Derrick L. Sauls

Elevated plasma homocysteine leads to alterations in fibrin clot structure and stability: Implications for the mechanism of thrombosis in hyperhomocysteinemia

Summary.  Elevated plasma homocysteine is associated with an increased risk of atherosclerosis and thrombosis. However, the mechanisms by which homocysteine might cause these events are not understood. We hypothesized that hyperhomocysteinemia might lead to modification of fibrinogen in vivo, thereby causing altered fibrin clot structure. New Zealand White rabbits were injected intraperitoneally (i.p.) every 12 h through an indwelling catheter with homocysteine or buffer for 8 weeks. This treatment raised the plasma homocysteine levels to about 30 µmol L−1 compared with 13.5 µmol L−1 in control rabbits by the end of the treatment period. The fibrinogen levels were 3.2 ± 0.6 in homocysteine‐treated and 2.5 ± 1.1 mg mL−1 in control rabbits. The reptilase time was prolonged to 363 ± 88 for plasma from homocysteine‐treated rabbits compared with 194 ± 48 s for controls (P < 0.01). The thrombin clotting time (TCT) for the homocysteine‐treated rabbits was significantly shorter, 7.5 ± 1.7 compared with 28.6 ± 18 s for the controls (P < 0.05). The calcium dependence of the thrombin clotting time was also different in homocysteinemic and control plasmas. Clots from plasma or fibrinogen of homocysteinemic rabbits were composed of thinner fibers than control clots. The clots formed from purified fibrinogen from homocysteine‐treated rabbits were lyzed more slowly by plasmin than comparable clots from control fibrinogen. Congenital dysfibrinogenemias have been described that are associated with fibrin clots composed of thin, tightly packed fibers that are abnormally resistant to fibrinolysis, and recurrent thrombosis. Our results suggest that elevated plasma homocysteine leads to a similar acquired dysfibrinogenemia. The formation of clots that are abnormally resistant to fibrinolysis could directly contribute to the increased risk of thrombosis in hyperhomocysteinemia.

Schedule and concentration-dependent induction of apoptosis in leukemia cells by nitric oxide

Nitric oxide (NO) has potent antiproliferative properties. In previous work we have shown that NO inhibits growth, induces differentiation and modulates gene expression in acute nonlymphocytic leukemia (ANLL) cells. The goal of this work was to determine whether the rate of NO delivery affected its growth inhibition of ANLL cells. We also wanted to determine whether the NO inhibition of ANLL cell growth is associated with the induction of apoptosis. We treated HL-60 and U937 cells with three compounds that generate the same amount of NO but at different rates. MAMA-NO, PAPA-NO and DETA-NO have half-lives of NO delivery of 2 and 30 min, and 20 h, respectively. The compound with the longest t½ of NO delivery (DETA-NO) was the most potent inhibitor of leukemia cell and colony growth. Furthermore, the NO-induced growth inhibition was associated with apoptosis in a rate and concentration-dependent fashion.

Elevated prothrombin level and shortened clotting times in subjects with type 2 diabetes

