Histamine Regulates Binding of Cross-Linked Fibrinogen to Endothelium: Role of Histaminylation in Vascular Biology

Abstract 3304

Recent studies have demonstrated that a wide variety of biogenic amines can be covalently cross-linked to intracellular and extracellular proteins. This “aminylation” reaction regulates several important reactions in vascular biology. Serotinylation of small GTPases recently was shown to regulate cell signaling events, platelet activation and promote vascular smooth muscle cell proliferation. The role of extracellular crosslinking of biogenic amines to plasma and extracellular matrix proteins is not well established and could play a role in altering transglutaminase reactions in vascular tissue. Tissue translutaminase (TGM-2) crosslinking of the a-C domain of fibrinogen has been shown to promote clustering of RGD domains, enhance binding of the cross-linked polymers to endothelial cells and promote cell adhesion. While Factor XIIIa prefers to crosslink fibrin, the TGM-2 molecule which is abundantly expressed by erythrocytes, endothelial and vascular smooth muscle cells does not show any preference for fibrin compared to fibrinogen. TGM-2 is a sulfhydryl rich calcium-dependent enzyme that could cross-link a variety of biogenic amines to fibrinogen. We investigated the preference of TGM-2 crosslinking of biogenic amines to fibrinogen in vitro and determined whether the crosslinking would modify the transglutaminase-dependent binding of fibrinogen to endothelial cells. We found histamine was the most effective primary amine inhibitor of the TGM2-mediated cross-linking reaction (Histamine > putrescine >>> serotonin, dopamine, noradrenaline). 1.25 mM histamine inhibited > 75% TGM2-mediated fibrinogen and fibrin cross-linking. The ability of TGM-2 crosslinked fibrinogen complexes to bind to confluent human umbilical vascular endothelial cell (HUVEC) was also studied. Free [¹²⁵I]-fibrinogen bound to endothelial cells with low affinity, however the binding was increased ∼7 fold when fibrinogen was cross-linked by TGM2. The increase in crosslinked fibrinogen binding was dependent on TGM2 and Ca⁺² concentration. Unlabeled crosslinked fibrinogen inhibited the binding by more than 85%. In contrast, unlabeled fibrinogen actually enhanced the binding 1.7 fold suggesting that fibrinogen and cross-linked fibrinogen formed multivalent complexes with cross-linked fibrinogens on the endothelial cell surface. When bound complex were eluted from HUVEC cells after binding experiments, >95% of bound materials were extensively crosslinked and could not enter a 5–15% polyacrylamide gel. In contrast, no high molecular weight material was eluted when non-crosslinked fibrinogen was used in the binding experiments. When fibrinogen was cross-linked in the presence of 8 to 500 micromolar of histamine, the binding was inhibited by ∼75 to 90 %, respectively. In summary, TGM-2 cross-linked fibrinogen showed enhanced binding to endothelial cells as previously reported for purified aC domains of fibrinogen. The binding of fibrinogen to endothelial cells is known to enhance endothelial cell adhesion and leukocyte transmigration, reactions that are involved in wound healing, angiogenesis and inflammation. The TGM-2 crosslinked fibrinogen/fibrinogen complexes may serve as proinflammatory, prothrombotic and proangiogenic factors in vivo. Histaminylation of fibrinogen by TGM-2 could provide a mechanism to regulate these vascular events. Fibrinogen could also serve to control local histamine function as only free histamine can bind to histamine receptors. Transglutaminase mediated histaminylation of fibrinogen could have multiple effects on acute and chronic inflammatory reactions.