Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Lifestyle Influences Changes in Fibrin Clot Properties Over a 10-Year Period on a Population Level.

Case-control and observational studies have provided a plausible mechanistic link between clot structure and thrombosis. We aimed to identify lifestyle, demographic, biochemical, and genetic factors that influence changes in total fibrinogen concentration and clot properties over a 10-year period in 2,010 black South Africans. Clot properties were assessed with turbidimetry and included lag time, slope, maximum absorbance, and clot lysis time. Linear mixed models with restricted maximum likelihood were used to determine whether (1) outcome variables changed over the 10-year period; (2) demographic and lifestyle variables, biochemical variables, and fibrinogen single-nucleotide polymorphisms influenced the change in outcome variables over the 10-year period; and (3) there was an interaction between the exposures and time in predicting the outcomes. A procoagulant risk score was furthermore created, and multinomial logistic regression was used to determine the exposures that were associated with the different risk score categories. In this population setting, female gender, obesity, poor glycemic control, increased low-density lipoprotein cholesterol, and decreased high-density lipoprotein cholesterol contributed to the enhanced progression to prothrombotic clot properties with increasing age. Alcohol consumption on the other hand, offered a protective effect. The above evidence suggest that the appropriate lifestyle changes can improve fibrin clot properties on a population level, decreasing cardiovascular disease risk and thus alleviate the strain on the medical health care system.

Activation of the Catalytic Activity of Thrombin for Fibrin Formation by Ultrasound.

The regulation of enzyme activity is a method to control biological function. We report two systems enabling the ultrasound-induced activation of thrombin, which is vital for secondary hemostasis. First, we designed polyaptamers, which can specifically bind to thrombin, inhibiting its catalytic activity. With ultrasound generating inertial cavitation and therapeutic medical focused ultrasound, the interactions between polyaptamer and enzyme are cleaved, restoring the activity to catalyze the conversion of fibrinogen into fibrin. Second, we used split aptamers conjugated to the surface of gold nanoparticles (AuNPs). In the presence of thrombin, these assemble into an aptamer tertiary structure, induce AuNP aggregation, and deactivate the enzyme. By ultrasonication, the AuNP aggregates reversibly disassemble releasing and activating the enzyme. We envision that this approach will be a blueprint to control the function of other proteins by mechanical stimuli in the sonogenetics field.

A unique group of scabies mite pseudoproteases promotes cutaneous blood coagulation and delays plasmin-induced fibrinolysis.

BACKGROUND: Scabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C-modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin. CONCLUSIONS/SIGNIFICANCE: The SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.

Fibrinogen function achieved through multiple covalent states.

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10-50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.

Atypical antipsychotic clozapine binds fibrinogen and affects fibrin formation.

Clozapine is an atypical antipsychotic used for the treatment of schizophrenia. The prescribed target daily doses may reach 900 mg. Literature studies report a connection between clozapine usage and thrombosis development. Our in vitro study aimed to provide insight into molecular bases of this observation, investigating clozapine binding to fibrinogen, the main plasma protein involved in hemostasis. Fibrinogen/clozapine interaction was confirmed by protein fluorescence quenching, with an affinity constant of 1.7 × 105 M-1. Direct interactions did not affect the structure of fibrinogen, nor fibrinogen melting temperature. Clozapine binding affected fibrin formation by reducing coagulation speed and thickness of fibrin fibers suggesting that in the presence of clozapine, fibrinogen may acquire thrombogenic characteristics. Although no difference in fibrin gel porosity was detected, other factors present in the blood may act synergistically with altered fibrin formation to modify fibrin clot, thus increasing the risk for development of thrombosis in patients on clozapine treatment. ORAC and HORAC assays showed that clozapine reduced free radical-induced oxidation of fibrinogen. All observed effects of clozapine on fibrinogen are dose-dependent, with the effect on fibrin formation being more pronounced.

A novel simultaneous clot-fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in haemorrhagic disorders.

Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0·63 µg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1-deficient (-def) and α2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.

Histopathological effects of modern topical sealants on the liver surface after hepatectomy: an experimental swine study.

The present study aimed to determine the impact of different sealant materials on histopathological changes to the liver surface after liver resection. Thirty-six landrace pigs underwent left anatomical hemihepatectomy and were assigned to a histopathological control group (HPC, n = 9) with no bleeding control, a clinically simulated control group (CSC, n = 9) with no sealant but bipolar cauterization and oversewing of the liver surface, and two treatment groups (n = 9 each) with a collagen-based sealant (CBS) or a fibrinogen-based sealant (FBS) on resection surface. After postoperative day 6, tissue samples were histologically examined. There were no significant differences in preoperative parameters between the groups. Fibrin production was higher in sealant groups compared with the HPC and CSC groups (both p < 0.001). Hepatocellular regeneration in sealant groups was higher than in both control groups. A significantly higher regeneration was seen in the FBS group. Use of sealants increased the degree of fibrin exudation at the resection plane. Increased hepatocellular necrosis was seen in the CBS group compared with the FBS group. The posthepatectomy hepatocellular regeneration rate was higher in the FBS group compared with the CBS group. Randomized studies are needed to assess the impact of sealants on posthepatectomy liver regeneration in the clinical setting.

Early morphological changes in tissues when replacing abdominal wall defects by bacterial nanocellulose in experimental trials.

Experimental trials were done on five dogs to explore if an anterior abdominal wall defect could be repaired using wet (99.9%), compact BNC membranes produced by the Мedusomyces gisevii Sa-12 symbiotic culture. The abdominal wall defect was simulated by middle-midline laparotomy, and a BNC membrane was then fixed to open aponeurotic edges with blanket suture (Prolene 4-0, Ethicon). A comparative study was also done to reinforce the aponeurotic defect with both the BNC membrane and polypropylene mesh (PPM) (Ultrapro, Ethicon). The materials were harvested at 14 and 60 days postoperative to visually evaluate their location in the abdominal tissues and evaluate the presence of BNC and PPM adhesions to the intestinal loops, followed by histologic examination of the tissue response to these prosthetics. The BNC exhibited good fixation to the anterior abdominal wall to form on the 14th day a capsule of loose fibrin around the BNC. Active reparative processes were observed at the BNC site at 60 days post-surgery to generate new, stable connective-tissue elements (macrophages, giant cells, fibroblasts, fibrin) and neocapillaries. Negligible intraperitoneal adhesions were detected between the BNC and the intestinal loops as compared to the case of PPM. There were no suppurative complications throughout the postsurgical period. We noticed on the 60th day after the BNC placement that collagenous elements and new capillary vessels were actively formed in the abdominal wall tissues, generating a dense postoperative cicatrix whose intraperitoneal adhesions to the intestinal loops were insignificant compared to the PPM graft.

An Evaluation of the Activated Partial Thromboplastin Time Waveform.

The activated partial thromboplastin time (APTT) waveform includes several parameters that are related to various underlying diseases. The APTT waveform was examined in various diseases. Regarding the pattern of APTT waveform, a biphasic pattern of the first or second derivative curve (DC) was observed in patients with hemophilia and patients positive for antiphospholipid (aPL) antibodies or coagulation factor VIII (FVIII) inhibitors. The time of the first and second DC and fibrin formation at 1/2 height were prolonged in patients with hemophilia, patients with inhibitors, patients positive for aPL, patients treated with anti-Xa agents, and patients with disseminated intravascular coagulation (DIC). These values all tended to decrease in pregnant women (at 28-36 weeks' gestation). The height of the second derivative peak 1 was significantly lower in patients with hemophilia, patients with FVIII inhibitors, patients positive for aPL, patients treated with anti-Xa agents, and patients with DIC; these values tended to be significantly higher in pregnant women. The height of the first DC was significantly lower in patients who were positive for FVIII inhibitors and was significantly higher in patients treated with anti-Xa agents and pregnant women. The height of the first and second DC was useful for the analysis of hemophilia, FVIII inhibitor, and aPL.

Recombinant factor VIIa addition to haemophilic blood perfused over collagen/tissue factor can sufficiently bypass the factor IXa/VIIIa defect to rescue fibrin generation.

