Platelet protein synthesis, regulation, and post-translational modifications: mechanics and function.

Dogma had been firmly entrenched in the minds of the scientific community that the anucleate mammalian platelet was incapable of protein biosynthesis since their identification in the late 1880s. These beliefs were not challenged until the 1960s when several reports demonstrated that platelets possessed the capacity to biosynthesize proteins. Even then, many still dismissed the synthesis as trivial and unimportant for at least another two decades. Research in the field expanded after the 1980s and numerous reports have since been published that now clearly demonstrate the potential significance of platelet protein synthesis under normal, pathological, and activating conditions. It is now clear that the platelet proteome is not a static entity but can be altered slowly or rapidly in response to external signals to support physiological requirements to maintain hemostasis and other biological processes. All the necessary biological components to support protein synthesis have been identified in platelets along with post-transcriptional processing of mRNAs, regulators of translation, and post-translational modifications such as glycosylation. The last comprehensive review of the subject appeared in 2009 and much work has been conducted since that time. The current review of the field will briefly incorporate the information covered in earlier reviews and then bring the reader up to date with more recent findings.

Cardiovascular serotonergic system: Evolution, receptors, transporter, and function.

The serotonergic system, serotonin (5HT), serotonin transporter (SERT), and serotonin receptors (5HT-x), is an evolutionarily ancient system that has clear physiological advantages to all life forms from bacteria to humans. This review focuses on the role of platelet/plasma serotonin and the cardiovascular system with minor references to its significant neurotransmitter function. Platelets transport and store virtually all plasma serotonin in dense granules. Stored serotonin is released from activated platelets and can bind to serotonin receptors on platelets and cellular components of the vascular wall to augment aggregation and induce vasoconstriction or vasodilation. The vascular endothelium is critical to the maintenance of cardiovascular homeostasis. While there are numerous ligands, neurological components, and baroreceptors that effect vascular tone it is proposed that serotonin and nitric oxide (an endothelium relaxing factor) are major players in the regulation of systemic blood pressure. Signals not fully defined, to date, that direct serotonin binding to one of the 15 identified 5HT receptors versus the transporter, and the role platelet/plasma serotonin plays in regulating hypertension within the cardiovascular system remain important issues to better understand many diseases and to develop new drugs. Also, expanded research of these pathways in lower life-forms may serve as important model systems to further our understanding of the evolution and mechanisms of action of serotonin.

The role of the red blood cell and platelet in the evolution of mammalian and avian endothermy.

This review presents evidence to support the hypothesis that the reduced O2 during the Permian/Triassic period was the impetus for the evolutionary selection of endothermic animals. The evolution of smaller red blood cells with greater surface areas along with increased: capillary density, capillary surface area, hematocrits, blood pressure, blood flow rates, and shear rates were critical for efficient gas exchange in endothermy. The evolution of the four-chambered mammalian/avian heart allowed for low pulmonary and high systemic blood pressure. It is proposed that hypoxia-induced angiogenesis led to increased vascularization in endothermic animals. The increased blood pressure, flow rates, and shear forces likely required changes in hemostatic mechanisms that were met in mammals by the evolution of anucleate platelets. The evolution of mammals and birds occurred in a parallel fashion with further genetic changes to anucleate RBCs/platelets occurring in mammals. Although it is possible that the evolution of endothermy in birds and mammals occurred as two independent events, it is more likely that a common ancestor developed genetic mutations that laid down the road map for parallel alterations of their cardiovascular system in response to environmental pressures. Model systems to support the proposed changes from ectotherm to endotherm were developed from published data. The evolutionary development of endothermy occurred over millions of years with a continuum of genetic alterations that involved skeletal, soft tissue, cardiovascular macrochanges along with numerous molecular alterations. Genetic signals and potential regulators for the evolutionary changes of endothermic blood cells from their bipotential stem cells are also proposed.

