Coagulant activity of leukocytes. Tissue factor activity.

Peritoneal leukocytes harvested from rabbits which have received two spaced doses of endotoxin have significantly greater (10-fold) coagulant activity than leukocytes from control rabbits. The coagulant activity accelerates the clotting of normal plasma and activates factor X in the presence of factor VII and calcium and is therefore regarded as tissue factor. A total of 40-80 mg tissue factor activity was obtained from the peritoneal cavity of single endotoxin-treated rabbits. In leukocyte subcellular fractions, separated by centrifugation, the specific tissue factor activity sedimented mainly at 14,500 g and above. The procoagulant activity was destroyed after heating for 10 min at 65 degrees C but was preserved at lower temperatures. Polymyxin B, when given with the first dose of endotoxin, reduced both the number of peritoneal leukocytes and their tissue factor activity by two-thirds. When given immediately before the second dose of endotoxin, polymyxin B had no inhibitory effect.

The stimulatory effect of platelets and platelet membranes on the procoagulant activity of leukocytes.

Leukocytes can generate procoagulant (tissue factor) activity when incubated with endotoxin. These studies were undertaken to determine whether platelets could influence the procoagulant activity generated by leukocytes. Intact or disrupted platelets (rabbit or human) enhanced the clot-promoting properties of rabbit leukocytes. The enhancing effect of human platelets on human leukocytes required the presence of human serum (devoid of factor VII and X activities). When platelets were incubated with endotoxin in the absence of leukocytes, no increase in their clot-promoting properties was discernible. However, a mixture of platelets, leukocytes, and endotoxin generated procoagulant activity which appeared rapidly and was fivefold greater than that produced by leukocytes incubated with endotoxin alone. The enhancement produced by platelets was even more pronounced if homogenates were used. The platelet effect was examined in more detail by the substitution of membranes, granules, and the "soluble" fraction for whole platelets in the test system. The stimulating activity was localized to the particulate fractions, i.e., membranes and granules. Prior treatment of platelet membranes with phospholipase C or gangliosides or by extraction of lipid resulted in loss of enhancing activity, whereas no inhibition was observed after exposure to neuraminidase or trypsin. It is proposed that platelets contribute a membrane lipoprotein surface which enhances the procoagulant activity generated by leukocytes in the presence of endotoxin. This mechanism may be involved in some of the clinical and pathologic manifestations of gram-negative sepsis with disseminated intravascular coagulation.

Enhancement of mononuclear procoagulant activity by platelet 12-hydroxyeicosatetraenoic acid.

Platelets induce generation of procoagulant tissue factor activity (TFa) by mononuclear leukocytes, and also enhance the TFa induced by endotoxin. Our present investigation demonstrated that arachidonic acid, which by itself had no effect on mononuclear TFa, greatly enhanced platelet-induced TFa. The effect was concentration dependent for both platelets and arachidonate (1-20 microM); other fatty acids tested were inactive. The enhancing effect of arachidonate was more pronounced if platelets were exposed to aspirin, suggesting lipoxygenase product involvement. Production of 12-hydroxyeicosatetraenoic acid (12-HETE) was demonstrated biochemically in aspirin-treated platelet/arachidonate/mononuclear cell preparations that generated high levels of TFa. The enhancing role of 12-HETE was verified as follows. Addition of platelet-derived or synthetic 12-HETE amplified endotoxin-induced TFa more than threefold. Other lipoxygenase products were inactive. Enhancement of mononuclear cell TFa by 12-HETE represents a newly described biological function for this eicosanoid in cell-cell interactions between platelets and mononuclear cells.

C5 chemotactic fragment induces leukocyte production of tissue factor activity: a link between complement and coagulation.