INTRODUCTION: Factor VIII (FVIII) or factor IX (FIX)-deficient haemophilic patients display deficits in platelet and fibrin deposition under flow detectable in microfluidics. Compared to fibrin generation, decreased platelet deposition in haemophilic blood flow is more easily rescued with recombinant factor VIIa (rFVIIa), whereas rFVIIa requires FXIIa participation to generate fibrin when tissue factor (TF) is absent. AIMS: Perfusion of haemophilic whole blood (WB) over collagen/TF surfaces was used to determine whether rFVIIa/TF was sufficient to bypass poor FIXa/FVIIIa function in blood from patients with haemophilia A and B. METHODS: Whole blood treated with high-dose corn trypsin inhibitor (40 µg mL-1 ) from seven healthy donors and 10 patients was perfused over fibrillar collagen presenting low or high TF (TFlow or TFhigh ) at wall shear rate of 100 s-1 . RESULTS: With WB from healthy controls, platelet deposition and fibrin accumulation increased as TF increased. Factor-deficient WB (1-3% of normal) displayed striking deficits in platelet deposition and fibrin formation at either TFlow or TFhigh . In contrast, mildly factor-deficient WB (14-32%) supported fibrin formation under flow on TFhigh /collagen. With either TFlow or TFhigh , exogenously added rFVIIa (20 nm) increased platelet deposition and fibrin accumulation in WB from factor-deficient patients (1-3% of normal) to levels commensurate with untreated healthy WB. CONCLUSION: The absence of FIXa/FVIIIa in patients with severe haemophilia results in deficits in fibrin formation that cannot be rescued by wall-derived TF ex vivo. The effects of rFVIIa on platelet adhesion and rFVIIa/TF can act together to reinforce thrombin generation, platelet deposition and fibrin formation under flow.

Tissue Plasminogen Activator as an Antiangiogenic Agent in Experimental Laser-Induced Choroidal Neovascularization in Mice.

PURPOSE: We investigate the antiangiogenic efficacy of tissue plasminogen activator (tPA) on experimental laser-induced choroidal neovascularization (CNV) in mice. METHODS: After CNV was induced by laser photocoagulation in 92 C57BL/6J wild-type mice, tPA (4 or 40 international units [IU]/µl) or PBS was injected intravitreally immediately after laser injury. Fluorescein angiography was performed on day 7 to grade CNV leakage. The CNV volume was measured by confocal microscopy in eyes enucleated 7 days after laser injury. Immunohistochemical studies were performed 3 days after laser injury to evaluate fibrin/fibrinogen and CD31 expression. The possible adverse effects of tPA were assessed by electroretinography (ERG) and histology on day 7. RESULTS: Intravitreal administration of tPA significantly suppressed CNV leakage and CNV volume in a dose-dependent manner (P < 0.01). Intravitreal injection of tPA suppressed fibrin/fibrinogen and CD31 expression in laser-induced lesions. Histologic examination and ERG showed no evidence of retinal toxicity in eyes injected with tPA. CONCLUSIONS: Intravitreal injection of tPA suppressed fibrin/fibrinogen expression and laser-induced CNV. The current results suggested that tPA may be a potential therapeutic adjuvant for treating CNV.

The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-ß) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-ß cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Polyphosphate as a haemostatic modulator.

Platelets are small anuclear cells that play a central role in haemostasis. Platelets become activated in response to various stimuli triggering release of their granular contents into the surrounding milieu. One of these types of granules, termed dense granules, have been found to contain polyphosphate (polyP) in addition to other inorganic biomolecules, such as serotonin, ADP, ATP, PPi. Individuals deficient in dense granules exhibit bleeding tendencies, emphasizing their importance in haemostasis. Platelet polyP is of a relatively defined size, approximately 60-100 phosphate monomers in length. These linear polymers act at various points in the coagulation and fibrinolytic systems thereby modulating the haemostatic response. Due to its highly anionic nature, polyP lends itself to being a natural activator of the contact system. The contact system functions in multiple pathways including coagulation, fibrinolysis, inflammation and complement. Activation of the contact system accelerates thrombin generation, the terminal enzyme in the coagulation cascade. PolyP also modulates factors further downstream in the coagulation cascade to augment thrombin generation. The net effect is increased fibrin formation and platelet activation resulting in faster clot formation. PolyP is incorporated into the forming clot thereby modifying the structure of the resulting fibrin network and its susceptibility to degradation by certain plasminogen activators. In conclusion, release of platelet polyP at the site of injury may facilitate clot formation and augment clot stability thereby promoting wound healing.

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of µ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.

Coagulation activation in sickle cell trait: an exploratory study.

