Abstract
Abstract 1150
During plasma coagulation the protease α-thrombin (αIIa) cleaves fibrinogen to form a fibrin clot. Conversion of prothrombin to αIIa is catalyzed by factor (f) Xa, and results in expression of two electropositive regions on αIIa designated anion binding exosites (ABE) I and II. ABE I is involved in fibrinogen binding. In the presence of fVa and phospholipid, fXa cleaves prothrombin preferentially after Arg320, generating the intermediate meizothrombin (MzIIa), which also expresses ABE I. MzIIa is rapidly converted to αIIa. αIIa can be converted to β-thrombin (βIIa) and γ-thrombin (γIIa), both of which are cleaved within ABE I, and have greatly reduced capacity to convert fibrinogen to fibrin. Physiologic functions for βIIa or γIIa are not established; however, both have been identified in clotting blood. αIIa up-regulates its own generation in plasma by converting fXI to the protease fXIa. Yun et al. (J Biol Chem 2003;278:48112) showed that amino acids in ABE I are required for optimal fXI activation in the presence of the polyanion dextran sulfate (DS). MzIIa also activates fXI, consistent with a role for ABE I in protease binding to fXI. Given the absence of ABE I in βIIa and γIIa, it seems reasonable to postulate these proteases would interact poorly with fXI.
In a clotting assay in which thrombin is added to plasma anticoagulated with citrate (low calcium), βIIa (12.5 nM) and γIIa (50 nM) did not induce clot formation, consistent with their low capacity to cleave fibrinogen. However, when plasma was recalcified to allow thrombin to form from endogenous prothrombin, both βIIa and γIIa induced clot formation. Recalcified plasma in the absence of βIIa or γIIa did not clot (800 sec observation period), indicating fibrin formation was βIIa/γIIa-dependent. Addition of an antibody to fXI prolonged the clotting time with βIIa, and prevented clotting with γIIa, suggesting βIIa and γIIa were activating fXI. In addition, with γIIa, a fXIIa inhibitor modestly prolonged clotting time, indicating the plasma contact phase was activated.
We studied fXI activation by thrombin using western blot. βIIa and γIIa activated fXI at approximately half the rate of αIIa, while MzIIa activated fXI ∼4 fold faster than αIIa. FXI activation by αIIa is greatly enhanced by DS. In the presence of DS, αIIa and βIIa activated fXI comparably, while results with γIIa were not informative because the protease does not interact well with DS. Importantly, fXI activation by αIIa was not affected by the ABE I blocking peptide hirugen, indicating ABE I is not required for fXI activation by thrombin.
While βIIa and γIIa were less effective fXI activators than αIIa and MzIIa in solution, significantly different results were obtained in a plasma thrombin generation assay. Here coagulation is initiated in fXII deficient plasma with thrombin (10 nM), and subsequent thrombin generation from endogenous prothrombin is monitored. The system is fXI-dependent, as a fXI antibody blocks thrombin generation. Prior work with this system indicates fXI is probably converted to fXIa by the thrombin added to initiate the process. Initiation of coagulation with αIIa and MzIIa resulted in comparable thrombin generation (∼250 nM). βIIa and γIIa, as well as recombinant αIIa with mutations in amino acids in ABE I induced thrombin generation ∼2-fold greater than for αIIa and MzIIa. We hypothesized this was due to the inability of fibrinogen to compete with fXI for binding to thrombin species lacking ABE I. Consistent with this, hirugen peptide enhanced αIIa initiated thrombin generation ∼4-fold.
Finally, we followed up on the observation that a fXIIa inhibitor prolonged time to γIIa-induced clot formation in recalcified plasma. In solution, γIIa, but not αIIa, βIIa, or MzIIa cleaves the contact factors fXII and PK. The cleaved proteases, in turn, are capable of cleaving chromogenic substrates, and have activity in a reciprocal fXII-PK activation assay.
Our studies show that ABE I is not required for thrombin-mediated activation of fXI, that thrombin species not fully expressing ABE I may be better than αIIa and MzIIa as initiators of fXI-dependent thrombin generation in plasma, and that γIIa can activate the plasma contact proteases. Taken as a whole, the data indicate forms of thrombin other than αIIa may contribute directly to feedback activation of fXI, and may represent a previously unrecognized link between coagulation and the contact system.
Disclosures:
No relevant conflicts of interest to declare.
PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED, ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES
