Excessive Thrombin Research Articles

Protein Z, an anticoagulant protein with expanding role in reproductive biology

Protein Z (PZ) is a vitamin K-dependent factor characterized by its homology to other vitamin K-dependent factors (factors VII, IX, and X, protein C and protein S), but lacks any enzymatic activity. Instead, PZ acts as a cofactor for the inhibition of factor Xa through the serpin PZ-dependent protease inhibitor (ZPI). PZ deficiency is associated with a procoagulant state, highlighted by excessive FXa secretion and thrombin production, and is linked with several thrombotic disorders, including arterial vascular and venous thromboembolic diseases. A role for the PZ-ZPI complex in the regulation of physiological pregnancy has been demonstrated, highlighted by the progressive elevation in PZ levels in the first trimester of gestation, which then steadily decline toward delivery. An association between altered plasma PZ concentrations and adverse pregnancy outcomes (recurrent miscarriage, stillbirth, preeclampsia, intrauterine growth restriction, and placental abruption) has been reported. The mechanism by which PZ deficiency leads to adverse pregnancy outcomes is not clear, but it is multifactorial. It may be attributed to the anti-PZ IgG and IgM autoantibodies, which apparently act independently of classical antiphospholipid antibodies (lupus anticoagulant, anticardiolipin, and anti-β2-glycoprotein I antibodies). PZ deficiency has also been reported to be constitutional, and a number of variants in the PROZ (PZ) gene and SERPINA10 (ZPI) gene are linked with specific adverse pregnancy complications. This review summarizes the relationship between adverse pregnancy outcomes and acquired and constitutional PZ-ZPI deficiency, in order to understand whether or not PZ deficiency could be considered as a risk factor for poor pregnancy outcomes.

Cascading into the Thrombin‐Thrombomodulin Complex

According to the Centers for Disease Control every year more than 60,000 Americans die of excessive blood clots or thrombophilia, and many more suffer from this disorder. Clotting disorders pose unique problems for women because of their impact on reproductive health and pregnancy. Thrombin, a protease that cleaves fibrinogen and activates platelets, plays a key role in the generation of blood clots. Several mechanisms prevent excessive thrombin activity. Thrombin that diffuses away from the clot binds to its receptor, thrombomodulin. Thrombomodulin‐bound thrombin can no longer generate blood clots. Instead, it cleaves and activates Protein C which shuts down further thrombin generation. The substitution Gln387Pro in thrombomodulin inhibits Protein C activation and interferes with this feed‐back inhibition. The Laconia SMART (Students Modeling A Research Topic) Team used 3D printing technology to model thrombin in association with thrombomodulin. Amino acid residues within the catalytic site (Ser195, His57, and Asp102) and the two substrate binding sites, exosite I (Lys36‐Arg77), and exosite II (Arg 93‐Lys240 of thrombin, involved in these interactions are highlighted. Understanding the relation between structure and function of the thrombin‐thrombomodulin complex may lead to new therapeutics for clotting disorders. This work was supported by a grant from NIH‐CTSA UL1RR031973.

Decidual cell expressed tissue factor promotes endometrial hemostasis while mediating abruption associated preterm birth

During human pregnancy, progesterone induced decidual cells protect against hemorrhage: 1) as endovascular trophoblast breech and remodel uterine blood vessels; and 2) in the third stage of labor following preterm and term delivery. De- cidual cells promote hemostasis through enhanced expression of tissue factor (TF), the primary initiator of hemostasis via thrombin generation, and plasminogen activator inhibitor-1, which inactivates tissue type plasminogen activator, the primary fibrinolytic agent. Abruptions (decidual hemorrhage) produce excess thrombin which acts as autocrine/paracrine inducer of decidual cell expressed matrix metalloproteinases and of neutrophil chemoattractant and activator, interleukin-8. The latter mediates aseptic abruption-related neutrophil infiltration. During abruptions, decidual cell and neutrophil-derived proteases effectively degrade the decidual and fetal membrane extracellular matrix to promote preterm premature rupture of the membranes and preterm delivery (PTD). Decidual cell-derived thrombin weakens the amniotic membrane and lowers decidual cell-expressed progesterone receptor levels by increasing phospho-ERK1/2 signaling. The resulting functional progesterone withdrawal accompanies PTD.

Elevated plasma factor VIII enhances venous thrombus formation in rabbits: Contribution of factor XI, von Willebrand factor and tissue factor

Elevated plasma levels of factor VIII (FVIII) are associated with increased risk of deep venous thrombosis. The aim of this study is to elucidate how elevated FVIII levels affect venous thrombus formation and propagation in vivo. We examined rabbit plasma FVIII activity, plasma thrombin generation, whole blood coagulation, platelet aggregation and venous wall thrombogenicity before and one hour after an intravenous infusion of recombinant human FVIII (rFVIII). Venous thrombus induced by the endothelial denudation of rabbit jugular veins was histologically assessed. Thrombus propagation was evaluated as indocyanine green fluorescence intensity. Argatroban, a thrombin inhibitor, and neutralised antibodies for tissue factor (TF), factor XI (FXI), and von Willebrand factor (VWF) were infused before or after thrombus induction to investigate their effects on venous thrombus formation or propagation. Recombinant FVIII (100 IU/kg) increased rabbit plasma FVIII activity two-fold and significantly enhanced whole blood coagulation and total plasma thrombin generation, but did not affect initial thrombin generation time, platelet aggregation and venous wall thrombogenicity. The rFVIII infusion also increased the size of venous thrombus 1 hour after thrombus induction. Argatroban and the antibodies for TF, FXI or VWF inhibited such enhanced thrombus formation and all except TF suppressed thrombus propagation. In conclusion, elevated plasma FVIII levels enhance venous thrombus formation and propagation. Excess thrombin generation by FXI and VWF-mediated FVIII recruitment appear to contribute to the growth of FVIII-driven venous thrombus.

Abruption-Induced Preterm Delivery Is Associated with Thrombin-Mediated Functional Progesterone Withdrawal in Decidual Cells

Plasma progesterone levels remain elevated throughout human pregnancy, suggesting that reduced reproductive-tract progesterone receptor (PR) initiates labor. Placental abruption and excess thrombin generation elicit preterm delivery (PTD). PR, glucocorticoid receptor (GR), and total and p-ERK1/2 in decidual cells (DCs) and interstitial trophoblasts (IT) were assessed via immunohistochemical staining in abruption-associated PTD versus gestational-age matched control placentas, and in cultured DCs incubated with estradiol (E2) ± medroxyprogesterone acetate (MPA) ± thrombin. Immunostaining for PR was lower in DC nuclei in abruption versus control decidua and was absent from ITs; GR was higher in IT than DCs, with no abruption-related changes in either cell type; p-ERK1/2 was higher in DCs in abruption than control decidua, with total ERK 1/2 unchanged. Immunoblotting of cultured DCs demonstrated strong E2, weak MPA, and intermediate E2+MPA mediated elevation of PR-A and PR-B levels, with constitutive GR expression. In cultured DCs, thrombin inhibited PR but not GR mRNA levels, reduced PR binding to DNA and [(3)H]progesterone binding to PR, and enhanced phosphorylated but not total ERK1/2 levels. Coincubation with a specific p-ERK1/2 inhibitor reversed thrombin-enhanced p-ERK1/2 and lowered PR levels. Thus, abruption-associated PTD is initiated by functional progesterone withdrawal, as indicated by significantly reduced DC nuclear expression of PR-A and PR-B. Functional withdrawal of progesterone results in increased p-ERK1/2, and is thus one pathway initiating abruption-associated PTD.

PAR‐1‐dependent and PAR‐independent pro‐inflammatory signaling in human lung fibroblasts exposed to thrombin

Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (∼0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation.

Thrombin generation post elective caesarean section: Effect of low molecular weight heparin

