Thrombin Activity Research Articles

Delayed administration of nafamostat mesylate inhibits thrombin-mediated blood–spinal cord barrier breakdown during acute spinal cord injury in rats

BackgroundNafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action.MethodsTime to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood–spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.ResultsThe optimal administration time window of nafamostat was 2–12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2–12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site.ConclusionsOur study provides preclinical information of nafamostat about the administration time window of 2–12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.

Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 μg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 μg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

Chelidonium majus WATER EXTRACT INITIATES PLATELET AGGREGATION AND INHIBITS FIBRIN POLYMERIZATION IN BLOOD PLASMA

Chelidonium majus is a well-known source of biologically active compounds. Most of them are alkaloids, which are used in researches and for tradition medicine. In this study, we explored the influence of C. majus crude total extract onto blood coagulation system in vitro, primary and secondary hemostasis. Aim. To study influence of C. majus extract onto blood coagulation process. Methods. Turbidimetry of blood plasma, APTT test with chromogenic substrates, and platelet aggregation were used in the work. Results. We demonstrated moderate stimulating effect of the extract on platelets (the rate of platelet aggregation increased up to 10%, followed by disaggregation). Extract also increased the rate of platelet aggregation stimulated by 12.5 mcM of ADP. We observed the increase in the plasma clotting time in the presence of the extract, that corresponded to the 274, 411, 685, 1370 mcg of dry extract, from 70 s in control to 80, 90, 170 and 180 s, respectively, in turbidimetry test of plasma stimulated by APTT-reagent. However, APTT test with the chromogenic substrate of thrombin (S2238) did not show significant influence of this plant’s extract on thrombin activity. Conclusions. Thus, we can conclude that anticoagulant activity of Chelidonium majus extract corresponded to the direct inhibition of fibrin polymerization.

Fibrin(ogen) Is Constitutively Expressed by Differentiated Intestinal Epithelial Cells and Mediates Wound Healing.

Fibrinogen is a large molecule synthesized in the liver and released in the blood. Circulating levels of fibrinogen are upregulated after bleeding or clotting events and support wound healing. In the context of an injury, thrombin activation drives conversion of fibrinogen to fibrin. Fibrin deposition contains tissue damage, stops blood loss, and prevents microbial infection. In most circumstances, fibrin needs to be removed to allow the resolution of inflammation and tissue repair, whereas failure of this may lead to the development of various disorders. However, the contribution of fibrinogen to tissue inflammation and repair is likely to be context-dependent. In this study, the concept that fibrin needs to be removed to allow tissue repair and to reduce inflammation is challenged by our observations that, in the intestine, fibrinogen is constitutively produced by a subset of intestinal epithelial cells and deposited at the basement membrane as fibrin where it serves as a substrate for wound healing under physiological conditions such as epithelial shedding at the tip of the small intestinal villus and surface epithelium of the colon as well as under pathological conditions that require rapid epithelial repair. The functional integrity of the intestine is ensured by the constant renewal of its simple epithelium. Superficial denuding of the epithelial cell layer occurs regularly and is rapidly corrected by a process called restitution that can be influenced by various soluble and insoluble factors. Epithelial cell interaction with the extracellular matrix greatly influences the healing process by acting on cell morphology, adhesion, and migration. The functional contribution of a fibrin(ogen) matrix in the intestine was studied under physiological and pathological contexts. Our results (immunofluorescence, immunoelectron microscopy, and quantitative PCR) show that fibrin(ogen) is a novel component of the basement membrane associated with the differentiated epithelial cell population in both the small intestine and colon. Fibrin(ogen) alone is a weak ligand for epithelial cells and behaves as an anti-adhesive molecule in the presence of type I collagen. Furthermore, the presence of fibrin(ogen) significantly shortens the time required to achieve closure of wounded epithelial cell monolayers and co-cultures in a PI3K-dependent manner. In human specimens with Crohn’s disease, we observed a major accumulation of fibrin(ogen) throughout the tissue and at denuded sites. In mice in which fibrin formation was inhibited with dabigatran treatment, dextran sulfate sodium administration provoked a significant increase in the disease activity index and pathological features such as mucosal ulceration and crypt abscess formation. Taken together, these results suggest that fibrin(ogen) contributes to epithelial healing under both normal and pathological conditions.

Mild acid hydrolysis on Fucan sulfate from Stichopus herrmanni: Structures, depolymerization mechanism and anticoagulant activity

Fucan sulfate (FS) from sea cucumbers possesses linear sequences with repeating units that differ principally in the pattern of sulfation and position of glycosidic linkage. FS from Stichopus herrmanni (ShFS) was preliminarily identified as the relatively simple structure {-3)-L-Fuc2S-(α1-}n. Herein, mild acid hydrolysis was employed on ShFS to yield 21 oligosaccharides. Analyses on their structures complemented the features of ShFS to refine its spectral signal assignments. Combining with the methylation analysis, unit L-Fuc2S4S was determined as the micro-component in its intact structure. The irregularity came from minor sulfation on O-4. Temperature and acid concentration were the critical parameters to the depolymerization and formation of oligosaccharides. Meanwhile, a three-step cleavage of the hydrolysis mechanism involving the partial O-2 de-sulfation and preferential cleavage between Fuc0S and Fuc2S4S was proposed. Bioactivity assays revealed that the Mw 15–16 kDa conferred the potent anticoagulation via inhibiting the thrombin activity mediated by heparin cofactor II.

Thrombin Activity in Rodent and Human Skin: Modified by Inflammation and Correlates with Innervation.

Thrombin is present in peripheral nerves and is involved in the pathogenesis of neuropathy. We evaluated thrombin activity in skin punch biopsies taken from the paws of male mice and rats and from the legs of patients with suspected small-fiber neuropathy (SFN). In mice, inflammation was induced focally by subcutaneous adjuvant injection to one paw and systemically by intraperitoneal lipopolysaccharides (LPS) administration. One day following injection, thrombin activity increased in the skin of the injected compared with the contralateral and non-injected control paws (p = 0.0009). One week following injection, thrombin increased in both injected and contralateral paws compared with the controls (p = 0.026), coupled with increased heat-sensitivity (p = 0.009). Thrombin activity in the footpad skin was significantly increased one week after systemic administration of LPS compared with the controls (p = 0.023). This was not accompanied by increased heat sensitivity. In human skin, a correlation was found between nerve fiber density and thrombin activity. In addition, a lower thrombin activity was measured in patients with evidence of systemic inflammation compared with the controls (p = 0.0035). These results support the modification of skin thrombin activity by regional and systemic inflammation as well as a correlation with nerve fiber density. Skin thrombin activity measurments may aid in the diagnosis and treatment of SFN.

Factor VII, EPCR, aPC Modulators: novel treatment for neuroinflammation

BackgroundInflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing increased inflammation, and activated protein C (aPC), inducing decreased inflammation. Modulation of the aPC-PAR1 pathway may prevent the neuroinflammation associated with PAR1 over-activation.MethodsWe synthesized a group of novel molecules based on the binding site of FVII/aPC to the endothelial protein C receptor (EPCR). These molecules modulate the FVII/aPC-EPCR pathway and are therefore named FEAMs—Factor VII, EPCR, aPC Modulators. We studied the molecular and behavioral effects of a selected FEAM in neuroinflammation models in-vitro and in-vivo.ResultsIn a lipopolysaccharide (LPS) induced in-vitro model, neuroinflammation leads to increased thrombin activity compared to control (2.7 ± 0.11 and 2.23 ± 0.13 mU/ml, respectively, p = 0.01) and decreased aPC activity (0.57 ± 0.01 and 1.00 ± 0.02, respectively, p < 0.0001). In addition, increased phosphorylated extracellular regulated kinase (pERK) (0.99 ± 0.13, 1.39 ± 0.14, control and LPS, p < 0.04) and protein kinase B (pAKT) (1.00 ± 0.09, 2.83 ± 0.81, control and LPS, p < 0.0002) levels indicate PAR1 overactivation, which leads to increased tumor necrosis factor-alpha (TNF-α) level (1.00 ± 0.04, 1.35 ± 0.12, control and LPS, p = 0.02). In a minimal traumatic brain injury (mTBI) induced neuroinflammation in-vivo model in mice, increased thrombin activity, PAR1 activation, and TNF-α levels were measured. Additionally, significant memory impairment, as indicated by a lower recognition index in the Novel Object Recognition (NOR) test and Y-maze test (NOR: 0.19 ± 0.06, -0.07 ± 0.09, p = 0.03. Y-Maze: 0.50 ± 0.03, 0.23 ± 0.09, p = 0.02 control and mTBI, respectively), as well as hypersensitivity by hot-plate latency (16.6 ± 0.89, 12.8 ± 0.56 s, control and mTBI, p = 0.01), were seen. FEAM prevented most of the molecular and behavioral negative effects of neuroinflammation in-vitro and in-vivo, most likely through EPCR-PAR1 interactions.ConclusionFEAM is a promising tool to study neuroinflammation and a potential treatment for a variety of neuroinflammatory diseases.

