Fibrinolytic Cascade Research Articles

Serum Proteomics in COVID-19 Patients: Altered Coagulation and Complement Status as a Function of IL-6 Level.

Over 5 million people around the world have tested positive for the beta coronavirus SARS-CoV-2 as of May 29, 2020, a third of which are in the United States alone. These infections are associated with the development of a disease known as COVID-19, which is characterized by several symptoms, including persistent dry cough, shortness of breath, chills, muscle pain, headache, loss of taste or smell, and gastrointestinal distress. COVID-19 has been characterized by elevated mortality (over 100 thousand people have already died in the US alone), mostly due to thromboinflammatory complications that impair lung perfusion and systemic oxygenation in the most severe cases. While the levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) have been associated with the severity of the disease, little is known about the impact of IL-6 levels on the proteome of COVID-19 patients. The present study provides the first proteomics analysis of sera from COVID-19 patients, stratified by circulating levels of IL-6, and correlated to markers of inflammation and renal function. As a function of IL-6 levels, we identified significant dysregulation in serum levels of various coagulation factors, accompanied by increased levels of antifibrinolytic components, including several serine protease inhibitors (SERPINs). These were accompanied by up-regulation of the complement cascade and antimicrobial enzymes, especially in subjects with the highest levels of IL-6, which is consistent with an exacerbation of the acute phase response in these subjects. Although our results are observational, they highlight a clear increase in the levels of inhibitory components of the fibrinolytic cascade in severe COVID-19 disease, providing potential clues related to the etiology of coagulopathic complications in COVID-19 and paving the way for potential therapeutic interventions, such as the use of pro-fibrinolytic agents. Raw data for this study are available through ProteomeXchange with identifier PXD020601.

Complement in sepsis-when science meets clinics.

Sepsis as life-threatening organ dysfunction caused by microorganisms represents a dreadful challenge for the immune system. The role of the complement system as major column of innate immunity has been extensively studied in various sepsis models, but its translational value remains in the dark. Complement activation products, such as C3a and C5a, and their corresponding receptors provide useful diagnostic tools and promising targets to improve organ function and outcome. However, a monotherapeutic complement intervention irrespective of the current immune function seems insufficient to reverse the complex sepsis mechanisms. Indeed, sepsis-induced disturbances of cross talking complement, coagulation, and fibrinolytic cascades lead to systemic 'thromboinflammation', ultimately followed by multiple-organ failure. We propose to reliably monitor the complement function in the patient and to re-establish the immune balance by patient-tailored combined therapies, such as complement and Toll-like receptor inhibition. Our working hypothesis aims at blocking the 'explosive' innate immune recognition systems early on before downstream mediators are released and the inflammatory response becomes irreversible, a strategy that we name 'upstream approach'.

Use of Intraoperative Tranexamic Acid and Wound Complications in Spine Surgery: A Retrospective Cohort Study.

Study DesignA retrospective cohort study.PurposeThis study aims to examine the effect of tranexamic acid (TXA) on postoperative wound healing in spine surgery.Overview of LiteratureTXA (Cyklokapron, Hexakapron) is a widely used anti-fibrinolytic drug that is shown to be effective in mitigating hemorrhage during and after surgery by competitively blocking plasminogen in fibrinolytic cascade. Plasminogen also plays a role in inflammatory and infectious diseases. The modulation of this role by TXA may influence the development of postoperative infectious complications.MethodsWe collected and reviewed the charts of 110 patients who underwent spine surgery at our academic center. We used multivariate regression analysis to assess the factors affecting surgical site infection (SSI).ResultsOf the 110 patients included in this study, 21 patients (19%) were categorized as having postoperative wound complications, 16 patients (14%) had deep or superficial wound infection, and five patients (4%) had wound dehiscence. Patients with a higher surgical invasiveness index score, longer surgeries, and older patients were found to be at risk for wound complications. TXA was determined not to be a direct risk factor for wound healing complications and SSIs.ConclusionsWe found no risk of wound healing complications and SSI directly attributable to preoperative and intraoperative treatment with TXA in spine surgeries.