Complement-activated human plasma causes generation of tissue factor in human leukocytes. This phenomenon appears to be related to the fifth component of complement (C5) as demonstrated by the use of C5 deficient-plasma and suppression of activity with antibody to C5. Isolation of the chemotactic factor from activated serum or trypsinization of purified C5 reproduces the phenomenon. These data provide evidence for a direct link between complement products and activation of the coagulation system. Because chemotactic peptides from C5 can be generated by a variety of enzymes, our findings suggest a relationship between complement, coagulation, and inflammation.

Rapid generation of thrombin by atheroma and platelets.

Rapid re-occlusion of an atheromatous vessel after angioplasty may occur through yet incompletely known mechanisms. Atheromatous plaque has been shown to contain tissue factor (TF) activity. When atheroma extracts (atheroma) and platelets are incubated together a powerful prothrombinase is rapidly generated, which neither platelets nor atheroma alone can generate. Large amounts of thrombin were generated in minutes by many atheroma-platelet mixtures. However in these mixtures, generation of factor (F)Xa activity was not enhanced, but was in fact decreased by platelet tissue factor pathway inhibitor (TFPI) activity. Leukocytes had no appreciable effect in these short-term experiments. Although levels of factor VII and FX in atheroma were extremely low, antibodies to each of these factors inhibited prothrombinase formation. So did an antibody to factor V. A FXa inhibitor, DX 9065a, was very effective in preventing prothrombinase generation. These findings may explain the rapid occlusion that has been observed after angioplasty and point to avenues of prevention.

Purification of human brain tissue factor.

Tissue factor (thromboplastin or Factor III), a glycoprotein cofactor, is required for Factor VII to express its catalytic activity, thereby initiating the extrinsic as well as intrinsic pathway of blood coagulation. Human brain tissue factor was purified 2,500-fold to 98% homogeneity from 2% Triton X-100 extraction of acetone dried brain powder with an overall yield of 36%. The method was based upon affinity chromatography utilizing the high affinity binding of tissue factor to Factor VII noncovalently complexed to immobilized anti-Factor VII-agarose beads. The apparent molecular weight of the purified tissue factor is 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point is 4.8-5.1 by column chromatofocussing and flat bed agarose isoelectric focussing.

Human atheromatous plaque extracts induce tissue factor activity (TFa) in monocytes and also express constitutive TFa.

Tissue factor activity (TFa) is a major activator of the coagulation cascade and may play a role in atheroma-induced thrombosis. Monocyte-macrophages (MO-MF) generate considerable quantities of TFa when stimulated by a variety of inducers. To test the hypothesis that MO could be induced by atheromatous plaque to generate TFa, plaque extracts obtained from patients with obstructive atheromatous disease were used. These extracts were also assayed for constitutive TFa. The constitutive activity was variable from extract to extract but could be very high, up to 250 U TFa. The TFa induced in MO could be also very high, up to 200 U (i.e. 1/5 of the TFa of full strength rabbit brain thromboplastin). These findings point to a major role for MO-MF TFa in the induction or thrombosis by atheromatous plaque.

Studies on the anticoagulant action of Aspilia africana.

An anticoagulant activity was identified and isolated from the leaves of a West African plant, Aspilia africana by gel filtration on Sephadex G-100. The anticoagulant factor had an apparent molecular weight of approximately 60,000 d. Upon incubation with plasma, it prolonged the partial thromboplastin time, prothrombin time, thrombin and reptilase time. The factor decreased the fibrinogen content of plasma as well as the activity of coagulation factors V, VIII and IX but not factor VII, X or XI activities. After incubation with fibrinogen, the thrombin clotting time was prolonged and the quantity of clottable fibrinogen reduced. The action on fibrinogen was characterized by sequential lytic breakdown of the A-alpha-chain and B-beta-chain, the gamma-chain being lysed last, after prolonged incubation. Benzamidine, Epsilon aminocaproic acid or soybean trypsin inhibitor did not impede lysis.

A streptokinase dependent plasma factor (SKDF) induces leucocyte tissue factor activity.

