Hiromichi Kitaguchi

Effect of proteases on plasminogen activator release from isolated perfused dog leg

Abstract Inducement of secondary fibrinolysis can be explained most simply by assuming that a substance newly formed in the process of blood coagulation acts on the endothelial cells and releases plasminogen activator (P. A. ) into the circulatory blood. In the present study, it was found that thrombin caused release of P.A. from isolated dog leg perfused with physiological solution. Following administration of purified thrombin to the isolated perfused dog leg, P.A. was released within 10 sec and reached its peak activity at 15∼30 sec. A dose response in P.A. activity released to thrombin was also detected to some extent (1 unit/ml∼50 units/ml). These results suggest that thrombin mediates secondary fibrinolysis. The effects on P.A. release of DFP-treated thrombin and TLCK-treated thrombin, which are inactive in fibrinogen-fibrin conversion, were examined. These chemically modified forms of thrombin did not release P.A. Furthermore, examination of the effects of acetylated thrombin revealed that it also did not cause P.A. release. These results indicate that P.A. is released from the vascular wall by thrombin so long as the latter maintains an intact active center of proteolysis.

Plasminogen activator releasing by the K+ rich solution

Abstract Our previous studies on plasminogen activator releasing from the vascular wall indicated that strong vasoconstrictors induced the plasminogen activator releasing accompanied by vasoconstriction. The present study was made to know the relation of vasoconstriction to the plasminogen activator releasing by perfusing of the isolated hind leg of dog with the K+ rich solution, and the following results were obtained: The plasminogen activator releasing was induced by the K+ rich solution in the presence of Ca++ ion, accompanied by vasoconstriction. Whereas, such activator releasing was not induced, in the absence of Ca++ ion, without vasoconstriction. These results indicate that there is a common process of the plasminogen activator releasing, which is joined together with the process of vasoconstriction.

Prostacyclin release from the coronary vascular wall by vasoactive substances.

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI2) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 microM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 microM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 microM acetylcholine, 25 microM noradrenaline, 25 microM isoproterenol, 10 microM adenosine triphosphate (ATP), 5 microM adenosine, 1 microM angiotensin II, 25 microM histamine or 1 microM serotonin was infused for 30 sec, no inhibitory activity of platelet aggregation was observed. Bradykinin (5 X 10(-9) approximately 5 X 10(-6) M) and purified thrombin (1 X 10(-9) approximately 1 X 10(-7) M) induced a dose-dependent release of PGI2 which was assayed using a radioimmunoassay for 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha).

Some properties of the tissue plasminogen activator from the pig heart.

Abstract A purified preparation of tissue plasminogen activator was obtained from acetone-dried pig heart powder, principally based on the methods described by Bachmann et al. (1) and Rickli & Zaugg (2). The preparation exhibited the following properties; (i) the molecular weight similar to egg albumin, (ii) the relative thermal stability at neutral pH, (iii) inhibition by EACA and t-AMCHA, (iv) inhibition by DFP and (v) stability to iodoacetamide and TLCK. An additional study indicated that the tissue plasminogen activator was well adsorbed to polyacrylonitrile fiber and eluted with aqueous ammonia.

Conversion of cultured monocytes/macrophages into endothelial-like cells through direct contact with endothelial cells.

When culturing human umbilical vein endothelial cells in a culture medium containing 4% human serum albumin, it was possible to maintain the epithelioid morphology and function for several months without subculturing. When coculturing endothelial cells and labeled monocytes/macrophages (Mo/Φ) that were collected from peripheral blood and allowed to engulf fluorescent latex beads, some Mo/Φ changed their shapes and became epithelioid cells that were indistinguishable from vascular endothelial cells. This transformation started within several hours of coculturing. At 7 days after the start of coculturing, more than half of the labeled cells were identified as endothelial-like cells morphologically. Furthermore, morphologically altered Mo/Φ did not express Mo/Φ-specific antigens, ie, the MHC Class II molecule and CD68, but expressed VE cadherin and vWF, which are specific antigens for endothelial cells, and labeled cells that changed into endothelial-like cells no longer engulfed fluorescent latex beads. This strongly suggests that peripheral blood monocytes differentiate into endothelial-like cells.

EFFECT OF VASOACTIVE AGENTS ON FACTOR VIII RELEASE FROM PERFUSED ISOLATED DOG LEG

Abstract The present study was carried out to determine whether factor VIII with procoagulant activity was released by vasoactive agents from blood vessels of dog hind leg perfused with artificial physiological solution. F. VIII procoagulant activity was measured by the one-stage method using F. VIII deficient plasma. When bradykinin, acetylcholine, isoproterenol or salbutamol was administered via the perfused femoral artery, a transient but steep rise in F. VIII procoagulant activity was clearly observed in the venous perfusate. On the other hand, when adrenaline or noradrenaline was administered, a characteristic long-lasting release of F. VIII was observed. The effects of angiotensin II and vasopressin were found to be far weaker than those of the other vasoactive agents. The above results indicate that F. VIII with procoagulant activity may be released from the blood vessels by vasoactive agents. Furthermore, two different patterns of F. VIII release were demonstrated, viz. the transient release induced by bradykinin, acetylcholine, isoproterenol or salbutamol, and the long-lasting release induced by adrenaline or noradrenaline.

Plasminogen activator activity of cultured endothelial cells derived from canine coronary vessel and human umbilical artery and vein

The present study was undertaken to determine which cells participate in plasminogen activator (PA) release from the vascular wall. Canine coronary vessel was confirmed to release PA when various agents were administered in perfusion experiments. Endothelial cells from the coronary vessel were then isolated and cultured for 2 days. PA activity was observed in lysates of the cultured cells, and the medium used for the cultivation was found to contain little PA activity. This suggests that the endothelial cells participate in PA release from the vascular wall. In addition, the PA synthesis was also studied in cultured endothelial cells from human umbilical artery and vein. Both contained little PA activity, indicating that vascular endothelial cells may vary in PA synthesis and release according to the vessel.