The Polymerization of Proteins: the Action of Thrombin on Fibrinogen

The Polymerization of Proteins: the Action of Thrombin on Fibrinogen

1339-P: Plasma Heparin Cofactor II Activity Is Inversely Associated with the Development NAFLD in Patients with Diabetes

Aims and Introduction: The prevalence and incidence of nonalcoholic fatty liver disease (NAFLD) have been increasing worldwide. Thrombin exerts various pathophysiological functions via activation of protease-activated receptors (PARs) , and activation of PARs promotes the development of NAFLD. Since heparin cofactor II (HCII) specifically inhibits thrombin action by binding to dermatan sulfate proteoglycans in various tissue matrices, we hypothesized that plasma HCII activity is associated with the development of NAFLD. Materials and Methods: Plasma HCII activity and clinical markers for assessment of hepatic fibrosis including Fibrosis 4 (FIB-4) index, NAFLD fibrosis score (NFS) , and aspartate aminotransferase to platelet ratio index (APRI) were determined in 322 Japanese patients with diabetes mellitus (183 males and 139 females; mean age, 66.0±11.3 years) . The relationships between plasma HCII activities and the clinical markers were statistically evaluated. Results: The mean plasma HCII activity in all participants was 94.0 ± 17.7%. Multivariate regression analysis with adjustments for confounding factors including age, sex, and serum albumin showed that plasma HCII activity independently contributed to reduction of the FIB-4 index (p &amp;lt; 0.0001) , NFS (p &amp;lt; 0.0001) , and APRI (p &amp;lt; 0.05) . Conclusions: Plasma HCII activity was independently and inversely associated with the clinical markers of hepatic fibrosis. The results suggest that HCII can serve as a novel therapeutic target for the development of NAFLD in patients with diabetes. Disclosure T.Hara: None. I.Endo: None. M.Matsuhisa: Research Support; Nissui , Sysmex Corp., Speaker’s Bureau; Astellas Pharma Inc., Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. M.Abe: None. K.Aihara: None. H.Yamagami: None. S.Masuda: None. Y.Mitsui: None. K.Kurahashi: None. S.Yoshida: None. T.Otoda: None. T.Yuasa: None. A.Kuroda: None.

In Vitro Antithrombotic, Hematological Toxicity, and Inhibitor Studies of Protocatechuic, Isovanillic, and p-Hydroxybenzoic Acids from Maclura tricuspidata (Carr.) Bur.

In blood coagulation, circulating platelets and coagulation factors are crucial for the primary process because thrombi are generated by fibrin clotting with fibrinogen, thrombin, FXIIIa, and platelet activation. Therefore, strategies to reduce the activity of key coagulation factors, or interfere with their functions and delay the activation of platelets can be used as important tools to suppress excessive blood clot formation and platelet hyperactivation. This study examined the antithrombotic activity and hematological toxicity of PA, IVA, and 4-HA isolated from M. tricuspidata (Carr.) Bur in several in vitro experiments and inhibitor assays. We found that PA, IVA, and 4-HA attenuated the formation of fibrin polymers/clots and degraded the blood clots. These compounds inhibited the activities of procoagulant proteases and fibrinoligase, and prolonged the coagulation time. There was a significant reduction in platelet function and ATP or serotonin levels in thrombin-activated platelets. An inhibitor study showed that PA exhibited a mixed inhibition type for thrombin, an uncompetitive inhibition type for FXa, and a non-competitive inhibition type for FXIIIa and IVA, while 4-HA exhibited an uncompetitive inhibition type for thrombin and non-competitive inhibition type for FXa and FXIIIa. These three compounds (up to 50 μg/mL) were not toxic to blood cells.

