Novel Use of Scanning Electron Microscopy for Detection of Iron‐Induced Morphological Changes in Human Blood

Abstract

Fibrinogen is key to the maintenance of hemostasis and is an acute phase protein that is part of the coagulation cascade of proteins. It plays a fundamental role in inflammation, particularly as indicator for a proinflammatory state and is a prominent marker for developing vascular inflammatory diseases. The ultrastructure of fibrin nets can be studied using scanning electron microscopy (SEM) with the addition of thrombin to plasma. In inflammatory conditions such as thromboembolic ischemic stroke and diabetes, the fibrin networks are changed to from dense matted fibrin deposits (DMDs) instead of typical netlike appearance. Similar DMDs can also be induced with the addition of FeCl(2) and FeCl(3). Importantly, the iron-induced DMDs look similar to those from patients with prothrombotic conditions. Excessive or misplaced tissue iron now is recognized to pose a substantial health risk. The current research therefore investigates the establishment of a laboratory fibrinogen model to study that might mimic fibrin fiber generation that is achieved using plasma from healthy and diseased individuals. Furthermore, to determine whether the addition of iron to purified fibrinogen will show DMDs and whether hydrophilic agents can prevent them. We conclude that SEM is a very effective tool for the visualization of circulatory consequences of the interaction of iron-induced hydroxyl radicals with human fibrinogen. Furthermore, this novel fibrinogen model provides a convenient method to study the interactions of the intramolecular and intermolecular hydrophobic forces responsible for the maintenance of the tertiary structure of native fibrin(ogen) and the prevention of iron-induced DMDs formation by hydrophilic agents.

Iron-enhanced coagulation is attenuated by chelation A thrombelastographic and ultrastructural analysis

“…We found that the thrombelastographic, kinetic sine quo non of iron was a decrease in the onset time of coagulation and an increase in the velocity of thrombus growth [18]. The SEM signature of thrombi exposed to this small concentration of iron [18] demonstrated the typical changes previously mentioned [1][2][3][4][5][6]. Thus, the purpose of this study was to evaluate the effects of deferoxamine-mediated chelation on ironexposed plasma with thrombelastographic and SEMbased analyses.…”

“…When blood or plasma is exposed to iron addition, characteristic changes in thrombus formation are observed, which include fusion of fibrin polymers, matting, and even sheeting of fibrin [1][2][3][4][5][6]. This scanning electron micrographic (SEM) signature has also been documented in thrombi obtained from patients with diseases involving chronic iron overload [1,2,7,8].…”

Carbon monoxide: Anticoagulant or procoagulant?

“…First, it has been demonstrated by Lipinski and Pretorius that exposure of purified fibrinogen to ferric ions demonstrated to generate hydroxyl ions without a redox reagent present resulted in aggregation of the fibrinogen [90]. Fenton chemistry may play a role in pathological thrombus formation in diabetes mellitus and other diseases with chronic iron overload [91], and exposure to iron results in SEM evidence of thrombus matting similar to that observed in smoking, diabetes mellitus and rheumatoid arthritis [92,93]. Given the findings of Orino [62] that both iron and heme bind to fibrinogen, it is entirely possible that there are scenarios wherein CO is the primary modulator of fibrinogen functional change (e.g., Nielsen et al 18 18 smoking) contrasted to situations involving both CO and iron modulating fibrinogen (e.g., hemolysis, engagement of HO-1 and release of CO and free iron).…”

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

“…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].…”