Kallikrein augments the anticoagulant function of the protein C system in thrombin generation

Background Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project. Methods Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG. Results Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 x 10(-8)). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A-IV, into plasma dose-dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP-TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor. Conclusions Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis

New insight into antiphospholipid syndrome: antibodies to \u3b22glycoprotein I-domain 5 fail to induce thrombi in rats

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of \u3b22glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analysed the in vivo pro-coagulant effect of two groups of 5 serum IgG each reacting selectively with domain 1 or domain 5 in LPS-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy and vascular deposition of \u3b22glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-\u3b22glycoproteinI antibodies served as controls. All the anti-domain 1 positive IgG exhibited potent pro-coagulant activity while the anti-domain 5 positive and the negative control IgG failed to promote blood clot and vessels occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound \u3b22glycoprotein I while able to interact with fluid-phase \u3b22glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit in vivo thrombogenic effect and the interaction of these antibodies with circulating \u3b22glycoproteinI suggest their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining really at risk patients for more appropriate treatments to avoid recurrences and disability

The effects of signal transducer and activator of transcription three mutations on human platelets

Involvement of signal transducer and activator of transcription 3 (STAT3) in inflammation is well known. Recently, a role for STAT3 in platelet activation and platelet production has been suggested. Platelets exhibit important immune functions and engagement of STAT3 in platelet physiology may link inflammation and hemostasis. This study investigated the effects of STAT3 loss-of-function mutations and single nucleotide polymorphisms (SNPs) in STAT3 on glycoprotein VI (GPVI)-mediated platelet activation and platelet numbers in humans. Two cohorts were studied. The first cohort concerned patients with STAT3 loss-of-function mutations. Platelet numbers were investigated in eight patients and GPVI-mediated platelet activation was functionally tested in four patients. Additional experiments were performed to investigate underlying mechanisms. The second cohort concerned 334 healthy volunteers and investigated the consequences of SNPs in STAT3 on GPVI-mediated platelet activation and platelet numbers. Platelet activation was lower in STAT3 loss-of-function patients at baseline and after stimulation of the GPVI receptor, reflected by decreased P-selectin expression. This was independent of gene transcription. Blockade of the adenosine di-phosphate (ADP) pathway resulted in a further decrease of P-selectin expression, particularly in STAT3 loss-of-function patients. In contrast, the SNPs in STAT3 did not influence GPVI-mediated platelet activation. Also, platelet numbers were not affected by STAT3 loss-of-function mutations, nor was there an association with the SNPs. In conclusion, STAT3 signaling does not seem to play a major role in thrombopoiesis. We confirm that STAT3 is involved in GPVI-mediated platelet activation in humans, independent of gene transcription. GPVI-mediated platelet activation is highly dependent on secondary ADP release. Our findings suggest that STAT3 modulation may affect inflammation, hemostasis, and their interaction.</p

Synergistic Effects of Hypofibrinolysis and Genetic and Acquired Risk Factors on the Risk of a First Venous Thrombosis

Frits Rosendaal and colleagues show that the combination of hypofibrinolysis with oral contraceptive use, immobilization, or factor V Leiden results in a risk of venous thrombosis that exceeds the sum of the individual risks

Haemostatic changes in urogenital schistosomiasis haematobium: A case-control study in Gabonese schoolchildren

In many tropical areas schistosomiasis is a major health problem causing hepatosplenic, intestinal or urogenital complaints. Hepatosplenic schistosomiasis mansoni is also characterized by blood coagulation abnormalities. Liver pathology plays a role in the development of haemostatic changes and the parasitic infection may directly affect coagulation. However, these contributing factors cannot be studied separately in hepatosplenic schistosomiasis infections. This pilot study provides insight in haemostatic changes in urinary schistosomiasis by studying coagulation parameters in schistosomiasis haematobium-infected Gabonese schoolchildren. Selection on urinary schistosomiasis patients without hepatosplenic complaints allows for the investigation of the direct effects of the parasite on haemostasis. Levels of von Willebrand Factor (VWF) antigen, active VWF and osteoprotegerin were elevated, indicating inflammation-mediated endothelial activation. In contrast to hepatosplenic schistosomiasis, thrombin-antithrombin complex and D-dimer levels were not affected. Despite its small sample size, this study clearly indicates that Schistosoma haematobium directly alters the activation status of the endothelium, without initiation of coagulation

Stress-induced expression is enriched for evolutionarily young genes in diverse budding yeasts

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology

Cleavage of von Willebrand Factor by Granzyme M Destroys Its Factor VIII Binding Capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF