Inhibition of thromboxane A2 synthesis in human platelets by coagulation factor Xa.

Factor Xa binds to platelets provided that factor Va is present on the platelet surface, an interaction that results in a striking acceleration of the conversion of prothrombin to thrombin. Thrombin then initiates fibrin formation, induces platelet aggregation, and stimulates the intraplatelet synthesis of thromboxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet-rich plasma increased the basal level of TXA2, measured as thromboxane B2, from less than 0.5 pmol per 10(8) platelets to (mean +/- SEM) 100 +/- 22 and 250 +/- 10 pmol per 10(8) platelets, respectively. Treatment of platelet-rich plasma with increasing concentrations of factor Xa (1-12 nM) prior to the addition of thrombin progressively inhibited the production of TXA2. Thrombin (9.6 nM), which produced 93% of the maximal formation of TXA2, was inhibited 70% by factor Xa (10 nM). To identify which of these steps in thromboxane synthesis was inhibited by factor Xa, platelets labeled with [14C]arachidonic acid were exposed to thrombin and products of prostaglandin synthesis were separated by thin-layer chromatography. In contrast to the inhibition of TXA2 synthesis, prostaglandin E2 and prostaglandin F2 alpha synthesis were not inhibited suggesting that neither phospholipase(s) nor cycloxygenase was involved. The inhibition of TXA2 formation by factor Xa could be reversed by increasing the molar ratio of thrombin to factor Xa to 5.5. Incubation of platelets with an IgG fraction of a human monoclonal antifactor V antibody, previously shown to inhibit factor Xa binding, was found to block factor Xa inhibition of TXA2 synthesis. The inhibition of TXA2 synthesis requires the presence of the active site serine of factor Xa and is not specific for TXA2 formation induced by thrombin because it is also demonstrable when the agonist is ADP. Further, factor Xa does not require additional plasma components for its action because its inhibitory effects are detected in gel-filtered platelets. The effect of factor Xa was evident at physiological (1.3 mM) calcium concentrations. These results indicate that factor Xa binding to platelets through factor Va not only stimulates thrombin formation but also has a countervailing effect by inhibiting TXA2 formation.     

Inhibition of the activation of Hageman factor (factor XII) by human vascular endothelial cell culture supernates.

The supernatant fluid (conditioned medium) of cultured human vascular endothelial cells inhibits activation of Hageman factor (factor XII), whether by ellagic acid, bovine brain sulfatides, or bismuth subgallate; inhibition appears to be a property of one or more proteins in the culture supernates. This phenomenon may contribute to maintaining the fluidity of circulating blood by inhibiting surface activation of the intrinsic pathway of coagulation.     

Mitogenic effects of coagulation factor XII and factor XIIa on HepG2 cells.

The structure of coagulation factor XII (Hageman factor), inferred from its DNA sequence, includes two epidermal growth factor (EGF)-homologous domains in its amino-terminal region. This suggests that factor XII may exhibit EGF-like activities. Reciprocal antigenic cross-reactivity between factor XII and EGF was shown by exposing purified human factor XII or mouse EGF to anti-mouse EGF or anti-human factor XII. Western blot analysis showed that anti-mouse EGF recognized intact factor XII at 80 kDa. Together, these results suggest that the EGF-homologous domains are accessible for anti-EGF binding in native factor XII. To determine whether factor XII has mitogenic activity, HepG2 or L cells (10(4) cells per well) were grown in serum-free medium in the presence or absence of factor XII or kaolin-activated factor XII (factor XIIa). Both factors XII and XIIa (6.0 micrograms/ml) enhanced cell proliferation by approximately 2-fold (P less than 0.001 and P less than 0.005, respectively). In contrast, L cells, which are not EGF target cells, were not affected by either factor XII or factor XIIa. Various doses of factor XII enhanced cell proliferation, [3H]thymidine incorporation, and [3H]leucine incorporation in HepG2 cells cultured under the same conditions. These data indicate that factor XII, like EGF, is a mitogen for HepG2 cells and suggest a possible autocrine role in the liver.     

Reconstituted collagen is not capable of activating factor XII but causes intrinsic coagulation by activating platelets.

Using a rheological technique to measure the coagulation of plasma in collagen-coated tubes, we studied the intrinsic coagulant activities of different types (I, III, IV and V) and structures of reconstituted collagen. Recalcified, platelet-free plasma (PFP) in contact with the collagen surface did not clot, irrespective of the type and structure of collagen. Coagulation of platelet-rich plasma (PRP) did occur, although the time of onset of coagulation was highly dependent on the type and structure of the collagen used. Coagulation of PRP occurred rapidly on a collagen surface consisting of highly ordered fibrils (banded structure) to which large numbers of aggregated platelets adhered with shape change. In contrast, initiation of coagulation was delayed in PRP after incubation with 5 mM dibutyrylcyclic AMP (DB-cAMP) for 50 min. Coagulation of PRP was completely suppressed after 30 min incubation with colchicine (5.0 mg/ml). This suggested that disruption of platelet microtubules completely suppressed the stimulation of platelets associated with the initiation of intrinsic coagulation on the collagen surface. We conclude that reconstituted collagen is not capable of activating factor XII. Initiation of coagulation of recalcified PRP in contact with the reconstituted collagen surface is caused by the activation of platelets.     

Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination.

DNA sequences encoding the hormone-binding domains of several steroid hormone receptors were fused in frame to the MyoD gene. When the gene for this chimeric protein was expressed in NIH 3T3 or 10T1/2 fibroblasts, these cells displayed hormone-dependent induction of myogenesis. Our experiments focused on cell lines expressing estrogen receptor-MyoD chimeras. Induction of these lines in the presence of estradiol and an inhibitor of protein synthesis, cycloheximide, resulted in the activation of the endogenous myogenin gene but did not activate the muscle-specific creatine kinase or cardiac alpha-actin gene. This result suggests that MyoD is not a "direct" activator of these downstream myogenic genes but must first activate myogenin as an intermediary. Once muscle is induced by estrogen receptor-MyoD the muscle phenotype is very stable and does not need the continued presence of estradiol for its maintenance.     

Inhibition of the activation of hageman factor (factor XII) by eosinophils and eosinophilic constituents

Several syndromes characterized by striking eosinophilia may be complicated by thrombosis. The experiments described indicate that, paradoxically, eosinophils and certain of their constituents inhibit the activation of Hageman factor (HF, factor XII). In earlier studies, suspensions of mixed types of granulocytes, other nucleated peripheral blood cells, and platelets inhibited activation of Hageman factor by ellagic acid, glass, and sulfatides. After these cells were sedimented by centrifugation, the supernatant fluids were also inhibitory. No attempt had been made earlier to distinguish among different granulocytic species. In the present study, suspensions of eosinophils and the supernatant fluid after eosinophils had been separated by centrifugation inhibited activation of Hageman factor by ellagic acid. The protein concentration of that amount of supernatant fluid that inhibited activation by about half was 16 μg/ml, approximately the same as had been described for suspensions of peripheral blood mononuclear cells. Activation of Hageman factor by ellagic acid was also inhibited by certain constituents of eosinophils, including eosinophil peroxidase, eosinophil major basic protein and eosinophil cationic protein. Inhibition was not specific for ellagic acid-induced activation of Hageman factor, as inhibition was also observed with sulfatide-induced activation. Inhibition was presumably related to neutralization of the negative charge of activators of Hageman factor. Thus, bismuth subgallate, a particulate activator of Hageman factor, was no longer effective after it had been exposed to eosinophil cationic protein. The observations reported here raise the question of whether in vivo eosinophils modulate certain of the defense reactions ascribed to Hageman factor. © 1993 Wiley-Liss, Inc.     

拉米夫定抑制乙型肝炎病毒X基因的转录与表达

目的 乙型肝炎病毒(HBV)X蛋白在HBV相关性肝癌的发生中有着重要作用,本研究旨在探讨拉米夫定对HBV X基因复制、转录及表达水平的影响。 方法 用聚合酶链反应法检测拉米夫定对转染X基因复制的影响,用逆转录-聚合酶链反应法检测拉米夫定对转染X基因mRNA的影响,用生物分子相互作用系统检测拉米夫定对X蛋白表达的影响,探讨了拉米夫定作用的量效和时效关系。 结果 拉米夫定组与对照组HBV X基因复制的目的基因条带吸光度(A)值分别是151.4±3.5和144.0±11.4,差异无统计学意义。4.36×104mol/L拉米夫定持续作用24 h能完全抑制X基因的转录,对照组与治疗组(16 h)目的条带的A值分别为243.9±9.0和133.2±7.8,P<0.01,其抑制效应随药物浓度增加和作用时间延长而增强。4.36×104mol/L拉米夫定持续作用16 h,使代表X蛋白表达的最大结合量值从353.3±15.9降至252.3±18.8,P<0.01。结论 拉米夫定不影响HepG2x中转染的X基因复制,但对X基因的转录和蛋白表达有明显抑制作用。     

Proteolytic processing of human coagulation factor IX by plasmin

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 microM) by 80% and the activity of F.IXa (4 microM) by 50% after 30 minutes at 37 degrees C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated-F. IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator-catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.     

Interaction of coagulation factor Xa with human platelets.

When human 125I-labeled Factor Xa is incubated with washed platelets, prothrombin, and Ca2+, a small amount of thrombin is formed which causes the platelet release reaction after a period of time that decreases as the Xa concentration is increased from 0.9 to 19 ng/ml. After a further lag period, the Xa binds reversibly to receptors on the platelet surface and rapid thrombin formation follows (3 units or 1 mug of thrombin formed per min per ng of Xa bound to 10(8) platelets). When platelets are treated with either htrombin (0.5 units/ml) or calcium ionophore A23187 prior to addition of Xa, binding begins immediately. Thrombin formation occurs at the platelet surface at rates that correlate with the amount of Xa bound. Dibutyryl cyclic AMP inhibits the release reaction, Xa binding, and rate of thrombin generation in parallel. The platelet Xa receptor is distinct from the previously described thrombin receptor and appears to be a protein because treatment of platelets with thrombin at 50 units/ml destroys Xa binding sites. The results suggest that specific receptors for Xa appear on the platelet surface after the release reaction occurs. The bound Xa catalyzes thrombin formation 1000-fold faster than does Xa added to reactions in which phospholipids are substituted for platelets.