Common fibrin deposition and tissue plasminogen activator downregulation in nasal polyps with distinct inflammatory endotypes

Common fibrin deposition and tissue plasminogen activator downregulation in nasal polyps with distinct inflammatory endotypes

Endothelial glycocalyx: clinical relevance and translational approach

Endothelial glycocalyx is an undefined structure covering the luminal layer of the vascular endothelium and consisting mainly of three elements: proteoglycans, glycosaminoglycans and glycoproteins. It has different functions, such as the regulation of vascular permeability to liquids and molecules; transduction of the mechanical forces of vascular tension; regulation of coagulation and fibrinolysis cascades; and protection of leukocyte, platelet and pathogen adhesion. The determinants of a glycocalyx lesion can be of several types―e.g., increased tensile forces; reactive oxygen (O ) species; increased plasma level of substances such as sodium (hypernatremia), glucose (hyperglycemia) and cholesterol (hypercholesterolemia); and pro-inflammatory molecules. Any of the above-mentioned noxas, alone or combined, injure the glycocalyx. Its dysfunction will be clinically expressed as endothelial dysfunction, increased vascular permeability, filtration of lipoproteins to the subendothelium, activation of coagulation, or increased adhesion of leukocytes and platelets to the endothelium.

An Overview of Novel Unconventional Mechanisms of Hematopoietic Development and Regulators of Hematopoiesis \u2013 a Roadmap for Future Investigations

Hematopoietic stem cells (HSCs) are the best-characterized stem cells in adult tissues. Nevertheless, as of today, many open questions remain. First, what is the phenotype of the most primitive “pre-HSC” able to undergo asymmetric divisions during ex vivo expansion that gives rise to HSC for all hemato-lymphopoietic lineages. Next, most routine in vitro assays designed to study HSC specification into hematopoietic progenitor cells (HPCs) for major hematopoietic lineages are based on a limited number of peptide-based growth factors and cytokines, neglecting the involvement of several other regulators that are endowed with hematopoietic activity. Examples include many hormones, such as pituitary gonadotropins, gonadal sex hormones, IGF-1, and thyroid hormones, as well as bioactive phosphosphingolipids and extracellular nucleotides (EXNs). Moreover, in addition to regulation by stromal-derived factor 1 (SDF-1), trafficking of these cells during mobilization or homing after transplantation is also regulated by bioactive phosphosphingolipids, EXNs, and three ancient proteolytic cascades, the complement cascade (ComC), the coagulation cascade (CoA), and the fibrinolytic cascade (FibC). Finally, it has emerged that bone marrow responds by “sterile inflammation” to signals sent from damaged organs and tissues, systemic stress, strenuous exercise, gut microbiota, and the administration of certain drugs. This review will address the involvement of these unconventional regulators and present a broader picture of hematopoiesis.

Impact of Bradykinin Generation During Thrombolysis in Ischemic Stroke.

Ischemic stroke is one of the leading causes of death and disability worldwide. Current medical management in the acute phase is based on the activation of the fibrinolytic cascade by intravenous injection of a plasminogen activator (such as tissue-type plasminogen activator, tPA) that promotes restauration of the cerebral blood flow and improves stroke outcome. Unfortunately, the use of tPA is associated with deleterious effects such as hemorrhagic transformation, symptomatic brain edema, and angioedema, which limit the efficacy of this therapeutic strategy. Preclinical and clinical evidence suggests that intravenous thrombolysis generates large amounts of bradykinin, a peptide with potent pro-inflammatory, and pro-edematous effects. This tPA-triggered generation of bradykinin could participate in the deleterious effects of thrombolysis and is a potential target to improve neurological outcome in tPA-treated patients. The present review aims at summarizing current evidence linking thrombolysis, bradykinin generation, and neurovascular damage.