IntroductionCaesarean section (CS) is a significant risk factor for venous thromboembolism.. Low molecular weight heparin (LMWH) is commonly used for thromboprophylaxis post emergency caesarean delivery. However, no consensus exists regarding LMWH thromboprophylaxis following elective caesarean section. Measures of thrombin formation may indicate the full anticoagulant activity of LMWH in this setting. Materials and methodsAnti-Xa, tissue factor pathway inhibitor (TFPI), thrombin anti-thrombin complex (TAT) and endogenous thrombin potential (ETP) were measured in twenty healthy women who received 4,500IU tinzaparin 6hours post CS (CS1), twenty women who received 4,500IU tinzaparin at 10–12hours post delivery (CS2) and twenty women post spontaneous vaginal delivery (SVD). ResultsPrior to initiation of LMWH, TAT levels at 6hours post delivery were significantly higher in the CS1 and CS2 groups than the SVD group (P<0.002); TAT levels were significantly reduced up to 24hours post LMWH treatment despite declining anti-Xa levels (P<0.001). In CS1, peak thrombin and ETP were significantly reduced following LMWH prophylaxis (P<0.0001; P<0.002) and reverted to pre-delivery levels 10hours post LMWH. TFPI levels mirror anti-Xa levels during the 24hours following LMWH treatment in CS1 group with peak levels coinciding with peak anti-Xa levels 4hours post injection. ConclusionIn women post caesarean section, anti-Xa levels do not reflect the full anticoagulant effects of LMWH. In-vivo thrombin production (TAT) is effectively reduced even when anti-Xa levels are negligible. LMWH thromboprophylaxis in this healthy cohort of patients appears to have a sustained effect in reducing excess thrombin production post elective caesarean section.

Characteristics of Fibrin Formation and Clot Stability in Individuals with Congenital Type IIb Protein C Deficiency

Many studies have shown that unregulated or excessive thrombin formation is potentially a cause of thrombosis; however, studies examining processes that contribute to fibrin stabilization in individuals predisposed to thrombosis are limited. In this study, we investigate a family with familial thrombosis via type IIb protein C (PC) deficiency. Using contact pathway inhibited whole blood, thrombin generation, fibrin clot formation and factor (f)XIII activation were monitored over time in control (n=5) and PC deficient (n=4, 34 – 69% PC by activity) subjects. The dynamics of thrombin generation varied significantly with the time required to reach the maximal rate of thrombin-antithrombin formation being much shorter in PC deficiency (5.8±0.4 minutes) than in controls (9.7±0.4 minutes; p<0.001). PC deficient clots were significantly heavier than control clots (p<0.001) and this difference could not be contributed exclusively to differences in fibrinogen levels between groups. FXIII was consumed faster in PC deficient subjects (23.7±2.0 nM/minute) than in controls (5.1±1.5 nM/minute; p<0.0001) suggesting increased fXIII activation and incorporation of fXIIIa substrates into the clot. In plasma, the clot lysis time was increased in PC deficiency by both TAFIa dependent and independent mechanisms. Since PC deficient clots are both denser and show a greater degree of resistance to fibrinolysis, these clots would likely resist fibrinolysis and potentiate fibrin deposition observed in thrombosis.

The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling

Increasing evidence links blood coagulation proteins with the regulation of acute and chronic inflammatory disease. Of particular interest are vitamin K-dependent proteases, which are generated as a hemostatic response to vascular injury, but can also initiate signal transduction via interactions with vascular receptors. The endothelial cell protein C receptor (EPCR) is a multi-ligand vitamin K-dependent protein receptor for zymogen and activated forms of plasma protein C and factor VII. Although the physiological role of the EPCR-FVII(a) interaction is not well-understood, protein C binding to EPCR facilitates rapid generation of APC in response to excessive thrombin generation, and is a central requirement for the multiple signal-transduction cascades initiated by APC on both vascular endothelial and innate immune cells. Exciting recent studies have highlighted the emerging role of EPCR in modulating the cytoprotective properties of APC in a number of diverse inflammatory disorders. In this review, we describe the structure-function relationships, signal transduction pathways, and cellular interactions that enable EPCR to modulate the anticoagulant and anti-inflammatory properties of its vitamin K-dependent protein ligands, and examine the relevance of EPCR to both thrombotic and inflammation-associated disease.

Decidual Hemostasis, Inflammation, and Angiogenesis in Pre-Eclampsia

Invasion of the decidua by extravillous trophoblasts (EVTs) is accompanied by thrombin generation from decidual cell (DC)-expressed tissue factor (TF). This TF protects against hemorrhage as EVTs breach capillaries and subsequently invade and remodel spiral arteries and arterioles. Pre-eclampsia (P-EC) is the world's leading cause of fetal and maternal morbidity and mortality. It is associated with decidual hemorrhage and maternal thrombophilias, which form excess thrombin from DCs, and with maternal infections and other inflammatory conditions that are associated with excess expression of the proinflammatory cytokines interleukin (IL)-1 β and tumor necrosis factor (TNF) α. In human first-trimester leukocyte-free DCs, (1) thrombin enhances expression of soluble fms-like tyrosine kinase-1 (sFlt-1), a potent inhibitor of angiogenesis; (2) thrombin, IL-1β and TNF-α increase monocyte-recruiting chemokine expression leading to a macrophage excess in the pre-eclamptic decidua. The pathogenesis of P-EC likely stems from shallow EVT invasion leading to impaired decidual vascular remodeling. The resulting reduced uteroplacental blood flow is associated with a hypoxic placenta, which appears to secrete excess sFlt-1 into the maternal plasma. A regulatory role for DCs in vascular remodeling is indicated because impaired decidual vascular remodeling could stem from an aberrant local antiangiogenic milieu elicited by excess sFlt-1 and/or macrophage-inhibited EVT decidual invasion.

Neutrophil and monocyte activation markers have prognostic impact in disseminated intravascular coagulation: In vitro effect of thrombin on monocyte CD163 shedding

Monocyte and neutrophil activation occur during microvascular disturbance of disseminated intravascular coagulation (DIC). This study investigated the diagnostic and prognostic value of circulating neutrophil elastase (NE) and neutrophil volume distribution width (NDW) as neutrophil activation markers and circulating soluble CD163 (sCD163) and monocyte volume distribution width (MDW) as monocyte activation markers in 168 patients suspected of having DIC. The sCD163 provided significant diagnostic value. The prognostic value of sCD163 was comparable to that of D-dimer, but was dependent on other coagulation markers. In vitro, thrombin significantly induced sCD163 from monocytes upregulated with IL-10 or dexamethasone. NDW was an independent and powerful prognostic marker. MDW and NE did not provide diagnostic and prognostic power. Excessive thrombin during ongoing DIC induces florid secretion of CD163; sCD163 might therefore be a potential diagnostic and prognostic marker for DIC. NDW, a convenient parameter measured by an automated hematology analyzer, may be an independent prognostic parameter for DIC.

High-shear- and-thrombin-inducible platelet adhesion and aggregation in patients undergoing percutaneous coronary intervention

Thrombin-generation and activation of platelets during percutaneous coronary intervention (PCI) play a key role for early thrombotic events. Heparin and bivalirudin are approved anticoagulants for PCI. We examined the specific effects of these anticoagulants on platelet adhesion and aggregation under high shear conditions, and the presence of excess thrombin. To simulate in vivo conditions that may precipitate a bleeding/thrombotic event, we added thrombin in vitro to blood samples from 89 stable patients who had been randomly assigned to receive heparin or bivalirudin for elective PCI and examined thrombin-inducible platelet adhesion and aggregation under high shear conditions. Platelet adhesion increased by 10% of baseline with heparin, but decreased by 20% with bivalirudin (p=0.0047). Thrombin-inducible platelet adhesion and size of aggregates was equally inhibited by heparin and bivalirudin. Thus, under high shear conditions and excessive thrombin generation as they occur in atherosclerotic vascular compartments and acute vascular syndromes, heparin and bivalirudin inhibit thrombin-induced platelet adhesion and aggregation to a similar extent, while they have opposite effects on platelet adhesion in the absence of thrombin.