Circular RNA (circRNA) was an important bridge in the switch from the RNA world to the DNA world.

The concept that life on Earth began as an RNA world has been built upon extensive experimentation demonstrating that many of the building blocks required for living cells could be synthesized in the laboratory under conditions approximating our primordial world. Many of the building blocks for life have also been found in meteorites indicating that meteors may have been a source for these molecules, or more likely, that they represent the chemical library present in most/all bodies in the universe after the big bang. Perhaps the most important support for the concept comes from the fact that some RNA species possess catalytic activity, ribozymes, and that RNA could be reverse transcribe to DNA. The thrust of numerous papers on this topic has been to explore how the available molecules on Earth, at its birth, gave rise to life as we know it today. This paper focuses more on a reverse view of the topic. The "how" molecular building blocks were synthesized is not addressed nor how the "first" RNA molecules were synthesized. We can clearly speculate on the variable environmental conditions and chemistry available on Earth billions of years ago. However, we can never truly replicate the changing conditions or know the chemical composition of Earth at the beginning of time. We can, however, confirm that over millions, perhaps billions of years the basic building blocks for life accumulated sufficiently to initiate evolution to an RNA world followed by our RNA/DNA world. Here we are attempting to take the information from our current knowledge of biology and by inference and extrapolation work backward to hypothesize biological events in the march forward from RNA to DNA. It is proposed that the primordial replicating RNA cell, the ribocyte, evolved from liposomes encompassing required reactants and products for "life" and that ribonucleopeptide complexes formed membrane pores to support bidirectional ion and molecular transport to maintain biological functions and osmolarity. Circular RNA, circRNA, is proposed as a critical stable RNA molecule that served as the genetic precursor for the switch to DNA and the replication of circRNA by a rolling circle mechanism gave rise to the RNA complexity required for the genetic functions of the cell. The replicating ribocyte would have required protein synthesis as well as RNA replication and a model for non-coded and primordial coded protein synthesis is proposed. Finally, the switch from the RNA to the DNA world would have involved the synthesis of an RNA:DNA hybrid prior to the formation of dsDNA. If the hybrid was a circular molecule that ultimately yielded a circular dsDNA molecule, it could predict that the primordial DNA cell would evolve into a bacterial cell with a single circular chromosome. One would hope that continued speculation of the origin of life will spur new directions of research that may never fully answer the questions of the past but add to our ability to regulate potentially harmful biological events in the present and in the future.

Intracellular matrix metalloproteinase-2 (MMP-2) regulates human platelet activation via hydrolysis of talin.

Matrix metalloproteinase (MMP) activity is generally associated with normal or pathological extracellular processes such as tissue remodelling in growth and development or in tumor metastasis and angiogenesis. Platelets contain at least three MMPs, 1, 2 and 9 that have been reported to stimulate or inhibit agonist-induced platelet aggregation via extracellular signals. The non-selective Zn+2 chelating MMP inhibitor, 1,10-phenanthroline, and the serine protease inhibitor, AEBSF, were found to inhibit all tested agonist-induced platelet aggregation reactions. In vitro analysis demonstrated that 1,10-phenanthroline completely inhibited MMP-1,2,and 9 but had little to no effect on calpain activity while the converse was true with AEBSF. We now demonstrate that MMP-2 functions intracellularly to regulate agonist-induced platelet aggregations via the hydrolytic activation of talin, the presumed final activating factor of glycoprotein (GP)IIb/IIIa integrin (the inside-out signal). Once activated GPIIb/IIIa binds the dimeric fibrinogen molecule required for platelet aggregation. The active intracellular MMP-2 molecule is complexed with JAK 2/STAT 3, as demonstrated by the fact that all three proteins are co-immunoprecipitated with either anti-JAK 2, or anti-STAT 3 antibodies and by immunofluorescence studies. The MMP-2 platelet activation pathway can be synergistically inhibited with the non-selective MMP inhibitor, 1,10-phenanthroline, plus a JAK 2 inhibitor. This activation pathway is distinct from the previously reported calpain-talin activating pathway. The identification of a new central pathway for platelet aggregation presents new potential targets for drug regulation and furthers our understanding of the complexity of platelet activation mechanisms.

