Activation of coagulation releases endothelial cell mitogens

Recent studies have indicated that endothelial cell function includes elaboration of growth factors and regulation of coagulation. In this paper we demonstrate that activated coagulation Factor X (Factor Xa), a product of the coagulation mechanism generated before thrombin, induces enhanced release of endothelial cell mitogens, linking these two functions. Mitogenic activity generated by cultured bovine aortic endothelial cells in response to Factor Xa included platelet-derived growth-factor-like molecules based on a radioreceptor assay. Effective induction of mitogens by Factor Xa required the integrity of the enzyme's active center and the presence of the gamma-carboxyglutamic acid-containing domain of the molecule. Factor Xa-induced release of mitogens from endothelium occurred in serum-free medium and was not altered by hirudin or antibody to Factor V, indicating that it was a direct effect of Factor Xa and was not mediated by thrombin. Elaboration of mitogenic activity required only brief contact between Factor Xa and endothelium, and occurred in a time-dependent manner. Generation of enhanced mitogenic activity in response to Factor Xa was unaffected by the presence of actinomycin D and was not associated with increased hybridization of RNA from treated cells to a v-sis probe. Release of mitogenic activity was dependent on the dose of Factor Xa, being half-maximal at 0.5 nM and reaching a maximum by 5 nM. Radioligand binding studies demonstrated a class of endothelial cell sites half-maximally occupied at a Factor Xa concentration of 0.8 nM. The close correspondence between the parameters of Factor Xa-induced mitogen release and Factor Xa binding suggests these sites may be related. When Factor X was activated on the endothelial cell surface by Factors IXa and VIII, the Factor Xa formed resulted in the induction of enhanced release of mitogenic activity. These data suggest a mechanism by which the coagulation system can locally regulate endothelial cell function and vessel wall biology before thrombin-induced release of growth factors from platelets.     

Activated platelets but not endothelial cells participate in the initiation of the consolidation phase of blood coagulation

To address the question of whether initiation of the consolidation phase of coagulation occurs on platelets or on endothelium, we have examined the interaction of coagulation factor XI with human umbilical vein endothelial cells (HUVEC) and with platelets. In microtiter wells factor XI binds to more sites in the absence of HUVEC (1.8 x 10(10) sites/well, K(D) = 2.6 nm) than in their presence (1.3 x 10(10) sites/well, K(D) = 12 nm) when high molecular weight kininogen (HK) and zinc are present. Binding was volume-dependent and abrogated by HUVEC or Chinese hamster ovary cells and was a function of nonspecific binding of HK to the artificial plastic surface. Factor XI did not bind to HUVEC or to HEK293 cell monolayers anchored to microcarrier beads. Activation of HUVEC resulted in von Willebrand's factor secretion, but factor XI binding was not observed. Only activated platelets supported factor XI binding in the presence of HK and zinc (K(D) = 8 nm, B(max) = 1319 sites/cell). Activation of factor XI was observed in plasma in the presence of platelets activated by the thrombin receptor activation peptide but not with activated HUVEC. These results support the concept that activated platelets, but not endothelial cells, expose a procoagulant surface for binding and activating factor XI, thereby initiating the consolidation phase of coagulation.     

Characterization of the interaction between factor Xa and bovine aortic endothelial cells

