Regulation of plasminogen activation on cell surfaces and fibrin.

The fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma hemostasis cofactors. Vascular endothelial cells (VECs) retain tissue-type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI-1), regulates fibrinolytic potential via inhibition of the VEC surface-bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C-terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin-activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI-1, TAFI, TM, and the fibrin cross-linking enzyme, factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed.

The plasminogen receptor, Plg-RKT, is essential for mammary lobuloalveolar development and lactation.

Essentials Plg-RKT-/- female mice give birth, but no offspring of Plg-RKT-/- female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg-RKT-/- mice are unknown. Plg-RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg-RKT is essential for lactogenesis and mammary lobuloalveolar development. SUMMARY: Background Lactational competence requires plasminogen, the zymogen of the serine protease, plasmin. Plg-RKT is a unique transmembrane plasminogen receptor that promotes plasminogen activation to plasmin on cell surfaces. Plg-RKT-/- mice are viable, but no offspring of Plg-RKT-/- female mice survive to weaning. Objectives We investigated potential lactational failure in Plg-RKT-/- mice and addressed causal mechanisms. Methods Fibrin accumulation, macrophage infiltration, processing of extracellular matrix components, effects of genetic deletion of fibrinogen, expression of fibrosis genes, and proliferation and apoptosis of epithelial cells were examined in lactating mammary glands of Plg-RKT-/- and Plg-RKT+/+ mice. Results Milk was not present in the stomachs of offspring of Plg-RKT-/- female mice and the pups were rescued by foster mothers. Although the mammary ductal tree developed normally in Plg-RKT-/- glands, lobuloalveolar development was blocked by a hypertrophic fibrotic stroma and infiltrating macrophages were present. A massive accumulation of fibrin was also present in Plg-RKT-/- alveoli and ducts. Although this accumulation was decreased when Plg-RKT-/- mice were made genetically heterozygous for fibrinogen, defects in lobuloalveolar development were not rescued by fibrinogen heterozygosity. Transcriptional profiling revealed that EGF was downregulated 12-fold in Plg-RKT-/- glands. Furthermore, proliferation of epithelial cells was not detectable. In addition, the pro-survival protein, Mcl-1, was markedly downregulated and apoptosis was observed in Plg-RKT-/- but not Plg-RKT+/+ glands. Conclusions Plg-RKT is essential for lactogenesis and functions to maintain the appropriate stromal extracellular matrix environment, regulate epithelial cell proliferation and apoptosis, and, by regulating fibrinolysis, preserve alveolar and ductal patency.

A Case of Successful Thromboelastographic Guided Resuscitation after Postpartum Hemorrhage and Cardiac Arrest.

Amniotic fluid embolism (AFE) is an unusual cause of life threatening peri partum hemorrhage (PPH). AFE resuscitation is often associated with renal and respiratory insufficiency, and a coagulopathy similar to disseminated intravascular coagulation (DIC). Resuscitation requires immediate recognition and limited use of crystalloid. We present a case of PPH caused by AFE with resultant cardiac arrest, renal and respiratory failure, and DIC-like coagulopathy, whose successful resuscitation was guided by perfusionist-directed serial thromboelastography (TEG). Viscoelastic tests (VET)s, including the TEG and rotational thromboelastometry (ROTEM), may provide more individualized blood component therapy (BCT) in the treatment of severe PPH associated with AFE as has been previously noted with trauma resuscitation in the literature. However, VET's efficacy is often limited by a lack of standardization, quality assurance norms, and consistent operator proficiency. We suggest that there may be a role for perfusionsts adept at utilizing TEG in the optimization of BCT and adjunctive hemostatic agents in severely hemorrhagic patients. This patient's successful resuscitation demonstrates the importance of resuscitation guided by the perfusionist or other medical professionals with expertise in TEG guided resuscitation and how the administration of specific blood products and hemostatic agents guided by the TEG can help optimize patient outcomes in comparison to traditional 1:1:1 packed red blood cells (PRBC) /fresh frozen plasma (FFP) /platelets ratios given to severely hemorrhaging patients.

Contributions of thrombin targets to tissue factor-dependent metastasis in hyperthrombotic mice.

BACKGROUND: Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. OBJECTIVE: Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TM(Pro) mice). METHODS: Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. RESULTS: TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TM(Pro) mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TM(Pro) mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TM(Pro) mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TM(Pro) mice. CONCLUSIONS: Metastasis in the hyperthrombotic TM(Pro) mouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells.

Two novel inhibitory anti-human factor XI antibodies prevent cessation of blood flow in a murine venous thrombosis model.

Coagulation factor XI (FXI) is a promising target for anticoagulation, because of its major role in thrombosis and relatively minor role in haemostasis. This implies that inhibition of FXI can prevent thrombosis without causing bleeding. It was our aim to investigate the antithrombotic properties of two novel inhibitory anti-human FXI antibodies (αFXI-175 and αFXI-203). The in vitro properties of both antibodies were analysed using standard clotting assays and calibrated automated thrombography. For the in vivo model we used FXI knockout mice, in which FXI plasma levels were restored with purified human FXI. Thrombosis was induced by applying ferric chloride to the vena cava inferior, after which time to occlusion was analysed. A tail bleeding assay was used to investigate the safety of both antibodies. Using calibrated automated thrombography, both antibodies inhibited thrombin generation initiated via the intrinsic pathway. In contrast, upon tissue factor (TF)-initiated thrombin generation, αFXI-203 did not inhibit thrombin generation, while αFXI-175 inhibited thrombin generation only at low concentrations of TF. In the murine thrombosis model, the vena cava inferior remained patent for 25 minutes (min) in mice treated with αFXI-175 and for 12.5 min in αFXI-203 treated animals, which was significantly longer than in placebo-treated animals (5 min, p<0.05). Neither antibody caused severe blood loss in a tail bleeding assay. In conclusion, the two inhibitory antibodies against FXI prevented cessation of blood flow in a murine thrombosis model without inducing a bleeding tendency.

Specific determinants of conantokins that dictate their selectivity for the NR2B subunit of N-methyl-D-aspartate receptors.

Conantokins are naturally-occurring small peptide antagonists of ion flow through NMDA/glycine activated-N-methyl-d-aspartate receptor (NMDAR) ion channels. One member of the conantokin family, conantokin (con)-G, a 17-residue peptide, is selective for NMDARs containing the N-methyl-d-aspartate receptor subunit 2 B (NR2B), whereas the homologous peptides, con-T and con-R, show broader selectivity for NR2 subunits. In this study, con-G, con-R, and con-T variants were chemically synthesized and employed to investigate their subunit selectivities as inhibitors of agonist-evoked ion currents in human embryonic kidney-293 (HEK-293) cells expressing various combinations of NMDAR subunits that contain NR1a or NR1b combined with NR2A or NR2B. Using truncation and point mutants, as well as chimeric conantokins, we determined that the N-terminus of con-G contains all the determinants for NR2B selectivity. With this information, a large number of (con) variants were synthesized and used to establish minimal sequence determinants for selectivity. Tyr at position 5 broadens the NR2 selectivity, and recovery of NR2B selectivity in Tyr5 peptides was achieved by incorporating Ala or Gly at position 8. NR2B selectivity in con-R can be conferred through deletion of the Ala at position 10, thereby shifting the γ-carboxyglutamate (Gla) from position 11 to position 10, where a Gla naturally occurs in con-G and con-T. The nature of the amino acid at position 6 is also linked to subunit selectivity. Our studies suggest that the molecular determinants of conantokins that dictate NMDAR subunit selectivity are housed in specific residues of the N-termini of these peptides. Thus, it is possible to engineer desired NMDAR functional properties into conantokin-based peptides.

The protein C pathway and pathologic processes.

