Expression Of Procoagulant Activity Research Articles

Emicizumab promotes factor Xa generation on endothelial cells

BackgroundUntil recently, the treatment of hemophilia A relied on factor (F)VIII replacement. However, up to one-third of patients with severe hemophilia A develop neutralizing alloantibodies that render replacement therapies ineffective. The development of emicizumab, a bispecific antibody that partially mimics FVIIIa, has revolutionized the treatment of these patients. However, the use of an activated prothrombin complex concentrate [FEIBA (Takeda)] to treat breakthrough bleeding in patients on emicizumab has been associated with thrombotic complications including a unique microangiopathy. ObjectivesWe hypothesized that the thrombotic complications observed with the combination of emicizumab and FEIBA might be due to excessive expression of procoagulant activity on the surface of endothelial cells. MethodsWe examined the ability of emicizumab to promote FX activation on endothelial cells using 2 cell culture models. ResultsWe found that endothelial cells readily support emicizumab-mediated activation of FX by FIXa. The level of FXa generation depends on the concentration of available FIXa. The addition of FEIBA to emicizumab increased FXa generation in a dose-dependent manner on endothelial cells in both models. The rate of FXa generation was further enhanced by endothelial cell activation. However, unlike emicizumab, we found limited FXa generation in the presence of FVIII(a), which followed a significant lag time and was not dependent on FIXa concentration under these conditions. ConclusionEmicizumab promotes FXa generation on the surface of endothelial cells, which is markedly enhanced in the presence of FEIBA. These findings demonstrate a potential mechanism for the thrombotic complications seen with the combined use of emicizumab and FEIBA.

Keypoints in Cells and Atherogenesis

Endothelial dysfunction is the initial event in the atherogenic process, resulting from several events, such as expression of leukocyte binding sites, production of growth factors, chemotactic and vasoreactive molecules, ability to oxidize low-density lipoprotein (LDL) and respond to oxidized lipoproteins, ability to express procoagulant activity, and modulation of vascular permeability. Thus, the endothelium, when subjected to different conditions and factors, plays an active role in the development of atherosclerotic plaque. The expression of several adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), E-selectin, vascular cell adhesion molecule-1 (VCAM-1), P-selectin in atherosclerotic plaques, mediating the interaction between endothelial cells and leukocytes, has been described. The modification of LDLs, in conjunction with other chemotactic factors produced by injured endothelial cells, recruit circulating monocytes into the subendothelial space, where they become macrophages. The proliferation of smooth muscle cells is an important event in the progression of arterial injury, occurring in 3 stages: smooth muscle cell replication still within the media layer; migration from the middle layer to the intima and proliferation within the intima. Apoptotic cells were evidenced in the atherosclerotic lesion, suggesting that apoptosis is part of the normal vascular healing process. The final stage of atherosclerotic lesion development is conversion of the fibrotic lesion to an advanced lesion, a lesion in which a thrombus forms as a result of plaque ulceration or intraplaque hemorrhage.

57-Year-Old Woman With Weakness and Word-Finding Difficulties

57-Year-Old Woman With Weakness and Word-Finding Difficulties

Thrombogenic Risk Induced by Intravascular Mesenchymal Stem Cell Therapy: Current Status and Future Perspectives

Mesenchymal stem cells (MSCs) are currently studied and used in numerous clinical trials. Nevertheless, some concerns have been raised regarding the safety of these infusions and the thrombogenic risk they induce. MSCs express procoagulant activity (PCA) linked to the expression of tissue factor (TF) that, when in contact with blood, initiates coagulation. Some even describe a dual activation of both the coagulation and the complement pathway, called Instant Blood-Mediated Inflammatory Reaction (IBMIR), explaining the disappointing results and low engraftment rates in clinical trials. However, nowadays, different approaches to modulate the PCA of MSCs and thus control the thrombogenic risk after cell infusion are being studied. This review summarizes both in vitro and in vivo studies on the PCA of MSC of various origins. It further emphasizes the crucial role of TF linked to the PCA of MSCs. Furthermore, optimization of MSC therapy protocols using different methods to control the PCA of MSCs are described.

Clinical Cellular Therapeutics Accelerate Clot Formation.