The mechanism and treatment progress of microthrombus in delayed cerebral ischemia after subarachnoid hemorrhage

Delayed cerebral ischemia (DCI) is a severe complication leading to death and disability after subarachnoid hemorrhage (SAH). Cerebral vasospasm (CVS) is generally believed as the main cause of DCI. Recently, increasing studies have demonstrated that SAH-induced activation of inflammation, coagulation or endothelial cells, and even the damage of fibrinolytic cascade may lead to the formation of microthrombus, which may eventually develop into DCI. Autopsy has confirmed the presence microthrombus. Therefore, understanding the mechanism of microthrombus and the role of microthrombus during DCI is crucial for the treatment and prognosis of SAH. Key words: Subarachnoid hemorrhage; Delayed cerebral ischemia; Microthrombus; Vasospasm

Biphasic activation of complement and fibrinolysis during the human nasal allergic response

Biphasic activation of complement and fibrinolysis during the human nasal allergic response

Prochemerin cleavage by factor XIa links coagulation and inflammation

AbstractChemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. Processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity. Contact phase activation of human platelet-poor plasma by kaolin led to cleavage of chem163S, which was undetectable in FXI-depleted plasma and markedly enhanced in platelet-rich plasma (PRP). Contact phase activation by polyphosphate in PRP resulted in 75% cleavage of chem163S. This cleavage was partially inhibited by hirudin, which blocks thrombin activation of FXI. After activation of plasma, levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared with a matched control group (91 ± 10 ng/mL vs 58 ± 3 ng/mL, n = 8; P < .01) and inversely correlated with the plasma FXI levels. Thus FXIa, generated on contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, providing a molecular link between coagulation and inflammation.

Fibrin(ogen) and neurodegeneration in the progressive multiple sclerosis cortex.

Neuronal loss, a key substrate of irreversible disability in multiple sclerosis (MS), is a recognized feature of MS cortical pathology of which the cause remains unknown. Fibrin(ogen) deposition is neurotoxic in animal models of MS, but has not been evaluated in human progressive MS cortex. The aim of this study was to investigate the extent and distribution of fibrin(ogen) in progressive MS cortex and elucidate its relationship with neurodegeneration. A postmortem cohort of pathologically confirmed MS (n = 47) and control (n = 10) cases was used. The extent and distribution of fibrin(ogen) was assessed and related to measures of demyelination, inflammation, and neuronal density. In a subset of cases (MS, n = 20; control, n = 10), expression of plasminogen activator inhibitor 1 (PAI-1), a key enzyme in the fibrinolytic cascade, was assessed and related to the extent of fibrin(ogen). Motor cortical fibrin(ogen) deposition was significantly over-represented in MS compared to control cases in all compartments studied (ie, extracellular [p = 0.001], cell body [p = 0.003], and neuritic/glial-processes [p = 0.004]). MS cases with high levels of extracellular fibrin(ogen) had significantly upregulated PAI-1 expression in all cortical layers assessed (p < 0.05) and reduced neuronal density (p = 0.017), including in the functionally-relevant layer 5 (p = 0.001). For the first time, we provide unequivocal evidence that fibrin(ogen) is extensively deposited in progressive MS motor cortex, where regulation of fibrinolysis appears perturbed. Progressive MS cases with severe fibrin(ogen) deposition have significantly reduced neuronal density. Future studies are needed to elucidate the provenance and putative neurotoxicity of fibrin(ogen), and its potential impact on clinical disability. Ann Neurol 2017;82:259-270.

Dysfunctional fibrinolysis and cerebral venous thrombosis

Cerebral venous thrombosis (CVT) is an uncommon neurological disease with high morbidity and mortality. Even after extensive thrombophilia screening, majority of the thrombosis cases remain with unknown etiology. Hypofibrinolysis due to acquired or congenital deficiencies or abnormalities in factors in the fibrinolytic cascade is a known cause of thrombosis at any site. In the present study 104 cases of radiologically confirmed CVT cases were investigated for the conventional thrombophilia along with factors in the fibrinolytic cascade to find a possible etiology for the clinical manifestation. Conventional thrombophilia markers which included PC, PS, AT and FVL mutation were detected in 16.3% of the patients. Approximately 19% cases had grossly elevated plasma PAI-1 levels. PAI-1 4G/4G genotype was found to be strongly associated with high PAI-1 levels. 2.9% cases had reduced tPA levels, 1.9% had plasminogen deficiency and 1.9% cases had increased alpha-2-antiplasmin levels. Along with conventional thrombophilia, dysfunctional fibrinolysis is found to be strongly associated with CVT. Understanding the role of risk factors is important for appropriate treatment of this serious disorder.

