Inhibition of thrombin by iopromide in vitro.

Iopromide is a nonionic, iodinated, monomeric, radiographic contrast agent used in various indications, including coronary angiography and visceral and peripheral arteriography. Nonionic contrast media have been postulated to increase thrombogenicity when compared with ionic contrast media. The goal of this study was to characterize the interaction of iopromide with thrombin, specifically to determine the rate, extent, specificity, and reversibility of the thrombin inhibition by iopromide, the integrity of the thrombin-iopromide complex, and the inhibitory potency of iopromide using a validated assay methodology. Iopromide was mixed with purified thrombin or pooled serum from healthy male and female donors. The final concentrations of iopromide in the presence of estimated physiologic concentrations of thrombin (1 nmol/L) were 0-184 mmol/L. After incubation for defined time intervals, the activity of thrombin was determined by adding substrate and measuring the absorbance of the generated chromophores at 405 nm. The possible inhibition of the protease trypsin by iopromide was investigated to evaluate the specificity of thrombin inhibition by iopromide. Iopromide was compared with Thromstop, a known thrombin inhibitor, to assess the relative potency of iopromide. The inhibition of thrombin by iopromide was immediate, rapidly reversible, and proportionate to the iopromide concentrations. The minimum inhibitory concentration of iopromide was 50 mmol/L. At the highest iopromide concentration tested, 184 mmol/L, the mean inhibition of thrombin activity was 44.5%. The mean concentration of iopromide associated with a 50% inhibition was 206 mmol/L. The inhibitory potency of iopromide was 4 x 10(6) times smaller than that of Thromstop. The inhibition of thrombin by iopromide is specific, because trypsin was not inhibited by iopromide. The results indicate that in vitro iopromide at clinically relevant concentrations partially inhibits thrombin activity. However, the in vitro model used does not consider other factors that may be relevant for the overall coagulation response in vivo.

Minimum specimen volume requirements for routine coagulation testing: dependence on citrate concentration.

We evaluated the effect of sample volume and citrate concentration on results of routine coagulation assays (prothrombin time [PT] and activated partial thromboplastin time [APTT]). The study was performed on samples obtained from healthy persons and patients receiving oral anticoagulant therapy. Standard evacuated tubes (3.2% and 3.8% sodium citrate) were filled to varying total sample volumes ranging from 3.0 to 5.0 mL, and results of routine coagulation tests were compared. Underfilling may significantly affect the APTT and PT, resulting in artifactual prolongation of results. This effect is most pronounced in samples drawn into 3.8% citrate. By using 3.8% citrate, there is a statistically significant difference in the results of PT assays in the samples less than 80% filled compared with those that are 100% filled. For APTT assays performed on samples drawn into 3.8% citrate, a statistical difference occurred at less than 90% filled. This effect was less pronounced when samples were drawn into 3.2% sodium citrate. We found no statistically significant difference in PT results from a 3.2% citrate tube between fill volumes of 60% and 100% and none for APTT results between fill volumes of 70% and 100%. This study further supports the recommendation to use 3.2% sodium citrate concentration, because 60% of the optimum filled volume for PT and 70% of the optimum filled volume for APTT are acceptable.

Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing.

The effects of 3.2% and 3.8% sodium citrate concentration on the results of routine coagulation assays (prothrombin time [PT] and activated partial thromboplastin time [aPTT]) were evaluated by means of two sets of reagents, one responsive and the other nonresponsive. Five groups were entered in the study: healthy volunteers; outpatients receiving stable oral anticoagulant therapy; and hospitalized patients receiving intravenous (i.v.) heparin therapy, both i.v. heparin and oral anticoagulant therapy, or no anticoagulant therapy. With use of nonresponsive PT and aPTT reagents, varying the citrate concentration has little clinical significance except in patients receiving i.v. heparin therapy. In contrast, when responsive PT and aPTT reagents are used, the concentration of sodium citrate anticoagulant has a significant effect on assay results. Eighteen percent of samples from patients receiving stable oral anticoagulant therapy demonstrated a change of less than 0.7 INR (International Normalized Ratio) units between citrate concentrations. Nineteen percent of patients receiving i.v. heparin therapy had a greater than 7-second difference when aPTT results were compared. These data demonstrate that citrate concentration affects the results of coagulation tests. On the basis of these data, it is recommended that 3.2% citrate be used for all coagulation tests.

Coagulation and fibrinolytic profiles in patients with severe pulmonary hypertension.