Clinical cellular therapeutics (CCTs) have shown preliminary efficacy in reducing inflammation after trauma, preserving cardiac function after myocardial infarction, and improving functional recovery after stroke. However, most clinically available cell lines express tissue factor (TF) which stimulates coagulation. We sought to define the degree of procoagulant activity of CCTs as related to TF expression. CCT samples from bone marrow, adipose, amniotic fluid, umbilical cord, multi‐potent adult progenitor cell donors, and bone marrow mononuclear cells were tested. TF expression and phenotype were quantified using flow cytometry. Procoagulant activity of the CCTs was measured in vitro with thromboelastography and calibrated thrombogram. Fluorescence‐activated cell sorting (FACS) separated samples into high‐ and low‐TF expressing populations to isolate the contribution of TF to coagulation. A TF neutralizing antibody was incubated with samples to demonstrate loss of procoagulant function. All CCTs tested expressed procoagulant activity that correlated with expression of tissue factor. Time to clot and thrombin formation decreased with increasing TF expression. High‐TF expressing cells decreased clotting time more than low‐TF expressing cells when isolated from a single donor using FACS. A TF neutralizing antibody restored clotting time to control values in some, but not all, CCT samples. CCTs demonstrate wide variability in procoagulant activity related to TF expression. Time to clot and thrombin formation decreases as TF load increases and this procoagulant effect is neutralized by a TF blocking antibody. Clinical trials using CCTs are in progress and TF expression may emerge as a safety release criterion. Stem Cells Translational Medicine 2018;7:731–739

Agonist-induced platelet procoagulant activity requires shear and a Rac1-dependent signaling mechanism

AbstractActivated platelets facilitate blood coagulation by exposing phosphatidylserine (PS) and releasing microvesicles (MVs). However, the potent physiological agonists thrombin and collagen poorly induce PS exposure when a single agonist is used. To obtain a greater procoagulant response, thrombin is commonly used in combination with glycoprotein VI agonists. However, even under these conditions, only a percentage of platelets express procoagulant activity. To date, it remains unclear why platelets poorly expose PS even when stimulated with multiple agonists and what the signaling pathways are of soluble agonist-induced platelet procoagulant activity. Here we show that physiological levels of shear present in blood significantly enhance agonist-induced platelet PS exposure and MV release, enabling low doses of a single agonist to induce full-scale platelet procoagulant activity. PS exposed on the platelet surface was immediately released as MVs, revealing a tight coupling between the 2 processes under shear. Using platelet-specific Rac1−/− mice, we discovered that Rac1 plays a common role in mediating the low-dose agonist-induced procoagulant response independent of platelet aggregation, secretion, and the apoptosis pathway. Platelet-specific Rac1 function was not only important for coagulation in vitro but also for fibrin accumulation in vivo following laser-induced arteriolar injury.

Decreased procoagulant phospholipids in patients treated by vitamin K antagonists

IntroductionThe stimulation of cells by thrombin is associated with the release of microparticles (MPs) from cell membranes. These MPs can express procoagulant activity. As vitamin K antagonists (VKA) decrease the generation of thrombin, we compared plasma procoagulant phospholipids (PPL) levels in patients with a previous history of venous thrombosis who were being treated with VKA and compared them with an untreated group. Materials and methodsPlasma PPL were measured using a factor Xa-based coagulation assay. sGPV, a marker of platelet activation by thrombin, was measured by ELISA. Platelet MPs were also evaluated using standard flow cytometric techniques. Ninety-six VKA-treated patients and 80 patients not undergoing VKA therapy were tested and the results compared. ResultsPPL activity was significantly reduced (p<0.0001) in VKA-treated patients compared with the untreated group. PPL were correlated with platelet and white blood cell count and with sGPV levels in the untreated group, but not in VKA-treated patients. PPL were correlated with fibrinogen levels in both groups, but not with C-reactive protein. Polymorphonuclear neutrophils (PMN) were significantly lower (p=0.01) in VKA-treated patients than in untreated patients. ConclusionThe difference between PPL levels in VKA-treated patients and patients without treatment could be related to the decrease in PMN count. It remains to be established if this decrease of PPL is directly related to the capacity of activated PMN to generate MPs, or indirectly by reducing the amount of pro-inflammatory cytokines or reactive oxygen species produced by PMN.

