Significant Procoagulant Activity Research Articles

Exploring the Hemostatic Effects of Platelet Lysate-Derived Vesicles: Insights from Mouse Models.

Platelet transfusion has various challenges, and platelet-derived extracellular vesicles have been reported to have more significant procoagulant activity than platelets themselves. Furthermore, platelet products derived from platelet-rich plasma and platelet lysates (PLs) have gained attention for their physiological activity and potential role as drug delivery vehicles owing to the properties of their membranes. We aimed to investigate the characteristics of the fractions isolated through ultracentrifugation from mouse-washed PLs and assess the potential clinical applications of these fractions as a therapeutic approach for bleeding conditions. We prepared PLs from C57BL/6 mouse-washed platelets and isolated three different fractions (20K-vesicles, 100K-vesicles, and PLwo-vesicles) using ultracentrifugation. There was a notable difference in particle size distribution between 20K-vesicles and 100K-vesicles, particularly in terms of the most frequent diameter. The 20K-vesicles exhibited procoagulant activity with concentration dependence, whereas PLwo-vesicles exhibited anticoagulant activity. PLwo-vesicles did not exhibit thrombin generation capacity, and the addition of PLwo-vesicles to Microparticle Free Plasma extended the time to initiate thrombin generation by 20K-vesicles and decreased the peak thrombin value. In a tail-snip bleeding assay, pre-administration of 20K-vesicles significantly shortened bleeding time. PL-derived 20K-vesicles exhibited highly potent procoagulant activity, making them potential alternatives to platelet transfusion.

Chemical Constituents and Coagulation Activity of Amygdalus persica L. Flowers

Amygdalus persica L., belongs to Rosaceae family, and its flowers are used as medicine and food. n-Butanol extract of A. persica flowers were isolated and purified with various column chromatographies, and the fourteen compounds, chlorogenic acid butyl ester (1), rutin (2), protocatechuic acid (3), caffeic acid (4), 5-O-coumarroylquinic acid methyl ester (5), kaempferol-3-O-neohesperidoside (6), quercetin-3-O-β-D-glucoside (7), 3,5-dicaffeoylquinic acid (8), quercetin-3-O-α-L-rhamnoside (9), 5-O-coumaroylquinic acid (10), kaempferol-3-O-α-L-rhamnoside (11), kaempferol-3-O-β-D-galactoside (12), D-glucitol (13), and multiflorin A (14), were identified by spectroscopic data and physical data. All the compounds except compound 2 were identified from A. persica flowers for the first time. The compounds were investigated for their coagulation activity by activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and bibrinogen (FIB) in vitro. The results of coagulation activity showed that rutin (2), caffeic acid (4), kaempferol-3-O-neohesperidoside (6), kaempferol-3-O-α-L-rhamnoside (11), and kaempferol-3-O-β-D-galactoside (12) exhibited significant procoagulant activity, while chlorogenic acid butyl ester (1) possessed anticoagulant activity in vitro.

Chemical Constituents and Coagulation Activity of Amygdalus persica L. Flowers

The ethyl acetate extract of Amygdalus persica L. flowers (family Rosaceae) was fractionated by silica gel and Sephadex LH-20 column chromatography, and, after recrystallization, oleanolic acid (1), ursolic acid (2), quercetin (3), kaempferol (4), hesperetin (5), naringenin (6), and kaempferol-3- O-glucoside (7) were isolated. Their identities were elucidated by spectral techniques and from their physicochemical properties. Compounds 3-7 were identified from A. persica flowers for the first time. All the compounds were evaluated for their procoagulant activity by determining their activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) in vitro. The coagulation activity results showed that oleanolic acid and ursolic acid could significantly prolong APTT and TT; quercetin could significantly shorten PT and TT; kaempferol and hesperetin could significantly shorten APTT, PT, and TT; and naringenin could significantly shorten APTT, PT, and TT and decrease the content of FIB compared with the blank group. All of the above revealed that quercetin, kaempferol, hesperetin and naringenin possessed significant procoagulant activity, while ursolic acid had anticoagulant activity in vitro.

Hemostatic Effect of Aminated Multiwalled Carbon Nanotubes/Oxidized Regenerated Cellulose Nanocomposites.