Cultured bovine aortic endothelial cells incubated with Factor Xa activate prothrombin. Factor V, synthesized by the endothelial cells, or plasma Factor V and calcium are required for the reaction. In the present study, it has been demonstrated that 125I-Factor Xa binds specifically to endothelial cells. In addition, the activation of prothrombin by Factor Xa and aortic endothelial cells has been further characterized. The binding of 125I-Factor Xa to endothelial cells was saturable and reversible. The equilibrium dissociation constant (Kd) for 125I-Factor Xa binding was 3.6 X 10(-9) M, with 39000 molecules bound per cell. 125I-Factor Xa, inactivated by diisopropylfluorophosphate did not bind specifically to endothelial cells, indicating that the active site of Factor Xa was required for binding. Factor Xa, but not activated protein C, competed with 125I-Factor Xa for binding. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of cell lysates indicated that the radiolabeled material that bound to the cells had electrophoretic mobility identical to Factors Xa alpha and Xa beta. Although Factor X partially inhibited the binding of 125I-Factor Xa, Factor Xa did not inhibit the binding of 125I-Factor X, indicating that the zymogen and enzyme bound to different receptors. The relationship of the 125I-Factor Xa binding which was measured in these studies to aortic endothelial cell prothrombin activation is unclear since an anti-Factor V IgG blocked prothrombin activation but not Factor Xa binding. Additionally, 125I-Factor Xa binds to nonvascular cells; these cells do not activate prothrombin in the presence of Factor Xa. Moreover, the calcium requirements for each reaction and the saturation curves of 125I-Factor Xa binding and prothrombin activation differ. Although these data do not exclude a relationship between Factor Xa binding and prothrombin activation, the binding of 125I-Factor Xa to aortic endothelium measured in these studies may be related to a separate cellular function. To further characterize prothrombin activation by Factor Xa and endothelial cells, the rates of thrombin generation by intact bovine aorta or endothelial cells derived from this tissue were compared and were found to be equivalent. These data indicate that vascular endothelium may serve as a physiologic surface for hemostasis.     

Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.     

A pathway of coagulation on endothelial cells

Although the endothelial cell is considered antithrombogenic, endothelium has recently been shown to participate in procoagulant reactions. Factor IX bound to specific endothelial cell sites can be activated by the intrinsic and extrinsic pathways of coagulation. Perturbation of endothelium results in induction of tissue factor which promotes factor VIIa-mediated activation of factors IX and X, thus initiating procoagulant events on the endothelial surface. Cell bound factor IXa, in the presence of factor VIII, promotes activation of factor X. The factor Xa formed can interact with endothelial cell factor V/Va, resulting in prothrombin activation. Thrombin then cleaves fibrinogen and a fibrin clot closely associated with the endothelial cell forms. The perturbed endothelial cell thus provides a focus of localized procoagulant events. This model suggests a simple endothelial-cell-dependent mechanism for initiation of coagulation at the site of an injured or pathological vessel.     

Functional prothrombinase complex assembly on isolated monocytes and lymphocytes

Isolated peripheral blood monocytes and lymphocytes interact with Factor Va and Factor Xa to form a functional catalytic complex which proteolytically activates prothrombin to thrombin. The kinetics of prothrombin activation were monitored continuously using the fluorescent, reversible thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, which displays enhanced fluorescence upon binding to thrombin. Incubation of monocytes or lymphocytes with prothrombin, the cofactor (Factor Va), and the enzyme (Factor Xa) in the presence of Ca2+ generated thrombin at rates/cell exceeding those previously obtained with either bovine or human platelets. The rate of thrombin generation by monocytes exceeded that of lymphocytes and increased as monocytes adhered to a surface. Monocyte prothrombinase activity appears to be mediated through interactions, whereby Factor Va forms a receptor for Factor Xa at the monocyte surface. Monocytes possess approximately 16,100 Factor Va binding sites with a dissociation constant (Kd) of 4 X 10(-11) M. In addition, isolated, well washed monocytes and lymphocytes, respectively, contain approximately 61,400 +/- 9,900 and 24,500 +/- 4,800 molecules of Factor V/cell as determined by radioimmunoassay. Bioassay data of mononuclear cell preparations paralleled the radioimmunoassay data. The Factor V associated with washed mononuclear cells appears to be intracellular and not membrane-associated. The release of Factor V, and perhaps other sequestered coagulation factors, by these immunoreactive cells at an inflammatory site, coupled with the ability of these cells to effect thrombin generation may explain the relationship between extravascular fibrin deposition and mononuclear cell accumulation in the pathogenesis of inflammatory lesions.     

The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure.

Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC extraction, poly(ethylene glycol) precipitation, and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000-fold relative to the starting plasma. The apparent molecular weight of the purified factor V was 439,000 +/- 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 239,000; alpha-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.     