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography

Thrombin, the final enzyme of blood coagulation, is a multifunctional serine protease also involved in the progression of cancer. Tumor cells may activate blood coagulation proteases through the expression of procoagulant activities. However, specific information about the thrombin generation potential of malignant tissues is lacking. In this study we applied a single global coagulation test, the calibrated automated thrombogram assay, to characterize the specific procoagulant phenotypes of different tumor cells. Malignant hematologic cells (i.e. NB4, HEL, and K562) or solid tumor cells (i.e. MCF-7 breast cancer and H69 small cell lung cells) were selected for the study. The calibrated automated thrombo-gram assay was performed in normal plasma and in plasma samples selectively deficient in factor VII, XII, IX or X, in the absence or presence of a specific anti-tissue factor antibody. Furthermore, cell tissue factor levels were characterized by measuring antigen, activity and mRNA expression. In normal plasma, NB4 induced the highest thrombin generation, followed by MCF-7, H69, HEL, and K562 cells. The anti-tissue factor antibody, as well as deficiencies of factors VII, IX and XII affected the thrombin generation potential of malignant cells to different degrees, allowing differentiation of the two different pathways of blood clotting activation - by tissue factor or contact activation. The thrombin generation capacity of NB4 and MCF-7 cells was tissue factor-dependent, as it was highly sensitive to inhibition by anti-tissue factor antibody and factor VII deficiency, while the thrombin generation capacity of H69, HEL and K562 was contact activation-dependent, as no thrombin was generated by these cells in factor XII-deficient plasma. This study demonstrates that the calibrated automated thrombogram assay is capable of quantifying, characterizing, and comparing the thrombin generation capacity of different tumor cells. This provides a useful tool for understanding the key factors determining the global pro-coagulant profile of tumors, which is important for addressing specific targeted therapy for the prevention of thrombosis and for cancer.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

AbstractEffective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

Allosteric activation of coagulation factor VIIa

Coagulation factor VIIa (FVIIa) is present at subnanomolar concentration and represents a small percentage of the total amount of FVII in the circulation. FVIIa is poised to initiate blood clotting when it encounters its pivotal cofactor tissue factor (TF) which becomes exposed to blood upon vascular rupture. The requirement for complex formation with TF in order for FVIIa to express procoagulant activity ensures thrombin and fibrin generation at the right time and place. Thus TF acts as a guardian of safety of paramount importance to blood coagulation by providing localization to the site of injury and at the same time inducing maturation of zymogen-like free FVIIa to the active cofactor-bound enzyme. This review gives an account of the accumulated knowledge about the structure, function and TF dependence of FVIIa to arrive at a plausible allosteric mechanism by which TF induces maturation of the active conformation of FVIIa.

Conserved Structural Motifs Cooperate to Maintain Blood Coagulation Factor V in An Inactive Procofactor State.

Abstract 850Blood coagulation factor V (FV) is a multi-domain protein which circulates as an inactive procofactor and has high structural homology with factor VIII. To express procoagulant activity, FV must be proteolytically processed within its central B-domain (836 residues) with thrombin being considered the key physiological activator. Following liberation of the B-domain (residues 710-1545), activated FV (FVa) functions as a cofactor for factor Xa within the prothrombinase complex and dramatically enhances the rate of thrombin generation. The central role which FVa assumes in prothrombinase indicates that its activation must be a key regulatory step in hemostasis. Although the proteolytic events that lead to the activation of FV have been well studied, the molecular mechanism by which B-domain release facilitates the procofactor to cofactor transition is not well understood. Recently, we have shown that in the absence of intentional proteolysis, deletion or substitution of discrete B-domain sequences drives the expression of procoagulant function (JBC, 282, 15030-9, 2007). Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids (963-1008) which is highly basic and well conserved, even though most of the B-domain has weak homology within the vertebrate lineage. In the current study, we examined if this basic B-domain region is sufficient to preserve FV as an inactive procofactor. To investigate this, the basic region (46 residues) was incorporated within the short B-domain of a previously characterized FV variant, FV-810. Factor V-810 has amino acids 811-1491 within the B-domain deleted and is a constitutively active cofactor, with functional properties equivalent to FVa. Using a PT-based clotting assay, purified prothrombinase assay, and direct fluorescent binding measurements with FXa-membranes we found that insertion of the basic region into FV-810 (inserted after residue 810) converted this cofactor-like species back to the procofactor-like state, despite >75% of the B-domain being absent. Next, using this new variant (FV+BR; B-domain of 201 residues), we assessed whether residual B-domain sequences within FV+BR contribute to maintaining FV in an inactive, procofactor state. Elimination of ∼100 residues on the N-terminal side of FV+BR was without functional consequence; that is, the procofactor state was maintained. In contrast, removal of B-domain sequences (∼50 residues, 30% of which are acidic) to the C-terminal side of the basic region shifted FV-810+BR from an inactive procofactor to an active cofactor. As expected, all purified FV derivatives exhibited full cofactor activity following treatment with thrombin. Together, these data show that B-domain sequences 963-1008 (basic region) appear to work in concert with the acidic C-terminal region of the B-domain (1492-1545) to keep FV in an inactive procofator state. These sequence elements appear to be necessary and sufficient as we were able to construct a FV variant with a B-domain length of only 103 amino acids that remarkably still had procofactor-like properties. Interestingly, these two regions of the B-domain (963-1008 and 1492-1545) are generally well conserved throughout the vertebrate lineage, while the remaining regions of the B-domain are not. We speculate that these B-domain sequences bind intramolecularly to heavy and/or light chain sequences thereby concealing critical binding sites on the FV molecule which govern the function of the active cofactor species. Disclosures:Camire:Wyeth: Patents & Royalties, Research Funding.