Role of the plasma cascade systems in ischemia/reperfusion injury of bone

Ischemia/reperfusion (I/R) injury has been extensively studied in organs such as heart, brain, liver, kidney, and lung. As a vascularized organ, bone is known to be susceptible to I/R injury too, but the respective mechanisms are not well understood to date. We therefore hypothesized that, similar to other organs, plasma cascade-induced inflammation also plays a role in bone I/R injury. Reperfusion injury in rat tibia was induced by unilateral clamping of the femoral artery and additional use of a tourniquet, while keeping the femoral vein patent to prevent venous congestion. Rats were subjected to 4h ischemia and 24h reperfusion. Deposition of complement fragment C3b/c and fibrin as well as expression of tissue factor (TF), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and E-selectin was detected by immunohistochemistry. In plasma, the levels of high mobility group box1 (HMGB1) were measured by ELISA. The total level of complement in serum was assessed by the CH50 test. Our results show that deposition of C3b/c was significantly increased with respect to healthy controls in cortical bone as well as in marrow of reperfused limbs. C3b/c deposition was also increased in cortical bone, but not in bone marrow, of contralateral limbs. Deposition of fibrin, as well as expression of PAI-1, was significantly increased in bone after ischemia and reperfusion, whereas expression of tPA was reduced. These differences were most prominent in vessels of bone, both in marrow and cortical bone, and both in reperfused and contralateral limbs. However, PAI-1, was only increased in vessels of reperfused cortical bone and there were no significant changes in expression of E-selectin. With respect to solid bone tissue, a significant increase of C3b/c and fibrin deposition was shown in osteocytes, and for fibrin also in the bone matrix, in both contralateral and reperfused cortical bone compared with normal healthy controls. A slight expression of TF was visible in osteocytes of the normal healthy control group, while TF was not present in the experimental groups. Moreover, CH50 values in serum decreased over time and HMGB1 was significantly increased in plasma of animals at the end of reperfusion. We conclude that ischemia and reperfusion of bone leads to activation of the complement and coagulation systems and a downregulation of the fibrinolytic cascade. In the acute phase, a vascular inflammation induced by activation of the plasma cascade systems also occurs in the bone. This is similar to I/R injury of other vascularized organs and tissues.

F376A/M388A-solulin, a new promising antifibrinolytic for severe haemophilia A.

Haemophilia is a major bleeding disorder due to a deficiency of procoagulant factor VIII (type A) or IX (type B). The treatment is substitutive and based on infusion of factor concentrates. Main limitations of this therapy are cost, short factor half-life and the development of inhibitors (up to 30% of severe HA patients). An important aggravating factor of haemophilia is due to a premature fibrinolysis, directing attention to the therapeutic potential of suitable antifibrinolytics. Thrombomodulin (TM) is a key player of the coagulation cascade by activating protein C (an inhibitor of thrombin generation, thus antagonizing coagulation) and of the fibrinolytic cascade by activating thrombin activatable fibrinolysis inhibitor TAFI (thus reducing fibrinolysis). Solulin is a soluble form of TM that shows both capabilities. Here, we developed a new generation of solulin variants (F376A-, M388A- and F376A/M388A-solulin) with a decreased ability to activate protein C and a conserved capacity to activate TAFI. We produced and characterized solulin variants in vitro. In addition, F376A/M388A-solulin was tested ex vivo, using blood samples of haemophilic A patients, with thromboelastography. The solulin variants (F376A, M388A and the double-mutant F376A/M388A) lost their abilities to activate protein C but are still capable to activate TAFI. Thrombelastography showed increased clot firmness and stability, that, as opposed to wild-type solulin, was maintained even at high concentrations of F376A/M388A-solulin (100 nm). In sum, these results open new opportunities for the development of specific medication for haemophilic patients.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

Bone marrow(BM) residing stem cells are mobilized from their BM niches into peripheral blood(PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells(SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast‑like stem cells(VSELs) and mesenchymal stem cells(MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate(S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer(CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas(BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting(FACS) we evaluated the number of BM-derived SCs circulating in PB: i)CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii)CD45-/CD105+/CD90+/CD29+ MSCs; iii)CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells(EPCs); and iv)CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells(HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor1(SDF‑1), vascular endothelial growth factor(VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

The effect of exercise on coagulation and fibrinolysis factors in patients with peripheral arterial disease.