We have investigated the covalent conjugation of aminated multiwalled carbon nanotubes (MWCNTNH₂)s with Oxidized Regenerated Cellulose (ORC) in order to enhance the hemostatic effect. The MWCNT-NH₂s were prepared by functionalization of pristine MWCNTs (pMWCNTs) using amine groups. Neat ORC gauze and MWCNT-NH₂s were reacted using glutamic acid as cross linking bridge. We investigated an amination of pMWCNTs as well as the dispersion of MWCNT-NH₂s in the ORC gauze as matrix and their interfacial interactions by SEM and FT-IR. The results revealed that relatively strong interaction exists between aminated MWCNTs and the ORC macromolecules. The hydrophilicity test results in the significant increment of water uptake of MWCNT-NH₂s/ORC composites with increasing the concentration of MWCNT-NH₂s in composite. The in-vitro procoagulation test shows that the MWCNT-NH₂s/ORC gauzes have significant procoagulant activity. The hemostatic evaluation of MWCNT-NH₂s/ORC composites on rabbits shows that the aminated MWCNTs increase the rate of blood stopping and hence they decrease the blood loosing from injured sites. Hemostatic evaluation indicates that the MWCNT-NH₂s/ORC gauze has a valuable hemostatic performance. The products of platelets release reaction, activated platelets glycoprotein and activated clotting enzymes were increased simultaneously. The mechanism of the hemostasis for MWCNT-NH₂s/ORC gauze is discussed.

Coagulatory active constituents of Malus pumila Mill. flowers

BackgroundThe flowers of Malus pumila Mill (Rosaceae) is rich in resources, but lack of medicinal value research. Chemical constituents of the same family M. pumila have coagulatory activity. Considering the coagulatory activity could be beneficial for various cardiovascular diseases, the aim of this study is to evaluate coagulatory active constituents of M. pumila flowers.MethodsChemical constituents of M. pumila flowers were isolated by various column chromatographies, and their coagulatory activity were evaluated by activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) in vitro.ResultsNine compounds were isolated from M. pumila flowers, and identified as kaempferol-3-O-β-d-glucopyranoside (1), kaempferol-7-O-β-d-glucopyranoside (2), kaempferol-3-O-α-l-arabinofuranoside (3), phloridzin (4), kaempferol (5), phloretin (6), β-sitosterol (7), lupeol (8) and pyracanthoside (9). Compounds 1–9 were isolated from the flowers for the first time, compounds 1, 2 and 9 were isolated from the genus for the first time. Compound 2 could significantly shorten APTT, TT and PT, but significantly decrease the content of FIB. Compound 3 could shorten PT. Compound 4 could significantly shorten TT and PT, but significantly decrease the content of FIB. Compound 5 shortened APTT. Compound 6 and 7 could significantly shorten APTT and PT. Compound 9 was able to prolong TT and decrease the content of FIB, but shorten PT.ConclusionsCompounds 2–7 possessed significant procoagulant activity in vitro, compound 9 had anticoagulant activity in vitro, which showed coagulation potential of compounds from M. pumila flowers, as a new source of bioactive molecules for therapeutic purposes.

Collagen/aminated MWCNTs nanocomposites for biomedical applications

Collagen (COL) is a biopolymer that widely used for a number of medical treatments especially haemostatic applications. To enhance the medical performance of COL and explore an alternative approach, its modification using Carbon Nanotubes is considered. To treat a valuable cross linking and chemical bonding, aminated MWCNTs (MWCNT-NH2)s, which introduced the aromatic amine on the side wall of MWCNTs, were jointed to the COL sponges (COLSP)s. To explore the origin of this behaviour, dispersion of MWCNT-NH2s on COLSPs and their interfacial interactions were investigated by SEM, FT-IR and XPS. The analytical results show a relatively strong interaction existed between aminated MWCNTs and the sidewall of COLSPs. The hydrophilicity test results revealed significant increment in water uptake of MWCNT-NH2s/COL composites with increasing the concentration of MWCNT-NH2s until 13 wt%. Additionally, the addition of aminated MWCNTs decreases the coagulation time of anti coagulated rabbit blood, indicating that the MWCNT-NH2s/COLSPs have significant pro-coagulant activity. Due to agglomeration of MWCNT-NH2s in solution, also, the surface limitation of sponges, increment in concentration of MWCNT-NH2s up to 14 wt% is not adequate. This study promises that the MWCNT-NH2s cross-linked COL fibres present mechanical strength significantly higher than that of neat COL fibres.