Atorvastatin and its collateral effects on microparticles

Microparticles (MPs) are vesicular fragments that bud off cells during either activation or apoptosis. MPs have been postulated to play an important role in thrombosis, in part because phosphatidylserine on MP surfaces can be a site for catalytic assembly of the prothrombinase complex (1). Additionally MPs, particularly those released from monocytes represent a rich source of circulating tissue factor, and they also express other surface proteins involved in the process of coagulation (2). Indeed, MPs have been found to be increased in blood of patients with various thrombotic and inflammatory disorders, including acute coronary syndromes, sickle cell disease, diabetes mellitus, thrombotic thrombocytopenic purpura, vasculitis, antiphospholipid antibody syndrome (1, 3–6). As MPs (at least at high concentrations) have been shown to be related to thrombotic and inflammatory diseases, the modulation of their production might have important therapeutic implications (7–9). In this respect, there is increasing clinical attention towards the potential of pharmacological agents to modulate both levels of circulating MPs and their expression of prothrombotic markers (10). Statins are widely used in clinical practice, being the cornerstone of the current management of atherosclerosis. The beneficial net effect of statins on the rate of cardiovascular complications has largely been related to their ability to improve lipid profile. However, statins also possess additional (i.e. pleio tropic) biological properties, which are likely to facilitate their clinical success. In the context of numerous lipid-independent effects of statins it was therefore natural to explore their impact on circulating MPs. However, statins have been reported to exert controversial effects on MP levels and activation markers. Whilst Tramontano et al. (11) have reported inhibition of release of endothelium-derived MPs in the presence of fluvastatin, Diamant et al. (12) found that simvastatin induced the release of endothelium-derived MPs. Of note, both studies were restricted by in vitro nature of the experiments and further research in the field was awaited. Nonetheless, a few studies reported the effects of lipid-lowering therapy in the expression of MPs markers in vivo (13). Peripheral arterial occlusive disease is a clinical condition linked to excessive platelet activation, thrombin generation and endothelial dysfunction (14). Platelets are pivotal mediators of various endothelial, thrombotic and inflammatory responses and therefore are keys player in the initiation and progression of atherothrombosis (15). Platelet-derived MPs (PMPs) express on their surface several markers of platelet activation such as CD62P (P-selectin), apart from tissue factor (CD142), and the platelet-specific proteins, such as integrin CD61 (glycoprotein IIIa). Moreover, recently a cell-free form of CD36, a scavenger receptor associated with plaque instability, has been found not to be truly soluble but a constituent of MPs (16). This finding hints at the fact that several activation markers might be shed in the form of circulating MPs. This could have an important effect on thrombus propagation, related both to direct effects of MPs on platelet function and to their ability to facilitate thrombin generation by presenting tissue factor and serving as an assembly site for the prothrombinase complex. In this issue of Thrombosis and Haemo stasis, Mobarrez et al. (17) carried out both in vivo and in vitro studies to explore the effects of atorvastatin on the expression of prothrombotic markers on PMPs and secondly, to analyse their possible effects of atorvastatin on the thrombin generation. These authors found that the expression of activation markers on PMPs and thrombin generation decreased with statin and aspirin treatment in patients with peripheral arterial occlusive disease, which again confirms the benefits of statins in prothrombotic diseases (18). Herein, the authors proposed PMPs as possible contributors of thrombin activation since the statinrelated reduction in PMP expression of tissue factor and P-selectin paralleled diminished thrombin formation. Of note, these effects were not seen in placebo group, thus providing the evidence that the findings are truly attributable to the pleiotropic effects of atorvastatin. In order to provide further support of this novel concept, the authors showed that thrombotic effects of MPs could be blocked by the addition of antitissue factor and antiphosphatidylserine polyclonal antibodies, thus confirming the capacity of MPs to reinforce thrombin generation. However, the present manuscript does not provide the proof per se of the direct implication (or causality) of PMPs in thrombin generation nor evidence of pathophysiological implication(s) of these processes in disease progression. Nonetheless, these findings again confirm the possibility that statins exert their beneficial effects acting beyond lipoprotein pathways. As part of these additional effects, the authors speculate that the lower antigenic expression on PMPs in patients treated with statins might reflect a reduced platelet activation which could also contribute subsequently to reduced thrombin generation, as previously shown (19). Moreover, an additional point seems to be worth emphasising. The main procoagulant activity provided by MPs is usually attributable to the negatively charged surface (20). However, MP phosphatidylserine density has not

Novel insights into molecular mechanisms of abruption-induced preterm birth

Preterm birth (PTB) complicates more than 12% of all deliveries. Despite significant research, the aetiology of most cases of PTB remains elusive. Two major antecedents of PTB, intra-amniotic infection and decidual haemorrhage (abruption), can exhibit dissimilar demographic and genetic predispositions, despite sharing common molecular and cellular pathways. The use of high-throughput, high-dimensional technologies reveals substantial crosstalk between the coagulation and inflammation pathways. Tissue factor, thrombin and cytokines are key mediators of this crosstalk. Abruptions are associated with excess thrombin generated from decidual-cell-expressed tissue factor. Although thrombin is a primary mediator of the coagulation cascade, it can also promote inflammation-associated PTB by enhancing expression of matrix metalloproteinase and neutrophil-chemoattracting and -activating chemokines. Here, we provide novel insights into the molecular mechanisms and pathways leading to PTB in the setting of placental abruption.

