WASP plays a novel role in regulating platelet responses dependent on alphaIIbbeta3 integrin outside-in signalling

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent -yet- actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by alphaIIbbeta3 integrin outside-in signalling, we questioned whether its function might be linked to integrin. Agonist-induced alphaIIbbeta3 activation (PAC-1 binding) was normal for patient platelets, indicating normal integrin inside-out signalling. Inside-out signalling (fibrinogen, JON/A binding) was also normal for wasp-deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp-deficient murine platelets, indicating decreased integrin outside-in responses. Another integrin outside-in dependent response, fibrin clot retraction, involving contraction of the post-aggregation actin cytoskeleton, was also decreased for patient platelets and wasp-deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp-deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro-coagulant response, was enhanced for WASP-deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro-aggregatory and pro-coagulant responses downstream of integrin outside-in signalling.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

The plasmin-antiplasmin system: structural and functional aspects

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and (2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and (2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor (2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor (2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.

Confocal imaging protocols for live/dead staining in three-dimensional carriers

In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Glue-enhanced excimer laser-assisted nonocclusive anastomosis: a laboratory investigation

The excimer laser-assisted nonocclusive anastomosis (ELANA) technique has been developed as a clinical effective technique to perform intracranial high-flow bypass without temporary occlusion of cerebral vessels in otherwise untreatable or high-risk cerebrovascular diseases. We experimentally tested the application of a nonabsorbable cyanoacrylate-based sealant with the ELANA technique.

Meningitis caused by Mycoplasma mycoides subspecies capri in a goat

A 2-year-old, female goat from Connecticut was submitted for necropsy with a 5-day history of pyrexia and intermittent neurologic signs, including nystagmus, seizures, and circling. Postmortem examination revealed suppurative meningitis. Histologic examination of the brain revealed that the meninges were diffusely infiltrated by moderate numbers of lymphocytes, macrophages, and fibrin, with scattered foci of dense neutrophilic infiltrate. Culture of pus and brainstem yielded typical mycoplasma colonies. DNA sequencing of the 16S ribosomal RNA gene revealed 99% sequence homology with Mycoplasma mycoides subspecies capri and Mycoplasma mycoides subspecies mycoides Large Colony biotype, which are genetically indistinguishable and likely to be combined as a single subspecies labeled M. mycoides subsp. capri. The present case is unusual in that not only are mycoplasma an uncommon cause of meningitis in animals, but additionally, in that all other reported cases of mycoplasma meningitis in goats, systemic lesions were also present. In the present case, meningitis was the only lesion, thus illustrating the need to consider mycoplasma as a differential diagnosis for meningitis in goats.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

A novel mechanism for hypofibrinolysis in diabetes: the role of complement C3

Impaired fibrin clot lysis is a key abnormality in diabetes and complement C3 is one protein identified in blood clots. This work investigates the mechanistic pathways linking C3 and hypofibrinolysis in diabetes using ex vivo/in vitro studies.

New developments in the area of factor XIII

To cite this article: Schroeder V, Kohler HP. New developments in the area of factor XIII. J Thromb Haemost 2013; 11: 234-44. Summary.  Coagulation factor (F)XIII is best known for its role in fibrin stabilization and cross-linking of antifibrinolytic proteins to the fibrin clot. From patients with congenital FXIII deficiency, it is known that FXIII also has important functions in wound healing and maintaining pregnancy. Over the last decade more and more research groups with different backgrounds have studied FXIII and have unveiled putative novel functions for FXIII. FXIII, with its unique role as a transglutaminase among the other serine protease coagulation factors, is now recognized as a multifunctional protein involved in regulatory mechanisms and construction and repair processes beyond hemostasis with possible implications in many areas of medicine. The aim of this review was to give an overview of exciting novel findings and to highlight the remarkable diversity of functions attributed to FXIII. Of course, more research into the underlying mechanisms and (patho-)physiological relevance of the many described functions of FXIII is needed. It will be exciting to observe future developments in this area and to see if and how these interesting findings may be translated into clinical practice in the future.

Biologically based strategies to augment rotator cuff tears

Lesions of the rotator cuff (RC) are among the most frequent tendon injuries. In spite of the developments in both open and arthroscopic surgery, RC repair still very often fails. In order to reduce the failure rate after surgery, several experimental in vitro and in vivo therapy methods have been developed for biological improvement of the reinsertion. This article provides an overview of the current evidence for augmentation of RC reconstruction with growth factors. Furthermore, potential future therapeutic approaches are discussed. We performed a comprehensive search of the PubMed database using various combinations of the keywords "tendon," "rotator cuff," "augmentation," "growth factor," "platelet-rich fibrin," and "platelet-rich plasma" for publications up to 2011. Given the linguistic capabilities of the research team, we considered publications in English, German, French, and Spanish. We excluded literature reviews, case reports, and letters to the editor.