Precisely co-delivery of protein and ROS scavenger with platesomes for enhanced endothelial barrier preservation against myocardial ischemia reperfusion injury

Following myocardial ischemia–reperfusion (MI/R), the endothelial barrier is heavily damaged. This is partially due to the accumulation of intracellular reactive oxygen species (ROS) in endothelial cells, leading to disruptive intercellular VE-Cadherin junctions and endothelial cells apoptosis. Systemic administration of drugs is limited by poor delivery and release at pathological sites, resulting in compromised therapeutic effect. Herein, by exploiting the inherent nature of platelets migrating to injured endothelium and local activation of thrombin, we fabricated functional platesomes (Fe@PLP-TR-A) to co-deliver angiopoietin-like 4 (ANGPTL4) and ROS scavenger Fe3O4 to injured endothelium in heart affected by MI/R. Thanks to the capability for multidrug loading and easy-modification feature of platesomes, we conjugated ANGPTL4 on the surface of platesomes with a thrombin responsive peptide and encapsulated Fe3O4 in the inner space. After systemic injection, Fe@PLP-TR-A successfully arrived at the endothelial cells at injured endothelium due to the platelet-mimetic adhesion effect of platesomes which was fabricated by fusing platelet membranes and lipid membranes. Following the cleavage of thrombin-responsive peptide, which was activated by injured endothelium, ANGPTL4 was released extracellularly and bound to the receptor on endothelial cells to prevent the disruption of VE-Cadherin junctions. Further, endo-lysosomal escape of Fe3O4 to cytosol exerted ROS scavenging effect and protected endothelial cells from apoptosis. VE-Cadherin maintenance and cell rescue together preserved the endothelial barrier and subsequently decreased leukocytes extravasation and intracardial hemorrhage, alleviated cardiomyocyte apoptosis and eventually improved cardiac function. This work demonstrates a practical platform for the treatment of reperfusion injury by enhancing targeted release of drugs to injured endothelium.

Gender differences in hemostatic potential in coronary artery disease

Abstract Funding Acknowledgements Type of funding sources: None. Background Clinical presentation and contribution of different risk factors in coronary artery disease (CAD) vary between genders. Women are known to be generally older, have more comorbidities and are more likely to present with atypical symptoms. Previous research suggests there are important gender differences in the pathophysiology of CAD. One of the important mechanisms for atherothrombotic events seems to be increased coagulation, for which differences in presentation between genders is not yet fully documented. Purpose Our aim was to investigate a possible correlation between gender and coagulation appraised by overall hemostatic, coagulation and fibrinolytic potentials (OHP, OCP and OFP) in patients with CAD. Methods Consecutive patients after a recent acute myocardial infarction (within 90 days from inclusion) had fasting blood samples withdrawn. OHP, OCP and OFP were determined using thrombin and recombinant tissue-type plasminogen activator by absorbance measurements at 405 nm in 1-minute intervals for 40 minutes. Areas under the curve were constructed for OCP and OHP with the obtained measurements, OFP was calculated as the difference between the two areas: OFP = [(OHP – OCP) / OCP] x 100 (%). Baseline data and cardiovascular risk factors profiles were collected. Proportions were compared using χ2 test, means were compared using t-test, medians were compared using Mann-Whitney U test, linear regression models were constructed for the multivariate analysis. Results We included 117 patients (mean age 56 ± 10 years), 23 (20%) were women. Arterial hypertension was present in 86 (73.5%), diabetes mellitus in 10 (8.5%), dyslipidemia in 75 (64.1%), family history in 46 (39.3%) patients, 54 (46.2%) were active smokers (within 2 years). There were no significant differences in mean age and risk factors presence between genders. Median OCP and median OHP were significantly higher in women (25.1 (interquartile range [IQR] 5.8) vs 21.8 (IQR 5.7), p=0.035; and 9.5 (IQR 4.3) vs 7.8 (IQR 3.1), p=0.032, respectively) while median OFP were similar. Female gender retained its significant predictive value after multivariate adjustment for age and traditional risk factors for OCP (R2 0.25; ANOVA for regression p&amp;lt;0.001; Beta 2.96 [0.61-5.31], p=0.014) and OHP (R2 0.24; ANOVA for regression p&amp;lt;0.001; Beta 2.04 [0.76-3.32], p=0.002). Conclusion In patients after acute myocardial infarction women seem to have a higher procoagulant state, as determined by OCP and OHP. Our findings suggest different pathophysiology of CAD in women, further research is needed to fully understand and address gender differences in CAD.