ROTEM and multiplate – a suitable tool for POC?

The early recognition of perioperative coagulation disorders is essential to identify non-surgical reasons of bleeding, to initiate appropriate haemostatic treatment and finally to reduce perioperative blood loss. Conventional laboratory coagulation tests include prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen levels and platelet counts. Although these parameters are frequently assessed, their value has been challenged at least in the setting of acute perioperative bleeding [1]. PT and aPTT are performed in an artificial ‘in vitro’ system, i.e. in plasma separated from whole blood, which is then warmed to 37 C and buffered to a pH of 7AE4. These standardized conditions often do not reflect the patient’s situation [2]. Plasmatic ‘in vitro’ tests merely reflect the time elapsing until the activation of thrombin, but provide only marginal information about the functional state of the coagulation system (e.g. platelet function, interaction of plasmatic coagulation factors with platelets and red blood cells, clot firmness, fibrinolysis). Routine coagulation tests are mostly performed in the central laboratory, i.e. remote from the operating theatre. As a consequence, test results are often not available in time or with a considerable delay [3]. Current devices designed for point-of-care (POC) monitoring of coagulation have been designed for the assessment of coagulation directly at the bedside, so that results could be available earlier. Moreover, POC tests are usually performed in whole blood, thereby comprising interactions between cellular components (i.e. platelets and red blood cells) and plasmatic coagulation factors. Commonly used POC devices for assessment of plasmatic coagulation and platelet function are listed in Table 1. This review focuses on rotation thrombelastography (ROTEM ) as a global coagulation test and multiple electrode aggregometry (MEA, Multiplate ) for assessment of platelet function. Rotation thrombelastometry (ROTEM )

Appropriate Use of D-dimer in Hospital Patients

D-dimer, the final product of plasmin-mediated degradation of fibrin-rich thrombi, has emerged as a simple blood test that can be used in diagnostic algorithms for the exclusion of venous thromboembolism. D-dimer also is used as a part of the diagnostic tests for disseminated intravascular coagulation, where excessive thrombin generation is the key pathophysiological factor. However, there are no robust data available at present on the use of this test to exclude venous thromboembolism in a hospital inpatient. Considerable confusion also exists among physicians about its appropriate use and interpretation in disseminated intravascular coagulation. This article focuses on the available evidence to guide the appropriate use of D-dimer in patients admitted to a hospital.

Changes in amniotic fluid concentration of thrombin–antithrombin III complexes in patients with preterm labor: Evidence of an increased thrombin generation

Objective. Preterm labor is associated with excessive maternal thrombin generation, as evidenced by increased circulating thrombin–antithrombin (TAT) III complexes concentration. In addition to its hemostatic functions, thrombin has uterotonic properties that may participate in the mechanism leading to preterm birth in cases of intrauterine bleeding. Thrombin also has a proinflammatory role, and inflammation is associated with increased thrombin generation. The aim of this study was to determine whether intra-amniotic infection/inflammation (IAI) is associated with increased amniotic fluid (AF) thrombin generation in women with preterm and term deliveries.Study design. This cross-sectional study included the following groups: (1) mid-trimester (n = 74); (2) term not in labor (n = 39); (3) term in labor (n = 25); (4) term in labor with IAI (n = 22); (5) spontaneous preterm labor (PTL) who delivered at term (n = 62); (6) PTL without IAI who delivered preterm (n = 59); (7) PTL with IAI (n = 71). The AF TAT III complexes concentration was measured by enzyme linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis.Results. (1) TAT III complexes were identified in all AF samples; (2) patients with PTL who delivered preterm, with and without IAI, had a higher median AF TAT III complexes concentration than those with an episode of PTL who delivered at term (p < 0.001, p = 0.03, respectively); (3) among patients with PTL without IAI, elevated AF TAT III complexes concentration were independently associated with a shorter amniocentesis-to-delivery interval (hazard ratio, 1.5; 95% CI, 1.07–2.1); (4) among patients at term, those with IAI had a higher median AF TAT III complexes concentration than those without IAI, whether in labor or not in labor (p = 0.02); (5) there was no significant difference between the median AF TAT III complexes concentration of patients at term with and without labor; (6) patients who had a mid-trimester amniocentesis had a lower median AF TAT III complexes concentration than that of patients at term not in labor (p < 0.001).Conclusions. We present herein a distinct difference in the pattern of intra-amniotic thrombin generation between term and preterm parturition. PTL leading to preterm delivery is associated with an increased intra-amniotic thrombin generation regardless of the presence of IAI. In contrast, term delivery is associated with an increased intra-amniotic thrombin generation only in patients with IAI.

