Gerald Soslau

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.

Seventeen adults received the antifibrinolytic drug tranexamic acid during cardiac surgery utilizing extracorporeal circulation (ECC). In 8 patients, drug administration began prior to skin incision (pre-ECC); infusions commenced after ECC and protamine administration in another 9 patients (post-ECC). Compared with the post-ECC group, the pre-ECC group exhibited less bleeding via mediastinal drains (420 vs. 655 mL/12 h median, P = 0.024), decreased frequency of the presence (greater than or equal to 10 micrograms/mL) of fibrin split products (P less than 0.05), and greater platelet dense granule content of adenosine diphosphate after surgery (15.47 vs. 4.05 nmoles/mg protein median, P = 0.021). Follow-up in vitro study of tranexamic acid inhibition of plasmin-induced platelet activation utilizing normal human platelet rich plasma and porcine plasmin revealed a 13-fold lower concentration of tranexamic acid for 50% inhibition when plasmin was preincubated with the drug (1.2 micrograms/mL, 95% CI = 1.13-1.60 micrograms/mL) compared to when platelet rich plasma was preincubated with the drug (16 micrograms/mL, 95% CI = 7.3-99. micrograms/mL). Plasmin inactivated with tranexamic acid retained its ability to inhibit thrombin-induced platelet activation, thus suggesting that tranexamic acid inhibits plasmin's catalytic activity and not its binding to platelets. Both clot lysis and platelet dysfunction may contribute to bleeding after ECC. Tranexamic acid blocks plasmin-induced partial platelet activation during ECC, thus preserving platelet function and promoting hemostasis after ECC.

Ethidium bromide intercalates between the bases of native DNA, resulting in several biological anomalies. The effects of ethidium bromide on the mitochondria of cultured mouse L cells were studied. At a concentration of 1 microg ethidium bromide/ml it was observed that concentrations of cytochromes a + a(3) and b decreased, a + a(3) more rapidly than b. In contrast, the concentration of cytochromes c(1) and c increased or remained the same as in control cells. Concomitant with the decrease of cytochromes a + a(3) and b was an enlargement of the mitochondria and a reduction in the cristae. The cristae that remained were abnormally organized. After prolonged treatment with ethidium bromide a second population of small, more normally organized mitochondria was apparent. These effects of ethidium bromide could be reversed.

Abstract The glycopeptides from various subcellular fractions isolated from control, BHK21/C13, and virus-transformed C13/ B4 baby hamster kidney cells have been compared by chromatography on columns of Sephadex G-50 in order to determine (a) whether changes in cell surface glycoproteins previously shown to occur upon viral transformation are also found in the glycoproteins of membranes associated with other subcellular membrane fractions and (b) whether the glycopeptides associated with the subcellular fractions were similar to those found on the surface membrane. Cells were grown in the presence of d-[14C]- or [3H]glucosamine or l-[14C]- or [3H]fucose. Fractions to be compared were mixed and digested with pronase, and the glycopeptides were fractionated by gel filtration. These studies showed that the alterations accompanying transformation seen in the surface membrane glycopeptides were also found in other subcellular fractions. These alterations consist of as marked increase in the amount of higher molecular weight material in the transformed cell. Neuraminidase treatment of this material diminishes or eliminates the difference between the normal and malignant patterns. The relative distribution of glycopeptides in the mitochondrial, nuclear, and rough endoplasmic reticular fractions differed in some respects from that of the surface membrane. Glycopeptides associated with the subcellular fractions were not due to contamination by surface membranes.