John A. Samis

Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit

The complete cDNA of 3.2 kb for rat calpain II large subunit has been constructed from library- and polymerase chain reaction-derived fragments, and sequenced. The cDNA encodes a protein of 700 amino acids having 93% sequence identity with human calpain II, and 61% identity with human calpain I. The gene possesses 21 exons, of which exons 3-21 have been mapped over 33 kb of the rat genome. A new phagemid expression vector was created from pT7-7 by insertion of the f1 origin and mutation of an NdeI to an NcoI site. Rat calpain II cDNA ligated into this vector expressed in Escherichia coli an 80 kDa protein identical in size to highly purified rat calpain II; this protein was specifically recognized on immunoblots by an affinity-purified anti-rat calpain II antibody. This is the second mammalian calpain II large subunit to be fully sequenced, and the first to be artificially expressed.

Functional Characterization of Recombinant Human Meizothrombin and Meizothrombin(desF1) THROMBOMODULIN-DEPENDENT ACTIVATION OF PROTEIN C AND THROMBIN-ACTIVATABLE FIBRINOLYSIS INHIBITOR (TAFI), PLATELET AGGREGATION, ANTITHROMBIN-III INHIBITION

Recombinant human prothrombin (rII) and two mutant forms (R155A,R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M−1·min−1) for inhibition by antithrombin III (AT-III) were 2.44 × 105 (rIIa), 6.10 × 104 (rMZa), and 1.05 × 105 (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

Aortic endothelial cell von Willebrand factor content, and circulating plasminogen activator inhibitor-1 are increased, but expression of endothelial leukocyte adhesion molecules is unchanged in insulin-dependent diabetic BB rats.

Endothelial cell injury has been implicated in the increased incidence of vascular disease associated with diabetes mellitus. In diabetic humans, elevated plasma von Willebrand Factor (vWF) has been interpreted as an indication of endothelial damage. In contrast, in an animal model of inherited insulin-dependent diabetes, the bio-breeding (BB) rat, plasma vWF levels did not differ from those in age-matched control rats during the first 7 months of diabetes although morphological evidence of mild aortic endothelial alteration or injury was observed. In the present study efforts have been made to define the endothelial alterations in BB diabetic rats compared to controls more precisely over this time period. Thus, adhesion molecules: intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were evaluated by in situ immunohistochemistry, vWF content was determined by biochemical analysis of aortic extracts and by quantitative immunohistochemistry, plasma vWF levels were measured by ELISA and vWF mRNA by RNAse protection assay. Neither age nor diabetic state significantly affected either the expression of adhesion molecules, or the levels of circulating vWF. Endothelial vWF content was significantly increased in the diabetic vessels, as observed by both approaches but the vWF mRNA content was not different from that in control vessels. Plasma plasminogen activator inhibitor (PAI-1) activity was significantly increased in diabetic animals. In conclusion, endothelial alterations in BB rats associated with diabetes, together with the raised plasma PAI-1 levels, promote the thrombogenic potential of the vessel wall, and are consistent with an increased risk for vascular disease.