergistic prolongation of the anticoagulant effect of heparin upon prothrombin time (PT). In the current investigation, it is seen that acetaldehyde, the primary metabolite of ethanol metabolism, reacts synergistically with protamine to effect a prolongation of PT beyond the individual effects of acetaldehyde and protamine on the PT. In an analogous study, the effect of polylysine (1-4K) and acetaldehyde upon activated partial thromboplastin time (APTT) was studied. It was observed that the polylysine (PL) prolonged APTT. When PL was preincubated with plasma at RT for 15 minutes, followed by a further 15 minutes with acetaldehyde, an additional prolongation time was observed. When acetaldehyde was preincubated with plasma prior to the addition of PL, a synergistic APTT was noted. When a PL-acetaldehyde mixture was preincubated prior to the addition to plasma, a drastic reduction in the prolongation of APTT was seen, suggesting that PL and acetaldehyde may detoxify one another by a Schiff base reaction under highly specific conditions.

It has been known that factor XIa (XIa) can react covalently with antithrombin III (ATIII) to form two complexes with ratios of 2ATIII:1XIa and 1ATIII:1XIa. In the hands of these investigators, the M.W.s, as measured by SDS-PAGE, were 265 kDa and 225 kDa, respectively. In this investigation it has been observed that the addition of 1 μg or 5 μg heparin (H) to 3.1 × 10-5 (mol ATIII for 5 minutes prior to the addition of 1.125 μ 10-5 μmol XIa for a 30-minute incubation led to an increase in the 265 kDa band of 113% and 223%, respectively. These results were statistically significant (p < .01). However, when H was premixed with XIa first, before the addition of AT III, statistical increases in the 265 kDa band were also seen (267% and 183%, respectively; p < .0005, n = 6). Protamine sulfate (PS) statistically significantly (p < .05) inhibited the formation of the 265 and 225 kDa XIa-ATIII complexes at the 5 μg PS level when premixed with ATIII or XIa, respectively, the decreases in the 265 kDa band being 39.1 and 34.4%, respectively for [(ATIII/PS) + XIA] and [(XIa/PS) + ATIII] mixtures, and 23.1 and 23.8% for the 225 kDa band with [(ATIII/PS) + XIa] and [(XIa/PS) + ATIII] mixtures. These results with PS indicate that PS inhibits complex formation between XIa and ATIII at the 2ATIII:1 XIa and 1ATIII:1 XIa levels, in contrast with its reported stimulation of complex formation between thrombin and ATIII.

This laboratory has studied the effect of 1 and 5 μg protamine sulfate (PS) (0.2 and 1.0 nmol) upon the formation of covalent complexes between (excess) factor IXa (5 μg) and ATIII (1.92 μg) under three sets of conditions: preincubation of IXa with PS for 15 minutes prior to the addition of ATIII; preincubation of ATIII with PS for 15 minutes prior to the addition of IXa; and mixing each of the reagents within 1-minute intervals. Complex formation was detected by SDS-PAGE and quantitated. A major band for the enzyme was observed at about 45 kDa, and a minor degradation product band of the enzyme was seen at about 31 kDa. A single ATIII band of about 55 kDa was also noted. Upon mixing enzyme with inhibitor, two bands of complexes were observed, at approximately 115 kDa and 100 kDa. It was suggested that the 100 kDa band was a degradation product of the 115 kDa band. Upon preincubation of IXa, ATIII, or both with PS prior to addition of ATIII, Ixa, or buffer to the mixtures, it was noted in all three cases, and in the presence of both levels of PS, that there was a decrease in the amounts of 115 kDa and 100 kDa complexes observable. The decrease was greater in the presence of 5 μg, suggesting inhibition of complex formation. Concomitantly, with the decrease in complex formation, an increase in unreacted ATIII was noted. At the same time a decrease in free IXa was observed commensurate with an increase in the IXa fragment. The latter corresponds most likely to a promotion of autolysis of factor IXa by the presence of PS, in agreement with other proteolytic stimulations reported from this laboratory upon addition of PS to other coagulation factors.