Alterations in expression of protein C (PC) pathway components have been identified in patients with active inflammatory disease states. While the PC pathway plays a pivotal role in regulating coagulation and fibrinolysis, activated PC (aPC) also exhibits cytoprotective properties. For example, PC-deficient mice challenged in septic/endotoxemic models exhibit phenotypes that include hypotension, disseminated intravascular coagulation, elevated inflammatory mediators, neutrophil adhesion to the microvascular endothelium, and loss of protective endothelial and epithelial cell barriers. Further, inflammatory bowel disease has been correlated with diminished endothelial PC receptor and thrombomodulin levels in the intestinal mucosa. Downregulated expression of the cofactor, protein S, as well as PC, is also associated with ischemic stroke. Studies to elucidate further the structural elements that differentiate the various functions of PC will serve to identify novel therapeutic approaches toward regulating these and other diseases.

Severe deficiency of coagulation Factor VII results in spontaneous cardiac fibrosis in mice.

Mice genetically modified to produce low levels (approximately 1% of wild-type) of coagulation FVII presented with echocardiographic evidence of heart abnormalities. Decreases in ventricular size and reductions in systolic and diastolic functions were found, suggestive of a restrictive cardiomyopathy and consistent with an infiltrative myopathic process. Microscopic analysis of mouse hearts showed severe patchy fibrosis in the low-FVII mice. Haemosiderin deposition was discovered in hearts of these mice, along with increases in inflammatory cell number, ultimately resulting in widespread collagen deposition. Significant increases in mRNA levels of TGFbeta, TNFalpha and several matrix metalloproteinases in low-FVII mice, beginning at early ages, supported a state of cardiac remodelling associated with the fibrotic pathology. Mechanistic time-course studies suggested that cardiac fibrosis in low-FVII mice originated from bleeding in heart tissue, resulting in the recruitment of leukocytes, which released inflammatory mediators and induced collagen synthesis and secretion. These events led to necrosis of cardiomyocytes and collagen deposition, characteristics of cardiac fibrosis. The results of this study demonstrated that haemorrhagic and inflammatory responses to a severe FVII deficiency resulted in the development of cardiac fibrosis, observed echocardiographically as a restrictive cardiomyopathy, with compromised ventricular diastolic and systolic functions.

A urokinase-type plasminogen activator deficiency diminishes the frequency of intestinal adenomas in ApcMin/+ mice.

The interaction of urokinase-type plasminogen activator (uPA) and its receptor, uPAR, on cell surfaces facilitates the generation of cell-bound plasmin, thus allowing cells to establish a proteolytic front that enables their migration through protein barriers. This complex also activates cell signalling pathways that influence cell functions. Clinical studies have identified uPA as an indicator of poor overall survival in patients with colorectal cancer. In the current study, a mouse model of colon cancer, Apc(Min/+), with an additional deficiency of uPA (Apc(Min/+)/Plau-/-) was used to determine the effects of uPA on tumour initiation and growth. Utilizing this model, it was found that the number of tumours was diminished in these mice relative to Apc(Min/+) mice, which correlated with the decreased leukocyte infiltration in the tumours. However, tumour growth was not impeded in Apc(Min/+)/Plau-/- mice, and proliferation and tumour vascularization were, in fact, enhanced in Apc(Min/+)/Plau-/- mice. These latter effects are consistent with a mechanism involving up-regulation of COX-2 expression and Akt pathway activation in Apc(Min/+)/Plau-/- mice. The results from this study suggest that uPA plays dual and opposing roles in regulating lesion development: one early, during the transition from normal epithelia to dysplastic lesions, and another later during tumour growth.

A coagulation factor VII deficiency protects against acute inflammatory responses in mice.

