Anticoagulants and inhibitors of platelet aggregation derived from leeches

Increased life expectancy is associated with aging populations in the developed countries, and we can expect an increased incidence of cardiovascular and inflammatory diseases and cancers. A priority for medical research is to reduce such morbidity. Leeches have been demonstrated to be a useful source of drugs to treat cardiovascular diseases, as they have evolved highly specific mechanisms to feed on their hosts by blocking blood coagulation. Powerful molecules acting at different points in the coagulation cascade or in the inhibition of platelet aggregation have been purified from these animals. Moreover, clinical trials confirm their potential to treat cardiovascular diseases.     

Guanylate cyclase from human blood platelets

Human blood platelets were disrupted by ultrasonication, and the guanylate cyclase activity was determined in the 105,000 g supernatant. The guanylate cyclase preparation obtained in the absence of dithiothreitol (DTT) was characterized by a nonlinear dynamics of cGMP synthesis during incubation at 37 degrees C. The use of 0.2 mM DTT during platelet ultrasonication stabilized the guanylate cyclase reaction and did not influence the enzyme activity. With a rise in DTT concentration up to 2 mM the guanylate cyclase activity diminished. Sodium nitroprusside stimulated the enzyme; this effect was enhanced in the presence of DTT. The maximum guanylate cyclase activity was revealed at 4 mM Mn2+ or Mg2+ and with 1 mM GTP. In the presence of Mn2+ the enzyme activity was higher than with Mg2+. The apparent Km values for GTP in the presence of 4 mM Mn2+ and Mg2+ was 30 and 200 microM, respectively. At GTP/cation ratio of 1:4 the Km values for Mn2+ and Mg2+ were nearly the same (249 and 208 microM, respectively). It was assumed that besides being involved in the formation of the GTP-substrate complex, Mn2+ exerts a strong influence on guanylate cyclase by oxidizing the SH-groups of the enzyme.     

Soluble factors differ in platelets derived from separate niches: a pilot study comparing the secretome of peripheral blood and bone marrow platelets

Background aimsPlatelet-rich plasma (PRP) and bone marrow aspirate are commonly used in orthobiologics for their anti-inflammatory, anabolic/regenerative and immunomodulatory characteristics via platelet degranulation and cell secretions. Although platelets are derived from megakaryocytes in the bone marrow, no attention has been paid to the potential benefits of bone marrow platelets and whether their contents differ from aging platelets in peripheral blood.MethodsIn the present study, leukocyte-poor peripheral blood-derived platelets in plasma (LPP) and leukocyte-poor bone marrow platelets in plasma (BMP) were prepared from six donors, activated with calcium chloride, incubated and sampled at day 0, day 3 and day 6. LPP and BMP are platelet preparations intended to evaluate the respective platelet secretomes but are not classified as conventional PRPs, as they are not concentrated to the extent necessary to meet the qualifying criteria. At each time point, 15 growth and immunomodulatory factors were quantitated in LPP and BMP: platelet-derived growth factor AA, basic fibroblast growth factor/fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, macrophage colony-stimulating factor, stem cell factor, vascular endothelial growth factor, tumor necrosis factor alpha, IL-1β, interferon gamma, IL-4, IL-10, IL-1 receptor antagonist protein, IL-12p40 and arginase-1.ResultsThe results illustrate that platelets derived from bone marrow have a unique secretome profile compared with those derived from peripheral blood, with significant differences in anti-inflammatory cytokines, which are associated with monocyte polarization.ConclusionsUltimately, bone marrow-derived platelets may be useful as a stand-alone orthobiologic or as an effective adjuvant to autologous cell therapies where anti-inflammatory and anabolic processes are desired, especially with respect to monocyte function.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Glutamate uptake in blood platelets from epileptic patients

Glutamate, the major excitatory amino acid neurotransmitter, is involved in epileptogenesis and initiation and spread of seizures. We studied glutamate uptake into blood platelets from patients with distinct epileptic syndromes: included were 20 patients with temporal lobe epilepsy and hippocampal sclerosis (TLE+HS), 20 with juvenile myoclonus epilepsy (JME) and 20 healthy volunteers matched for age and sex. The affinity of glutamate for the transporters was highest in patients with TLE+HS, but the maximal velocity of transport was highest in controls. There were no differences in the plasma levels of glutamate. Carbamazepine (CBZ), valproate (VPA) and lamotrigine (LTG) did not affect the uptake in vitro. The alterations observed in the uptake of glutamate in TLE+HS patients may reflect an up-regulated uptake of glutamate in the brain.     

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].     

Adenosine and blood platelets

Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A₂ subtypes (A_2A or A_2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A₂ subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A₂ adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.     

Activation time of blood platelets

Summary Blood platelets react rapidly in their hemostatic function. Determination of the reaction or activation time of individual platelets is difficult because it requires that physical and/or chemical effectors of activation are nonlimiting. Analysis of experimental conditions shows that the best estimate of mean activation time comes fromin vivo measurements. Thus, the constancy of height-to-length ratio of a growing thrombus, and its change with flow rate, provide additional evidence for the activation time hypothesis.Dedicated with admiration and affection to the memory of Walther Wilbrandt, a very dear friend.     

Met-enkephalin immunoreactivity in blood platelets

Picogram amounts (50–150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin in IR) were detected by radioimmunoassay in human, rat and rabbit platelets. Characterization of this material by thin-layer chromatography, gel filtration chromatography and high-pressure liquid chromatography indicated that it behaves identically with synthetic met-enkephalin. No high molecular weight met-enkephalin IR could be detected in the platelet extracts, even after trypsin hydrolysis, using two antisera which are able to recognize some of the putative met-enkephalin precursors present in the adrenal gland or striatum. $\text{In vitro}$, thrombin released platelet met-enkephalin in IR concomitantly with 5-hydroxytryptamine (5-HT), suggesting a common subcellular localization, i.e. the 5-HT storing organelles, for met-enkephalin IR and the amine. $\text{In vivo}$, platelet met-enkephalin IR in the Sprague-Dawley rat was affected neither by adrenalectomy nor by hypophysectomy. Thirteen- and 18-week-old spontaneous hypertensive rats (SHR) had lower platelet concentrations of met-enkephalin in IR than age matched normotensive Wistar-Kyoto rats.