Komorowicz, Erzsébet

SummaryThe relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using spectrophotometric kinetic analysis of the competition between fibrin and synthetic peptide substrates for the proteases, turbi-dimetric assay for fibrin dissolution and gel-filtration of the partially degraded fibrin. When the substrate is fibrin polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 × 105 M-1·s-1 for plasmin, 1.9 × 105 M-1·s-1 for miniplasmin, 5.0 × 104 M-1·s-1 for PMN-elastase and 2.2 × 103 M-1·s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6 for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive crosslinking of fibrin decreases the catalytic action of all studied proteases, but no change in their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at physiological concentration) is also crosslinked to fibrin, the most efficient fibrinolytic enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity lysine binding site in the N-terminal kringle domain of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to newly exposed lysine residues. These results provide a quantitative basis for the evaluation of fibrinolytic efficiency and support the concept of synergistic fibrinolysis.

Atherosclerosis manifests as a systemic disease with near global involvement of the named segments of the arterial tree. Acute thrombotic arterial occlusion, however, is not equally distributed. To evaluate intra-individual regional differences in arterial thrombogenicity, we compared 111 In-platelet deposition in porcine carotid and femoral arteries after a standardized crush injury. Within the unidirectional flow conditions of elastic carotid arteries, platelet deposition was more than 3-fold higher compared with predominantly muscular femoral arteries with triphasic arterial flow. To determine the influence of rheology on platelet deposition after crush injury, carotid arteries were transplanted into the femoral position and compared with the paired native carotid and femoral arteries. Similarly, femoral arteries transposed to the carotid position were compared with the paired native carotid artery. In each of these experiments, arterial transposition to a new anatomic location imparts a predilection for platelet deposition indigenous to the new location. In the controlled environment of two high-shear thrombin–independent and –dependent flow chambers, porcine carotid and femoral arterial substrates were indistinguishable from one another with respect to platelet deposition. Regional differences in arterial hemodynamics may account for substantial differences in thrombosis arising from deep arterial injury.