Recent epidemiologic data suggest that sickle cell trait (HbAS; AS) is a risk factor for venous thromboembolism. We conducted an exploratory study of healthy subjects with AS under baseline conditions to determine whether a chronic basal hyperactivation of coagulation exists, and if so, what mechanism(s) contribute to this state. Eighteen healthy AS individuals were compared to 22 African-American controls with a normal haemoglobin profile (HbAA; AA) and 17 patients with sickle cell disease (HbSS; SS). Plasma thrombin-antithrombin complexes and D-dimer levels were elevated in AS relative to AA patients (P = 0·0385 and P = 0·017, respectively), and as expected, were much higher in SSversusAA (P < 0·0001 for both). Thrombin generation in platelet poor plasma was indistinguishable between AA and AS subjects, whereas a paradoxical decrease in endogenous thrombin potential was observed in SS (P ≤ 0·0001). Whole blood tissue factor was elevated in SS compared to AA (P = 0·005), but did not differ between AA and AS. Plasma microparticle tissue factor activity was non-significantly elevated in AS (P = 0·051), but was clearly elevated in SS patients (P = 0·004) when compared to AA controls. Further studies in larger cohorts of subjects with sickle cell trait are needed to confirm the results of this preliminary investigation.

Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity.

The ability of cellular fibronectin, found in the vessel wall in a fibrillar conformation, to regulate platelet functions and trigger thrombus formation remains largely unknown. In this study, we evaluated how parietal cellular fibronectin can modulate platelet responses under flow conditions. A fibrillar network was formed by mechanically stretching immobilised dimeric cellular fibronectin. Perfusion of anticoagulated whole blood over this surface resulted in efficient platelet adhesion and thrombus growth. The initial steps of platelet adhesion and activation, as evidenced by filopodia extension and an increase in intracellular calcium levels (419 ± 29 nmol/l), were dependent on integrins α5ß1 and αIIbß3. Subsequent thrombus growth was mediated by these integrins together with the GPIb-V-IX complex, GPVI and Toll-like receptor 4. The involvement of Toll-like receptor 4 could be conveyed via its binding to the EDA region of cellular fibronectin. Upon thrombus formation, the platelets became procoagulant and generated fibrin as revealed by video-microscopy. This work provides evidence that fibrillar cellular fibronectin is a strong thrombogenic surface which supports efficient platelet adhesion, activation, aggregation and procoagulant activity through the interplay of a series of receptors including integrins α5ß1 and αIIbß3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.

Laboratory evidence of disseminated intravascular coagulation is associated with a fatal outcome in children with cerebral malaria despite an absence of clinically evident thrombosis or bleeding.

BACKGROUND: A procoagulant state is implicated in cerebral malaria (CM) pathogenesis, but whether disseminated intravascular coagulation (DIC) is present or associated with a fatal outcome is unclear. OBJECTIVES: To determine the frequency of overt DIC, according to ISTH criteria, in children with fatal and non-fatal CM. METHODS/PATIENTS: Malawian children were recruited into a prospective cohort study in the following diagnostic groups: retinopathy-positive CM (n = 140), retinopathy-negative CM (n = 36), non-malarial coma (n = 14), uncomplicated malaria (UM), (n = 91), mild non-malarial febrile illness (n = 85), and healthy controls (n = 36). Assays in the ISTH DIC criteria were performed, and three fibrin-related markers, i.e. protein C, antithrombin, and soluble thrombomodulin, were measured. RESULTS AND CONCLUSIONS: Data enabling assignment of the presence or absence of 'overt DIC' were available for 98 of 140 children with retinopathy-positive CM. Overt DIC was present in 19 (19%), and was associated with a fatal outcome (odds ratio [OR] 3.068; 95% confidence interval [CI] 1.085-8.609; P = 0.035]. The levels of the three fibrin-related markers and soluble thrombomodulin were higher in CM patients than in UM patients (all P < 0.001). The mean fibrin degradation product level was higher in fatal CM patients (71.3 µg mL(-1) [95% CI 49.0-93.6]) than in non-fatal CM patients (48.0 µg mL(-1) [95% CI 37.7-58.2]; P = 0.032), but, in multivariate logistic regression, thrombomodulin was the only coagulation-related marker that was independently associated with a fatal outcome (OR 1.084 for each ng mL(-1) increase [95% CI 1.017-1.156]; P = 0.014). Despite these laboratory derangements, no child in the study had clinically evident bleeding or thrombosis. An overt DIC score and high thrombomodulin levels are associated with a fatal outcome in CM, but infrequently indicate a consumptive coagulopathy.

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome.

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6(-/-) mice after vascular injury. HPS6(-/-) platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5'-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6(-/-) mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype.