Bound Thrombin from Crushed Clots Is Composed of α-Thrombin and the N-Terminal Regions of α- and γ-Chains of Fibrinogen

Abstract

We aimed at clarifying the structural characteristics of the bound thrombin that is liberated by mechanical breakdown of fibrin clots. Fibrin clots were prepared with bovine thrombin and rabbit fibrinogen, and were crushed mechanically with a glass rod. The supernatant of the crushed clots was subjected to immunoaffinity chromatography to isolate the bound thrombin. Western blotting analysis revealed that the bound thrombin could be reacted with both antithrombin and antifibrinogen under unreduced conditions. SDS-PAGE under reduced conditions revealed that there were three bands, two of which were found to be the N-terminal fragments of the α- and γ-chains of fibrinogen. The bound thrombin could be dissociated into three distinct fibrin fragments and bovine α-thrombin when denatured by 8 M urea. Thus, the bound thrombin liberated from crushed clots is a stable complex between bovine α-thrombin and fibrin fragments of the N-terminal regions of rabbit α- and γ-chains.

RNAi Targeting Embryonic Myosin Heavy Chain Isoform Inhibited Bound Thrombin-Induced Migration of Vascular Smooth Muscle Cells

“…Preparation of bound thrombin was carried out as previously described [15, 20]. In brief, a mixture of 0.5 ml of bovine α-thrombin (500 U/ml) and 2.5 ml of rabbit fibrinogen (1.5 mg/ml in serum-free DMEM) was incubated for 15 min at 37°C to allow a clot to form.…”

“…We have reported some unique biological and physicochemical characteristics of bound thrombin [14,15,16], including platelet aggregation, complex with fibrin fragments. We also demonstrated that bound thrombin enhanced the phenotype conversion of VSM cells towards a synthetic type by upregulating mRNA expressions of SMemb and plasminogen activator inhibitor type 1 (PAI-1) by the comparative reverse transcription polymerase chain reaction (RT-PCR) method [17].…”