Chemical constituents and coagulation activity of Agastache rugosa

BackgroundIn the Chinese traditional medicine, plant of Agastache rugosa (Fisch. & C.A. Mey.) Kuntze (A. rugosa) has been used to treat nausea, vomiting and dispel damp. However, currently, few reports about the chemical constituents, especially the non-volatile components of A. rugosa are available.MethodsThrough separation with various column chromatographies to elucidate the chemical constituents of A. rugosa, the biological activities of the major constituents were investigated. The extracts and main constituents of A. rugosa were evaluated for their anticoagulant effects by assaying the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) in vitro.ResultsSeven known compounds (namely compounds 1–7) were isolated from the aerial parts of A. rugosa. They were identified as methyl hexadecanoate (1), β-sitosterol (2), acacetin (3), ursolic acid (4), apigenin (5), protocatechuic acid (6) and tilianin (7), respectively. Compounds 1 and 6 were isolated from the genus Agastache for the first time, and compound 4 was obtained from the plants for the first time. The results showed that the extract of A. rugosa had a significant procoagulant activity by shortening the time of PT (P < 0.001) and increasing FIB content (P < 0.001), as compared with Vitamin K1. While its major constituents acacetin and tilianin exhibited significant anticoagulant activities by prolonging the times of PT, APTT, TT and reducing FIB content (P < 0.001), as compared with blank control group.ConclusionsThe total extract of A. rugosa possessed significant procoagulant activity, while its main components, acacetin and tilianin possessed significant anticoagulant activities. Further investigation should be pursued to find out the bioactivity components responsible for the procoagulant action of the plant.

Moringa oleifera Lam.: Protease activity against blood coagulation cascade

Background:The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR).Materials and Methods:Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates.Results:Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity.Conclusions:These findings support the traditional usage of M. oleifera extracts for wound healing.

Platelet activation in the pathogenesis of obesity and vascular disease

The pathogenesis of atherosclerotic disease is complex, and very topical, as clearly highlighted in a recent position paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis and Vascular Biology in this journal, which deals with atherosclerotic plaques and their ‘stabilisation’ per se (1). A number of associations have been observed between lipid abnormalities, markers of metabolic syndrome and platelet activation (1–5). In recent years, many studies have explored associations for platelet activation markers to gain insights on the pathogenesis of obesity and atherosclerotic disease (6–13). In this issue of Thrombosis and Haemo stasis, Csongradi et al. report on a large cross-sectional cohort study that explored relationships for platelet activation markers in obese subjects, including those with and without common co-morbidities for atherosclerotic disease (14). Key findings of their study were that platelet and plasma P-selectin levels, and the plasma levels of platelet microparticles, were associated with carotid intimal medial thickness and a variety of other vascular risk factors, including fasting plasma glucose, insulin levels and triglycerides (14). These observations imply that the extent of atherosclerotic disease, and the metabolic derangements associated with obesity and atherosclerotic disease, influence the generation of platelet microparticles. This possibility is supported by a recent study that showed weight loss-induced improvements in insulin resistance, among women with obesity, reduced markers of platelet activation (6). While association studies cannot determine causation, they are important to developing new hypotheses on the pathogenesis of atherosclerotic disease. Platelet microparticles have attracted much interest both experimentally and clinically (15–19). Indeed, given their significant procoagulant activity (18, 19), their elevation in pathogenic states could contribute to the progression of vascular disease and acute thrombotic events. Nonetheless, uncertainties remain about whether observed associations between elevated levels of platelet-derived microparticles with prothrombotic, metabolic and inflammatory parameters are correlative or causative, and how much platelet microparticle generation, and other aspects of platelet activation, contribute to the pathogenesis of atherosclerotic disease in obese individuals. The “independent” associations that Csongradi et al. found between platelet-derived microparticle levels with body mass index, and between soluble P-selectin and fibrinogen, and platelet P-selectin and both carotid intimal medial thickness and PAI-1 (14) provide further evidence that platelet activation is influenced by the complex metabolic and inflammatory parameters that participate in the pathogenesis of atherosclerotic disease. Despite these advances, uncertainties still remain about whether measuring platelet activation markers has a role in diagnostic or therapeutic decision making for obese and non-obese individuals who have or do not have evidence of atherosclerotic disease. This still remains an exciting and expanding field of research.