Activated coagulation during open and endovascular abdominal aortic aneurysm repair

OBJECTIVE: The study was conducted to determine activation of coagulation in patients undergoing open and endovascular infrarenal abdominal aortic aneurysm repair (EVAR). METHODS: In a prospective, comparative study, 30 consecutive patients undergoing open repair (n = 15) or EVAR (n = 15) were investigated. Blood samples to determine fibrinopeptide A, fibrin monomer, thrombin-antithrombin complex, and D-dimer were taken up to 5 days postoperatively. Routine hematologic and hematochemical parameters as well as clinical data were collected. RESULTS: Both groups showed comparable demographic variables. Operating time was longer in open repair (249 +/- 77 minutes vs 186 +/- 69 minutes, P < .05). Perioperatively elevated markers of coagulation were measured in both groups. Fibrinopeptide A levels did not differ significantly between the groups (P = .55). The levels of fibrin monomer and thrombin-antithrombin complex were significantly higher in patients undergoing EVAR (P < .0001), reflecting increased thrombin activity and thrombin formation compared with open surgery. The D-dimer level did not differ significantly between the groups. These results were also valid after correction for hemodilution. CONCLUSION: These data suggest increased procoagulant activity in EVAR compared with open surgery. A procoagulant state may favor possible morbidity derived from micro- and macrovascular thrombosis, such as in myocardial infarction, multiple organ dysfunction, venous thrombosis and thromboembolism, or disseminated intravascular coagulation.

Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results

OBJECTIVE: To compare four different implantation modalities for the repair of superficial osteochondral defects in a caprine model using autologous, scaffold-free, engineered cartilage constructs, and to describe the short-term outcome of successfully implanted constructs. METHODS: Scaffold-free, autologous cartilage constructs were implanted within superficial osteochondral defects created in the stifle joints of nine adult goats. The implants were distributed between four 6-mm-diameter superficial osteochondral defects created in the trochlea femoris and secured in the defect using a covering periosteal flap (PF) alone or in combination with adhesives (platelet-rich plasma (PRP) or fibrin), or using PRP alone. Eight weeks after implantation surgery, the animals were killed. The defect sites were excised and subjected to macroscopic and histopathologic analyses. RESULTS: At 8 weeks, implants that had been held in place exclusively with a PF were well integrated both laterally and basally. The repair tissue manifested an architecture similar to that of hyaline articular cartilage. However, most of the implants that had been glued in place in the absence of a PF were lost during the initial 4-week phase of restricted joint movement. The use of human fibrin glue (FG) led to massive cell infiltration of the subchondral bone. CONCLUSIONS: The implantation of autologous, scaffold-free, engineered cartilage constructs might best be performed beneath a PF without the use of tissue adhesives. Successfully implanted constructs showed hyaline-like characteristics in adult goats within 2 months. Long-term animal studies and pilot clinical trials are now needed to evaluate the efficacy of this treatment strategy.

The impact of ozone treatment on enamel physical properties

PURPOSE: To assess the effects of the highly reactive molecule of ozone on sound enamel physical properties and its effects on sealing ability. METHODS: The effect of ozone on sealant tag length, microleakage and unfilled area proportion were evaluated on intact and prepared sound molar fissures. Microhardness, contact angle and acid resistance tests were performed on ground sound smooth surfaces. The samples were treated with ozone for 40 seconds (HealOzone). Control samples were treated with air (modified HealOzone) or left untreated. RESULTS: No statistically significant difference was observed between the control and ozone treated samples in all tests. Prepared fissures exhibited no unfilled areas and a statistically significantly lower microleakage compared to intact fissures. Ozone was shown to dehydrate enamel and consequently enhance its microhardness, which was reversible.

Anticipatory cognitive stress appraisal and the acute procoagulant stress response in men

OBJECTIVE: Acute mental stress elicits blood hypercoagulability. Following a transactional stress model, we investigated whether individuals who anticipate stress as more threatening, challenging, and as exceeding their coping skills show greater stress reactivity of the coagulation activation marker D-dimer, indicating fibrin generation in plasma. METHODS: Forty-seven men (mean age 44 +/- 14 years; mean blood pressure [MBP] 101 +/- 12 mm Hg; mean body mass index [BMI] 26 +/- 3 kg/m(2)) completed the Primary Appraisal Secondary Appraisal (PASA) scale before undergoing the Trier Social Stress Test (combination of mock job interview and mental arithmetic task). Heart rate, blood pressure, plasma catecholamines, and D-dimer levels were measured before and after stress, and during recovery up to 60 minutes poststress. RESULTS: Hemodynamic measures, catecholamines, and D-dimer changed across all time points (p values <.001). The PASA "Stress Index" (integrated measure of transactional stress perception) correlated with total D-dimer area under the curve (AUC) between rest and 60 minutes poststress (r = 0.30, p = .050) and with D-dimer change from rest to immediately poststress (r = 0.29, p = .046). Primary appraisal (combined "threat" and "challenge") correlated with total D-dimer AUC (r = 0.37, p = .017), D-dimer stress change (r = 0.41, p = .004), and D-dimer recovery (r = 0.32, p = .042). "Challenge" correlated more strongly with D-dimer stress change than "threat" (p = .020). Primary appraisal (DeltaR(2) = 0.098, beta = 0.37, p = .019), and particularly its subscale "challenge" (DeltaR(2) = 0.138, beta = 0.40, p = .005), predicted D-dimer stress change independently of age, BP, BMI, and catecholamine change. CONCLUSIONS: Anticipatory cognitive appraisal determined the extent of coagulation activation to and recovery from stress in men. Particularly individuals who anticipated the stressor as more challenging and also more threatening had a greater fibrin stress response.