Iron enhances generation of fibrin fibers in human blood: Implications for pathogenesis of stroke

Abstract

Stroke is associated with the intracerebral formation of fibrin clots which may lead to irreversible brain damage. Thrombolytic therapies employ a variety of natural and/or recombinant plasminogen activators to initiate fibrinolytic degradation of cerebral thrombi. However, such therapies when installed beyond 4‐ to 6‐h window, may fail to achieve the expected outcome. This is due to the hydroxyl radical‐induced modification of fibrin(ogen) molecules rendering them refractory to fibrinolytic degradation, but no cause of the increased free radical generation in stroke was offered. Here, we show by means of electron microscopy that iron ions added to human blood dramatically enhances fibrin fibers formation with thrombin, and significantly delays fibrinolysis during spontaneous clotting of native blood. Iron ions caused the appearance dense matted fibrin deposits, similar, if not identical, to those observed in plasma of patients with stroke. These results may explain a known relationship between thrombotic diseases and the increased body concentrations of free iron and/or hemoglobin derivatives. We conclude that any action resulting in the inhibition of hemostatic abnormalities, as well as in the reduction of body free iron and scavenging of hydroxyl radicals (e.g., by polyphenols) can potentially prevent pathological reactions associated with consequences of stroke. Microsc. Res. Tech. 75:1185–1190, 2012. © 2012 Wiley Periodicals, Inc.

Unusual clotting dynamics of plasma supplemented with iron(III)

“…Free radicals are known to affect coagulation and fibrinolysis, and free radical scavengers normalize these processes (32) as was evident from results of our experiments with DMSO. It was reported that hydroxyl radical-induced modification of fibrin(ogen) molecules makes them resistant to fibrinolytic degradation (33 , and degradation of fibrinogen and fibrin by plasmin activated by tPA (34)(35)(36). However, in that experiment we identified some residual but not lysed clots, which may be explained by the presence of fibrinogen molecules resistant to fibrinolytic degradation, as described by Lipinski et al (33).…”

Iron and carbon monoxide enhance coagulation and attenuate fibrinolysis by different mechanisms

“…Sample composition consisted of 332 ml of plasma; 3.6 ml of PBS or FeCl 3 (10 mmol/l final concentration); 3.6 ml of dH 2 O or CORM-2 (100 mmol/l final concentration); and 20 ml of 200 mmol/l CaCl 2 . These concentrations of FeCl 3 [2,3] and CORM-2 [8,12] were chosen as they have been demonstrated to maximally enhance coagulation in our plasma and whole blood systems. Plasma sample mixtures were placed in a disposable cup in a computercontrolled thrombelastograph hemostasis system (Model 5000; Haemoscope Corp., Niles, Illinois, USA), with addition of CaCl 2 after a 3-min incubation period as the last step to initiate clotting.…”

“…The four conditions were as follows: 10 ml PRP without additions and 5 ml thrombin (10 U/ml), mixed and incubated for 3 min; 10 ml PRP with 5 ml FeCl 3 (250 mmol/l final concentration) added and then addition of 5 ml thrombin (10 U/ml) mixed and incubated for 3 min; 10 ml PRP after 1% addition (v/v) of CORM-2 (10 mmol/l final concentration in PRP, suspended in PBS with final concentration of 0.1% dimethyl sulfoxide; PRP was then incubated for 5 min) with addition of 5 ml of thrombin (10 U/ml), mixed and incubated for 3 min; and 10 ml PRP after simultaneous 1% addition (v/v) of CORM-2 (10 mmol/l final concentration in PRP, incubated for 5 min) with 1% addition (v/v) of FeCl 3 (333 mmol/l final concentration), and then addition of 5 ml thrombin (10 U/ml), mixed and incubated for 3 min. The concentration of FeCl 3 for condition 2 was used for simplicity, as there is little difference in thrombus ultrastructure with concentrations between 6 mmol/l and nearly 4 mmol/l [3,6]. The concentration of CORM-2 used was smaller than that used in the viscoelastic studies to maximize detection of more subtle changes in thrombus ultrastructure, without and with a lower concentration of FeCl 3 .…”

“…HO-1 activity is also increased systemically in inflammatory disorders associated with thrombophilia, such as diabetes mellitus [21] and rheumatoid arthritis [22]. Critically, SEM-based investigations demonstrated that the fibrin matrix formed from blood obtained from individuals with diabetes mellitus [23] or rheumatoid arthritis [24] was very similar to that observed when normal blood was exposed to ferric chloride [2][3][4][5][6][7]. In a complementary fashion, using a viscoelastic methodology validated to detect carbon monoxide-mediated hypercoagulability in the setting of tobacco smoking [25], it was determined that a patient with a clotted ventricular assist device and hemolysis had carbon monoxide-mediated hypercoagulability, and upregulation of HO-1 was documented by increased carboxyhemoglobin concentrations [26].…”

“…First, it was posited, and then determined that iron modified fibrinogen, which enhanced coagulation and attenuated fibrinolysis as documented by spectrophotometric and scanning electron micrographic (SEM) techniques [1][2][3][4][5][6][7]. Second, it was serendipitously discovered that carbon monoxide enhanced fibrinogen-dependent coagulation via an associated heme(s) group and attenuated fibrinolysis via upregulation of a 2 -antiplasim activity and downregulation of plasmin activity via enzyme-associated heme(s) [8][9][10][11][12][13][14].…”