Procoagulant Properties of Bare and Highly PEGylated Vinyl-Modified Silica Nanoparticles

Undesired alterations of the blood clotting balance may follow the intravascular injection of nanotherapeutics/diagnostics. Here, we tested the procoagulant activity of synthetic amorphous silica (SAS) and organically modified silica (ORMOSIL) nanoparticles (NPs) and whether a high-density polyethylene glycol coating minimizes these effects. Hageman factor- and tissue factor-dependent activation of human blood/plasma coagulation, and binding to human monocytes, endothelial cells and platelets were quantified in vitro using naked and PEGylated ORMOSIL-NPs. Their effects were compared with those of SAS-NPs, present in many industrial products, and of poly(lactic-co-glycolic acid)- and small unilamellar vesicles-NPs, already approved for use in humans. Both SAS-NPs and ORMOSIL-NPS presented a significant procoagulant activity. However, highly PEGylated ORMOSIL-NPs were particularly averse to the interaction with the soluble factors and cellular elements that may lead to intravascular blood coagulation. Stealth, highly PEGylated ORMOSIL-NPs with a poor procoagulant activity can be used as starting blocks to design hemocompatible nanomedical-devices.

Formation of Procoagulant Platelet Derived Microparticles through Platelet GPIb-Interaction with Von Willebrand Factor under High Shear Stress.

We describe here novel findings of the mechanism of initial platelet contact with immobilized von Willebrand factor (VWF) under high shear stress and how this leads to the formation of procoagulant platelet derived microparticles. In a parallel plate perfusion chamber whole blood was perfused over multimeric VWF or dimeric VWF A1 domain at shear rates between 2,000 s−1 and 40,000 s−1. Platelet attachment to VWF always occurred through glycoprotein Ibα receptors located in discrete adhesion points (DAPs), i.e. few limited membrane areas of 0.05 to 0.1 μm2 that arrested the platelets on the surface. The ongoing flow translocated such anchored platelets downstream, thus pulling membrane tethers from the intact and unstimulated platelet. Tethers could remain connected with the platelet body or be eventually severed, which occurred preferentially at shear rates above 6,000 s−1. Depending on the length of the severed membrane fragment they represented either isolated tethers or microparticles (arrowheads; see Figure below), the latter defined by a diameter of 50 to 100 nanometers. The shear rate threshold for microparticle formation was between 6,000 s−1 and 10,000 s−1. Immuno-fluorescence and immuno-electron microscopy showed glycoprotein Ibα clustered in DAPs of microparticles and tethers, i.e. the contact sites with the surface immobilized VWF. The microparticles also exhibited tissue factors on their surface and showed significant procoagulant activity measured by thrombin generation. We propose that after GPIbα anchoring to VWF in flowing blood passive mechanical pulling of membrane from platelets may generate platelet derived microparticles that can potentially support thrombogenesis. [Display omitted]

DETERMINATION OF TUMOR CELL PROCOAGULANT ACTIVITY BY SONOCLOT™ ANALYSIS IN WHOLE BLOOD

Coagulation activation in cancer may be due to expression of procoagulant activity (PCA) by tumor cells. Some PCA activate coagulation, while others influence platelet aggregation. Thus, clotting time assessments of PCA may not reflect the potential for hypercoagulability. We therefore studied the effect of various malignant and non-malignant cells on whole blood coagulation using the Sonoclot™ Analyzer. Five human (HT29 colon, J82 bladder, MCF-7 breast, BT-474 breast and A375 malignant melanoma) and three rodent (MC28, WEHI-164 and Neuro2a) cell lines were used. Rat thymocytes and peritoneal macrophages and human endotoxin-stimulated mononuclear cells and umbilical vein endothelial cells (HUVEC) were used as non-malignant controls. All tumor cells markedly shortened the recalcification time of citrated blood and the most potent (HT29 and J82) also increased clot rate ( P<0.01). The breast cancer cells MCF-7 and BT-474 expressed only weak PCA and did not affect clotting rate. None of the non-malignant cells significantly affected clotting time or rate in whole blood. J82 and HT29 cells grown in serum-rich media shortened the recalcification time of both normal and FVII-deficient plasmas. However, cell grown in serum-free conditions had significantly less PCA in FVII-deficient plasma. We conclude that the Sonoclot™ Analyzer is useful for determining cellular PCA; significant PCA is a feature of malignant cells and cells grown in medium containing serum supplements cannot be reliably evaluated for PCA. Copyright © 1996 Elsevier Science Ltd

Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras.