Restricted accessAbstractFirst published March 2006Factor Ixa-Atiii Complex Formation is Inhibited by Protamine SulfateA.S. Brecher and A.R. MoonView all authors and affiliationsVolume 54, Issue 2_supplhttps://doi.org/10.1177/108155890605402s13

This investigation explored the effect of glycosaminoglycans upon the initial enzyme in the intrinsic blood coagulation pathway, namely, Factor β--XIIa, and its interaction with antithrombin III (ATIII). Primary and secondary complexes of β--XIIa with ATIII were observed by SDS-PAGE and Western blot analysis utilizing a polyclonal human ATIII antibody to identify the complexes which were subsequently quantitated. With 3.15 μg β-XIIa: 3.15 μg ATIII pre-incubations for 15 min at RT, it was noted that 7.4 and 1.7% of the total protein in the SDS-PAGE lane was present as primary and secondary complexes, respectively, where the 2° complex is believed to be a proteolytic degradation product of the 1° complex. In the presence of a prior ATIII/12.6 μg heparin pre-incubation, the 1° complex increased to 34.1% of the protein in the lane, while the 2° complex increased to 12.1%. Upon replacing heparin with dermatan sulfate (chondroitin sulfate B), an increase of the 1° complex to 19.2% was noted whereas the 2° complex increased to 4.8%. Chondroitin sulfates A and C, by and large, exhibited no major changes in the 1° and 2° complex formation. These data indicate that both heparin and dermatan sulfate exhibit anticoagulant effects in the intrinsic blood clotting system and suggest that disorders in glycosaminoglycan production and degradation may be factors to consider in coagulopathies.

The comparative effects of glycosaminoglycans and acetaldehyde (AcH)--glycosaminoglycan (GAG) mixtures upon Factor Xa- (FXa) and Factor X-deficient plasma (FXDP) have been studied by activated partial thromboplastin time (APTT) studies. Heparin at 0.025, 0.030, 0.04, and 0.05 U statistically prolonged the APTT when pre-incubated with FXa at 37 degrees C for 3 min prior to addition to FXDP and subsequent addition of Ca2+. Upon addition of 0.25, 0.375, and 0.5 microg heparin-6000 (H6k) to FXa, significant increases in APTT were observed. Similarly, profound increases in APTT were observed when 0.5, 0.75, and 1.0 microg heparin-3000 (H3k) was added to FXa. The chondroitin sulfates (CSA, CSB, CSC) had far less impact upon APTT with the FXa-FXDP system. In examining the effects of AcH-GAG mixtures upon the clotting factor, it was observed that 44.3 and 443 mM AcH synergistically prolonged the APTT in a statistically significant manner regardless of the order of premixing the three components. Hence, AcH may play a role in prolonging APTT in alcoholics. It synergistically prolonged APTT in concert with GAGs and FXa at the AcH levels used in this study. The effect of the GAGs upon FXDP is far less than its effect upon FXa.

BACKGROUND:The affect of acetaldehyde-treated heparin on thrombin activity has been investigated using factor II-deficient human plasma. METHODS:It was observed that 0.021 units of heparin exerts a marked inhibition of thrombin activity (1.03 units) as measured by clotting times, prolonging the clotting times from 9.6 +/- 0.1 seconds to 24.8 +/- 0.1 seconds. However, when the heparin is preincubated with 447 mmol/L acetaldehyde at RT for 30 minutes prior to mixing with thrombin, a clotting time in excess of 200 seconds is observed. Clotting times remain elevated with heparin-acetaldehyde mixtures of 89.4, 17.9, 3.6, and 0.72 mmol/L acetaldehyde, with corresponding clotting times of > 200, 156.0 +/- 2.1, 81.6 +/- 1.0, 38.8 +/- 0.6 seconds, respectively. At 140 mumol/L acetaldehyde-heparin mixtures, the clotting time was 17.0 +/- 2.0 seconds. RESULTS:These data support the hypothesis from this laboratory that acetaldehyde-modified heparin enhances coagulation time. They further indicate that thrombin is targeted by the acetaldehyde-treated heparin. Heparin-acetaldehyde mixtures also reacted with plasma prior to the addition of thrombin to modestly prolong coagulation time. Similarly, but more effectively, thrombin/heparin mixtures increased the clotting time of acetaldehyde-exposed plasma. These data further suggest the possibility that reactions of acetaldehyde and heparin are not restricted to those with thrombin, and that they may extend to other blood factors/proteins. CONCLUSIONS:The amount of heparin (0.021 units) required to substantially affect clotting time of thrombin (1.03 units) is substantially lower than that required to prolong clotting of 0.1 mL of whole plasma (0.36 units), by an order of magnitude. It is inferred that heparin may interact with numerous cationic proteins or proteins with cationic domains in blood plasma, among them being the clotting factors.

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.