Upregulation of the activated Factor VII (FVIIa)/Tissue Factor complex, downregulation of natural anticoagulation pathways, and inhibition of fibrinolysis, are major contributors to coagulopathies associated with acute inflammation. Provision of FVIIa, and consequent downstream coagulation-related proteases, also stimulates further inflammatory changes, which can result in disseminated intravascular coagulation. Thus, the potential protective effects in vivo of a genetic-based reduction in FVII levels have been investigated in a murine model of acute inflammation, namely lipopolysaccharide (LPS)-induced lethal endotoxaemia. Mice with a total FVII deficiency do not survive the neonatal period. Therefore mice expressing low levels of FVII (FVII(tTA/tTA)), producing sufficient amounts of FVII for survival (approximately 5% of wild-type (WT) FVII), were employed to investigate in vivo pathways involved in the crosstalk between coagulation, inflammation, and survival, consequent to administration of a lethal dose of LPS. The FVII(tTA/tTA) mice presented with reduced mortality, coagulation, and inflammatory responses in comparison with similarly treated WT mice after administration of LPS. The attenuated inflammatory responses in FVII(tTA/tTA) mice were associated with downregulation of Egr-1 signalling. Administration, in vivo, of specific inhibitors of FXa and thrombin demonstrated that the inflammatory responses were unaltered in WT mice, but further reduced in FVII(tTA/tTA) mice. Therefore, a FVII deficiency enhances survival from lethal endotoxaemia both through attenuation of inflammatory responses that result directly from reduced FVIIa levels, and, indirectly, from downregulation of coagulation proteases downstream of the FVII-dependent cascade.

Delayed inflammatory responses to endotoxin in fibrinogen-deficient mice.

Severe inflammation leads to haemostatic abnormalities, such as the development of microvascular thrombi. As a result, ischaemia-related downstream organ damage can occur. The present study demonstrates that mice with a total deficiency of fibrinogen (Fg(-/-)) present with altered responses to challenge with Gram-negative lipopolysaccharide (LPS). Early survival in response to continuous LPS challenge was increased in Fg(-/-) mice and histological findings indicated that this improvement correlated with a lack of fibrin deposition in organs. Neutrophils appeared early in the lungs of challenged wild-type (WT) mice, but occurred in Fg(-/-) mice at later times. This delayed response in Fg(-/-) mice was confirmed by studies that showed a strong dependence on Fg of binding of neutrophils to endothelial cells in the presence of LPS. While cytokines were also elevated in both WT and Fg(-/-) mice, their levels were generally lower at early times in this latter group. The time course of MIP-2 expression correlated with the occurrence of pulmonary leakage after LPS challenge, which was delayed in Fg(-/-) mice. These results suggest that fibrin(ogen) plays a role as an early mediator in the cross-talk between coagulation and inflammation.

A central role for plasminogen in the inflammatory response to biomaterials.

The inflammatory response to implanted biomaterials severely limits their deployment in patients. Plasminogen has been shown to play a central role in cell migration, and therefore could regulate this inflammatory response. We sought to determine if plasminogen influences recruitment of inflammatory cells to a biomaterial implanted into plasminogen-deficient (Plg(-/-)) mice. Small disks of polyethylene terephthalate, a material used in vascular grafts, were surgically implanted into the peritoneum of wild-type and Plg(-/-) mice. Recruitment of neutrophils and monocytes/macrophages into the peritoneum and onto the disks was measured, primarily at 18 h. Monocyte/macrophage recruitment was markedly blunted in Plg(-/-) mice compared with wild-type mice. Unexpectedly, neutrophil recruitment was also markedly decreased in the Plg(-/-) mice. While recruitment of leukocytes into the peritoneum was plasminogen-dependent, the adhesion of the emigrating cells to the implants was not. In contrast, adhesion but not recruitment was reduced in fibrinogen-deficient mice. Reconstitution of Plg(-/-) mice with intravenous or intraperitoneal plasminogen differentially restored monocyte/macrophage and neutrophil recruitment. Tranexamic acid, an inhibitor of the lysine binding sites of plasminogen, suppressed leukocyte recruitment in wild-type mice, but aprotinin, a plasmin inhibitor, did not. Plasminogen exerts a marked influence on both neutrophil and monocyte/macrophage recruitment to implanted biomaterials. This role is distinct from that of fibrinogen, and the two inflammatory cell types use plasminogen in different ways. Plasminogen represents a therapeutic target for controlling the inflammatory response to implanted materials.

Activated protein C attenuates coagulation-associated over-expression of fibrinolytic activity by suppressing the thrombin-dependent inactivation of PAI-1.

Several activated coagulation factors have been reported to enhance fibrinolysis by neutralizing plasminogen activator inhibitor type 1 (PAI-1) activity. We evaluated the physiological relevance of this mechanism using the euglobulin clot lysis time (ECLT) assay in the presence and absence of Ca2+, which is controlled by PAI-1 and mimics physiological thrombolysis. We found that the ECLT (18.5 +/- 0.6 h) was shortened by Ca2+ (5 mm) (6.6 +/- 0.1 h). A significant difference was observed in thrombin generation by the presence of Ca2+ in the euglobulin fraction. Prothrombin was almost fully converted to thrombin within 15 min in the presence of Ca2+, whereas essentially no conversion was observed without Ca2+. The presence of activated protein C (aPC) suppressed thrombin generation, and attenuated the shortening of ECLT in a dose-dependent manner, an effect enhanced by phospholipid and protein S. In the absence of Ca2+, aPC did not prolong the ECLT. After addition of biotin-labeled recombinant PAI-1 to the euglobulin fraction, PAI-1 was cleaved to lower molecular weight forms only in the presence of Ca2+. This cleavage did not occur in the presence of aPC, suggesting that thrombin was the catalyst for PAI-1 cleavage. The cleavage and inactivation of PAI-1 by generated thrombin is proposed to be responsible for the shortening of ECLT by Ca2+ and for coagulation-associated over-expression of fibrinolysis. Under such conditions, aPC appeared to suppress thrombin generation and to normalize highly activated fibrinolysis.

Endogenous plasmin converts Glu-plasminogen to Lys-plasminogen on the monocytoid cell surface.

Recently, we showed that localization of Glu-plasminogen on cell surfaces enhances its conversion to Lys-plasminogen by exogenous plasmin. This leads to stimulation of plasminogen activation because Lys-plasminogen is the preferred substrate on cell surfaces. Here, we show that Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell-derived. Therefore, we tested whether endogenous monocytoid plasminogen could provide a source of plasmin to convert cell-associated Glu-plasminogen to Lys-plasminogen because plasmin is the only enzyme known to effect this reaction. We used a recombinant human plasminogen mutant, [D(646)E]Pg, which can be cleaved by plasminogen activators, but cannot catalyze the generation of Lys-plasminogen. Upon incubation with either THP-1 or U937 monocytoid cells, 35 and 38%, respectively, of the cell-bound ligand was converted to Lys-[D(646)E]Pg. Trasylol, alpha2-antiplasmin, and an anticatalytic antiplasminogen monoclonal antibody decreased Lys-[D(646)E]Pg formation to < 5% on monocytoid cells, consistent with a plasmin-dependent mechanism. Plasminogen was detected in these cells by Northern blotting and RT-PCR. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that this enzyme is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

In utero transplantation of wild-type fetal liver cells rescues factor X-deficient mice from fatal neonatal bleeding diatheses.

Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting fetal liver cells from FX+/+, Rosa26 fetuses into midgestation embryos derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and approximately 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinatally similar to untreated FX-deficient mice. The plasma levels of FX in the rescued 16-week-old FX-/- mice were approximately 1-6% of wild-type levels. beta-Galactosidase-staining cells derived from the donor Rosa26 fetal liver cells were detected in 47% of the livers of adult mice. In addition, donor-derived cells were also recovered in the bone marrow, spleen, lung, and occasionally in the brain and testis. These results suggest that in utero cell transplantation could be an effective therapeutic strategy to treat pathologies resulting from the deficiency of hepatic-expressed factors.