Iron levels found in hemochromatosis patients inhibit γ-thrombin-induced platelet aggregation.

Hemochromatosis is a common genetic disorder of iron metabolism. The increase in systemic iron associated with hemochromatosis can negatively affect every system in the body, with potentially fatal implications. Little is known about the effects of iron overload on hemostasis and platelet activation. While performing platelet aggregation studies on hemochromatotic blood samples, it was discovered that the increased serum iron levels associated with this disease almost completely inhibited γ-thrombin-induced platelet aggregation. Further studies were conducted with samples derived from control and hemochromatotic individuals to determine the effects of iron on both α- and γ-thrombin-induced platelet aggregations. It was found that γ-thrombin-induced platelet aggregations were strongly inhibited by the direct binding of iron to these enzymes. α-Thrombin activates platelets at multiple receptor sites including the protease-activated receptor-1 (PAR-1), and at high concentrations, at protease-activated receptor-4 (PAR-4), while γ-thrombin selectively activates platelets at PAR-4. γ-Thrombin activity was significantly more sensitive to toxic levels of iron than α-thrombin in the activation of the PAR receptors. Also, the hydrolysis of a synthetic peptide by α-thrombin was unaffected by iron while γ-thrombin was strongly inhibited. These results indicate that the iron-thrombin complex strongly regulates the activity of γ-thrombin while has little or no effect on α-thrombin-induced platelet aggregation or hydrolytic activity. It is unknown, at this time, what specific clinical implications iron inhibition of γ-thrombin may have, but it is very possible that other conditions caused by hemochromatosis could be exacerbated by the inability of platelets to aggregate normally.

Acinetobacter sp. HM746599 isolated from leatherback turtle blood.

A newly described bacterial isolate, Acinetobacter sp. HM746599, has been obtained from leatherback sea turtle hatchling blood. The implication is that the hatchling was infected during development in the egg, which is substantiated by other studies to be reported by us in the future. The 16S rRNA gene sequence of the bacterium (GenBank accession number: HM746599) showed the greatest similarity to the identified species, Acinetobacter beijerinckii (97.6-99.78%) and Acinetobacter venetianus (99.78%). Acinetobacter sp. HM746599 are gram-negative, rod-shaped coccobacilli and are hemolytic/cytotoxic to human and sea turtle red blood cells (RBCs). Hemolysis is not the result of any detectable soluble toxin. Acinetobacter beijerinckii and A. venetianus hemolyze sheep RBCs while Acinetobacter sp. HM746599 does not, and unlike A. venetianus, the growth of Acinetobacter sp. HM746599 and A. beijerinckii is not supported by l-arginine. Many Acinetobacter species, especially hemolytic ones, are pathogenic to immunologically compromised humans and it is possible that, in addition to sea turtles, this bacterium might also be a danger to susceptible humans who handle infected hatchlings. The bacteria are available from CCUG (Culture Collection, University Gothenburg, Göteborg, Sweden) and from NRRL (Agricultural Research Service Culture Collection, Peoria, IL).

Inhibition of gamma-thrombin-induced human platelet aggregation by histone H1subtypes and H1.3 fragments.