STUDY OBJECTIVES: Although in situ thrombosis is a prominent finding in lung vessels from patients with primary and secondary pulmonary hypertension, to our knowledge, plasma coagulation factors that might contribute to a hypercoagulable state have not been fully investigated. We hypothesized that the local coagulation environment in the lung vasculature is important to progression if not initiation of pulmonary hypertension. DESIGN: Quasi-experimental cross-sectional design with concurrent controls. SETTING: Referral clinics and inpatient services of a University Hospital and a Veterans Administration Medical Center. PARTICIPANTS: To investigate the role of plasma coagulation factors in severe pulmonary hypertension, we sampled plasma from patients with primary pulmonary hypertension, patients with pulmonary hypertension secondary to a discernible etiology, and normal adult control subjects. RESULTS: We detected abnormalities of the thrombomodulin/protein C anticoagulant system, evidenced by a decrease in soluble thrombomodulin, in patients with primary pulmonary hypertension. In the patients with primary pulmonary hypertension, we found impaired fibrinolytic activity, with a rise in the fibrinolytic inhibitor plasminogen activator 1 and elevated euglobulin lysis time. Lower fibrinolytic activity correlated with high mean pulmonary artery pressure. In contrast, in patients with secondary pulmonary hypertension, von Willebrand factor antigen and fibrinogen levels were increased, and fibrinolytic activity decreased. CONCLUSIONS: Different patterns of coagulation and fibrinolytic abnormalities are apparent in plasma from patients with primary and secondary pulmonary hypertension. Although we are unable to address causality with this study, we speculate that abnormalities of these coagulation mechanisms may initiate or play a role in perpetuation of pulmonary hypertension.

Correlation of antiphospholipid antibodies and protein S deficiency with thrombosis in HIV-infected men.

Antiphospholipid antibodies (aPL) and free protein S (PSF) deficiency have been associated with clinical thrombosis. Previous reports described a high prevalence of these abnormalities in HIV-infected individuals, but suggested there was little associated clinical thrombosis. A cohort of 74 HIV-infected men were studied for aPL, PSF deficiency and the development of thrombosis. aPL, predominantly anticardiolipin antibodies (aCL), were detected in 86% and PSF deficiency in 33%. While 42% of men with aPL also had low PSF levels, there was no correlation between aCL titres or most measures of aPL and PSF levels. However, a strong correlation was noted between a subset of aPL that reacted to phosphatidylethanolamine by hexagonal array assay and low PSF levels. There was no significant correlation between aPL, PSF deficiency and clinical features (medication use, opportunistic infection, CD4 cell count) of HIV in 60 patients for whom clinical information was available. The overall incidence of thrombosis in this group was 18%, and thrombosis developed in 6.6% of those followed prospectively over a median follow-up of 12 months. Development of thrombosis was not significantly correlated with aPL or PSF deficiency, but the high prevalence of these abnormalities may necessitate larger study groups to determine the risk associated with these coagulation changes. Study of a larger group with careful analysis of subsets of aPL, especially those associated with low PSF levels, and longer clinical follow-up could identify the HIV-infected individuals at risk for thrombosis.

Contribution of plasma proteinase inhibitors to the regulation of activated protein C in plasma.

Activated protein C (APC), a serine protease, is regulated in plasma by protease inhibitors. This study was undertaken to determine the role of the major plasma inhibitors in regulating APC in plasma. Kinetic analysis and specific immunoassays for APC-inhibitor complexes were used to determine the inhibitors that form complexes with APC. Of the eight plasma inhibitors investigated, four interact with APC: protein C inhibitor (PCI), alpha 1-proteinase inhibitor (PI), alpha 2-antiplasmin (AP) and C1 esterase inhibitor (C1 Inh). The second order rate constants are: 1.3 x 10(4) M-1 s-1 (PCI); 15 M-1 s-1 (PI); 410 M-1 s-1 (AP); and < 6 M-1 s-1 (C1 Inh), with a relative effectiveness of each inhibitor to inactivate APC in plasma: 49:36:15: < 1, respectively. PCI, PI and AP are the major inhibitors of APC in plasma. Low concentrations of APC will be inhibited by PCI with PI and AP playing a secondary role. However, as increasing APC is generated, PI and AP begin to play more important roles as the PCI is consumed.

Activated protein C is not regulated by alpha-2-macroglobulin in plasma.

Alpha-2-macroglobulin (a2M) is a wide spectrum plasma inhibitor which functions by a unique mechanism and is a secondary inhibitor of coagulation and fibrinolytic enzymes. Human activated protein C (APC) is the central enzyme of a major regulatory system of coagulation and fibrinolysis. APC is primarily regulated (inhibited) by a specific plasma inhibitor. We undertook this study to investigate the role of a2M as a secondary inhibitor of APC. APC did not interact with a2M by any of the known mechanisms of interaction. APC failed to bind and form the classic proteinase-a2M complex as seen with similar serine proteases, thrombin and trypsin. APC also failed to cleave the a2M molecule. Experiments, using purified APC and either purified a2M or plasma, failed to demonstrate APC binding to a2M in gel filtration chromatography. No enzymatic activity of APC or radiolabeled APC was demonstrated in the a2M peak. Using an immuno-enzymatic assay (Harpel, J Biol Chem 260:4257, 1985) for an a2M and enzyme complex, the amount of APC bound to a2M was less than 3% of the added APC (non-specific binding only); whereas in similar experiments with thrombin, 75-86% of the added trypsin or thrombin bound. These data demonstrate that APC is one of a small number of unique serine proteases that do not interact with a2M. The absence of sequence homology between the a2M 'bait region' and the APC substrate cleavage sequences appear to be the reason APC is not inhibited by a2M.