Peripheral arterial disease is a widely prevalent atherosclerotic occlusive disorder. Symptoms commence with exercise-induced pain in the lower extremities, known as claudication. Despite the fact that exercise has been shown to improve fibrinolytic profile some patients, the effect of exercise on coagulation and fibrinolysis cascades in claudicants has not been comprehensively defined. Literature search in English language yielded 13 studies of exercise on claudicants, including 420 patients. Claudicants tend to have a higher coagulation activity at rest compared to healthy individuals, a trend that persists even after exercise. Post-exercise coagulation activity of claudicants is increased when compared to their respective baseline levels, but it is so in a non-consistent manner. From the available data, it has been suggested that claudicants have a functional and effective fibrinolytic mechanism in place, operating continuously at a relatively higher activity level compared to healthy individuals. Fibrinolysis seems to be activated by exercise; a positive outcome with a prolonged effect as shown by a few of the studies. A final conclusion whether coagulation or fibrinolysis activity is affected mostly by exercise type and intensity in claudicants could not be answered. All conclusions regarding the effect of exercise on the coagulation and fibrinolysis mechanisms should be taken under cautious consideration, due to the limited number of studies, the small number of patients and the different exercise strategies employed in each study. Further randomized studies with similar exercise protocols could provide safer conclusions in the future.

Plasmin as a complement C5 convertase

Both the complement and coagulation/fibrinolytic systems are essential components of the host defense system that evolved early in the evolution of vertebrates (Krem and Di Cera, 2002). While the coagulation and fibrinolytic systems are necessary for maintenance of the integrity of the vasculature, these systems also respond to infections, and together with the complement system, play an important role in innate and adaptive immunity. There are two pathways in mammals that initiate coagulation and three that initiate complement while fibrinolysis commences in response to the presence of a fibrin clot (Bajic et al., 2015, Smith et al., 2015). The intrinsic or contact pathway for initiation of coagulation and the alternative (properdin) and lectin pathways that initiate complement activation are activated by specific properties of some surfaces such as bacterial or yeast cell walls. Cross talk between the various systems is apparent with several well-described interactions such as that between C4-binding protein (C4BP) that regulates complement by inhibiting the classical initiation pathway and protein S, the latter being a cofactor for activated protein C, a regulator of coagulation and inflammation. There are in vitro and in vivo data that suggest coagulation cascade enzymes such as thrombin could cleave C5, carrying out the same reactions as the C5 convertase (Huber-Lang et al., 2006). The cleavage products, however, did not directly generate C5a (Krisinger et al., 2012). A new study (Foley et al., 2016) shows in a venous stasis model that C5a correlates strongly with clot weight implying that pathways initiated during clot formation lead to C5 cleavage. In contrast, there is a very weak correlation with thrombin–antithrombin complexes, suggesting that thrombin is not directly cleaving C5. They then investigated the ability of thrombin, factor Xa and plasmin to cleave C5 by measuring formation of C5a by ELISA. Plasmin, the effector enzyme in the fibrinolytic cascade, was the only enzyme that was able to cleave complement C5 at a similar rate as the canonical C5 convertase formed from complement components. The resultant C5b can assemble into the next stage of the terminal pathway, C5b,6, showing that the C5b generated by plasmin cleavage is functional in assembling the membrane attack complex. The hypothesis that plasmin can act as a C5 convertase was tested in an arterial model in which plasminogen was activated by tissue plasminogen activator (tPA). They found that the levels of C5a were increased, consistent with plasmin activating C5. C5 activation with consequent generation of C5a and the membrane attack complex is a feature of many diseases such as arthritis, sepsis and acute lung injury. Defining the extent of plasmin's role in C5 cleavage outside of its role in thrombotic diseases would clarify if this reaction is important in other contexts. The possibility of exploiting this mechanism as a therapeutic target would need to be approached carefully because of the critical role plasmin plays not just in fibrinolysis but also in tissue repair and inflammatory cell migration (Syrovets et al., 2012). The idea that plasmin is a bona fide C5 convertase has several implications for the links between complement and coagulation. Fibrinolysis occurs after coagulation and therefore, the C5 cleavage products will be present during clot resolution as well as clot formation and progression. However, C5a is a major chemoattractant and activator for neutrophils, leading to elaboration of procoagulant neutrophil extracellular traps (NETs) (Yousefi et al., 2009). Leukocytes attracted to the thrombus can contribute to lysis by secretion of plasminogen activators, thus the precise balance between the prothrombotic and fibrinolytic factors may depend on the disease setting. This study focused on the role of plasmin in C5 cleavage in thrombotic scenarios, but complement plays a key role in eliminating infections. In some infectious diseases such as Yersinia, fibrinogen deficiency leads to decreased survival while plasminogen deficiency increases survival (Degen et al., 2007). The survival advantage conferred by plasminogen deficiency is effectively eliminated in fibrinogen/plasminogen double deficient mice. Thus formation of fibrin prevents the spread of the disease and fibrinolysis promotes it. The Yersinia virulence factor, Pla, can activate plasminogen and many other bacteria express plasminogen activators. It will be important to investigate the role of plasmin in activating the terminal pathway of complement under these circumstances. Foley et al. (Foley et al., 2016) only consider the forward reactions, but these cascades are tightly regulated. The rate of plasminogen activation is closely controlled by the availability of tPA as well as by plasminogen activator inhibitor-1 while plasmin activity is regulated by alpha-2 antiplasmin. Interestingly, carboxypeptidase B2 (TAFI), which can be activated from its zymogen by either thrombin bound on thrombomodulin or by plasmin (Morser et al., 2010), both reduces activation of plasminogen as well as inactivating C3a and C5a, thereby functioning as a regulator of both the enzyme cleaving C5 and one of the products, C5a.