Various inducers endow human leucocytes with a procoagulant activity of tissue factor type. We have observed a novel plasma factor which in combination with streptokinase induces powerful leucocyte procoagulant activity. This streptokinase dependent factor (SKDF) is present in normal plasma or serum albeit quantitatively different in individual donors. The generation of tissue factor activity as a function of streptokinase-plasma complex shows a specific and saturable sigmoidal dose-response curve. The Hill plot shows a straight line with Hill coefficient, H = 2.2, suggesting a strong positive cooperativity for the binding of this streptokinase-plasma complex to the leucocyte surface receptor for the signal transduction leading to the biosynthesis of tissue factor apoprotein. It also suggests that the leucocyte surface receptor for streptokinase-plasma complex differs from that for endotoxin lipopolysaccharides. SKDF is of apparent high molecular weight. It does not appear to be an antibody to streptokinase since its level does not correlate with the level of antibodies to streptokinase, and it does not correlate with the antistreptolysin titre. Furthermore, SKDF does not bind to protein A. It has a narrow pH range of stability, and is destroyed at 56 degrees C, or at freeze-drying, Urokinase, another plasminogen activator, or plasmin were unable to activate SKDF to induce the leucocyte procoagulant activity. SKDF may play a role in thrombolytic therapy.

Lipid A as the biologically active moiety in bacterial endotoxin (LPS)-initiated generation of procoagulant activity by peripheral blood leukocytes.

Preparations of rabbit or human leukocytes, when incubated with bacterial endotoxins (lipopolysaccharides, LPS) are stimulated to generate a procoagulant-tissue factor activity (TFa). As LPS has been shown to consist of specific repeating oligosaccharide side chains (O-antigen) linked to a central polysaccharide core region that is, in turn, linked to the lipid region of the molecule (lipid A), we have examined the biochemical requirement of the LPS necessary for generation of TFa. Using preparations of LPS from mutant strains of bacteria, which contain varying amounts of polysaccharide in relation to lipid A, we have demonstrated that activity is associated with the lipid A region of the LPS molecule. These observations have been confirmed using isolated lipid A, which is a potent stimulator of TFa, as well as a native protoplasmic polysaccharide that is both devoid of lipid A and without detectable TFa stimulatory activity. Modification of LPS by treatment with mild alkali abrogated its capacity to stimulate TFa generation. In addition, such altered preparations of LPS partially inhibit the stimulatory effect of native LPS. Similarly, treatment of LPS (or lipid A) with the antibiotic polymyxin B substantially inhibited the stimulatory effect of LPS.

Trial of heparin and cyclophosphamide (NSC-26271) in the treatment of lung cancer.

Preliminary reports have suggested that heparin may enhance the effectiveness of chemotherapy for lung cancer. The VALCSG initiated a study to evaluate further the efficacy and feasibility of administering a heparin and chemotherapy regimen. Treatment consisted of continuous iv heparin infusion for 48--72 hours followed by iv administration of cyclophosphamide (1100 mg/m2) and continued treatment with heparin and warfarin. Only one of the 19 patients (5%) with nonresectable or metastatic lung cancer showed evidence of objective tumor response. This indicated no benefit from the addition of heparin. The study also revealed some of the limitations and problems of such a treatment program.

Factors influencing the release of procoagulant-tissue factor activity from leukocytes.

Leukocytes stimulated with endotoxin generate a procoagulant (tissue factor) activity. Part of this procoagulant activity is released rapidly into the surrounding milieu, with maximum release occurring within 30 min incubation. Optimal release of procoagulant activity requires the presence of Ca ions in a concentration of 2.5 mM, a pH of 7-8, and a temperature of 37 degrees C. The released procoagulant activity is TFa as determined by the two-stage assay and the ability of an antibody to tissue factor to neutralize the procoagulant activity. The release of TFa is not parallel to LDH release. Release of TFa is inhibited by vinblastine and cytochalasin B.