In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID‐19

Study objectiveEndothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells.MethodsCitrate plasma was sampled as part of the Pa‐COVID‐19 cohort study (ethics approval EA2/066/20) from patients with severe COVID‐19 (high flow O2 or mechanically ventilated; WHO severity score: 5‐7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell‐substrate impedance sensing (ECIS), immunofluorescence for endothelial VE‐cadherin and F‐actin, western blot analyses of PAR‐1 cleavage, and real‐time Ca2+ imaging. Plasma from healthy donors served as control.ResultsCOVID‐19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti‐coagulant with thromboprotective function were decreased. COVID‐19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca2+ influx in response to COVID‐19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC‐067047).ConclusionHere, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID‑19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID‐19.

Low COAT platelets are frequent in patients with bleeding disorders of unknown cause (BDUC) and can be enhanced by DDAVP

Low COAT platelets are frequent in patients with bleeding disorders of unknown cause (BDUC) and can be enhanced by DDAVP

Design of variants of human fibroblast growth factor 1 with enhanced stability and cell proliferation activity

Human Fibroblast growth factor 1 (hFGF1) is a member of FGF1 subfamily which play significant biological roles, including cell proliferation, cell differentiation, and wound healing. Under physiological temperatures, hFGF1 has low thermodynamic stability and susceptible to the action of proteases. Circular dichroism, fluorescence spectroscopy, and other related studies reveal that reversal of charge on R136 by acidic amino acids (Glu) drastically increases the enzymatic resistance to thrombin, thermal stability, and cell proliferation activity of the growth factor. Interestingly, introduction of R136D mutation shows that the protein stability and cell proliferation activity is similar to the wild‐type protein. Microsecond molecular dynamics studies suggest that length of the side chain of acidic amino acid at position 136 is critical to confer stability to the protein. Detailed analysis of the results will be presented.

Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI

AbstractImpairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbβ3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.

Pre-operative Hemostatic Status in Dogs Undergoing Splenectomy for Splenic Masses

Portal system thrombosis is a rare but potentially fatal complication of splenectomy in dogs. The mechanism behind development of post-operative portal system thrombosis is unclear but may include alterations of portal blood flow following surgery, acquired hypercoagulability and endothelial dysfunction. The aim of the study was to evaluate hemostatic biomarkers in hemodynamically stable (heart rate <130 beats/min, blood lactate < 2.5 mMol/L) and non-anemic (hematocrit >35%) dogs prior to splenectomy for splenic masses. Our hypothesis was that this population of stable dogs would have pre-existing laboratory evidence of hypercoagulability unrelated to shock, bleeding, anemia, or other pre-operative comorbidities. Pre-operatively, abdominal ultrasonography was performed and blood was collected for platelet enumeration, prothrombin time (PT), activated partial thromboplastin time (aPTT), kaolin-activated thromboelastography (TEG), fibrinogen, von Willebrand factor activity (vWF:Ag), antithrombin and thrombin-antithrombin complex (TAT). Histopathological diagnosis and 30-day survival were recorded. None of the 15 enrolled dogs had pre-operative sonographic evidence of portal system thrombosis. Three of fifteen dogs were thrombocytopenic, three had thrombocytosis, three were hyperfibrinogenemic, one had low vWF:Ag, three had mild prolongations of PT and none had abnormal aPTT. Based on the TEG G value, 13/15 dogs were hypercoagulable (mean ± SD 13.5 ± 5.4 kd/s). Antithrombin deficiency was identified in 9/15 dogs (mean ± SD 68.7 ± 22.7%) with 5/9 having concurrently elevated TAT suggesting active thrombin generation. No dogs developed portal system thrombosis and all achieved 30-day survival. Pre-operative hypercoagulability was recognized commonly but its association with post-operative thrombosis remains undetermined.

