Serum contents of immunoreactive pancreatic secretory trypsin inhibitor and seminal plasma acrosin-trypsin inhibitor in polytraumatized patients

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RGD-containing Peptides Inhibit Fibrinogen Binding to Platelet αIIbβ3 by Inducing an Allosteric Change in the Amino-terminal Portion of αIIb

To determine the molecular basis for the insensitivity of rat alpha(IIb)beta(3) to inhibition by RGD-containing peptides, hybrids of human and rat alpha(IIb)beta(3) and chimeras of alpha(IIb)beta(3) in which alpha(IIb) was composed of portions of human and rat alpha(IIb) were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of alpha(IIb)beta(3) was measured. These measurements indicated that sequences regulating the sensitivity of alpha(IIb)beta(3) to RGDS are located in the seven amino-terminal repeats of alpha(IIb). Moreover, replacing the first three or four (but not the first two) repeats of rat alpha(IIb) with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human alpha(IIb) with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing alpha(IIb)beta(3) regardless whether the alpha(IIb) in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of alpha(IIb)beta(3) to RGD-containing peptides are located in the third and fourth amino-terminal repeats of alpha(IIb). Because RGDS binds to both human and rat alpha(IIb)beta(3), the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding

Relative contributions of stromal interaction molecule 1 and CalDAG-GEFI to calcium-dependent platelet activation and thrombosis: STIM1 and CalDAG-GEFI in platelet activation

Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store-operated calcium entry (SOCE) in platelets. We previously reported the Ca2+-sensing guanine nucleotide exchange factor CalDAG-GEFI as critical molecule in Ca2+ signaling in platelets

Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts

Background: The extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 59-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites. Principal Findings: In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in singlestranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine’s presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 39 end of ten and twenty base oligomers. GNC rich regions in dsDNA are known to cause a decrease in the enzyme’s nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these GNC rich regions. Thus the presence of a guanine tract per se impairs the enzyme’s functionality, possibly due to the tract’s bulky nature and preventing efficient progression through the active site. Conclusions: This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

© 2016 by The American Society of Hematology. Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk

Platelet Factor 4 Activity against P. falciparum and Its Translation to Nonpeptidic Mimics as Antimalarials

SummaryPlasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential

Association of homocysteine and methylene tetrahydrofolate reductase (MTHFR C677T) gene polymorphism with coronary artery disease (CAD) in the population of North India

The implications of the methylene tetrahydrofolate reductase (MTHFR) gene and the level of homocysteine in the pathogenesis of coronary artery disease (CAD) have been extensively studied in various ethnic groups. Our aim was to discover the association of MTHFR (C677T) polymorphism and homocysteine level with CAD in north Indian subjects. The study group consisted of 329 angiographically proven CAD patients, and 331 age and sex matched healthy individuals as controls. MTHFR (C677T) gene polymorphism was detected based on the polymerase chain reaction and restriction digestion with HinfI. Total homocysteine plasma concentration was measured using immunoassay. T allele frequency was found to be significantly higher in patients than in the control group. We found significantly elevated levels of mean homocysteine in the patient group when compared to the control group (p = 0.00). Traditional risk factors such as diabetes, hypertension, smoking habits, a positive family history and lipid profiles (triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol), were found significantly associated through univariate analysis. Furthermore, multivariable logistics regression analysis revealed that CAD is significantly and variably associated with diabetes, hypertension, smoking, triglycerides and HDL-cholesterol. Our findings showed that MTHFR C677T polymorphism and homocysteine levels were associated with coronary artery disease in the selected population

Fine Tuning of Globin Gene Expression by DNA Methylation

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the γ(A) and γ(G) genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control γ(A) methylation during development. DNA methylation causes a 20-fold repression of γ(A) both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress γ gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of γ-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate γ-globin expression in patients with β-hemoglobinopathies