Inherited platelet disorders: thrombocytopenias and thrombocytopathies.

Platelets play an important role in normal haemostasis halting blood flow immediately after injuries through four fundamental different mechanisms: adhesion, aggregation, secretion, and expression of procoagulant activity. First, in the presence of vascular damage, platelets adhere to the connective tissue and, particularly when the damage occurs in vessels with a low shear rate, to subendothelial collagen, fibronectin and laminin. On the other hand, when damage occurs in regions with a high shear rate, platelet adhesion requires the presence of subendothelial von Willebrand factor (VWF) and specific platelet receptors, such as the glycoprotein Ib/IX/V (GPIb/IX/V) complex. In any case, following initial adhesion, platelets aggregate to complete the formation of a solid haemostatic plug. Platelet aggregation requires stimulation by agonists such as ADP, thrombin, collagen, or epinephrine, as well as the presence of calcium or magnesium ions and specific plasma proteins (fibrinogen, VWF and the glycoprotein IIb/IIIa (GPIIb/IIIa) complex). Platelet stimulation results in the generation of intracellular second messengers that convey the stimulus back to the platelet surface, exposing protein binding sites on GPIIb/IIIa. Fibrinogen (or VWF) then binds to GPIIb/IIIa and cross-links adjacent platelets to produce platelet aggregates, following platelet secretion and the elaboration of platelet procoagulant activity. In platelet disorders caused by a reduction in the number of platelets (thrombocytopenia) (Table I) or by platelet function defects (thrombocytopathies) (Table II), bleeding generally occurs immediately after injury, primarily in the skin, mucous membranes, nose, and gastrointestinal and urinary tracts and, generally, does not involve joints and muscles. Congenital disorders are uncommon and sometimes may be misdiagnosed with acquired disorders, which are much more frequently encountered in clinical practice. The differential diagnosis between congenital or acquired platelet disorders is often very complex and requires medical experience and a careful assessment of the patient’s personal and family history of bleeding episodes. A lifelong bleeding diathesis may suggest a congenital platelet disorder, which is often first recognised during childhood, but an onset in adulthood does not exclude a congenital defect. Because technological progress in laboratory practices and greater information about platelet functions have increased the specificity and accuracy of diagnosis of platelet disorders, a large number of cases has been identified recently. In this review, we describe current knowledge on congenital platelets disorders with regards to granule packaging, signalling and platelet coagulant function, and hereditary thrombocytopenias in order to improve information available for research and clinical practice. Table I Classification of thrombocytopathies

Human peripheral blood endothelial progenitor cells synthesize and express functionally active tissue factor

Introduction Endothelial progenitor cells are circulating cells able to home to sites of vascular damage and to contribute to the revascularization of ischemic areas. We evaluated whether endothelial progenitor cells synthesize tissue factor, a procoagulant protein also involved in angiogenesis. Materials and Methods Endothelial progenitor cells were obtained from the peripheral blood mononuclear fraction of normal donors and cultured in endothelial medium supplemented with specific growth factors. The procoagulant activity expressed by cells disrupted by freeze-thaw cycles was assessed by a one stage clotting assay. Tissue factor mRNA expression was evaluated by RT-PCR. Results Endothelial progenitor cells do not express procoagulant activity in baseline conditions. However, lipopolysaccharide induces the expression of procoagulant activity. The effect is dose-dependent and reaches statistical significance at 100 ng/mL lipopolysaccharide. Inhibition with an anti-tissue factor antibody and amplification of cDNA with primers based on the tissue factor sequence confirm the identity of this activity with tissue factor. The kinetics of tissue factor expression by endothelial progenitor cells is identical to that of human umbilical vein endothelial cells showing maximal activity within 4 hours, and then decreasing; in contrast, tissue factor expression by mononuclear cells lasts for longer times. Both 5,6-dichloro-beta D-ribofuranosyl-benzimidazole and cycloheximide prevented the expression of procoagulant activity. Stimulation of endothelial progenitor cells with tumor necrosis factor-α did not elicit any detectable procoagulant activity. Conclusions Endothelial progenitor cells can be stimulated by lipopolysaccharide to synthesize tissue factor. This protein might be involved in thrombotic phenomena and might contribute to endothelial progenitor cells related neovascularization.