Coagulation factor V, an integral component of the prothrombinase complex, possesses two C-type domains at the carboxyl-terminal end of the molecule. Homologous C-type domains are present in factor VIII as well as several non-coagulation proteins. Deletion of the second C-type domain of factor V results in the loss of procoagulant activity and the ability to bind phosphatidylserine. We now report the effect of substitution of all or a portion of the C2 domain of factor V with the corresponding regions of factor VIII or the human breast carcinoma protein BA46. Substitution of the entire domain with a heterologous C2 domain does not restore significant procoagulant activity, although smaller, exon-size substitutions do result in chimeras with partial activity (approximately 10% of factor Va). Using chimeras with partial substitutions, we determined that the amino-terminal region of the domain is involved in binding to phosphatidylserine. In contrast, the central region of the domain is not involved in phosphatidylserine binding, but an antibody binding at or near this site inhibits procoagulant activity, suggesting that this region is involved in a separate function. Lastly, the molecular basis for the light chain doublet, which is important in the expression of full procoagulant activity, is located within the carboxyl-terminal region of the C2 domain.

Extravascular coagulation and fibrinolysis in murine lung inflammation induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Diffuse pulmonary inflammation in interstitial lung diseases is associated with increased coagulation in the extravascular spaces of the lung. We hypothesized that conditions favoring coagulation over fibrinolysis in the lung are related to inflammation. Pulmonary coagulation and fibrinolysis were studied in two strains of mice susceptible or resistant to the development of lung inflammation in response to the mycobacterial cell wall glycolipid trehalose-6,6'-dimycolate (TDM). Susceptible animals treated with TDM intravenously develop well-organized collections of mononuclear cells in the lung parenchyma referred to as granulomas in this report. More granulomas were found in the susceptible ICR mice than in the resistant A/J mice after intravenous administration of TDM (7 +/- 1 granulomas/mm2 versus 1 +/- 0.3 granulomas/mm2, p = 0.005). Granuloma formation was associated with increased lung procoagulant activity (PCA) measured in bronchoalveolar lavage (BAL) cell lysates from susceptible mice. In contrast, TDM-resistant A/J mice challenged with TDM did not have a significant BAL cell PCA response, but expressed several-fold greater levels of lung BAL fluid plasminogen activator activity (PAA) than ICR mice. To examine the role of coagulation in the TDM pulmonary inflammatory response, susceptible C57Bl/10SnJ mice were anticoagulated by oral administration of warfarin prior to challenge of TDM; these mice developed fewer pulmonary granulomas than TDM-treated mice without warfarin treatment (2.6 +/- 0.5 granulomas/mm2 versus 6.5 +/- 0.8 granulomas/mm2, p < 0.001) but had similar BAL cell PCA and lung inflammatory changes as measured by lung weights and BAL cellularity.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of factor Xa in determining the procoagulant activity of whole-blood clots.

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.

INDUCTION AND REGULATION OF MONOCYTE PROCOAGULANT ACTIVITY

Monocyte (MO) procoagulant activity (PCA) is induced by various stimuli including allogeneic stimulation, immunocomplexes, and bacterial products. Antigen-antibody complex stimulation therefore represents a pathway for MO PCA induction. Activation of MO PCA has been demonstrated in immunocomplex disease and could represent a major pathology in transplanted immunocomplex disease patients. Stimulation of monocytes via their FcRI receptor has been demonstrated to induce TNF and PGE2. This report demonstrates that stimulation of the high-density FcRI receptor-bearing (FcRI+) MO by resetting with anti-Rh coated erythrocytes also induces significant PCA levels (P less than 0.001). Muramyl dipeptide (MDP), a Gram-positive bacterial cell wall analogue, further increased PCA levels in the FcRI stimulated MO subpopulation (P less than 0.003). Although increased PCA levels were also induced in the FcRI- MO subpopulation by MDP (P less than 0.003), the FcRI+ MO responded with much greater levels of PCA and PGE2 (P less than 0.001). Greater PCA levels in the FcRI-positive MO subpopulation may indicate that stimulation of MO through their FcRI represents a different pathway from allogenic PCA activation, which can be augmented by subsequent bacterial challenge. A novel inhibitory effect of IL-4 on MO PCA induction is also demonstrated. IL-4 downregulated MO PCA levels either after isolation stimulation (55 +/- 19%), FcRI stimulation (57 +/- 12%), or FcRI plus MDP stimulation (60 +/- 13%). PCA and PGE2 levels were concomitantly downregulated by IL-4 both in the FcRI-stimulated, FcRI+ and in the MDP-stimulated FcRI- MO subpopulations. Since indomethacin blocked MDP induced MO PGE2 production without affecting MO PCA levels, PGE2 production is not required for FcRI-stimulated PCA induction.

