Cellular Procoagulant Activity Dictates Clot Structure and Stability as a Function of Distance From the Cell Surface

Abstract

Background-Thrombin concentration modulates fibrin structure and fibrin structure modulates clot stability; however, the impact of localized, cell surface-driven in situ thrombin generation on fibrin structure and stability has not previously been evaluated. Methods and Results-Human fibroblasts were incubated with factors Xa, Va, prothrombin and fibrinogen, or plasma.Fibrin formation, structure, and lysis were examined using laser scanning confocal microscopy and transmission electron microscopy. In situ thrombin generation on the cell surface produced clots with a significantly denser fiber network in a 10-m region proximal versus distal to (40 to 50 m) the cell surface. This morphology was not altered by addition of integrin-blocking RGDS peptide and was not apparent in clots made by exogenous thrombin addition, suggesting that spatial morphology was dictated predominantly by localized thrombin generation on the fibroblast surface. The fibrin network lysed more rapidly distal versus proximal to the cell surface, suggesting that the structural heterogeneity of the clot affected its fibrinolytic stability. Conclusions-In situ thrombin generation on the cell surface modulates the three-dimensional structure and stability of the clot. Thrombus formation in vivo may reflect the ability of the local cell population to support thrombin generation and, therefore, the three-dimensional structure and stability of the fibrin network. (Arterioscler Thromb Vasc Biol. 2008;28:2247-2254.)Key Words: coagulation Ⅲ fibrin clot structure Ⅲ thrombin generation Ⅲ fibrinolysis A fter vascular injury, thrombin-catalyzes the enzymatic conversion of soluble fibrinogen to an insoluble fibrin network. The fibrin network stabilizes the primary platelet plug, enabling it to withstand the rigors of blood flow during wound healing. A growing number of studies suggest that abnormal fibrin structure makes clots overly stable or friable during this process, contributing to an individual's risk of thrombosis and embolism. [1][2][3][4] Mechanisms of fibrin production and clot assembly have been elucidated primarily from studies in which a specific amount of thrombin is added to purified fibrinogen. These studies have shown that the thrombin concentration present at the time of fibrin gelation influences fibrin clot structure. [5][6][7][8][9] Low thrombin concentrations produce porous clots composed of thick fibrin fibers, whereas high thrombin concentrations produce clots composed of a dense network of thin fibers. Additional studies have correlated fibrin architecture with the resistance of a clot to mechanical and fibrinolytic disruption. 8 -11 Clots composed of a dense network of thin fibrin fibers are more resistant to fibrinolysis compared to clots composed of a porous network of thick fibers. In vivo, however, thrombin generation is a dynamic process in which the concentration of thrombin actively changes during the reaction course in accord with the local conditions. Importantly, and in contrast to in vitro experiments initiated by the ...

Confocal Microscopy as Useful Tool for Studying Fibrin-Cell Interactions

“…Our results confirmed previous work performed by Jerome et al [17]. Other authors have found that the clot organization was related to the thrombin generated on the cell surface, and when they added exogenous thrombin no change was observed in the fibrin morphology [19].…”

“…This fibers arrangement proximal to the cells is lost in the presence of antibodies against α v and/or β 3 integrin subunits [17] or the synthetic RGD peptide [18]. In contrast, other studies have concluded that the fibrin structure near the cell surface was related to the thrombin generated on the cells surface, and in their experiments the addition of RGD peptide did not affect the fibrin structure [19,20]. They did not add antibodies against the integrin subunits that would be more conclusive.…”

Fibrinogen α and γ genes and factor VLeiden in children with thromboembolism: results from 2 family-based association studies

“…29,30 We note in this context that the fXIII Leu34 variant, which has been identified as a protective trait in thrombotic disorders, produces similar alterations in fibrin structure as high levels of ␥Ј fibrin. 29 In vitro experiments with purified fibrinogen suggest that an increased rate of thrombin generation, as probably occurs in FV Leiden carriers, may override the biochemical differences between the structures of Ala312 and Thr312 fibrin, or ␥A/␥A and ␥Ј/␥A fibrin, [31][32][33][34] providing a theoretical explanation how the FV Leiden mutation might mitigate the risk conferred by FGA-H1/FGG-H2.…”

Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin

“…14 Fibrin structure is influenced by platelets. [15][16][17][18] The platelet surface is a regulator of procoagulant activity and local thrombin concentration, parameters that have been shown to dictate clot structure. 18 Platelets interact directly with fibrin by the ␣IIb␤3 integrin that alters fibrin structure and susceptibility to lysis.…”

“…[15][16][17][18] The platelet surface is a regulator of procoagulant activity and local thrombin concentration, parameters that have been shown to dictate clot structure. 18 Platelets interact directly with fibrin by the ␣IIb␤3 integrin that alters fibrin structure and susceptibility to lysis. [15][16][17] When stimulated, platelets release polyphosphate (polyP) from the dense granules.…”