Evidence of a Pivotal Role for the Distal Part of the Complement Cascade in the Diurnal Release of Hematopoietic Stem Cells Into Peripheral Blood.

We found that diurnal activation of the three evolutionarily ancient proteolytic cascades in peripheral blood (PB), namely, the complement, coagulation, and fibrinolytic cascades, late at night or in the early morning hours, precedes the diurnal release of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into PB in wild-type mice. Moreover, activation of the distal part of the complement cascade (ComC), involving cleavage of the fifth component (C5), seems to play a crucial role in pharmacological mobilization of HSPCs. In order to shed more light on the role of diurnal rhythms in the egress of HSPCs, we studied diurnal changes in the number of circulating HSPCs in C5-deficient mice and did not observe diurnal changes in the number of these cells circulating in PB in C5(-/-) animals. Based on this finding, we conclude that activation of the distal part of the ComC, C5 cleavage, and release of C5a and desArgC5a are required in executing the diurnal release of HSPCs from BM into PB. Moreover, the fact that C5(-/-) mice still displayed normal activation of the coagulation and fibrinolytic cascades indicates that, of all the proteolytic cascades, the ComC is the dominant player regulating diurnal egress of HSPCs.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions.

Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression.

Tissue regeneration during wound healing or cancer growth and progression depends on the establishment of a cellular microenvironment. Mesenchymal stem cells (MSC) are part of this cellular microenvironment, where they functionally modulate cell homing, angiogenesis, and immune modulation. MSC recruitment involves detachment of these cells from their niche, and finally MSC migration into their preferred niches; the wounded area, the tumor bed, and the BM, just to name a few. During this recruitment phase, focal proteolysis disrupts the extracellular matrix (ECM) architecture, breaks cell-matrix interactions with receptors, and integrins, and causes the release of bioactive fragments from ECM molecules. MSC produce a broad array of proteases, promoting remodeling of the surrounding ECM through proteolytic mechanisms. The fibrinolytic system, with its main player plasmin, plays a crucial role in cell migration, growth factor bioavailability, and the regulation of other protease systems during inflammation, tissue regeneration, and cancer. Key components of the fibrinolytic cascade, including the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are expressed in MSC. This review will introduce general functional properties of the fibrinolytic system, which go beyond its known function of fibrin clot dissolution (fibrinolysis). We will focus on the role of the fibrinolytic system for MSC biology, summarizing our current understanding of the role of the fibrinolytic system for MSC recruitment and the functional consequences for tissue regeneration and cancer. Aspects of MSC origin, maintenance, and the mechanisms by which these cells contribute to altered protease activity in the microenvironment under normal and pathological conditions will also be discussed.