Platelet Tissue Factor: Expression of Pro-Coagulant Activity depends on Gpibα Activation and Signaling through Lyn-Mediated Phosphorylation. A Platelet-Based Model of Hemostasis

Human platelets synthesize and store functionally silent tissue factor (TF) which has procoagulant activity (PCA) after platelet activation. We now explored the location of inactive and active forms of TF and mechanisms of activation. We reported that resting, non-permeabilized human platelets express scant surface TF, which was strikingly enhanced and co-localized with GPIbα after stimulation (Blood 2007; 109:5242). The externalization of TF was confirmed by immunoprecipitation (Ip) from biotinylated membranes before and after platelet activation. Moreover, TF and GPIbα co-precipitated in Ip assays and both glycoproteins were prominently found in lipid rafts (Lr) domains. TF-dependent PCA, assessed by FXa generation, was negligible or absent in resting, leukocyte-free platelet preparations. Interestingly, FVII was found in platelet membranes (western blot) and no exogenous FVIIa was needed to trigger TF-dependent FXa generation from washed platelets. Platelet responses to activation (TF-dependent PCA, platelet aggregation and secretion) depended on the agonist used. With 1IU/mL VWF + 1.2 mg/mL Ristocetin (Ris) PCA increased 5 to 10-fold 2 min after stimulation and platelets formed large aggregates with null 14C-serotonin secretion. In contrast, both platelet aggregation and secretion were normal 2 min after activation with 1IU thrombin, 10μM TRAP or 2μg/mL collagen; and PCA induced by thrombin was only ≈1/2 of that elicited by VWF+Ris, whereas 2 min after TRAP or collagen stimulation no PCA induction was detected. Removal of membrane cholesterol (by methyl-β-cyclo-dextrin) or disruption of Lr (with filipin III) abolishes the VWF-Ris-induced PCA. Lr's from resting platelets contain TF of Mr ≈60kDa. Five min after activation an increase in Lr's TF was observed, but its Mr depended on the agonist: species of ≈47kDa and ≈60kDa were found after VWF-Ris and TRAP activation, respectively. Given that GPIbα signals through Lyn, member of the Src family, inhibition of signaling with PP2 resulted in 80% fall of VWF-Ris-induced PCA. Ip assays revealed that Lyn co-precipitated with both GPIbα and TF in VWF-Ris activated, but not resting platelets. The phosphokinase activity of Lyn on TF was tested. A polyclonal antibody raised against the phosphorylated (Ser253/Ser258) cytoplasmic domain of TF (Dr. W. Ruf, La Jolla) recognized membrane TF only in activated, not in resting platelets. These findings indicate that VWF-induced activation of GPIbα, subsequent signaling with Lyn-induced serine phosphorylation, along with a change of TF to a ≈47kDa species, triggers human platelets PCA. The previously described role of phospho-disulphide isomerases (PDI) in TF activation through SH groups oxidation was also explored. TF and PDI co-precipitated in resting and activated platelet membranes and antagonizing PDI with bacitracin inhibited TF-dependent PCA. After platelet activation and labeling with MPB, a sulfhydryl-specific probe, TF protein (with PCA) was detected on the plasma membrane, denoting presence of reduced thiols. Furthermore, platelet incubation with phenylarsine oxide, a blocking reagent of vicinal SH groups, or HgCl2, a potent oxidant of thiol groups, had no effect on platelet PCA. Thus, it seems unlikely that TF activation depends on SH oxidation. Finally, we found that platelet TF was sufficient to speed up the clotting of plasma. In fact, clotting time of PRP (2 × 108 platelets mL−1) incubated with ionophore A23187 (2 min, 37°C) and then re-calcified, was 59 ± 6 sec, whereas clotting time in re-calcified PRP without previous activation was 137 ± 19 sec (n=19, p<0.0001). Taken together, our results highlight the crucial role of platelets, not only in assembling clotting complexes and reactions on their surface, but also providing enough TF to trigger the whole process. This novel, comprehensive understanding of hemostasis, (“platelet-based hemostasis model”), unifies primary and secondary hemostasis around the platelets, which would be able to synchronize and modulate the times of both processes ensuring a confined thrombin generation and adequate deposit of fibrin when and where it is needed. It also emphasizes the self-sufficiency of intravascular components to carry out both normal hemostasis and thrombus formation. In this context, platelet PCA may become a central pharmacological target for preventing or managing bleeding and thrombotic disorders.

Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists

Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species, and (2) determine the profiles of gene expression initiated utilizing these adaptor molecules. Monocytes were used to establish concentration response curves for Escherichia coli lipopolysaccharide (LPS; TLR4 ligand) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam 3CSK 4; TLR2 ligand) based on expression of procoagulant activity (PCA) and production of tumor necrosis factor-alpha (TNF-α); effects of polyinosine–polycytidylic acid (Poly I:C; TLR3 ligand) were determined by quantifying expression of mRNA for interferon-beta (IFN-ß). Expression of genes associated with the MyD88- (TNF-α, IL-1ß, IL-6 and IL-10) and TRIF-dependent pathways (IFN-ß, IP-10, RANTES and TRAF1) were measured at intervals spanning 20 h. LPS and Pam 3CSK 4 induced significantly higher expression of TNF-α, IL-1ß, and IL-10 than did Poly I:C. Poly I:C induced significantly higher expression of IFN-ß, IP-10 and RANTES than did either the TLR2 or TLR4 ligands. High concentrations of E. coli LPS did not significantly increase expression of genes associated with the TRIF-dependent pathway. The results of this study suggest that equine monocytes utilize a common intracellular pathway in response to TLR2 and TLR4 ligands, but a distinct pathway in response to TLR3 ligands.

A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide

Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1 ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30 pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3 h and peaked at 6 h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.

Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen

Introduction Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role. Material and methods Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation. Results Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced. Conclusions Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.

The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response.

In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium ([Na+](i)), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in [Na+](i). An increase in [Na+](i) and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.

Antiphospholipid antibody effects on monocytes

Although the presence of autoantibodies is known to increase the risk of thrombosis in the antiphospholipid syndrome, the mechanism by which these antibodies exert their effects is poorly understood. Several studies suggest that autoantibody-mediated dysregulation of monocytes is one pathobiologic mechanism of this disease. Recent studies have focused on extra- and intracellular interactions involved in monocyte activation and expression of procoagulant activity. Agents specifically targeting monocyte activation and activity may provide a novel and efficacious approach that is safer than current antithrombotics.

Cyclosporine enhances platelet procoagulant activity

Clinical use of cyclosporine (CsA) was suggested to be associated with an increased risk of thromboembolic complications. The molecular mechanisms underlying these effects remain unresolved. We tested the hypothesis that CsA may produce platelet procoagulant activity due to its interaction with the platelet plasma membrane. To verify this hypothesis the possible relationship between platelet morphology, exposure to platelet phosphatidylserine (PS) and platelet procoagulant activity (measured as phospholipid-dependent thrombin generation) was studied. It was found that CsA (1-100 microg/ml) potentiates collagen-evoked platelet procoagulant response. Platelets treated in vitro with CsA (20-200 microg/ml 20-60 min) expressed procoagulant activity. The CsA-induced platelet procoagulant response was both dose- and time-related and weaker than that produced by collagen. Flow cytometry studies revealed that CsA treatment results in a left shift (decrease) in the forward and side scatter of the entire platelet population. The shift was unimodal, dose-dependent and less pronounced than that elicited by collagen. Using flow cytometry and fluorescein isothiocyanate-labelled annexin V as a probe for PS, we demonstrated an increased binding of this marker to a CsA-treated platelet population. CsA-evoked PS-expression was dose- and time-dependent and smaller than that produced by collagen. CsA, at concentrations similar to those affecting platelet procoagulant response, released lactate dehydrogenase from platelets. These observations indicate that the thrombogenic properties of CsA may result from the alteration of lipid organization in platelet plasma membrane, leading to externalization of PS and accelerated thrombin generation.