Disparate mechanisms of induction of procoagulant activity by live and inactivated bacteria and viruses

This study describes the dose response, time course, and lymphocyte requirements of procoagulant activity (PCA) induction following stimulation of thioglycolate-elicited BALB/c peritoneal macrophages with live and inactivated bacteria (Bacteroides fragilis, Escherichia coli, and Staphylococcus aureus) and murine hepatitis virus type 3 (MHV-3). Induction of PCA by MHV-3 was significantly more rapid and the maximal PCA achieved was significantly greater than by the three bacterial species studied. In relation to induction of PCA by bacteria, the PCA response was more rapid and of greater magnitude with S. aureus and E. coli than with B. fragilis. MHV-3 induced an augmented PCA response at all concentrations of virus studied in a dose-dependent fashion, whereas higher titers of live bacteria (greater than 10(7) CFU/ml) inhibited PCA, suggesting the production of an inhibitory factor. Significant PCA induction was observed when macrophages were incubated with bacteria or virus in the absence of lymphocytes. At low titers of B. fragilis (10(3) CFU/ml), addition of lymphocytes greatly augmented PCA production, whereas at higher titers (10(7) CFU/ml), the addition of lymphocytes only slightly augmented the PCA response. In contrast, MHV-3 induction of PCA was enhanced by the addition of lymphocytes at all concentrations of virus studied, suggesting a lymphocyte-dependent process. Heat-inactivated bacteria were as effective as live bacteria in inducing PCA, suggesting that induction of PCA by bacteria requires only a bacterial surface component. In contrast, UV-inactivated MHV-3 did not induce PCA, suggesting that viral replication is a necessary step in PCA induction. These results suggest that the cellular and metabolic requirements for induction of PCA differ among viral and bacterial pathogens and may partly explain their differences in pathogenicity.

Activation/inactivation of human factor V by plasmin

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.

Activation/Inactivation of Human Factor V by Plasmin

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.

Induction of monocyte-macrophage procoagulant activity by transformed cell lines.

Human peripheral blood monocytes and mature macrophages were found to produce significant procoagulant activity (PCA), identified as tissue factor, on exposure to a variety of human (K562, HL60, Raji) and murine (TU5, NS-1) transformed cell lines. The monocyte procoagulant response was vigorous, generating PCA to a level nearly comparable to the response to endotoxin, a known stimulant for monocyte PCA. The response was rapid and could be fully elicited, in a dose-dependent fashion, within 4 hr with HL60 and Raji cell lines and within 14 hr with K562, TU5, and NS-1 cells. The monocyte PCA-inducing activity was found to reside in the membrane fraction of transformed cells. Other transformed human (Laz 509, Laz 221, Laz 156, U937, CEM) and murine (L1210, P815, TLX9, WEHI 164) cell lines had little, if any, activity. The induction of monocyte PCA by transformed cells most probably was not due to an allogeneic signal, as 1) the K562 and HL60 cell lines were potent PCA inducers despite the lack of class II histocompatibility antigen expression, whereas Laz 156, which did express HLA antigens, was ineffective; 2) mouse peritoneal macrophages responded with the production of strong PCA to the syngeneic transformed cell lines TU5 and NS-1. The monocyte-macrophage procoagulant response to transformed cell lines appeared to be independent of T lymphocytes. Indeed, monocytes purified on the basis of reactivity with monoclonal antibody Mo2 and sorting or depleted of contaminating T cells by anti-T3 antibody and complement responded similarly to conventional monocyte preparations. The production of tissue factor by monocyte-macrophages in response to exposure to some tumor cells may represent a mechanism whereby blood coagulation is activated in malignancy.