Allosteric effects potentiating the release of the second fibrinopeptide A from fibrinogen by thrombin.
Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.
Shainoff
John R
JR
Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA. j.shainoff@csuohio.edu
Smejkal
Gary B
GB
DiBello
Patricia M
PM
Sung
Shen-Shu
SS
Bush
Leslie A
LA
Di Cera
Enrico
E
D000495:Allosteric Site; D001665:Binding Sites; D002794:Choline / Q000737:chemistry / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005340:Fibrinogen; D012964:Sodium; D013917:Thrombin; D002794:Choline