Eosinophilia and Thrombophilia in Churg Strauss Syndrome: A Clinical and Pathogenetic Overview

During the past decade, there has been an increased description of Churg Strauss syndrome (CSS) characterized by vascular occlusions possibly linked to the thrombogenic potential of the eosinophil that is poorly appreciated. The purpose of this overview is 3-fold: the first to evaluate the available prevalence of thrombosis in Churg Strauss series, the second to demonstrate that any vascular district may be affected, and the third to describe the pathogenesis of thrombosis in CSS. A Pubmed, EMBASE, and Google search of CSS series from 1951 to date revealed a prevalence of arterial occlusion ranging between 3.1% and 18.7% and a prevalence of venous occlusion between 5.8% and 30%, whereas a specific survey for venous thromboembolism in CSS yielded a prevalence of 8.1%. Eosinophils store and release tissue factor as well as other cationic proteins: the former initiates coagulation while the latter inhibits natural anticoagulant activity and activate platelets eventually culminating in excessive thrombin generation and clot formation. In addition, antineutrophil cytoplasmic antibodies may shift the endothelial lining to proadhesive and prothrombotic surface. It is hoped that the review will represent a basis to foster novel research on this topic.

Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interleukin-8 synergistically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding.

Evidence of maternal platelet activation, excessive thrombin generation, and high amniotic fluid tissue factor immunoreactivity and functional activity in patients with fetal death

Objective. Fetal death can lead to disseminated intravascular coagulation or fetal death syndrome. However, currently it is not clear what are the changes in the coagulation system in patients with a fetal death without the fetal death syndrome. This study was undertaken to determine: (1) whether fetal death in the absence of fetal death syndrome is associated with changes in hemostatic markers in maternal plasma and amniotic fluid; and (2) whether maternal hypertension or placental abruption are associated with further changes in the hemostatic profile of these patients.Methods. A cross-sectional study included the following: (1) determination of changes in markers of coagulation and platelet activation in patients with a normal pregnancy (n = 71) and patients with fetal demise (FD) without disseminated intravascular coagulation (n = 65); (2) determination of the amniotic fluid (AF)–tissue factor concentration and activity, as well as the concentrations of thrombin–antithrombin III (TAT) complexes in patients with a normal pregnancy (n = 25) and those with a FD (n = 36) who underwent amniocentesis. Plasma and AF concentrations of TAT complexes and TF (an index of thrombin generation), as well as maternal plasma concentrations of sCD40L (a marker of platelet activation), tissue factor pathway inhibitor (TFPI) and prothrombin fragments (PF) 1 + 2 (also an indicator of in vivo thrombin generation) were measured by ELISA. TF and TFPI activity were measured using chromogenic assays.Results. (1) patients with FD without hypertension had a higher median maternal plasma sCD40L concentration than normal pregnant women (P < 0.001); (2) patients with FD had a higher median maternal plasma TAT III complexes than women with a normal pregnancy (P < 0.001); (3) the median AF–TF concentration and activity were higher in the FD group than in the normal pregnancy group (P < 0.001 for both); (4) patients with preeclampsia and FD had a higher median maternal plasma immunoreactive TF concentration than both normotensive patients with FD and women with normal pregnancies (P < 0.001 and P = 0.001, respectively); (5) the median plasma TF activity was higher in patients with preeclampsia and FD than that of women with normal pregnancies (P = 0.003); (6) among patients with a FD, those with placental abruption had a higher median AF–TAT complexes concentration than those without abruption (P = 0.0004).Conclusions. Our findings indicate that: (1) mothers with a FD have evidence of increased in vivo thrombin generation and platelet activation than women with normal pregnancies; (2) patients with a FD and hypertension had a higher degree of TF activation than those with fetal death but without hypertension; (3) the AF of women with a FD had a higher median TF concentration and activity than that of normal pregnant women. AF can be a potential source for tissue factor and it participates in the development of fetal death syndrome in patients with a retained dead fetus.