Human platelets are differentially activated by varying concentrations of alpha-thrombin or by beta- and gamma-thrombin via three thrombin receptors, PAR-1, PAR-4 and GPIbalpha.It is likely that the development of a normal or abnormal hemostatic event in humans is dictated, in part, by the selective activation of these receptors. The ability to differentially inhibit these thrombin receptors could, therefore, have clinical significance. We have previously demonstrated that histone H1 selectively inhibits the PAR-4 receptor. In the current study we investigated whether five subtypes of the H1 molecule or fragments of the H1.3 subtype differentially inhibited the PAR-4 receptor. PAR-4 inhibition by all H1 subtypes was saturated at 1 uM with no statistical difference observed with the five H1 subtypes tested. Of the five fragments generated from the H1.3 molecule only one had significant inhibitory activity against PAR-4. The C-terminal fragment, N.1, generated by the proteolysis of the parent molecule by Asp-N endoproteinase (Aeromonas proteolytica) at the single aspartate residue, showed the same level of PAR-4 inhibition as the intact H1.3 at 1 uM concentrations. Removal of two N-terminal amino acids (Asp-Val as determined by MALDI analysis) from the N.1 fragment further enhanced its inhibitory activity. These studies may help to develop specific drugs to differentially inhibit the platelet thrombin receptors.

Cytokine mediated proliferation of cultured sea turtle blood cells: morphologic and functional comparison to human blood cells.

Blood cells from three different sea turtle species were cultured for approximately 3 weeks in nutrient medium supplemented with recombinant human cytokines known to induce terminal maturation of human hematological stem cells. Cultured turtle erythrocytes were translucent, approximately 10x larger than human erythrocytes, contained a single fluorescent inclusion body, contained nuclear epsilon (embryonic) globin proteins, and, absent of organelles while fresh cells contained few, but well defined mitochondria. Cells with basophilic cytoplasm and in all stages of proliferation were observed in cytokine-supplemented cultures and appeared to possess active protein synthesis. Cultured thrombocytes aggregated in response to agonists for at least 8 days, post-collection, contained P-selectin in the nucleus of 6 day cultured cells which appeared to be released after activation with collagen, and after 6 days had no organelles or open canalicular-like system (OCS) while freshly isolated cells demonstrated few, if any organelles but had a well developed OCS. The response of turtle cells to apparently homologous but unnatural human cytokines and the sustained biological properties of thrombocytes identify this suspension culture system as a powerful tool to explore the evolution of cell types and molecular components of hematopoiesis and hemostasis.

Effect of low and high dose melagatran and other antithrombotic drugs on platelet aggregation.

BACKGROUND: Thrombin-induced aggregation of human platelets can be completely inhibited by melagatran, the bioactive form of ximelagatran, an oral direct thrombin inhibitor. METHODS: The potential of melagatran to differentially inhibit alpha- and gamma-thrombins was tested with a synthetic thrombin substrate. Washed human platelets were also employed to determine if melagatran differentially inhibited alpha- and gamma-thrombin-induced platelet aggregation at distinct platelet thrombin receptors. In vitro studies were conducted with washed human platelets to determine thrombin-induced aggregation responses in the presence of varying doses of the anti-thrombotic drugs: melagatran, argatroban, heparin, and hirudin. RESULTS: Melagatran rapidly inhibits the hydrolysis of a thrombin chromogenic substrate within 0-1 min with alpha-, beta- and gamma-thrombin being equally inhibited by high dose melagatran while alpha-thrombin was significantly more sensitive at low doses. The maximum level of melagatran inhibition of alpha- and gamma-thrombin-induced platelet aggregation requires platelets to be pre-incubated with the drug for 10-30 min. Melagatran appears to have no direct effect on the PAR-1 receptor. It does appear to have a direct effect on the GPIbalpha thrombin receptor activity as well as the PAR-4 receptor. Inhibition of platelet aggregation is dose dependent, however, at low melagatran doses (0.01-0.04 nM) platelets aggregate at significantly (P < 0.05) higher levels. The lower the level of thrombin-induced aggregation that was observed with control samples (aggregations from 10% to 39%), corresponded with a higher observed melagatran-induced stimulation with drug-treated platelets. The range of stimulation varies between several hundred percent at approximately 10% aggregation to around 20% at about 20-39% aggregation. Preliminary studies indicate that this in vitro stimulatory effect is abrogated in platelets derived from volunteers who took aspirin (81 mg/day) for 7 days. Three other anti-thrombotic drugs, argatroban, heparin and hirudin, were tested with low drug levels but none were found to consistently stimulate the reaction. CONCLUSIONS: These results indicate that melagatran acts as both a direct thrombin inhibitor and indirectly by some interaction with the platelet membrane. While melagatran has been withdrawn from clinical use, its ability to differentially inhibit gamma-thrombin/PAR-4 versus alpha-thrombin/PAR-1 at low doses may warrant it, or less toxic analogs to be used in the future for as yet unknown disease states involving PAR-4.

Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood.

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.

Comparison of functional aspects of the coagulation cascade in human and sea turtle plasmas.

Functional hemostatic pathways are critical for the survival of all vertebrates and have been evolving for more than 400 million years. The overwhelming majority of studies of hemostasis in vertebrates have focused on mammals with very sparse attention paid to reptiles. There have been virtually no studies of the coagulation pathway in sea turtles whose ancestors date back to the Jurassic period. Sea turtles are often exposed to rapidly altered environmental conditions during diving periods. This may reduce their blood pH during prolonged hypoxic dives. This report demonstrates that five species of turtles possess only one branch of the mammalian coagulation pathway, the extrinsic pathway. Mixing studies of turtle plasmas with human factor-deficient plasmas indicate that the intrinsic pathway factors VIII and IX are present in turtle plasma. These two factors may play a significant role in supporting the extrinsic pathway by feedback loops. The intrinsic factors, XI and XII are not detected which would account for the inability of reagents to induce coagulation via the intrinsic pathway in vitro. The analysis of two turtle factors, factor II (prothrombin) and factor X, demonstrates that they are antigenically/functionally similar to the corresponding human factors. The turtle coagulation pathway responds differentially to both pH and temperature relative to each turtle species and relative to human samples. The coagulation time (prothrombin time) increases as the temperature decreases between 37 and 15 degrees C. The increased time follows a linear relationship, with similar slopes for loggerhead, Kemps ridley and hawksbill turtles as well as for human samples. Leatherback turtle samples show a dramatic nonlinear increased time below 23 degrees C, and green turtle sample responses were similar but less dramatic. All samples also showed increased prothrombin times as the pH decreased from 7.8 to 6.4, except for three turtle species. The prothrombin times decreased, to varying extents, in a linear fashion relative to reduced pH with the rate of change greatest in leatherbacks>green>>loggerhead turtles. All studies were conducted with reagents developed for human samples which would impact on the quantitative results with the turtle samples, but are not likely to alter the qualitative results. These comparative studies of the coagulation pathway in sea turtles and humans could enhance our knowledge of structure/function relationships and evolution of coagulation factors.

Differential activation and inhibition of human platelet thrombin receptors by structurally distinct alpha-, beta- and gamma-thrombin.

The development of drugs to neutralize the action of thrombin has to date focused on the alpha form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active alpha-thrombin which may be further hydrolyzed to beta- and gamma-thrombin. While all three forms of the enzyme retain catalytic activities, only alpha-thrombin is presumed to be physiologically important. The beta- and gamma-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that beta- and gamma-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173-21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. Beta-thrombin, like gamma-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the beta-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For beta-thrombin and gamma-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that alpha-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that alpha-thrombin may be rapidly converted to beta-thrombin and/or gamma-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by alpha-, beta- and gamma-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits alpha-thrombin , is a slightly weaker inhibitor of beta-thrombin and a very weak inhibitor of gamma-thrombin -induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, alpha(1)-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only beta- and gamma-thrombin , primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like beta- and gamma-thrombin , activates PAR-4 and is also inactive with TRAP-4 desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to gamma-thrombin than to alpha-thrombin. The analysis of the crystalline structures of alpha-, gamma-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.