The partial thromboplastin time [1] and its successor, the activated partial thromboplastin time (aPTT) [2], have long been used to detect coagulation factor deficiencies and monitor replacement therapy in patients at risk of bleeding. More recently, a correlation has been reported between short aPTT values and the risk of thrombosis [3,4], as well as the risk of recurrence in patients who have already suffered a thromboembolic event [5]. High factor (F)VIII or IX levels can contribute, but are not the only determinants of a short aPTT [6]. An elevated level of prothrombin has also been associated with thrombosis [7], and elevated levels of prothrombin lead to increased thrombin generation in an in vitro model of hemostasis [8]. Thus, it seems likely that elevated prothrombin levels could contribute both to thrombotic risk and to a shortening of the aPTT. Insulin resistance and type 2 diabetes mellitus (DM) are associated with an increased risk of atherothrombotic events [9], as well. In order to determine whether the levels of prothrombin or other plasma clotting factors might play a role in the thrombotic tendency in diabetes, we assayed clotting times, and prothrombin, antithrombin, fibrinogen and plasminogen activator inhibitor-1 (PAI-1) levels in subjects with or without type 2 DM. The prothrombin time (PT) and aPTT assays were performed in the Durham Veterans Affairs Medical Center Clinical Hematology Laboratory on an STA analyzer (Diagnostica Stago, Asnières, France) using Neoplastin (ISI 1.3) and PTT Automate reagents, respectively. The reference range for the PT International Normalized Ratio (INR) is 0.89–1.20 and for the aPTT it is 23.5–35.1 s. Levels of prothrombin, fibrinogen and PAI-1 were determined by ELISA. The subjects were the first 40 of 81 subjects recruited into a trial on the effects of muscadine grape juice on parameters related to glycemic control and cardiovascular risk [10]. This study was conducted under a protocol approved by the Institutional Review Board of North Carolina State University. The details of this study, including subject characterization have been published previously [10]. Blood samples were collected before and after a period during which some of the subjects consumed wine or grape juice. Blood samples collected before the dietary intervention were used to assess coagulation parameters. Subjects were provisionally classified as DM based on their report of having been so diagnosed by a physician. They were subsequently reclassified based on fasting glucose, insulin and glycated hemoglobin (HbA1C) levels. Selfclassification into DM or control groups was verified for all except three subjects, who were excluded from the analysis because the fasting glucose, insulin and HbA1C levels did not allow them to be unambiguously classified. Data from a total of 10 male and 9 female controls, and 8 male and 10 female diabetic subjects were available for analysis. Informed consent was obtained from all participants. We found that DM subjects had shorter values than controls for the aPTT (25.6 ± 3.7 vs. 29.3 ± 3.4 s; P 1⁄4 0.006) and PT assays (11.3 ± 0.5 vs. 11.9 ± 0.6 s; P 1⁄4 0.005). While the differences were small in absolute terms, they were highly statistically significant. In addition, the distribution of the values was different in controls and diabetics. Only the distributions of aPTT values are shown in the top panel of Fig. 1, but the distribution of PT values looked very similar. In addition, male controls tended to have a shorter mean aPTT than females, but the difference did not reach statistical significance. PT values were not different for male and female control subjects. DM subjects also had significantly higher prothrombin levels than controls (1.8 ± 1.2 vs. 1.0 ± 0.3 units mL; P 1⁄4 0.04), and male controls had higher prothrombin levels than female controls (1.22 ± 0.48 vs. 0.83 ± 0.29 units mL; P 1⁄4 0.03). There was also a striking tailing of prothrombin values to the high side of the distribution curve in the diabetic subjects, as shown in the lower panel of Fig. 1. In the diabetic subjects there was a significant inverse correlation between the prothrombin level and the PT and aPTT (r 1⁄4 )0.4 for females and )0.55 for males), which was not found in the control groups. This suggests that the elevated prothrombin level in diabetics is an important contributor to the shortened clotting times. There was no significant difference in fibrinogen, antithrombin or PAI-1 levels between the groups. We believe that the difference in prothrombin level could be responsible for shortening of both the PT and the aPTT. However, because the PT clots so much more rapidly than the Correspondence: Maureane Hoffman, Laboratory Service (113), Durham Veterans Affairs Medical Center, 508 Fulton St, Durham, NC 27705, USA. Tel.: +1 919 286 6925; fax: +1 919 286 6828; e-mail: maureane@ med.unc.edu

Homocysteinylated fibrinogen forms disulfide-linked complexes with albumin☆

We have shown that homocysteinemic rabbits have altered fibrinogen that forms fibrin clots with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with amines and introduce a new sulfhydryl group into proteins. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We have shown that in vitro reaction of Hcys thiolactone with human fibrinogen (Hcys-fibrinogen) alters fibrinogen function in a manner similar to that in homocysteinemic rabbits. Several naturally-occurring mutations that introduce a new cysteine into fibrinogen are associated with clinical thrombosis due to increased resistance of clots to fibrinolysis. In those cases the new cysteine mediates disulfide formation between the mutant fibrinogen and albumin. We now report that Hcys-fibrinogen similarly forms disulfides with albumin in vitro, specifically through sites in its D-domain. However, fibrin clots formed from Hcys-fibrinogen-albumin show a similarly reduced ability to support plasminogen activation and a similar resistance to fibrinolysis as clots formed from Hcys-fibrinogen. Thus, fibrinogen-albumin conjugates may result from N-homocysteinylation of fibrinogen in vivo. However, there is no evidence that conjugation to albumin further impairs fibrinogen function above the defect induced by homocysteinylation of critical lysines. Similar to the utility of glycated hemoglobin as a marker for the deleterious effects of hyperglycemia, the level of fibrinogen-albumin complexes might possibly be a clinically useful marker for the level of homocysteine-associated damage in vivo.