Structural studies on the murine granulocyte colony-stimulating factor

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein hemopoietic growth factor which regulates the production of granulocytes and macrophages. Reversed-phase microbore high-performance liquid chromatography was employed to purify a number of tryptic and Staphylococcus aureus V8 proteinase peptides generated from approximately 400 pmol G-CSF purified from medium conditioned by lungs from mice previously injected with endotoxin. N-Terminal amino-acid sequence analyses were performed on the parent polypeptide and on four tryptic peptides and one Staphylococcus aureus V8 protease peptide, yielding 68 unique amino-acid assignments; this corresponds to approximately 38% of the molecule.     

Interaction of factor VIII-von Willebrand Factor with phospholipid vesicles.

The interaction of Factor VIII-von Willebrand Factor with phospholipid vesicles has been studied by using sucrose-density-gradient ultracentrifugation. When purified Factor VIII-von Willebrand Factor was run alone. Factor VIII activity and Factor VIIIR-Ag sedimented together to the lower half of the tube. Addition of phosphatidylserine/phosphatidylethanolamine vesicles at concentrations above 250 microgram/ml resulted in complete separation of Factor VIII activity and Factor VIIIR-Ag, the former appearing with the phospholipid on the top of the tube and the latter sedimenting as before. This separation was obtained even in the presence of proteinase inhibitors. Activation of Factor VIII-von Willebrand Factor by thrombin resulted in formation of a slow sedimenting component containing essentially all the Factor VIII activity, whereas the Factor VIIIR-Ag sedimented towards the bottom of the tube as before. The thrombin-induced Factor VIII activity was strongly bound to phospholipid vesicles as determined by density-gradient centrifugations at various Factor VIII concentrations and low concentrations of phospholipid. Based on certain assumptions a dissociation constant of 2.5 nM was calculated, a mechanism for the formation in vivo of the Factor X-activator complex is suggested.     

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha(2-8) polysialic acid. 1) Amino-acid sequence of the light (L-) chain, kappa-isotype

Here we report the complete amino-acid sequence of the anti-alpha(2-8)polysialic acid antibody mAb735 light chain. The sequence was determined after digestion of the reduced and carboxymethylated L-chain with trypsin, SV-8 proteinase, and Asp-N proteinase, isolation of the generated peptides by RP-HPLC and characterization of these fragments by sequence analysis, amino-acid analysis and/or plasma desorption mass spectrometry. According to Kabat et al. the variable region belongs to the V kappa-II subgroup, whilst according to Hum et al. it belongs to the V kappa-1B subgroup. With the exception of proline at position 46, the sequence from position 1 to 95 is identical to the translated DNA sequence of a V kappa-germline gene segment previously reported.     

Identification of a factor IX/IXa binding protein on the endothelial cell surface

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.     

Immunohistochemical studies of human tissues with antibody to factor Xa

Summary Factor Xa is a serine proteinase which functions principally at coagulation cascades. Factor Xa-like immunoreactivity has been examined in several human organs. Antibodies to the Factor stained macrophages in some tissues examined, including microglia in the brain white matter. They also stained epithelial cells in the nose, bronchus and duodenum. Some brainstem neurons, such as those in the oculomotor nucleus, substantia nigra and pontine nucleus, were also positive for the Factor. As reported by others, these results suggest that factor Xa may have pleiotrophic functions. Furthermore, the prefential localization to epithelium in the nose and bronchus is interesting in view of the previous notion that several viruses targeting the respiratory tract require factor Xa-like cellular proteinases for their replication and spread.     

