Functional properties of carbohydrate-depleted tissue plasminogen activator.

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose).

Peritoneal fibrinolysis: evidence for the efficiency of the tissue-type plasminogen activator.

Blood shed into a closed peritoneal cavity is incoagulable. We have investigated this poorly understood phenomenon in animal experiments. Nonthrombogenic femoral vein-peritoneal cavity shunts were established in five dogs and 10 ml/kg blood admixed with 125I-dog fibrinogen was rapidly drained into the peritoneal cavity.

Effects of moxalactam on blood coagulation and platelet function.

Bleeding complications have occasionally been reported in clinical trials of moxalactam therapy for debilitated and/or malnourished patients. Complications that occur secondary to hypothrombinemia are readily corrected by administration of 5-10 mg of vitamin K. In a few instances, the bleeding complications occurred secondary to suppression of platelet function.

Effects of benoxaprofen and indomethacin on platelet function and biochemistry.

Benoxaprofen did not impair platelet function or platelet prostaglandin synthesis in vivo or in vitro in contrast to indomethacin, a known prostaglandin synthesis inhibitor. Platelet cyclic AMP levels were not changed by either drug in vivo or in vitro. The results of routine tests for hemostatic function (template bleeding time and blood clotting assays) were not changed by either drug.

Sensitivity and specificity of plasma serine protease chromogenic substrates.

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160, S-2238, S-2222 and S-2251 and Chromozym TH were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha-thrombin, and bovine factor Xa. S-2160, S-2238, and chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All three substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin.

Serine protease specificity for peptide chromogenic substrates.

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin.

Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation.

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and "early" fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory.

Enhancement of blood coagulation by soluble fibrin complexes.

We have detected a species of soluble fibrin complexes with significant biological properties. Agarose gel chromatography of normal plasma or purified fibrinogen previously incubated with small amounts of thrombin revealed the presence of a species of high molecular weight soluble fibrin complexes, which contained only small quantities of fibrinogen by immunological assays but which exhibited enhanced sensitivity to thrombin. In addition, these complexes substantially shortened the thrombin-clotting time of normal plasma and enhanced the resistance of normal plasma to heparin action.

Studies on the prekallikrein (kallikreinogen)--kallikrein enzyme system of human plasma. I. Isolation and purification of plasma kallikreins.

By measurement of its arginine esterase activity, plasma kallikrein was purified from fresh frozen ACD plasma. The steps involved alcohol fractionation, isoelectric precipitation, and carboxymethyl (CM) Sephadex and DEAE cellulose chromatography. Three enzymatically active fractions were finally isolated and termed plasma kallikreins I, II, and III; they represented purifications of 970,320- and 590-fold, respectively.

Studies on the prekallikrein (kallikreinogen)--kallikrein enzyme system of human plasma. II. Evidence relating the kaolin-activated arginine esterase to plasma kallikrein.

Evidence is presented in this paper that the kaolin-activated arginine esterase of plasma is related to plasma kallikrein activity. Such a relationship is based on studies that (1) establish a constant ratio of esterase activity on various synthetic substrates for the kaolin-activated arginine esterase, purified kallikrein(s), and preparations obtained during the fractionation procedure; (2) exclude other known plasma and tissue arginine esterases; (3) confirm the requirement for factor XII in the activation of the enzyme precursor; and (4) show similarities in behavior between the plasma esterase and purified kallikrein(s) toward a variety of inhibitors. Based on this probable identification, evidence is provided that the concentration of active factor XII determines the rate of activation of plasma kallikreinogen, and that the activation may be blocked by polybrene.