Thrombin induces morphological and inflammatory astrocytic responses via activation of PAR1 receptor

Spinal cord injury (SCI) will result in the significant elevation of thrombin production at lesion site via either breakage of blood-spinal cord barrier or upregulated expression within nerve cells. Thrombin-induced activation of the protease activated receptors (PARs) evokes various pathological effects that deteriorate the functional outcomes of the injured cord. The cellular consequences of thrombin action on the astrocytes, as well as the underlying mechanism are not fully elucidated by far. In the present study, SCI model of rats was established by contusion, and primary astrocytes were isolated for culture from newborn rats. The expression levels of thrombin and PAR1 receptor at lesion sites of the spinal cord were determined. The primary astrocytes cultured in vitro were stimulated with different concentration of thrombin, and the resultant morphological changes, inflammatory astrocytic responses, as well as PAR1-activated signal pathway of astrocytes were accordingly examined using various agonists or antagonists of the receptor. Thrombin was found to reverse astrocytic stellation, promote proliferation but inhibit migration of astrocytes. Furthermore, the serine protease was shown to facilitate inflammatory response of astrocytes through regulation of MAPKs/NFκB pathway. Our results have provided the morphological evidence of astrocytic reactivity in response to thrombin stimulation and its neuroinflammatory effects following SCI, which will be indicative for the fundamental insights of thrombin-induced neuropathology.

The role of thrombin in haemostasis.

Thrombin is a multifunctional serine protease generated in injured cells. The generation of thrombin in coagulation plays a central role in the functioning of haemostasis. The last enzyme in the coagulation cascade is thrombin, with the function of cleaving fibrinogen to fibrin, which forms the fibrin clot of a haemostatic plug. Although thrombin primarily converts fibrinogen to fibrin, it also has many other positive regulatory effects on coagulation. Thrombin has procoagulant, inflammatory, cellular proliferation and anticoagulant effects. In coagulation system, thrombin has two very distinct roles. Firstly, it acts as a procoagulant when it converts fibrinogen into an insoluble fibrin clot, activates factor (F) XIII, activates thrombin activatable fibrinolysis inhibitor (TAFI) and activates FV, FVIII and FXI. Thrombin also enhances platelet adhesion by inactivating a disintegrin and metalloprotease with thrombospondin type1 motif (ADAMTS13). However, when thrombin activates protein C, it acts as an anticoagulant. A natural anticoagulant pathway that supplies regulation of the blood coagulation system contains protein C, which is the key component. This is accomplished by the specific proteolytic inactivation of FV and FVIII. In this review, the multiple roles of thrombin in the haemostatic response to injury are studied in addition to the cofactors that determine thrombin activity and how thrombin activity is thought to be coordinated.

Direct thrombin inhibitor-bivalirudin improved the hemocompatibility of electrospun polycaprolactone vascular grafts

Synthetic artificial vascular grafts are hampered by their low patency rate in substituting small-caliber arteries (inner diameter <6 mm) because of their poor hemocompatibility . As compared to the heparin, bivalirudin (BV), a direct thrombin inhibitor, is a more reliable anticoagulant. In this study, BV-loaded polycaprolactone (PCL) grafts (PCL-BV) were fabricated by electrospinning. The loading rate of BV was approximately 87%. The PCL-BV grafts achieved the sustained release of BV for up to 30 days in vitro , but mainly released in the first 15 days. Scanning electron microscopy (SEM) and mechanical testing revealed that the BV loading did not affect the structure and mechanical properties of PCL grafts. In vitro coagulation analysis showed that the loading of the BV reduced thrombin activity, prolonged hemocompatibility parameters (activated partial thromboplastin time (APTT), prothrombin time (PT) and thromboplastin time (TT)), reduced blood coagulation, and inhibited the adhesion and activation of fibrinogen (FIB) and platelets. The assessment of vascular grafts in a rabbit arteriovenous (AV)-shunt ex vitro and carotid artery implantation in vivo indicated that BV significantly improved the antithrombogenicity and patency of PCL grafts. Overall, these results suggested that the incorporation of BV into electrospun grafts would be an effective approach to improve the hemocompatibility and patency rate of synthetic vascular grafts.