A prothrombinase complex of mouse peritoneal macrophages

Addition of prothrombin to mouse peritoneal macrophages in vitro resulted in the formation of a thrombin-like enzyme, as demonstrated by use of the luminogenic peptide substrate S-2621. The prothrombinase activity was sedimented by high-speed centrifugation following homogenization of the cells and was abolished by treatment of the cells with the nonionic detergent Triton X-100 at 0.02% concentration. Moreover, the activity was drastically reduced by maintaining cultures in the presence of warfarin and, presumably due to competitive substrate inhibition, by adding S-2222, a chromogenic peptide substrate for Factor Xa. These findings suggest that prothrombin cleavage is catalyzed by Factor Xa at the macrophage surface. The generated thrombin was inhibited by antithrombin, and this reaction was accelerated by heparin with high affinity for antithrombin but not by the corresponding oligosaccharides composed of 8-14 monosaccharide units. Such oligosaccharides which are capable of accelerating the inactivation of Factor Xa by antithrombin, inhibited thrombin formation from prothrombin in the macrophage cultures, presumably by promoting inactivation by antithrombin of Factor Xa in a prothrombinase complex. Activation of the macrophage coagulation system, as proposed to occur in certain inflammatory conditions, thus may be modulated at various levels by heparin, or heparin oligosaccharides, released from mast cells.     

Vascular smooth muscle cells synthesize,secrete and express coagulation factor V

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [³⁵S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor X_a-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.     

Effect of FXIII on monocyte and fibroblast function.

Factor XIII is a plasma transglutaminase that participates in the final stage of the coagulation cascade. Thrombin-activated FXIII (FXIIIa) catalyzes the formation of covalent crosslinks between gamma-glutamyl and epsilon-lysyl residues on fibrin molecules to yield the mature clot. In addition to its role in hemostasis, FXIIIa was previously shown by us to stimulate endothelial cells to exhibit pro-angiogenic activity. In this work, we studied the effect of FXIIIa on other cells that participate in angiogenesis and tissue repair, such as monocytes and fibroblasts. FXIIIa significantly enhanced migration and proliferation, and inhibited apoptosis of monocytes and fibroblasts. Similar to our previous observations with endothelial cells, the stimulating effect of FXIIIa on monocytes and fibroblasts was elicited via its binding to alpha (v)beta (3) integrin leading to cJun upregulation and TSP-1 downregulation. Since monocytes and fibroblasts are essential components of the tissue repair process, the results of this study, together with the proangiogenic activity of FXIIIa, further substantiate a significant role of FXIII in tissue repair.     

Activation of the coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells and their extracellular matrix. The role of flow and factor IX/IXa

Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR1). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR1-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR1-specific agonists and inhibitors were used to demonstrate that PAR1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR1 and not PAR2. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.     

Antibody and immune complexes induce tissue factor production by human endothelial cells

Patients with systemic lupus erythematosus (SLE) have an increased incidence of arterial and venous thromboses. The mechanism by which thromboses develop in these patients is unknown. We had previously observed that the sera of patients with SLE contain antibodies and immune complexes that can bind to endothelial cells. Because endothelial cells can synthesize tissue factor, a potent activator of coagulation, we studied the effect of IgG complexes and sera from patients with SLE on the production of tissue factor by these cells. Human umbilical venous endothelial cells incubated with heat-aggregated IgG (HA-IgG) (0.5 to 4.0 mg) elaborate procoagulant activity in a dose-dependent manner. All procoagulant activity was found in the particulate cell fraction, and none was secreted into the medium. Maximum expression of procoagulant activity required 6 to 8 hr, and its production was totally inhibited by the addition of cyclohexamide or actinomycin D. The presence of gel-filtered platelets augmented production of procoagulant activity by endothelial cells stimulated by HA-IgG. Endothelial cell procoagulant activity was not inactivated by diisofluoropropylphosphate, required the presence of Factor VII for its expression, and was neutralized by a specific anti-tissue factor antibody. Endothelial cells incubated with sera from 14 of 16 patients with SLE produced increased amounts of tissue factor compared with 21 normal sera (p less than 0.025). Fractions of two SLE sera containing monomeric IgG, IgA, or IgM, as well as fractions containing IgG complexes, each stimulated endothelial cells to produce more tissue factor than similar fractions prepared from two normal sera. These studies demonstrate that endothelial cells will produce the procoagulant tissue factor after exposure to anti-endothelial cell antibodies or IgG-containing immune complexes. The production of tissue factor by endothelial cells at sites of immune vascular injury may play a role in the development of thromboses in patients with SLE.     

Cyclic peptide analogs of 558–565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.